Sx^t^

UNITED STATES DEPARTMENT OF THE INTERIOR

Stewart L. Udall, Secretary

James K. Carr, Under Secretary

Frank P. Briggs, Assistant Secretary for Fish and Wildlife

FISH AND WILDLIFE SERVICE, Clarence F. Pautzke, Commissioner

Bureau of Commercial Fisheries, Donald L. McKernan, Director

THE AMERICAN OYSTER

Crassostrea virginica Gmelin

By Paul S. Galtsoff

Fishery Bulletin of the Fish and Wildlife Service

VOLUME 64

UNITED STATES GOVERNMENT PRINTING OFFICE • WASHINGTON . 1964

For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C., 20402 - Price S2.75 (Paper Cover)

CONTENTS

Chapter Page

Preface Ill

I. Taxonomy 1

II. Morphology and structure of shell 16

III. The ligament 48

IV. General anatomy 65

V. The mantle 74

VI. The labial palps Ill

VII. The gills 121

VIII. The adductor muscle 152

IX. Transport of water by the gills and respiration 185

X. The organs of digestion and food of the oyster 219

XI. The circulatory system and blood 239

XII. The excretory system 271

XIII. The nervous system 281

XIV. Organs of reproduction : 297

XV. Eggs, sperm, fertilization, and cleavage 324

XVI. Larval development and metamorphosis 355

XVII. Chemical composition 381

XVIII. Environmental factors affecting oyster populations 397

Subject and author index 457

THE AMERICAN OYSTER CRASSOSTREA VIRGINICA GMELIN

By Paul S. Galtsoff, Fishery Biologist
Bureau of Commercial Fisheries

CHAPTER I
TAXONOMY

Page

Taxonomic characters - ..- 4

Shell 4

Anatomy.- 4

Sex and spawning. 4

Habitat 5

Larval shell (Prodissoconch). 6

The genera of living oysters 6

Genus Ogtrea 6

Genus Crassostrea. 7

Genus Pyaiodonie 7

Bibliography... 14

The family Ostreidae consists of a large number
of edible and nonedible oysters. Their distribution
is confined to a broad belt of coastal waters within
the latitudes 64° N. and 44° S. With few excep-
tions oysters thrive in shallow water, their vertical
distribution extending from a level approximately
halfway between high and low tide levels to a
depth of about 100 feet. Commercially exploited
oyster beds are rarely found below a depth of 40
feet.

The name "Ostrea" was given by Linnaeus
(1758) to a number of mollusks which he described
as follows:

"Ostrea. Animal Tethys, testa bivalvis in-
aequivalvis, subaurita. Cardo edentulus and
fossula cava ovata, striisque lateralibus transversis.
Vulva anusve nuUus." The name Tethj-s (from
Greek mythology and also refers to the sea)
applies to the type of marine animals, living either
witliin the shells or naked, that Linnaeus listed
under a general name "Vermes" which includes
worms, mollusks, echinoderms, and others. The
translatioiKjf Linnaeus' diagnosis reads as follows:
Shell bivalve, unequal, almost ear-shaped. Hinge
tootldess, depression concave and oval-shaped,
with transverse lines on the sides. No vulva or
anus.

XuTK. — Approved for publication April 24, 1904.
FISHERY bulletin: VOLUME 64, CHAPTER I

This broad characterization included a number
of genera such as scallops, pen shells (Pinnidae),
limas (Limidae) and other mollusks which ob-
viously are not oysters. In the 10th edition of
"Systema Naturae," Linnaeus (1758) wi'ote:

"Ostreae non omnes, imprimis Pectines, ad
cardinem interne fulcis transversis numerosis
parallelis in utraque testa oppositis gaudentiquae
probe distinguendae ab x\rcis poly pie pto(jinglymis,
cujus dentes numerosi alternatim intrant alterius
sinus." i.e., not all are oysters, in particular the
scallops, which have many parallel ribs running
crosswise inward toward the hinge on each shell
on opposite sides; these should properly be dis-
tinguished from Area polyleptoginylymis whose
many teeth alternately enter between the teeth
of the other side.

In the same publication the European flat
oyster, Ostrea edulis, is described as follows:
"Vulgo Ostrea dictae edulis. 0. testa semi-
orbiculata membranis imbricatis undulatis:
valvula altera plana integerrima." i.e., commonly
called edible oyster; shell semicircular, outer valve
with wavy grooves; the other small valve com-
pletely flat. With a minor change Linnaeus'
diagnosis is repeated by Gmelin (1789).

Lamarck (1801) restricted the family Ostreidae
to the species of the genus Ostrea which was
characterized by him as follows: Adhering shell,
valves unequal, irregular, with divergent beaks
which with age become very unequal; the upper
valve becomes misplaced. Hinge without teeth.
Ligament half internal, inserted in the cardinal
lunule of the valves; the lunule of the lower
vah-e and the beak grow with age and sometimes
reach great length.

Great confusion in the usage of the generic
name of living oysters resulted, however, from

1

0 3

Centimeters

Figure 1. — Gryphaea arcuala, Jurassic fossil. Specimen
No. 283 from the Museum of Comparative Zoology,
Harvard University. Dimensions: height S.5 cm.,
depth 3.5 cm.

Liuniirck's (1801) allocation of e.xtinct and recent
species of oysters to the genus Gnjphaea. Under
this generic name, which was published with a
diagnosis, Lamarck included nine nominal species,
some of which at the time of publication were
"nomina nuda" since they lacked diagnosis, but
other species were validated by citation of biblio-
graphical references (Hemming, 1951). Lamarck
had not designated the type species, and a selec-
tion of "types" was made by Anton (1839), who
designated fossil Gnjphara arcvafa (fig. 1) as type.
Dall (1898) and Anthony (1906) also selected G.
arcuata as type. A living species, Gryphaea aniju-
lata, was included by Lamarck (1801) but without
a diagnosis.

Lanuirck (1819) further confused the nonuMi-
clature of the genus (ktrea when he again describcil
the genus Gryphaea. A longer list of species
included common oyster of Portugal and Medi-
terranean, Gryphaea angulafa, this time, however,
with a diagnosis (fig. 2). It was assumed l)y
Children, Grey, Fisher, Tryon, Stoliczka, and
Sacco (quoted from Dall, 1898: 671-GS8) that
G. aiKjulata is Lamarck's type species, an ojiinion
entirely without foundation. Anatomical and
embryological studies have demonstrated tliat
G. angulata has no characteristics of the fossils
Gryphaeidae. The species is simply another type
of oyster similar to the American species \('.
virginica), with a slightly twisted beak which only

remotely resembles the curved beak of the
Gryphaea.

Oysters are frequently found so closely adhering
to the substratum that their shells faithfully re-
produce the configurations and detailed structures
of the objects upon which they rest. For instance,
under the name of (ktrea tuherculata, Lanrarck
(1819) described a shell from the Timor Sea
(Netherland Indies) grown on a coral of the family
Astraeidae; this particular shell repeated the
tubercles and other structural elements of the
coral upon which it was resting. Other speci-
mens of the same species, but grown on a smooth
surface, were listed as different species. 0.
haliotidea of Australia, another Lamarckian species
assumed a shape of the abalone shell to which it
was attached. Oysters adhering to the shells of
Trochus maculatus repeated the granular structure
of this gastropod (Smith, 1878), while those grown
on branches of mangrove trees usually formed a
groove between the folds of the shell facing the
branch while the same species attached to the
trunk of the tree did not develop such structure
(Gray, 1833). 0. equestris found growing on
naWgation buoys (Galtsoff and Merrill, 1962) re-
peated the configurations of bolts and shells of
barnacles upon which they happened to attach
themselves.

The influence of other factors of the envu-on-
ment on the shajje and sculjjture of oyster shell
has been reported by many investigators who
noticed that specimens growing in calm water on
flat surfaces have a tendency to acquu-e a round
shape and to have poorly developed umbones. On
soft bottom and overcrowded reefs the same species
tend to form long and slender, laterally compressed
bodies wath hookhke umbones. Lamy (1929)
oljserved tliat oysters attached to a pebble or shell
and, therefore, slightly raised above the bottom,
had deep lower valves, more or less radially ribbed.
This type of structure, according to Lamy, offered
greater resistance to dislodgment by currents or
wave action.

Since Lamarck's inclusion of a hving estuarine
species, G. angulata, in one genus with the fossil
Jurassic and Cretaceous Gryphaeas was not ac-
ceptable to nuxny biologists, the question was
submitted for ruling by the International Commis-
sion on Zoological Nomenclature. The retention
of the name "Gryphaea" and the designation of G.
angulata as the type species of the genus Gryphaea
was favored by the "majority" of European zool-

FISH AND WILDLIFE SERVICE

Centimeters

Figure 2. — (a) Crassosirea angulata, Arcachon, France. Dimensions: lieight 'J cm., length 5 cm.
(b) C. virginka, Brownsville, Tex. Dimensions: height 8.5 cm., length 4.5 cm.
Note similarity of the two forms.

ogists(Ranson, 1948a) who requested the Interna-
tional Commission to suppress the name ' ' Gryphaea"
(Lamarck 1801) as applied to fossil species and to
vahdate tlie name "Gryphaea." (Lamarck 1819)
which included the living oysters. The American
zoologists (Gunter, 1950) were in favor of retaining
the name "Gryphaea" for fossil forms.

The findings of the Liternational Commission,
published as Opinion 338 on March 17, 1955, are as
follows:

Gryphaea Lamarck, 1801, is available for the
purposes of the Law of Priority and has as its type
species the Mesozoic Fossil species G. arcuata
Lamarck, 1801, by selection by Anton (1839),
and not G. angulata Lamarck, 1801, which was
selected by Children in 1823, this latter name
being a nomen ntidmn (not having been published

with an indication for the recent species to which
it is applicable until 1819).

"This nominal species Gryphaea angulata La-
marck, 1819, is not the type species of any nominal
genus, but the generic name Crassosirea Sacco,
1897, is available for use for that species by those
specialists who regard it as congeneric with Ostrea
viryinica Gmelin, (1790) (the type species of
Crassosirea Sacco) and who do not refer both species
to the genus Ostrea Linnaeus, 1758."

The names Gryphaea Lamarck, 1801, Cras-
sosirea Sacco, 1897, arcuata Lamarck, 1801 {Gry-
phaea) and angulata Lamarck, 1819 {Gryphaea)
were placed on the Official Lists of Generic Names
and Specific Names respectively and the nomen
nudum angulata Lamarck, 1801 {Grypnaea) was
placed on the Official Luie.x of Rejected and Invalid
Names in Zoology.

TAXONOMY

TAXONOMIC CHARACTERS

According to the view shared today by all
specialists on pelecypod taxonomy, the genus
Ostrea (in a broad sense), as characterized by
Lamarck, comprises several groups of oysters of
the family Ostreidae sufficiently different to be
considered as separate genera or subgenera
(Lamarck, 1819; Thiele, 1935). There is, how-
ever, no general agreement about the validity
of various genera and species. A uniform system
of classification of oysters is lacking, and for the
separation of genera and species various authors
use the characters of different categories: namely,
shape and structure of shell, anatomy, sex and
spawning, habitat, and structure of the larval
shell (prodissoconch).

SHELL

In spite of great variability, certain shell charac-
ters are generally constant although they may
be obscured in grossly distorted specimens. Two
characters of this category are important: the
cavity of the valves and the structure of the shell.

The lower valve is usually deep and cup-shaped
with a depression or recess of greater or lesser
extent near the hinge area. The upper valve
may be flat or curved and slightly bulging near
the hinge.

The oyster shell consists of extremely thin
outer periostracum, the median prismatic layer,
which is well developed on the flat (right) valve,
the inner calcitic ostracum that constitutes the
major part of the shell thickness, and hypostracum ,
a very thin layer of aragonite (orthorhombic
CaCOs) pad under the place of attachment of the
adductor muscle.' The prismatic layer of Ostrea
edulis is confined to the intricate brown scales
of the flat valve, while among the Australian
oysters only one species, 0. angasi Sowerby, has
a well-developed prismatic layer (Thomson, 1954).
In the genus Pycnodonte the shell is peculiarly
vacuolated (Ranson, 1941). The white patches
or so-called "chall^y deposits" in the shells of
many oysters are not significant as taxonomic
characters.

Size, shape, curvature, and proportion of the
beak, i.e., the pointed (dorsal) end of an oyster
shell, are useful generic characters, but Wke other
parts of the shell they are variable and cannot be
entirely depended upon for identification.

The sculpture of the shell may be useful for

' The author is prateful to H. B. Stenzel for the information on aragonite
in oyster shells.

recognizing some species (0. (Alectryonia) mego-
don, fig. 3) with valves reinforced by a number of
prominences or folds (also called ribs, ridges, or
flutings by various authors) which end in the
crenulations at the edge of the shell. In American
oysters this character varies greatly depending on
local conditions but is rather constant in 0.
eqtiestrin (Galtsoff and MerrUl, 1962).

The position of the muscle scar and its outline
differs in various species and, therefore, is used
as a taxonomic character.

ANATOMY

Anatomical characters are of limited usefulness
to malacologists who have to base their identifica-
tion primarily on shells alone. Consequently the
anatomical characteristics have been ignored by
the majority of taxonomists. Some of the ana-
tomical differences are, however, important for
the separation of the genera. Thus, the presence
of the promyal chamber separates the Ostrea, in
which this feature is absent in sufficiently studied
species, 0. edulis, 0. lurida, 0. equestris, from
Crassostrea in which the chamber is well developed.

Size of the gill ostia, large in the larviparous
species and relatively small in oviparous ones, is
of generic significance. The relation of the rectum
and the heart is of importance since in the genus
Pycnodonte the ventricle is penetrated by the
rectum, a unique feature not found in other
Ostreidae.

Convolution of the edge of the mantle with
three folds in the majority of the species and only
two in some Japanese species (Hirase, 1930) has
been mentioned by some investigators as a
specific character. The existence of two or
three folds may be significant, but other characters
such as ridges of the mantle, pigmentation of the
tentacles and their size and spacing are variable
and, in my opinion, have no taxonomic value.

SEX AND SPAWNING

On the basis of sexual habits, oysters fall into
two distinct categories of ncniincubatory (or ovi-
parous) species, {Crassostrea spp.) i.e., those in
which tlie eggs discharged into the water are
fertilized outside the organism ; and the incubatory
or larviparous species {Ostrea spp.) in which
fertilization takes place in the gill cavity, and
the larvae are incubated and discliarged after
having reached an advanced stage of develop-
ment. Incubatory oysters, as for example 0.
edulis, 0. lurida, and 0. equestris, are bisexual

FISH AND WILDLIFE SERVICE

-L

Centimeters

Figure 3. — O. (Alectryonia) megodon. Pearl Islands, Panama. Dimensions: height 17 cm., length 16 cm.

(hermaphroditic). The gonad of a bisexual oyster
produces eggs and sperm simuUaneously, but the
relative cjuantity of sex cells of one or another
type alternates periodically from male to female
and vice versa.

The sexes of nonincubatory (oviparous) oj'sters
{C. nrginica, C. gigas, C. angulata) are separate.
Instances of hermaphroditism in this group are
very rare. The sexes are, however, unstable and
once a year a certain percentage of oysters change
their sex. This change takes place after spawning
during the indifferent pliase of gonad development.
Alternation of sex (discussed in ch. XV) has been
studied in detail only for a few species.

HABITAT

Salinity, turbidity, and depth of water are
frecjuently mentioned in the brief statements
that accompanj' the descriptions of various

species. Ecological data are, however, of no
help to classification. With the exception of a
few commercial species, which have been more
adequately studied, little is known about the
environmental requirements of the populations
of other species. In a general way it can be
stated that C. virginica, C. gigas, and probably C.
angulata are more tolerant to diluted sea water
than are 0. lurida and 0. edulis. The two latter
species survive better in more saline and less
turbid environment. Geographical distribution of
0. equestris suggests the preference of this species
to the waters of full oceanic salinity (about 35°/oo).
The same is true for many tropical oysters living
along the continental shores and in the lagoons
of oceanic islands where the salinity of water
changes but little or remains constant throughout
the year. Tolerances of these tropical species to
lowered salinity have not been studied.

TAXONOMY

LARVAL SHELL (PRODI SSOCONCH)

The difficulty in identifying a species by its
shell led Ranson (1948b) to base the classification
of oysters entirely on the features of a "definite"
prodissoconch, i.e., the shell of a fully developed
larva. He claims that distinctive crenulations of
prodissoconchs are sufficient for the separation of
species and that these specifically larval charac-
ters can be detected in well-preserved adult shells
and even in fossils. In a brief paper comprising
only 6 incomplete pages of text and 35 pages of
drawings of 34 species of oyster larvae Ranson
(1960) summarizes the basic idea of his classi-
fication. He states that a lamellibranch larval
shell passes through two distinct stages, the first
one is a "primitive" prodissoconch with un-
differentiated hinge and the second phase, which
he calls "definite" prodissoconch, is characterized
by the development of hinge teeth. At the first
phase all lamellibranchs have similar prodisso-
conchs, but at the second phase the hinge becomes
dift'erentiated. This makes it possible to dis-
tinguish the families and the genera. He main-
tains, without giving substantiating evidence,
that the general shape of a definite prodissoconch
is absolutely constant even if the size of the
larva varies and that each species of oysters can
be recognized by the shape of its larval shell
and its structural characteristics.

Ranson's system of classification recognizes the
following three genera of oysters: Pycnodonte
{Pycnodonta in Ranson's spelling), Crassostrea,
and Osirea. His diagnoses of the prodissoconchs
of these genera are given in the following section
of this chapter. Unfortunately, the diagnoses
of the larvae of 34 species of oysters studied by
Ranson are lacking, and the prodissoconchs are
shown only by diagrammatic drawings, some of
which are reproduced in chapter XVI of the book.
My attempts to locate the prodissoconchs on the
shells of fully grown C. inrginica (from 3 to 8
years old) were not successful. On a few occasions
the prodissoconchs were faintly visible, but the
structure of the hinge, and the number and
location of hinge teeth could not be detected.
Final decision regarding the possibility of identi-
fj'ing adult oysters by their larval characters must
wait, however, until Ranson's system is given a
fair trial by malacologists. His suggestion that
identification can be made by observing spat
attached to the shell of the adult is not valid
because in many places several species of oysters

live together in the same locality and the larvae
settle indiscriminately on any shell or other object
available at the time of setting.

THE GENERA OF LIVING OYSTERS

There is an obvious need for a complete taxo-
nomic revision of the family Ostreidae. This
revision should cover all the principal species of
living oysters and must be supplemented by
morphological, anatomical, and ecological observa-
tions which at present are available only for a
few commercially utilized species. In the absence
of these data for the large majority of species of
living oysters, it is at present impossible to propose
a logical taxonomical system for the family.

Opinions vary regarding the number of genera of
living oysters of the family Ostreidae. Stenzel
(1947) recognizes 12 valid generic names, some
of which, as was shown by Gunter (1950), are
synonymous. The latter author admits the
existence of three definite genera {Ostrea, Cras-
sostrea and Pycnodonte) and three others {Den-
dostrea Swainson, Aledryonia Fisher de Wald-
heim, and Striostrea Vialov) of doubtful validity.

Ranson (1941, 1948b, 19G0) merges the three
doubtful genera of Gunter in Ostrea and recognizes
only the three definite genera listed above. This
opinion, based primarily on structure of pro-
dissoconchs, is shared by Thomson (1954) and is
supported by the evidence accumulating from
morphological and biological data. It can be,
therefore, stated with a certain degree of assurance
that on the basis of present knowledge, the living
Ostreidae comprise three genera: namely, Ostrea
Linnaeus; Crassostrea Sacco; and Pycnodonte
Fisher de Waldheim. The genus Lopha, named
by Bolton in 1798, without definition and described
as a subgenus only in 1898 by Dall (1898), is
undoubtedly a synonym of Ostrea. The dis-
tinctive feature upon which this genus was founded
was the sharply crenulated nature of the shell
margin, a very poor distinguishing character.

The three genera of oysters can be defined as
follows:

Genus Ostrea Linne, 1758. Genotype: O. edulis L.

Shell subcircular; lower valve shallow, not
recessed under the hinge; upper valve flat, opercu-
lar, sometimes domed; muscle scar subcentral.
Promyal chamber absent. Gill ostia relatively
large. Incubatory.

6

FISH AND WILDLIFE SERVICE

Prodissoconch with long hinge; two denticles
at each end, the anterior pair frequently reduced;
the ligament is internal at the level of the hinge,
at the center, and between the center and anterior
end (Ranson, 1960, fig. 350, ch. XVI).

Genus Crassostrea Sacco, 1897. Genotype: C. virginica
Gmelin

Shell very variable, usually elongated; lower
valve cuplike, deep, and recessed under the hinge;
upper valve flat, opercular. Muscle scar dis-
placed in dorsolateral direction. Large promyal
chamber on the right side of the body. Gill
ostia and eggs relatively small. Nonincubatory.
Excellent illustrations of the species can be found
in the monographs of Lister (1685) and Chemnitz
(1785).

This genus includes species formerly known as
0. virginica, 0. gigas, and G. angulata. The
separation of the cuplike oysters (Crassostrea)
from the flat ones (Ostrea) is justified because of
the anatomical differences (promyal chamber,
size of the ostia) and spawning habits. The name
"Crassostrea" (Sacco, 1897) is validated in ac-
cordance with the rules of the International Com-
mission on Zoological Nomenclature (1955).

Valves of the prodissoconch unequal; hinge with
two teeth at each end; internal anterior ligament
extends beyond the hmge (Ranson, 1960). (Fig.
348, ch. XVI).

Genus Pynodonte Fisher de Waldheim, 1835. Genotype :
P. radiata F. de W.

Shells large and heavy; lower valve slightly
recessed under hinge; both shells lack sculpture
except for sharp crenulations along the tip; hinge
very broad. Inner sides of valves chalky white
or greenish; row of small denticles along the edges
of valves on both sides of the hinge; muscle scar
white, elevated on a shelf like projection; the ad-
ductor muscle is oval in outline and rounded on
the hinge side; the gonads are bright orange; the
ventricle of the heart surrounds the rectum, which
runs posteriorly beyond the adductor muscle
almost to the junction of the mantle. Non-
incubatory.

Valves of prodissoconch equal; hinge with five
teeth arranged over the entire length of it; internal
anterior ligament, immediately after the hinge;
10 small denticles at the edge of each valve an-
teriorly to the ligament (Ranson, 1960).

The following species of living oysters are known
from the coastal waters of the continental United
States and from the State of Hawaii:

C. virginica Gmelin, Eastern oyster, Atlantic
oyster. This is the principal edible oyster of the
Atlantic and Gulf Coasts of the U.S.A. (fig. 4).
Its range of distribution extends from the Gulf of
St. Lawrence to the Gulf of Mexico and the West
Indies. The species was introduced in the waters
of San Francisco Bay, Puget Sound, Willapa Bay,
and Oahu Island but failed to establish itself,
although occasionally single specimens can be
found in these waters.

The right (upper) valve smaller than the left.
The beaks elongated and strongly curved. The
valve margins straight or only slightly undulating.
The muscle scar usually deeply pigmented. The
adductor muscle located asymmetrically, well
toward the posterodistal border. Large promyal
chamber on the right side. Nonincubatory, dis-
charging eggs and sperm directly into the water.

Adults vary from 2 to 14 inches in height
(dorsoventral direction) depending on age and en-
vironment. Shape, sculpture, and pigmentation
of inner side of the shell and along the edges of the
mantle and tentacles vary greatly.

Crassostrea rhizophorae Guilding. Light, thin,
foliaceous, and deeply cupped shell with smaller
flat upper valve fitting to the lower one (fig. 5).
The inner margins straight and smooth with con-
siderable purple coloration especially around the
left valve. The beaks twisted dorsally. Muscle
scar near the dorsal margin. Promyal chamber
present. Nonincubatory, discharging eggs and
sperm directly into the water.

Similar to C. virginica from which it differs by
the following characters: lower left valve is less
plicated than in virginica; the muscle scar is more
rounded and often unpigmented. (Prodissoconch
shown in fig. 349, ch. XVI).

Adults may reach 4 inches in height. Fre-
quently attached to the aerial roots of the man-
grove Rhizophora mangle. Inhabits Caribbean
region including Puerto Rico and Cuba where the
species is commercially exploited.

Crassostrea gigas Thunberg, Japanese oyster,
Pacific oyster (fig. 6) Cuplike shells of large size
with coarse and widely spaced concentric lamellae
and coarse ridges on the outside; shells usually
much thinner than those of C. virginica. Upper
(right) valve flat and smaller than lower (left)

TAXONOMY

a

Centimeters

FiGDRE 4. — C. virginica.

Wellfleet Harbor, Mass. Dimensions: height 9.5 cm., length 8 cm.
(b) inside of upper (right) valve.

(a) lower (left) valve;

valve. Interior surface white, often with faint
purpHsh stain over the muscle scar or near the
edges. Large promyal chamber on the right side.
Edges of the mantle deeply pigmented. Non-
incubatory, discharging a very large number of
eggs and sperm directly into the water. Intro-
duced from Japan into the waters of British
Columbia, western states of the United States,
and Alaska (Ketchikan). Small number of speci-
mens of C. gigas were at various times planted in
Mobile Bay, Ala., and in Barnstable Bay, Mass.

Highly variable. Typical C. gigas is a long,
straplike oyster. The form C. laperousi (con-
sidered by Japanese malacologists as a separate
species) has round, highly ridged shells.

C. commercialis (Iredale and Roughley), Sydney
rock oyster, commercial oyster. This Australian
species (fig. 7) was imported to Hawaii about
1925-28 and planted along the shores of the
western end of Kaneohe Bay, in Oalm Island. In
1930 several of the imported specimens were ex-
amined by the author and found to be ripe and
spawning. During World War II the small popu-
lation of this species was destroyed by dredging
operations.

Valves markedly unequal and variable. The
left valve deep and cup-shaped, recessed under the
hinge, slightly fluted; the edge weakly crenulated.
The upper valve flattened. Inner side of valves
chalky white, frequently with bluish or creamy
markings on the upper valve; muscle scar usually
not pigmented. Edges of valves with small denti-
cles extending about half way around the valve.
Sexes are separate; nonincubatory.

Usually grows to 3 to 4 inches in height, but
cultivated specimens have been reported to reach
10-inch size. Normal range of distribution New
South Wales and Queensland; frequently found
in the intertidal zone attached to rocks, sticks,
and shells.

C. rimdaris Gould (fig. 8). This Japanese
species has been planted in waters of Puget Sound
with the shipments of seed of C. gigas. In Japan
tlie oyster is known as "suminoegaki" (Hirase,
1930).

The shell orbicular; strong and large, adult
specimens reaching 6 inches in height. Left,
lower, valve slightly concave, upper valve shorter
and flat. The left valve with generally indistinct
lamellae of pale pink color with radiating striae.
The lamellae of the right valve are thin and al-

8

FISH AND WILDLIFE SERVICE

Centimelers

Figure o. — C. rhizophorae. On mangrove roots, Florida Keys. Dimensions: height 7 cm., lengtii 3.7 cm. (a) exterior;

(b) interior of the shell.

most smooth, sometimes covered with tubular pro-
jections. The color of the right valve is cream
buff with many radial chocolate bands; their
arrangements greatly variable. Muscle scar, situ-
ated near the center or a little dorsally, is white,
occasionally with olive-ochre spots. Margin of
the mantle is dark violet; the tejitacles are
arranged in two rows; those of the outer row are of
irregular size; the imier tentacles in a single row
are slender.

The species, described from China by Gould,
occurs in Ariake Bay and in the bays of Okayama
Prefecture, Japan. It has established itself in
Puget Sound.

Ostrea edulis Linne (fig. 9). This European
flat oyster is the type species of the genus Ostrea.
Shell round or oval; left valve larger and deeper,
slightly- bulging with 20 to 30 ribs and irregular

concentric lamellae. Upper valve smaller, flat,
without ribs, with numerous concentric lamellae.
Beaks poorly developed. Ligament consists of
three parts; the middle part is flat on the left
valve and forms a projection on the right valve.
Muscle scar is eccentrically located, unpigmented.
Promyal chamber absent. Ostia and eggs rela-
tively large. Hermaphroditic and incubatory
mollusk, discharging eggs into the gill cavity.
Small numbers of European flat oysters were
introduced several years ago into the coastal
waters of Maine in Boothbay Harbor where they
survived and reproduced themselves. At present
the population is too small to be used commer-
cially. Recently the stock of European oysters
in Maine waters was increased by planting seed
raised from eggs fertilized and developed in the
Bureau of Commercial Fisheries Biological Labora-

TAXONOMY

J L

Centimeters

FiGunE 6. — C. gigas. Adult oyster grown in Willapa Harbor, Wash., from seed imported from Japan. Dimensions:
height 14 cm., length 9 cm. (a) exterior of lower (left) valve; (b) interior of upper (right) valve.

0 3

Centimeters

Frii-re 7. — C. roiiitiifrciaHs. Kaneohe Bay, Oahu Island, Hawaii. Dimensions: height 7 cm., length 4.S cm. (a) exterior

of lower (left) valve; (b) interior of upper (right) valve.

10

FISH AND WILDLIFE SERVICE

Cent i meters

Figure S. — C. rivularis. Puget Sound. Introduced from Japan with the seed of C. gigas. Dimensions: height 10.7
cm., length 10.5 cm. (a) exterior of lower (left) valve; (b) interior of upper (right) valve.

Centimeters

Figure 9. — O. edutis, European flat oyster introduced from Europe to Boothbay Harbor, Maine. Dimensions: height
9 cm., length 11 cm. (a) exterior of the left (lower) valve; (b) interior of the right (upper) valve.

tory at Milford, Conn. Prodissoconch shown in
fig. 350 ch. XVI.

0. lurida Carpenter (fig. 10). Shells from 2 to
3 inches in height, with coarse concentric lines.
Inner side of valves usuallj^ olive-green. Promyal
chamber absent. Hermaphroditic, incubatory,

eggs retained in the gill cavity until fully de-
veloped larvae are formed and discharged into the
water. Inhabits the tidal waters of the Pacific
Coast of North .America from Alaska to Lower
California. (Prodissoconch shown in fig. 351,
ch. XVI.).

TAXONOMY

11

0

Centimeters

Figure 10. — 0. lurida from Pugpt Sound. Left (a) and
right (b) valves. Height 5.5 cm.; length 3 cm.

0. equestris Say, horse oyster, crested oyster
(fig. 11). This small, noncommercial oyster of
the South Atlantic states. Gulf of Mexico, and
West Indies is often mistaken by laymen for
young C. virginica. Its average size is about 2
inches, but occasionally specimens measuring up
to 3K inches in height are found. Say (1834),
who described the species, hsts the following
identifying characters: Shell small, with trans-
verse wrinkles, and more or less deeply and
angularly folded longitudinally; ovate-triangular,
tinted with violaceous; lateral margins near the
hinge with from 6 to 12 denticulations of the
superior valve; superior valve depressed but
slightly folded; inferior valve convex, attached by
a portion of its surface, the margins elevated,
folds unequal, much more profound than those of
the superior valve; hinge very narrow, and curved
laterally and abruptly.

The shape of the oyster shell is very variable
depending on crowding and type of substratum.
The most significant combuiation of features by
which 0. equestris can be distinguished from small
C. virginica and from 0. frons are as follows: a
rather higli vertical and crenulated margin of tlie
lower valve; off-center position of the adductor
nmscle scar; a dull greenish color of the interior
surface; and the presence of a single row of denti-
cles of the upper valve with the corresponding
depression on the lower valve (GaltsofF and Merrill,
1962). The number of denticles and their size

very variable. Hanson (1960) does not include
0. equestris in the drawings of prodissoconchs
reproduced in his publication. A rough sketch of
the prodissoconch of 0. equestris, not showing the
details of hinge structure, is given by Menzel
(1953).

This incubatory species frequently occurs in
large numbers on commercial oyster beds in associ-
ation with C. virginica. It thrives in waters of
high salinity (35 %o) but has been found in regions
where salinity is about 20 to 25°/oo. The north-
ernmost boundary of distribution established by
Merrill is about half way between Delaware and
Chesapeake Bays, (37°31' N., 73°18' W.) at the
depth of 60 fathoms.

0. frons Linne (fig. 12). Small shells of 1 to 2
inches in height with radial plicated sculpture
and sharply folded margins. Valves closely set
with minute denticles almost around the entire
circumference. Muscle scar near the hinge.
Beaks slightly curved. Interior white, exterior
usually purple-red. Frequently attached to
branches or roots of mangrove trees by a series of
hooked projections. Connnon in Florida, Louisi-
ana, and the West Indies. Was found attached
to navigation buoys off Port Royal, S.C., and off
Miami Harbor, Fla. (personal communication of
A. S. Merrill).

0. pennollis Sowerby (fig. 13). This small
oyster lives commensally, completely embedded
in sponges with only the margins of the valves
visible. The species is common along the west
coast of Florida, north of Tarpon Springs, where
it is constantly associated with tlie sponge Stellata
grnbi (fig. 14). Rarely exceeds 1.5 inches in size.
Surface of valves soft and silky. Beaks twisted
back into a strong spiral. Inner margins with
numerous small denticles. Nonedible species.
North Carolina to Florida and the West Indies.
Pycnodonte hyotis Linne. This species dredged
from 300 feet of water off Palm Beach was de-
scribed by McLean (1941) as 0. thomasi, nova
species, but identified by Ranson as Pycnodonte
hyotis. It is characterized by the peculiar foam-
like appearance of its shell structure, particularly
at the margins. Shells circular, 3 to 4 inches in
diameter.

Fycnodonte is frequently found on navigation
buoys off Key West, Fla., and near the entrance
to Miami Harbor (personal communication of
A. S. Merrill).

12

FISH AND WILDLIFE SERVICE

a

J

0 2

Centimeters

Figure 11. — O. equeslris. Left (a) and right (b) valves. 5 cm. in height.

TAXONOMY

733^851 O — 64 2

13

0 r- .■ . 3

Centimeters

Figure 12. — 0. Jrons. Key Biscayne, Florida. Natural
size.

0 3

Centimeters

FiGUKE 13. — 0. per mollis.- West of Andote Light, Tarpon
Springs, Fla., at 6 fathoms. Dimension.?: height 3.5
cm., length 3 cm.

0 ^ . 3

Centimeters

Figure 14. — O. pcrmolh's embedfled in sponge Stellaia gruhi-

BIBLIOGRAPHY

Anthony, R.

1906. Etude monographique des Aetheriidae. An-
nales de la Socicte Royale Zoologique de Belgiquc,
tome 41, pp. 322-428.
Anton, H. E.

1839. Verzeichnis der Conchylien, welche sich in der

Sammlung von Hermann Eduard Anton befindcn.
Heraasgegeben von demBesitzer, Halle, xvi + 110 pp.
Carcelles, Alberto.

1944. Catdlogo de los moluscos marines de Puerto
Quequ^n (Repiiblica Argentina). Revista del
Museo de La Plata, Neuva Serie, Secci6n Zoologia,
tomo 3, pp. 233-309.
Chemnitz, Joh. Hieron.

1785. Neues systematisches Conchylien-Cabinet.
8th Band, Niirenberg, Bauer und Raspe, 372 pp.,
taf. 70-102.
Contreras, Francisco.

1932. Datos para el estudio de los ostiones mexi-
canos. Anales del Institute de Biologia, Uni-
versidad de Mexico, tomo 3, No. 3, pp. 193-212.
Dall, William H.

1898. Tertiary fauna of Florida. Supcrfamily O.s-
tracea. Transactions of the Wagner Free Insti-
tute of Science of Philadelphia, vol. 3, part IV,
pp. 671-688.

1914. Notes on West American oysters. Nautilus,
vol. 28, No. 1, pp. 1-3.
DouviLL^, Henri.

1898. Sur la classification phylogcnique des Lamelli-
branches. Comptes Rendus Hebdomadaires des
Seances de I'Acad^mie des Sciences, tome 126,
pp. 916-919.

1910. Observations sur les Ostr^id^s, origine et
classification. Bulletin de la Society G^ologique
de France, serie 4, tome 10, pp. 634-645.

1912. Classification des Lamellibranches. Bulletin
de la Societe Geologique de France, s^rie 4, tome
12, pp. 419-467.
Galtsoff, Paul S., and Arthur S. Merrill.

1962. Notes on shell morphology, growth, and dis-
tribution of Oslrea equestris Say. Bulletin of
Marine Science of the Gulf and Caribbean, vol. 12,
No. 2, pp. 234-244.
Gmelin, J. F.

1789. Caroli a Linnc Systema Naturae, torn I, pars
VI, pp. 3315-3334. 13th ed. (Lugduni, Apud
J. B. Delamolliere, 1789).
Gray, John Edward.

1833. Some observations on the economy of mol-
luscous animals, and on the structure of their
shells. Philosophical Transactions of the Royal
Society of London for the year 1883, part 1, vol.
123, pp. 771-819.
Gunter, Gordon.

1950. The generic status of living oysters and the
scientific name of the common American species.
American Midland Naturalist, vol. 43, No. 2,
pp. 438-449.

1951. The species of oysters of the Gulf, Caribbean
and West Indian region. Bulletin of Marine
Science of the Gulf and Caribbean, vol. 1, No. 1,
pp. 40-45.

Hedgpeth, Joel W.

1954. A problem in oyster taxonomy. Systematic
Zoology, vol. 3, pp. 21-25, 45.
Hemming, Francis.

1951. On an application, the grant of which would
require that the name "Gryphaea" Lamarck, 1801,

14

FISH AND WILDLIFE SERVICE

should be suppressed, under the plenary powers,
thus validating the name "Gr3phaea" Lamarck,
1819. (Class Pelecypoda). Bulletin of Zoological
Nomenclature, vol. 2, parts 6/8, pp. 239-240.

HlHASB, ShINTARO.

1930. On the classification of Japanese oysters.
Japanese Journal of Zoology, vol. 3, pp. 1-65.
Ihbhing, H. von.

1902. Historia de las ostras argentinas. Anales del
Museo Xacional de Historia Natural de Buenos
Aires, tomo 7 (series 2, tome 4), pp. 109-123.
International Commission on Zoological Nomen-
clature.

1955. Opinion 338. Acceptance of the Mesozoic
Fossil species Gryphaca arcuata Lamarck, 1801, as
the type species of the nominal genus Gryphata
Lamarck, 1801, and addition of the generic name
Gryphaea Lamarck, 1801 (Class Pelecypoda) to the
Official List of Generic Names in Zoology. Opin-
ions and Declarations rendered by the Inter-
national Commission on Zoological Nomenclature,
vol. 10, part 5, pp. 125-180.
Ieedale, Tom.

1949. Western Australian molluscs. Proceedings
of the Royal Zoological Society of New South
Wales for the year 1947-48, pp. 18-20.
Lamarck, J. B. P.

1799. Prodrome d'une nouvelle classification des
coquilles, comprcnant une redaction appropriee des
caractferes generiques, et I'etablissement d'un
grand nombre de genres nouveaux. M^moires de
la Socicte d'Histoire Naturelle de Paris, vol. 1,
pp. 63-91.
1801. Systeme des animaux sans vertebres. Crape-
let, Paris, 432 pp.
1819. Histoire naturelle des animaux sans vertebres.
Tome 6, No. 1, pp. 200-220. A. Belin, Paris.
Lamy, Ed.

1929. Revision des Ostrea vivants du Museum Na-
tional d'Histoire Naturelle de Paris. Journal de
Conchyliologie, serie 4, tome 27, vol. 73, pp. 1-46;
ibid. 71-108; ihid. pp. 133-168; ihid. pp. 23.3-275.
Linnaeus, C.

1758. Systema Naturae, Editio Decima, Reformata:
Tomus 1, pp. 696-700. Holmiae, Impensis direct,
Laurentii Salvii.
Lister, M.

1685. Historiae sive Synopsis methodicae Conehy-
liorum querum omnium picturae, ad vivum, deline-
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S. & A. Lister Figuras 1 in. 6 Pt. (in'"2 vol.) Fol.
Londini, 1685-92 (-97).
McLean, Richard A.

1941. The oysters of the Western Atlantic. Academy
of Natural Sciences of Philadelphia, Notulae
Naturae, No. 67, pp. 1-14.
Menzel, R. Winston.

1953. The prodissoconchs and the setting behavior
of three species of oysters. National Shellfisheries
Association, 1953 Convention Papers, pp. 104-112.

Nicol, David,

1954. Malacology. — trends and problems in pelecypod
classification (the supergeneric categories). Journal

of the Washington Academy of Sciences, vol. 44,
No. 1, pp. 27-32.
Ranson, Gilbert.

1941. Les especes actuelles et fossiles du genre
Pycnodonta F. de W. I. Pycnodonla hyolis (L.).
Bulletin du Museum (National) d'Histoire Natur-
elle, Paris, serie 2, tome 13, pp. 82-92.

1948a. Gryphaea angulata Lmk. est I'espece "type"
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1948b. Prodis.soconques et classification des OsMidis
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Naturelle de Belgique, tome 24, fascicule 42, pp.
1-12.

1960. Les prodissoconques (coquilles larvaires) des
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1950. The identification and classification of lamelli-
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1897. I moUuschi dei terreni terziarii del Piemonte e
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Say, Thomas.

1834. American conchology, or descriptions of the
shells of North America. 7 parts, 258 pp., 68 plates
col. 8"*, 1830-34. School Press, New Harmony,
Ind.

Smith, Edgar A.

1878. Descriptions of five new shells from the island
of Formosa and the Persian Gulf, and notes upon a
few known species. Proceedings of the Zoological
Society of London, 1878, pp. 728-733.

SowERBY, George Brettingham.

1870-71. Monograph of the genus Oslraea. In
Reeve, Conchologia Iconica, vol. 18, 33 plates with
letter press. London.

Stenzel, H. B.

1947. Nomenclatural synopsis of supraspecific groups
of the family Ostreidae. (Pelecypoda, MoUusca).
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Thiele, J.

1935. Handbueh der systematischen Weichtierkunde,
Band 2, pp. 813-814. Gustav Fischer, Jena.

Thomson, J. M.

1954. The genera of oysters and the Australian
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VlALOV, O. S.

1936. Sur la classification des hultres. Comptes
Rendus (Doklady) de I'Academie des Sciences de
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ViLELA, HeRCULANO.

1951. Ostras de Portugal. Revista da Faculdade de
Sciencias Universidade de Lisboa, 2a serie, C —
Ciencias Naturais, vol. 1, fascicule 2, pp. 291-306.

TAXONOMT

15

CHAPTER II
MORPHOLOGY AND STRUCTURE OF SHELL

Page

Appearance and principal axes _ _ 16

Dimensions. ___ _ __ 'jO

Shape of shells _ 21

Growth rings and growth radii _ _ _ 26

Changes In the direction of principal axes of shell. 27

Dimensional relationships of shell 29

Shell area 30

Chalky deposits _ __ 32

Chambering and blisters 35

Structure of shell 36

Organic material of the shell 37

Muscle attachment 41

Chemical composition 43

Bibliography 45

APPEARANCE AND PRINCIPAL AXES

The body of the oyster is covered with two
calcareous valves joined together by a resilient
ligament along the narrow hinge line. The valves
are slightly asymmetrical. The left one is larger
and deeper than the right one, which acts as a lid.
Under normal conditions the oyster rests on the
left valve or is cemented by its left valve to the
substratum. The difference between the riglit
(flat) and left (cuplike) valve is to a certain degree
common to all the species of oysters which have
been sufficiently studied. Orton's (1937) state-
ment with reference to Ostrea edulis that: "In
life the flat or right valve usually rests on the sea
bottom and is often referred to as the lower one"
is an obvious oversight.

In C. virginica the left valve is almost always
thicker and heavier than the right one. When
oysters of this species are dumped from the deck
of a boat and fall through water they come to rest
on their left valves. I observed this many times
while planting either small oysters not greater
than 2 inches in height, or marketable adults of 5
to 6 inches. In the genus Ostrea the difference
between the two valves is not great, it is greater
in the genus Crassostrea, and extremely pronounced
in the oyster of uncertain systematic position
from Australia which Saville-Kent (1S9.3) has
called "Ostrea mordax var. cornucopiaeformis." -

- I am indebted to H. B. Stenzel for calling my attention to this species and
for several suggestions regarding the morphological terminology used in this
chapter.

16

The oyster is a nearly bilaterally symmetrical
mollusk with the plane of symmetry passing be-
tween the two valves parallel to their surfaces.
In orienting any bivalve it is customary to hold
it vertically with the narrow side uppermost (fig.
15). The narrow end or ape.x of the sheU is called
the umbo (plural, umbos or umbones) or beak.
A band of horny and elastic material, the ligament
(fig. 16) joins the valves at the hinge on which
they turn in opening or closing the shell.

In many bivalves the hinge carries a series of
interlocking teeth, but these structures are absent
in the family Ostreidae. The hinge consists of the
following parts: a projecting massive structure
within the right valve, the buttress, according to
Stenzel's terminology, supports the midportion of
the ligament and fits the depression on the left
valve. The tract made by the buttress during
the growth of the shell along the midportion of the
ligamental area is the resilifer. On the left valve
the resilifer is the tract left on the depression.
The central part of the ligament is called resilium.

The pointed end of the valve or the beak repre-
sents the oldest part of a shell. In old individuals
it reaches considerable size (fig. 17). The beaks
are usually curved and directed toward the
posterior end of the mollusk although in some
specimens they may point toward the anterior.
In the majority of bivalves other than oysters
the beaks usually point forward. The direction
and degree of curvature of the beaks of oysters as
well as their relative proportions vary greatly
as can be seen in figure IS, which represents
different shapes found in old shells of C. virginica.
Very narrow, straiglit, or slightly curved beaks of
the kind shown in figure 18-1 are usually formed
in oysters which grow on soft, muddy bottoms.
Extreme development of this type can be seen in
the narrow and slender oysters growing under
overcrowded conditions on reefs (fig. 19). Other
forms of beaks (fig. IS, 2-4) cannot be associat-
ed with any particular environment. In fully

FISHERY bulletin: VOLUME 64, CHAPTER II

Centimeters

Figure 15. — Blue Point oyster (C. virginica) from Great South Bay, Long Island, N.Y. The size of this 5-year-old
oyster is about 10 x 6.6 cm. (4x3 inches). The shell is strong and rounded; its surface is moderately sculptured.
Left — outside surface of left valve. Right — inner surface of right valve. Small encircled area under the hinge on
the inner surface of right valve is an imprint of Quenstedt's muscle.

Centimeters

Figure 16. — Cross section below the hinge of an adult C.
virginica. Left valve at bottom, right valve at top of
the drawing. The buttress of the right valve fits the
depression on the left valve. The two valves are connect-
ed by a ligament (narrow band indicated by vortical
striations) which consists of a central part (resilium)
and two outer portions. Slightly magnified. r.V. —
right valve; bu. — buttress; dc. — depression or furrow on
left valve (l.v.); lig. — ligament.

grown C. virginica the pointed end of the upper
(flat) valve is always shorter than that of its
opposite member (fig. 17). The angle between
the two beaks determines the greatest extent to
which the valves can open for feeding or respira-
tion and is, therefore, of significance to the oyster.

If the oyster shell is oriented in such a way that
both of its valves are visible and the beaks point
up and toward the observer, the flat valve with a
shorter, convex resilifer is the right one and the
cuplike valve with the longer concave resilifer
is the left one. The dorsal margin of the oyster
is the beak or hinge side, the ventral margin the
opposite. If viewed from the right (flat) valve
with the hinge end pointing away from the ob-
server the anterior end of the oyster is at the
right side of the valves and the posterior is at the
left.

The posterior and anterior parts of the oyster
shell may also be identified by the position of the
muscle impression, an oval-shaped and highly
pigmented area marking the attachment of the
adductor muscle on the inner side of each valve.

MORPHOLOGY AND STRUCTURE OF SHELL

17

Centimeters

Figure 17. — Side view of a very old and large C. virginica from Stony Creek, Conn. Notice the curvature of the beak,
the depressed resilifer on the lower valve and the protruding rcsilifer on the upper one. The angle between the beaks
determines the maximum movement of the upper valve. Dimensions: height — 25.5 cm. (10 inches) and width^ — 6.4
cm. (2.5 inches).

The muscle impression is asymmetrically located
closer to the posterior end of the valve. This
area of the attachment of the adductor muscle
has been called the "muscle scar." Some mala-
cologists prefer to use the expression "muscle
impression" or "area of attachment" (Stenzel,
personal communication) because the word "scar"
usually means the mark left by healing of an
injury. The proposed change in terminology
does not seem to be desirable because the name
"muscle scar" has been so well established in
scientific and popular writings that its abandon-
ment may cause confusion.

The three principal dimensions of bivalves, in-
cluding oj-sters, are measured in the following
manner (fig. 20): height is the distance between
the umbo and the ventral valve margin; length is
the maximum distance between the anterior and
posterior margin measured parallel with the
hinge axis; and width is the greatest distance
between the outsides of the closed valves measured
at right angles to the place of shell commissure.

In manj' popular and trade publications on
shellfish the word "length" is used instead of
"height", and tlie word "width" is employed to
designate the length of the oysters. To avoid
confusion the scientific rather than popular
terminology is used throughout the text of this
book.

The shape of oyster shells and their proportions
are higlily variable and, therefore, are, in some
cases, of little use for the identification of species.
The variability is particularly great in the species
of edible oysters {C. virginica, C. gigas, C. angulata,

and C. rhizophorae) that have a wide range of
distribution, thrive on various types of bottom,
and are tolerant to changes in salinity and turbidity
of water. Certain general relationships between
the shape of the oyster shell and the environment
are, however, apparent in C. virginica. Oj'sters
growing singly on firm bottom have a tendency
to develop round shells ornamented with radial
ridges and foliated processes (figs. 4, 15). Speci-
mens living on soft, muddy bottoms or those
which form clusters and reefs are, as a rule,
long, slender, and sparsely ornamented (figs.
19,21).

The thickness and strength of the valves of
C virginica are higldy variable. Shells of oysters
grown under unfavorable conditions are often
thin and fragile (Galtsoff, Chipman, Engle, and
Calderwood, 1947). Likewise, so-called "coon"
oysters from overcrowded reefs in the Carolinas
and Georgia are, as a rule, narrow and have light
shells (fig. 19). Heavy and strong shells are not
typical for any particular latitude. They can be
found on hard, natural bottoms throughout the
entire range of distribution of C. virginica. I have
in my collection shells from Prince Edward Island,
Cape Cod, Delaware Bay, Louisiana, and Texas
which in shape and strength of valves are in-
distinguishable from one another. Sometimes
the growth of shells in length (in anteroposterior
direction) equals or exceeds the growth in height.
Such specimens, one from Texas and one from the
waters of Naushon Island off the Massachusetts
coast, were found in stickv mud. As can be seen

18

FISH AND WILDLIFE SERVICE

0

Centimeters

Figure 18. — Four shapes of beaks on left valves of old oysters, C. virginica. 1 — narrow, short and almost straight;
2 — strongly curved to the posterior; 3 — of medium width, pointed forward; 4 — very broad and slightly curved to the
posterior.

from figure 22, the sliells are almost identical in
shape and size.

Oysters are frequently marketed under specific
brands or trade names such as Blue Points (fig.
15), Cotuits, Chincoteagues, and others which

imply the existence of local varieties different
in size and shape of shells. There is no e\adence,
liowever, to substantiate this claim. So-called
"Blue Points" characterized by round shape,
strong shell, and medium size may be found.

MORPHOLOGY AND STRUCTURE OF SHELL

19

UMBO

HINGE AXIS

WIDTH

Figure 19. — Several generations of oysters, C virginica,
growing vertically on muddy bottom of Altaniaha
Sound, Ga. Notice the very long and narrow beak of
the lowermost shell.

Figure 20. — Diagram showing the correct method of
measuring the height, length, and width of oyster shells.

for instance, in any part of the coast where
oysters grow singly on hard bottom and are not
crowded. As a matter of fact, in past years
"Blue Points" sold in retail stores actually were
taken from the Chesapeake Bay and North
Carolina. This is also true for "Cotuits" and
other popular brands.

That the shape of oysters cannot be associated
with any particular geographical location is best
shown by the fact that all the kinds represented
in trade, including long and narrow "coon"
oysters which are regarded as being typical for
the tidal areas of the South Atlantic States, are
found in various bays and estuaries of Cape
Cod, Mass. The only shell character that
appears to be associated with the geographical
distribution of the species is the pigmentation
of the interior surfaces of the valves. In North
Atlantic oysters the inner surface is unpigmented
or very lightly pigmented (outside of the place
of attachment of the adductor muscle), while
in South Atlantic and Gulf oysters the dark
brown or reddish pigmentaton of the valves is
more pronounced.

DIMENSIONS

Oysters {C. virginica) of marketable size usually
measure from 10 to 15 cm. (4 to G inches) in height;
depending on the place of origin an oyster of this
size may be 3, 4, or 5 years old.

As a rule, oysters do not stop growing after
reaching certain proportions but continue to
increase in all directions and, consequently, may
attain considerable size. Such old and very large
oysters are usually found on grounds undisturbed
by commercial fishing. The largest oyster in my
collection was found in the vicinity of Boothbay
Harbor, Maine. Its dimensions were as follows:

20

FISH AND WILDLIFE SERVICE

J_

_L

_L

_L

Centimeters

Figure 21. — Shells of C. gigas (left) and C. virginica (right) grown on soft, muddy bottom. Note the remarkable simi-
larity in the shape, size, and sculpture of the two species of oysters. The C. gigas was obtained from the northern
part of Puget Sound and the C. virginica from Georgia. The shells of the two species can be distinguished by the
absence of pigmentation of the muscle impression in C. gigas and by its lighter shell material.

height — 20.6 cm. (8.1 inches); height of left and
right beak — 5.5 cm. (2.1 inches) and 4.5 cm.
(1.75 inches) respectively; length of shell — 9.7
cm. (3.8 inches); maximum width (near the
hinge) — 6.5 cm. (2.6 inches). The total weight
was 1,230 g., the shell weighing 1,175 g., the meat
35.8 g., and the balance of 19.2 g., representing
the weiglit of sea water retained between the
valves. Apparently the largest oyster recorded
in American literature is the giant specimen from
the Damariscotta River, Maine, reproduced in
natural size by Ingersoll (1881, pi. 30, p. 32).
Tliis shell is 35.5 cm. (14.3 inches) in lieiglit and
11 cm. (about 4.4 inches) in length.

SHAPE OF SHELLS

The shells of many gastropods and bivalves are
spiral structures in which the convolutions of the
successive whorls follow a definite pattern. Tiie
spiral plan is frequently accentuated by ridges,

furrows, spines and nodides, or by pigmented
spots which repeat themselves with remarkable
regularity. A spiral structure is not restricted to
mollusk shells. As a matter of fact, it is very com-
mon throughout the animal and plant kingdom as
well as in architecture and art. Examples of a
great variety of spirally built organisms and
structures are given in the beautifully illustrated
books entitled "Spirals in nature and art" and
"Curves of life" (Cook 1903, 1914). As the title
of the second book implies. Cook is inclined to
attach some profound significance to the kind
of curves found in animal and plant forms. This
view, inherited from the philosophers of the 18th
and 19th centuries, considers the spiral organic
structures as a manifestation of life itself. The
influence of this philosophy persisted among some
scientists until the thirties of the present century.
It can be found, for instance, as late as 1930 in the
writings of a French physiologist, Latrigue (1930)

MORPHOLOGY AND STRUCTURE OF SHELL

21

Centimeters

Figure 22. — Two left shells of C. virginica grown on sticky mud. On the left side is the oyster from Karankawa Reef in
Matagorda Bay, Tex.; on the right is the oyster from Hadley Harbor, Naushon Island, near Woods Hole, Mass.
The dimensions of the Texas oyster are 1.3 by 11.5 cm. (.5.1 by 4.5 inclies) and for the Hadley Harbor oyster 15.5 by
14.5 cm. (6.1 by 5.7 inches).

who in the book, "Biodynamique generale," at-
tributes mysterious and not well-defined meaning
to the "stereodynamics of vital vorte.x." These
speculations contributed nothing to the under-
standing of the processes which underlie the for-
mation of shells and other organic structures.

In the earlier days of science the geometric
regularity of shells, particularly that of gastropods,
had been a favored object for mathematical
studies. Properties of curves represented by the
contours of shells, as well as those seen in horns,
in flower petals, in the patterns of distribution of
branches of trees, and in similar objects, were
carefully analyzed. An e.xcellent review of this
chapter of the history of science is given in a well-
known book "On growth and form" (Tliompson,
1942) in which the reader interested in nuithe-
matics and its application to the analysis of organic
forms will find many stimulating ideas.

Among the array of curves known in matlie-
matics, the kind most fretiuently encountered in
the shells of mollusks is the logarithmic or equi-
angular spiral (fig. 23). The latter name refers to
one of its fundamental characteristics, described
by Descartes, namely, that the angle between

tangent PG (fig. 23) and radius vector OP is con-
stant. Another property of tliis curve which may
be of interest tt) biologists is the fact that distances
along the ctu've intercepted by any radius vector
are proportional to the length of these radii.
D'Arcy Thompson showed that it is possible to
apply the mathematical characteristics of curves
to the interpretation of the growth of those shells
which follow the pattern of a logarithmic spiral.
According to his point of view, growtli along the
spiral contour is considered as a force acting at any
point P (fig. 23) which may be resolved into two
components PF and PK acting in directions per-
pendicular to each other. If the rates of growth
do not change, the angle the resultant force, i.e.,
the tangent PG, makes with the radius vector re-
mains constant. This is the fundamental property
of the "equiangular" (logarithmic) spiral. The
idea forms the basis of Huxley's (1932) hypothesis
of tlie interaction of two differential growth ratios
in tlie bivalve shells and also underlies Owen's
(1953) concept of tlie role of the growth compo-
nents determining the shape of the valves. [
.\nother important characteristic nf the growth "
of l)ivalves pointed out by Thompson is tliat

22

FISH AND WILDLIFE SERVICE

Figure 23. — Logarithmic or equiangular spiral. Expla-
nation in text.

increase in size is not accompanied by any change
in shape of the shell; the proportions of the latter
remain constant, and the shell increases only in
size (gnomonic growth). This general rule holds
true for many free-moving gastropods and bi-
valves. It is not, however, applicable to sessile
forms like oysters, in which the shape of the shell
changes somewhat with size, particularly at the
early stages of growth, and is greatly modified
by contact with the substratum upon which the
niollusks rest. The plasticity and variability of
attached forms are probably associated with their
inability to escape the eflfects of proximate
environment.

The contour of oyster shell may be either circular
(young C. virginica, 0. edulis) or elongated and
irregular. Spiral curvature may be noticed,
however, on a cross section of the lower (concave)
valve cut along its height perpendicular to the
hinge. The curve can be reproduced by covering
the cut surface with ink or paint and stamping
it on paper. The upper valve is either flat or
convex.

The curvature of bivalve shells is sometimes
called conchoid. The term may be found in

general and popular books dealing with bivalve
shells, but the author who introduced it in scien-
tific literature could not be traced. The Greek
word "conchoid", derived from "conch" — shell
and "eidos" — resembling or similar to, implies
the similarity of the cinwe to the contour of a
molluscan shell.

The curve is symmetric with respect to the 90°
polar axis (fig. 24). It consists of two branches,
one on each side of the fixed horizontal line CD
to which the branches approach asymptotically
as the curve extends to infinity. The curve,
known us conchoid of Nicomedes, is constructed by
drawing a line through the series of points P and
Pi which can be found in the following way: from
the pole 0 draw a line OP which intersects the
fixed line CD at any point Q. Lay off segments
QP=QPi = b along the radius vector OP. Repeat
the process along the radii originating from the pole
O and draw the two branches of the curve by
joining the points. The cmwe has tliree distinct
forms depending on whether "a" (a distance OQ
from the pole to the point of intersection of the
polar axis with the fixed line CD) is greater, equal
to or less than b. The formula of the cm-ve if
b<a, is r = a sec ^±b, where r is the locus of the
equation and sec 6 is secant of the vectorial angle B.
Sporn (1926) made a detailed mathematical
analysis of the conchoid curve and considered
that the curvatures of bivalve shells conform to
this geometrical type. Lison (1942) rejected this
conclusion as not supported by observations and
experimental evidence. He quite correctly stated
that Sporn 's work deals exclusively with abstract
mathematical analyses of curves which in reality
are Tiot those found in molluscan shells. If one
cuts a bivalve shell at any angle to the plane of
closure of the valves, one obtains the curved
lines of the two valves (fig. 25) which only remotely
resemble the conchoid of Nicomedes and touch

Figure 24. — Construction of the conchoid curve of
Xicomedes. Explanation in text.

MORPHOLOGY AND STRUCTURE OF SHELL

23

Figure 25. — Cross section of two valves of Cardium.
The similarity with the conchoid in figure 24 is super-
ficial.

each other at the ends. The two branches of
conchoid (C and D) join together only in infinity

(fig. 24).

Lison pointed out that the shape of the shell
may be considered as a whole series (ensemble)
of arches, the curvatures of which are described
by logarithmic spirals of the same parameter which
have a common origin at the umbo. The latter
is their common pole. The arches terminate at
the edge of the valve. The contour of the valve
edge, frequently called the "generating curve",

is usually confined to one plane parallel to the
plane of opening and closing of the shell.

Among many spirals that can be drawn on the
surface of a shell only one is completely confined
to a single plane. This spiral was called by Lison
the "directive spiral"; its plane is the "directive
plane" of the shell. A& other spirals which can
be easily noticed on the shell surface as ridges,
furrows, or as pigmented bands deviate to the
right or left depending on which side of the
directive plane they are located (fig. 26).

By mathematical analysis of the curved surfaces
of various bivalve species Lison arrived at the
general equation ^ of a valve. He observed that
by itself such an equation may not be helpful to
biologists unless it can be used for comparing the
shape of the individuals of the same species or in
maldng comparison between the different species.
Lison stated that in practice it is not necessary to
make the involved mathematical computations.
It is sufficient to compare certain "natural"
characteristics of shells, namely, the du-ective
plane described above, the plane of closure of
valves (or connnissure plane), and the angle of

3 General equation of a valve as given by Lison (1939) is as follows:
CT=(ropx; w=w„+«; z = ZoeP'* in whicli p is a constant and ao, wo, and z„
are the functions which express on cylindrical coordinates the form of tlie
free edge of the valve when the directive plane is located within the xy and
the origin of the coordinates is at the umbo. (Translation by Paul S.
Galtsoff.)

Figure 26. — Directive plane of scallop shell, Peclen, viewed from hinge end 2a, and from the broad side 2b. Tin.

arrows indicate the directive plane. (After Lison, 1939.)

24

FISH AND WILDLIFE SERVICE

incidence. The plane of closure of the valves
originates at the umbo and passes between the
edges of the two opposing valves when they are
closed and touching each other. The angle of
incidence, as defined by Lison, is the angle between
the plane of closure and the directive plane. In
round and symmetrical shells of scallops, pearl
oysters, and other bivalves the directive plane is
perpendicular to the plane of closure and the
angle of incidence is 90° (fig. 26). In the shells
of CanUiim orbita, the directive plane forms an
acute angle of 81 ° and is much smaller in elongated
shells such as Fimbria fimbriata and Trapezium
ohlomjnm. The comparison between the shells
can easily be made by recording the contours at
the free margins of the valves and determining
the angle of incidence.

To determine the shape of logarithmic spiral of
the valve the shell may be sawed along the direc-
tive plane (fig. 27) and the section oriented with
the umbo O at lower left. If Si and S2 are respec-
tive lengths of the two radii the value of para-
meter p can be computed by using the fundamental
equation of logarithmic spiral,

„ log. Si— log. S2

(logarithms in this equation are natural, to base e) .

In resume, Lison attempted to prove that the
form of the shell in which the generating curve is
confined to one plane is determined by three
conditions: (1) the angle of the dii'ective spiral,
(2) the angle of incidence, and (3) the outline of
the generating curve.

Further attention to the problem of the shape
and formation of the bivalve shell was given by
Owen (1953). In general he accepted Lison's
conclusions and stated that "the form of the valves
should be considered with reference to: (a) the
outline of the generative curve, (b) the spiral angle
of the normal axis, and (c) the form (i.e., plani-
spiral or turbinate-spiral) of the normal axis."
The normal axis is considered by Owen with
reference to: (1) the umbo, (2) the margin of the
mantle edge, and (3) the point at which the great-
est transverse diameter of the shell intersects the
surface of the valves. Thus, it can be seen from
this statement that Owen's "normal axis" does
not coincide with Lison's directive plane except
in bilaterally symmetrical valves (fig. 28). Ac-
cording to Owen's view, the direction of growth
at any region of the valve margin is the result of
the combined effect of three different components:

(a) a radial component radiating from the umbo
and acting in the plane of the generating curve,

(b) a transverse component acting at right angles
to the plane of the generating curve, and (c) a
tangential component acting in the plane of the
generating curve and tangentially to it. The
turbinate-spiral form of some bivalve shells is due
to the presence of the tangential component which
in plani-spiral shells may be absent or inconspicu-
ous. Likewise, the transverse component may be
greatly reduced or even absent in the valve.
Thus, from this point of view the great variety of
shell forms may be explained as an interaction of
the three components (fig. 29). Owen's point of

Figure 27. — Valve of a shell sawed along the directive a.xis
describes a plane logarithmic spiral. According to
Lison (1942). OM — radius vector; T — tangent;
0 — umbo; V — angle between the two radii.

poslenor

Figure 28. — Comparison of directive plane of Lison with
normal axis (Owen). A — shell not affected bj' tangen-
tial component; B — shell affected by tangential com-
ponent.

MORPHOLOGY AND STRUCTURE OF SHELL

25

view is basically similar to Huxley's l^vpothesis
(1932) of differential length growth and width
growth of moUuscan shells. Owen correctly
points out an error in Lison's interpretations that
the lines of equal potential activities involved in
the secreting of shell material at the edges of the
valves are parallel to each other. This is obvi-
ously not the case since all lines of growth of the
lamellibranch shell radiate from a common node
of minimum growth near the umbo. For this
reason the comparison of bivalves can be more
conveniently made by using radial coordinates as
has been shown by Yonge (1952a, 1952b).

The mathematical properties of shell surfaces
are of interest to the biologist because they may
provide clues to understanding the quantitative
aspects of the processes of shell formation. It can
be a priori accepted that any organism grows in
an orderly fashion following a definite pattern.
The origin of this pattern and the nature of the
forces responsible for laying out structural ma-
terials in accordance with the predetermined
plan are not known. The pattern of shell
structure is determined by the activities at the
edge of the shell-forming organ, the mantle. At
the present state of our knowledge it is impossible
to associate various geometrical terms which
describe the shape of the shell with concrete
physiological processes and to visualize the
morphogenetic and biochemical mechanisms in-
volved in the formation of definite sculptural and
color patterns. The solution of this problem will

be found by experimental and biochemical studies
wliich ma_y supply biological meanings to abstract
matliematical concepts and equations. Experi-
mental study of the morphogenesis of shells
oft'ers splendid opportunities for this type of
research.

GROWTH RINGS AND GROWTH RADH

Nearly 250 years ago Reaumur (1709) discovered
that shells grow by the accretion of material
secreted at their edges. Since that time this
important observation has been confirmed by
numerous subsequent investigations. The rings
on the outer surfaces of a bivalve shell, frequently
but incorrectly described as "concentric", rep-
resent the contours of the shell at dift'erent ages.
Rings are common to all bivalves but are partic-
ularly pronounced on the flattened shells of
scallops, clams, and fresh-water mussels. De-
pending on the shape of the shell, the rings are
either circular or oval with a common point of
origin at the extreme dorsal side near the umbo
(figs. 30 and 31). The diagrams clearly show
that the rate of growth along the edge of the
shell is not uniform. It is greater along the
radius, AD, which corresponds to the directive
axis of Lison, and gradually decreases on both

Figure 29. — Normal axis and the two growth components
in the shell of scallop. LS — plane perpendicular to the
plane of the generating curve; X — turning point of the
concave side of the shell shown at right; M and O — aux-
iliary radii; P — transverse component; R — radial com-
ponent; UY — normal axis. From Owen (1953).

Figure 30. — Diagram of a circular bivalve shell of the
kind represented in Perten, Anomia, and young C.
virginica. Radii extending from the umbo to the
periphery of the generating curve are proportional to
the rate of growth at the edge of a circular shell
Radius AD corresponds to the directive axis of Lison.

26

FISH AND WILDLIFE SERVICE

Figure 31. — Diagram of a shell of adult C. virginica.
Radii extend from the umbo to the periphery of the
generating curve. The principal axis AGF shows the
change in the direction of growth at G. The length of
radii is proportional to the rate of shell growth at the
edge.

sides of it along growth radii AC, AB, and Ad,
ABi.

Circular shells in C. virginica may be found only
in very young oysters (fig. 32a). Within a few
weeks after setting the shell becomes elliptical,
and as elongation (increase in height) continues
the principal vector of growth shifts to one side
(fig. 32b).

A series of curves noticeable on round shells
(fig. 32) clearly illustrate the differential rate of
growth along the periphery of the valve, whicli
increases in size without altering in configuration.
Tliompson (1942) found an interesting analogy
between tills type of growth, radiating from a
single focal point (the umbo), and the theorem
of Galileo. Imagine that we have a series of
planes or gutters originating from a single point
A (fig. 30) and sloping down in a vertical plane

MORPHOLOGY AND .STRUCTURE OF SHELL

at various angles along the radii AB, AC, ACi,
and ABi which end at the peripheiy of a circle.
Balls placed one in each gutter and simultaneously
released will roll down along the vectors B, Bi, C,
Ci, and D. If there is no friction or other form
of resistance, all the balls will reach the peripheiy
at the same time as the ball dropping vertically
along AD. The acceleration along any of the
vectors, for instance, AB, is found from the
formula t^ = 2/g AD where t is time and g is
acceleration of gravity.

A similar law, involving a more complex
formula, applies to cases in which the generating
curve is nearly elliptical, for instance, in the
shells of adult oysters. The rate of growth at
different sectors of the periphery of the shell
obviously has nothing to do with the acceleration
of gravity, but the similarity between the length
of the radii which represent the rate of growth
along a given direction of the shell and the accelera-
tion along the vectors in the theorem of Galileo
is striking. It appears reasonable to expect that
the Galileo formula may be applicable to the
physiological process taking place near the edge
of the valve. One may assume, for instance, that
the rate of physiological activities is afTected by
the concentration of growth promoting sub-
stances or by enzymes involved in the calcification
of the shell and that these factors vary at different
points of tlie mantle edge in conformity with
Galileo's formula. Experimental exploration of
the possibilities suggested by mathematical paral-
lelism may be, therefore, profitable in finding the
solution to the mysteiy of the formation of shell
patterns.

CHANGES IN THE DIRECTION OF
PRINCIPAL AXES OF SHELL

Tlie principal axes of shells of C. virghvica are
not as permanent as they are in clams, scallops,
and other bivalves in which the shape of the
valves remains fairly constant and is less afl'ected
by environment than in the oyster. The plasticity
of oysters of the species Crassasfrea is so great
that their shape cannot be determined geometri-
cally (Lison, 1949). This inability to maintain
a definite shape is proba])ly the result of the
sedentary living associated with complete loss
of the power of locomotion.

In some species of oysters the shells are circular
or nearly circular. In such cases the ratio of
the heiglit of tlie valve to its length is ecjual to

27

a

0

_L

0.5
Centimeters

1.0

Figure 32. — Two small C. virginica growing attached to tar paper. Ma.ximum dimension of shell: a — n.S5 cm.

b — 1.0 cm. At b the principal axis curves to the left.

1.0, as, for instance, in C. rimilaris (fig. 8) and
0. (Alectryonia) megodon Hanley (fig. 3) (Olsson,
1961). Oysters of the latter species from the
Pacific Coast of Central and South America grow
singly, in vertical position, cemented to the rocks
by their left valves. The specimens 1 collected
on Pearl Islands, Gulf of Panama, measured 17
to IS cm. in height and 16 to 17 cm. in length.
The European flat oyster, 0. edulis (fig. 9) usually
forms rounded shells in which the length exceeds
the iieight. Small, noncommercial species, O.
sandwicliensifi of the Hawaiian Islands and 0.
mexicana from the Gulf of Panama, are almost
circular witli the tendency to extend in lengtli
rather than in height. Crowded conditions under
which these species thrive attached to rocks in a
narrow tidal zone greatly obscure and distort tlie
shape of their shells.

Small C. virginica growing singly on fiat surfaces
without touching each other are usually round
(fig. 32). In a random sample consisting of 100
single small oysters (spat about 6 weeks old)
varying from 5 to 15 nun. in height and growing
on tar paper, the height/length ratio varied from
0.6 to 1.2. Xearly half of them (49 percent) were
perfectly numd (H/L ratio=l); in .30 percent the
ratio was less than 1 ; and in 21 percent the length
exceeded the height.

In snuill single oysters less than 10 mm. in
height the principal (normal) axis of growth, is
clearly marked. All other radii synunetrically
oriented on both sides of the principal axis are
indicated by the pigmented bands on the surface
of the shell. The newly deposited shell, dis-
cernible at the periphery of the oyster, forms a
band which is wider at the ventral edge of the
shell and slightly narrows anteriorly and pos-
teriorly (fig. 32a). With the growth of the oyster
its principal axis is shifted to the side, curves, and
is no longer confined to one plane. The curvature
of the valve becomes a turbmate-spiral. Grad-
ually the oyster becomes slightly oval-shaped
and asymmetrical.

The change in the direction of tlie principal
axis of growth is not associated with the environ-
ment since it takes place only in some of the
oysters growing under identical conditions. Oc-
casionally oysters are formed in which the pig-
mentation along the principal axis is so pronounced
that the dark band which marks its position may
be mistaken for an artifact (fig. 33) while the
secondary axes are not visible. The shells of
adult C. nnjinica usually curve slightly to the
left (if tiie f)yster is placed on its left valve and
viewed from above). Frequently, however, in-
verted specimens are found in whicli the growth

28

FISH AND WILDLIFE SERVICE

Centimeters

[CURE 33. — Principal axis of growth of a C. virginica
from Chatham, Mass., is deeply marked by a pigmented
band.

has shifted into the opposite direction (fip;. 34).
The "norniid" oyster (the right side of tlie figure)
is curved to the left while in the inverted specimen,
shown on the left of the figure, the shell curves
to the right. Such "right-handed" oj'sters are
probably common in all oyster populations since
they were found in Texas, Chesapeake Bay,
Narragansett Baj', and Great Bay, X.H. In
every other respect the inverted specimens are
normal and had typicaUv cupped left valves with
well-developed grooved beaks. There is no evi-
dence that inversion was caused by jnechanical
obstruction or some unusual position on the
bottom.

Complete inversion in bivalves was described
by Lamy (1917) for Lncina, Chnma. and several
species of the subgenus Goodallia (fain. Astartidae).
It consisted in the appearance of structures, typical
for the right valve, on the left valve and vice
versa. In the case of C. inrginica the structural
elements remain unaffected and the inversion is
limited to the contours of the valves.

MORPHOLOGY AND STRUCTURE OF SHELL
733-851 0—64 3

The once established principal axis of growth
does not always remain unchanged. Occasionally
old oysters are found in which the direction of
growth had undergone sudden changes of about
90°. The change shown in figure 35 took place
when both oysters were about 6 to 7 years old.

The instability of the principal axis of growth
may be even more pronounced. My collection
has an oyster {C. inrginica) found on the banks of
a lagoon near Galveston, Tex., in which the princi-
pal axis, clearly indicated by pigmented bands on
the surface of the valves, changed its direction at
the end of each growing period. The resulting
zigzag line is clearly visible in the specimen (fig.
36).

DIMENSIONAL RELATIONSHIPS OF
SHELL

Shape of a bivalve sliell is often expressed as
a ratio between its height and length or by some
other numerical index. Lison (1942) pointed out
that the shape of an oyster shell cannot be ex-
pressed in precise geometrical terms, presumably
because of its great variability. The "index of
shape" determined as a ratio of the sum of height
and width of a shell to its length was used by
Crozier (1914) in studying the shells of a clam,
Dosrnia discus. For the moUusks ranging from 2
to 7 cm. in length collected near Beaufort, N. C.
this index varied from 1.24 to 1.28 indicating that
the increase of the species in height and width was
directly proportional to the increase in length.
Such regularity is not found in tlie sliells of adult
C. inrginica taken at random from commercially
exploited bottoms. For tlie entire range of
distribution of this species in the Atlantic and
Gulf states the index of shape varied from 0.5 to
1.3. The histogram (columns in figure 37) shows
nearly normal frequency distribution with the
peak of frequencies at 0.9. No significant dif-
ferences were found in the index of shape in the
nortliern and southern populations of oysters
examined separately. Tlie boundary between the
two groups was arbitrarily drawn at the Virginia-
North Carolina line. The two cm'ves connecting
the frequency points on figure 37 indicate that in
the southern population the index of shape ex-
tends from 0.5 to 1.3, while in the northern oysters
it varies from 0.6 to 1 .2. The difference is probably
not very significant, but it may be due to a greater
percentage of wild oysters on commercially ex-
ploited natural bottoms of the southern states.

29

_L_

Centimeters

Figure 34. — Left valves of the two large C. inrginica from Narragansett Bay, R.I. On the right is a "normal" oyster;
its shell curves to the left. On the left side is an inverted oyster; its shell curves to the right.

Most of the oysters from tlie North Atlantic and
Chesapeake states were taken from bottoms on
which oysters are regularly planted for cultivation.
There are no significant differences in the mean,
mode, and median of the two groups (table 1).
Contrary to the conditions found by Crozier in
Dosinia discus, the "index of shape" of C i^ir(iij}ica
is highly variable.

SHELL AREA

Information regarding the approximate area
of an oyster shell of known height may be useful
to oyster growers who want to determine in ad-
vance what percentage of tlie bottom area set

Table 1. — Index of shape (heiiihl+ width) of oi/siers taken by

length
commercial fishery

Locality

Mean

Standard
deviation

Mode

Median

Northern grounds

0.87
0.87

0.05
0.02

0.94
0,94

0.09

0.9

aside for planting will be covered by oysters of
known size. Since the oystermen usually know
the number of oysters of various sizes needed to
make up a bushel, the information given below
may be used in determining in advance whether
the area of the bottom is sufficient to provide space
for their additional growth.

It is self-evident that the area of the valve in-
creases proportionally tc the increase in its linear
dimensions. For determining the area a piece of
thin paper was pressed against the inner surface
of the right (flat) valve and the outlines were
drawn with pencil. The area was measured with
a planimeter. The outlines of small shells were
placed over graph paper and the number of milli-
meter squares counted.

The relationship between the height and shell
area (fig. 38) is represented by an exponential
curve of a general type y=ax'' which fits many
empiriciil data. The y in the fonnula is the shell
area, and the x is the height. The parabolic
nature of the curve is demonstrated by the fact

30

FISH AND WILDLIFE SERVICE

_l_

Centi meters

Figure 35. — Two upper (right) shells of old C. virginica from Chesapeake Bay (left) and Matagorda Bay, Tex. (right).
The direction of growth changed suddenly about 50° to the left in the Chesapeake oyster and about 75° to the right in
the Texas oyster.

that the log/log plot (fig. 39) fits a straight line.
The numerical values of factors a and b were
found to be equal to 1.25 and 1.56 respectively.
The formula reads, therefore, y=1.25x'^^ As
a convenience to the reader who may be interested
in finding directly from the curve the average area
occupied by a shell of a given height, the data
computed from the equation can be read from the
curve in figure 38. The measurements are given
both in centimeters and inches. The data refer
to the random collection of live oysters from the
coastal areas between Prince Edward Island,
Canada, and the eastern end of f^ong Island
Sound (table 2).

The relationship between the height and area
ol the upper valve of C. virginica is in agreement
with the findings of other investigators (New-
combe, 1950; Nomura, 1926a, 1926b, 1928) wlio
concluded that in several marine and fresh-water
bivalves and gastropods the dimensi'inal relation-

ships can be adequately e.xpressed by the formula
of heterogenic growth, y = bx''. According to No-
mura's (1926a) interpretation of the growth of the
clam Meretrix meretrir, the constant b in this
fornmla represents the eff'ect of the environment
while k is a factor of differential growth. No-
mura's conclusions may be applicable to otlier bi-
valves, and if confirmed by further studies tliis
method may become useful for quantitative de-

Table 2. — Height and shrlt area of northern oijsters com-
puted 1)1/ ii.itni/ thi eejiiation ;/ = l.Sox'-'''^

Height

.\rea

Cm.

InctLex
1.97
2.30
3.15
3.94
4.72
.■i. .11
H,30
7.09
7.87

1.5.4
20. .5
32.3
4.5.4
CO. 3
7fi. 7
94. .5
113. .5
133. S

/«.■■

fi ___

s

3. 1.S

in

7 04

12..

9.3.5

14

11.9

Ifi . .

14 0

18

17.fi

20

■'0 7

MORPHOLOGY AND STHrCTIRF, OF SHELL

31

Centimeters

Figure 36. — Shell of an adult C. virginica showing periodic
changes in the direction of the principal axis of growth.
Note the zigzag line of pigmented bands in the middle of
the valve. Actual dimensions: S.5 by 6 cm. (3.25 by 2.5
inches).

terminations of tlie effect of local conditions on
growth and shape of shells.

CHALKY DEPOSITS

The glossy, porcelainlike inner surlacc of an
oyster shell is frequently marred hv irregularly
shaped white spots wliicli consist of soft and
porous material of different appearance and text-
ture than the surrounding shell suhstance. These
areas are called "chalky deposits". They are
very common in C. nrginica and 0. edulis. Since
the first record of their presence in edible oysters
made by Gray (1833) they have been mentioned
frequently by many biologists. Recent review
of the literature on the sul)ject is given l)v K<ir-
ringa (1951).

Tlie e.xact location of chalky deposits is <if
interest since some si)eculations regarding their
role and origin are basefl on the position they occu-
py on the shell. Orton and Amirtlialini;aiii
(1927) assumed that chalky material is foiined in
the i)laces where the mantle loses contact uilli
the shell. No experimental evidence in support

I

UJ

m

0 WWsA/v-

05 0.6 0,7 0.8 0,9 tO I I \.2 \ i
INDEX OF SHAPE OF C, virginica

Figure 37. — Histogram of the distribution of the index of
shape (height + width) of shells of C. virginica from the

length
Atlantic Coast. Frequency distribution of the index of
Nortli Atlantic oysters (open circles) and South Atlantic
oysters (points) are shown by two separate curves.

of this explanation was presented by the authors
or by Ranson (1939-41), who fully accepted tlie
theory without making additional studies and
stated positively that chalky deposits are formed
wherever there is a local detachment of the mantle
from the valve.

( 'onsidering the possibility that the mantle may
be more easily detached from the valve if the
oyster is placed with its lower (cuplike) side upper-
most, K<irringa (19.")!) made a simple field ex-
periment. In one tray he placed 25 metlium
sized (>. t'l/iilis in their normal position, with their
cupped valves undermost; the other tray contained
an equal number of oysters resting on their flat
valves. At the end of the growth season he oli-
served no significant differences in the deposition
of shell material in the oysters of the two groups.

To determine whether chalky deposits are
foi-mcd in |)laces of partial detachment of the
mantle, 1 performed the following e\])eriment:
Small pieces of thin plastic about 1 cm.'' were bent
as shallow cups and introduced between tlie
mantle and the shell of ('. rircjirnca. In 10
oysters the cups were inserted with the concave
side facing the mantle, in anotiier 10 oysters the
position of the cup was reversed, i.e., the concave
side faced the valve. The oysters were kept for

32

FISH AND WILDLIFE SERVICE

HEIGHT IN INCHES

6 8 10 12 14 16 18 20

HEIGHT IN CENTIMETERS

55 days in running sea water in the laboratory.
During this time they fed actively and had con-
siderable shell growth along the margin of the
valves. After their removal from the shells the
cups were found to be covered with hard calcite
deposits on the sides facing the mantles. No
chalky material was found on cups or on the sur-
face of valves adjacent to the area of insertion.
On the other hand, conspicuous chalky areas
were formed along the edge of the shell in places
where the opposing valves were in close contact
with each other (fig. 40). It is clear from these
observations that the detachment of the mantle
from the inner surface of the shell does not result
in the deposition of chalky material and that such
deposits may be laid in the narrowest space of
shell cavity where the two valves touch each other.
Suggestions that challiy deposits result from
secondary solution of calcium salts of the shell
(Pelseneer, 1920) or that their formation is
somehow related to the abundance of calcareous
material in the substratum (Ranson, 1939-41,

Figure 38. — Shell area in cm.' plotted against height of
shells in cm. Inch scales are on top and on the right.

100
90
80
70
60

'% 50
o

< 40
UJ

q:
<

30

20

1

J 1 I I I I I I I I I I I I III

4 5 6 7 8 9 10

HEIGHKcm)

20

30

Figure 39. — Logarithmic plot of shell area against shell
height.

zA. d.

Centimeters

Figure 40. — Chalky deposits (ch. d.) on the newly
formed shell at the edge of the valve, and near the
muscle attachment.

MORPHOLOGY AND STRUCTURE OF SHELL

33

1943) are not supported by evidence. The inner
surface of bivalve shells may become slightly
eroded due to the increased acidity of shell liquor
when the mollusk remains closed for a long time,
but the erosion is, however, not localized; it
occurs over the entire shell surface. As to the
effect of the abundance of lime in the substratum
on the formation of chalky deposits, one must
remember that the concentration of calcium salts
dissolved in sea water is fairly uniform and that
calcium used for building of shells is taken
directly from the solution (see p. 103). Under
these conditions the abundance of calcium car-
bonates in bottom deposits cannot have any
effect on the formation of shell.

Chalky areas of shell do not remain unchanged.
They become covered by hard substance and in
this way they are incorporated in the thickness
of the valves (fig. 41).

Korringa's theory (1951) that the oyster
deposits chalky material ". . . when growing
older, in its efforts to maintain its efficiency in
functioning" and that ". . . where possible the
oyster always uses soft porous deposits when
cjuite a lot of shell volume has to be produced . . ."
is based on the assumptions: (1) that chalky
deposits most frequently develop in the area
posterior to the muscle attachment, (2) that the
layers of chalky material are more numerous in
cupped than in flat oysters, (3) that in the area
of the exhalant chamber (in the postero ventral
quadrant of the shell) the oyster attempts to
decrease the distance between the two valves by
rapid deposition of shell material, and (4) that
chalky material is used by the oyster "as a measure
of economy, as a cheap padding in smoothing out
the shell's interior." The validity of these

assumptions with reference to C. piniinica was
tested by studing the relative frequency of the
occurrence of challvy deposits on the left and
right valves and by estimating the extent of
these deposits in different parts of the valves.
Tlie collection of shells studied for this purpose
compi-ised several hundred adult specimens from
various oyster beds along the Atlantic and Gulf
coasts. For determining the distribution of
chalky areas the inner surface of the valves was
arbitrarily divided into four quadrants shown in
figure 42 and designated as follows: A — dorso-
posterior; B — dorsoanterior; C — ventroposterior;
and D — ventroanterior. The following five classes
corresponding to the degree of the development
of chalky deposits in each quadrant were
established:

No deposits within the quadrant 0

1 to 25 percent of the area covered with

deposits 1

26 to 50 percent of the area covered witli

deposits 2

51 to 75 percent of the area covered with

deposits 3

76 to 100 percent of the area covered with

deposits 4

With a little practice it was easy to select the
correct class by visual examination. The first
question was whether there is an.y difference in
the frequency of occurrence and extent of chalky
deposits on right and left valves. For this
purpose the entire surface of the valve was exam-
ined and classified. Chalky deposits were
found as often on the right as on the left valve
of C. inrginica. This is shown in table 3 which
suniniarizps tlie observations made on 472 shells
collected at random at oyster bottoms along the

_L

J_

_L

Centimeters

Figure 41. — Left valve of an old C. virginica cut along the principal axis of growth. Chalky areas on both sides of the
hypostracum (dark platform for the attachment of the adductor muscle) are enclosed in the thin layers of hard
crystalline material. Hinge on the right. Xatural size.

34

FISH AND WILDLIFE SERVICE

Figure 42. — Four arbitrary quadrants of the inner surface
of shell used for estimating the distribution and extent
of clialky deposits.

Atlantic Coast from Long Island Sound to Georgia.
Nearly one-half of the total number of valves
examined (48 percent of left and 53 percent of
right valves) were free of the deposits. (The
percentage of oysters without chalky deposits
was not determined because in many shells of the
collection the valves had separated and could
not be arranged in pairs.) In about 25 percent
of the total number of shells the chalky deposits
cover less than one-quarter of the valve area.
Larger deposits occurred in diminishing number
of shells; those covering more than three-quarters
of available space (class 4) comprised less titan
3 percent of the total number examined.

There was no particular area on the valve
surface where chalky deposits were formed more
often than in any other place. The differences
in the frequency of their occurrence in different
quadrants of a valve were not significant.

In 0. edulis, according to Korringa, chalky
deposits form more often in deep (cupped) shells

Table 3. — Percent of valves of C. viruinica with chalky
deposits

Item

Area of valve covered by chalky deposits

Class 1

(1-25

percent)

Class 2

(26-50

percent)

Class 3

(51-75

percent)

Class 4
(76-100
percent)

25.9
24.9

13.0
12.1

9.8
8.4

2.8

Right valve

1.5

MORPHOLOGY AND ST

RUCTU

RE OF

SHELL

than in flat ones and can be found principally in
the area in front of the cloaca, cjuadrant V accord-
ing to our terminology. i\o such differences in
the place of formation or in the type of shell
could be observed in C. virginica.

From the observations on oysters of Prince
Edward Island, Medcof (1944) concluded that
chalky deposits are normal parts of shells and
that they have "functional importance" in pre-
serving "a size relationship between meats and
shell cavity" and hi regulating "the curvature of
the inner face of the shell throughout the oyster's
life." There could be no argument about the
first conclusion that chalky deposits are normal
parts of the oyster shell. The fact that they
appear during the first weeks of the oyster's life
confirms this statement. The second conclusion
that they preserve the curvature of tlie shell is
unpossible to prove without careful study of a
large number of shells. In comparing the con-
tours of the shells of New England and Chesapeake
Bay oysters with and without chalky deposits,
I failed to notice any significant difference between
the two groups.

Japanese investigators (Tanaka, 1937, 1943)
found great variability in tlie distribution of
chalky deposits in C (jigas and C. futamiensis.
Large porous areas may be found in the shells
of these species near tlie anus, in front of the
labial palps, or near the gonads. There seems
to be no evidence that they occur primarily in
one particular place of the valve. These obser-
vations agree with my observations on C. rirginica.

CHAMBERING AND BLISTERS

The French word "chambrage" or chambering
has been used by European biologists to describe
shallow cavities, mostly in the cupped valves of
0. edulis. The cavities are usuallj' filled with sea
water and putrified organic material. In the
museum specimens these spaces are dry and filled
with air. Sometimes only one chamber is found,
but occasionally an entire series of cavities may
be present. The chambers may be invaded by
tube-forming annelids living in the oj'ster (Houl-
bert and Galaine, 1916a, 1916b). The successive
layers of shell material in the chamber are not in
contact with each other but surround an empty
space. This gives the impression that the body
of the oyster had shrunk or retracted and occupies
only a small portion of shell space. This view is
generally accepted by European oyster biologists

35

(Korringa, 1951 ; Orton, 1937; Orton and Amirtlm-
lingain, 1927; Worsnop and Orton, 1923), wlio
agree that chambering is caused by the shrinkage
of the body, withdrawal of shell-forming organ,
and deposition of partitions. Salinity changes
were suggested by Orton as one of tlie principal
causes of chambering, and shrinkage due to
spawning was also considered by Korringa as a
probable factor. These conditions have not been
reported for C. mrginica. I did not find any
evidence that chambers or blisters in the American
oyster are associated with shrinkage or other
body changes.

It is interesting to add that some taxonomists
of the middle of the past century (Gray, 1S33;
Laurent, lS39a, lS39b) were so puzzled by the
presence of chambers that they compared cham-
bered oyster with Nautilus and even suggested
the possibility of some family relation between tlie
latter genus and Ostrta!

An interesting sliell structure consisting of a
series of cluimbers near the hinge end is found in
the Panamanian oyster, 0. iridescens. The loca-
tion of chambers and the regularity at wiiich tiiey
are formed as the shell grows in height can be seen
in figure 43 representing a longitudinal section of
the valve made at a right angle to the hinge.
This type of chambering is obviously a part of a
structural plan of tlie shell and is not a result of an
accidental withdrawal of the oyster body or of an
invasion by commensals. Arch-forming septae
of the chambers apparently contribute to the
strength of the hinge and at the same time require

relatively small amounts of building material.
Wliat advantage 0. Iridescens obtains from this
type of structure is of course a matter of specu-
lation.

Ciianibers found in C. rirgiiiica consist of
irregular cavities containing mud or sea water.
Sucli formations are called blisters. Blisters can
be artificially induced b}' inserting a foreign
object between the mantle and the shell (see p. 105).
They are also caused by the invasion of shell
cavity by P(di/d<ira (see p. 422) or by perforations
of the siiell by boring sponges and clams (p. 420).

STRUCTURE OF SHELL

For more than a Imndred years the structure of
the molluscan sliell was an object of research by
zoologists, mineralogists, and geologists. Several
reviews of the voluminous literature (Biedermann,
1902a, 1902b; Bciggild, 1930; Cayeu.x, 1916; Haas,
1935; Korringa, 1951; Schenck, 1934; Scldoss-
berger, 1.S56) deal with the problem from different
points of view. Recently these studies have been
extended by the use of X-ray and electron micro-
scope. The methods, especially those of electron
microscopy, opened entirely new approaclies par-
ticularly witli reference to the structure of the
organic constituents of the shell (Gregoire, 1957;
Gregoire, Duchateau, and Florkin, 1950, 1955;
Watabe, 1954).

Terminology of molluscan shells is somewhat
confusing depending whether the emphasis is
placed on morphological, crystallographical, or
mineralogical properties. The names of different

0

J I

Centimeters

Figure 43. — Shell of (>. iridrsrcrm put at right angle to the hinge. Note a series of empty chambers at the hinge area.

Specimen from the Gulf of Panama.

36

FISH AND WILDLIFE SERVICK

layers of shell described in this chapter are those
which are found in more recent biological publi-
cations (Korringa, 1951; Leenhardt, 1926).

The shell of the oyster consists of four distinct
layers: periostracum, prismatic laj-er, calcite-
ostracum, and hypostracum. The periostracum
is a film of organic material (scleroprotein called
conchiolin), secreted by the cells located near the
very edge of the mantle. The periostracum is
very poorly developed in C. virginica and cannot
be found in old shells. It covers the prismatic
layer which can be best studied by removing from
the edge of an oyster a small piece of newly formed
shell. Microscopic examination reveals that the
prismatic layer is made of single units shown in
figure 44. Each prism consists of an aggregate of
calcite crystals (Schmidt, 1931) laid in a matrix
of conchiolin which after the dissolution of mineral
constituents in weak hydrocliloric acid retains the
general configuration of the prisms (fig. 45). The
double refraction of the walls of empty prisms is
pronounced and causes slight iridescence notice-
able under the microscope. In a well-formed

0

Millimeters

0.5

Figure 44. — Prismatic layer at earlier stages of calcifica-
tion. C. virginica.

MORPHOLOGY AND STRUCTURE OF SHELL

layer the prisms are wedge-shaped and slightly
curved (fig. 46). Conchiolin adiiering to the
prisms can be destroyed by boiling in potassium
hydroxide solution and the prisms separated
(Schmidt, 1931). Their shape and size are very
variable.

The optical axes of the prism are, in general,
perpendicular to the plane of the prismatic layer,
but in places they are irregularly inclined toward
it.

Calcite-ostracum, called also a subnacreous
layer (Carpenter, 1844, 1847), makes up the major
part of the shell. The layer consists primarily of
foliated sheets of calcite laid between thin mem-
branes of conchiolin. The separate layers are
irregularly shaped with their optical axes in ac-
cidental position (B0ggild, 1930). In a polished,
transverse section of the shell of C. virginica the
folia are laid at various angles to the surface
(fig. 47). This layer is frequently interrupted by
soft and porous chalky deposits (upper two layers
of fig. 47) which appear to consist of amorphous
material. It can be shown, however, that chalky
deposit is formed by minute crystals of calcite
oriented at an angle to the foliated lamellae of the
hard material.

Hypostracum is a layer of shell material under
the place of the attachment of the adductor muscle.
In the shells of C. virginica the layer is pigmented
and consists of aragonite (orthorhombic calcium
carbonate, CaCOs).

For many j^ears oyster shells were considered to
be composed entirely of calcite (B0ggild). Re-
cently Stenzel (1963) has discovered that on each
valve of an adult f. virginica aragonite is present
as padding of the muscle scar, in the imprint of
Quenstedt's muscle, and in the ligament.

As the oyster grows the adductor muscle in-
creases in size and shifts in the ventral direction.
The new areas of attachment become covered
with aragonite while the older, abandoned parts
are overlaid with the calcite. The progress of
the muscle from hinge toward the ventral side can
be clearly seen on a longitudinal section of the
shell where it can be easUy distinguished by its
darker color and greater hardness of the secreted
material (fig. 48).

ORGANIC MATERIAL OF THE SHELL

After the removal of mineral salts of the shell by
weak acids or by chelating agents, such as sodium
versenate, the insoluble residue appears in the

37

0 0.3

Millimeters

FiouRE 45. — Photomicrograph of a thin pioco of prismatic layer after the dissolution of calcium carbonate in weak acid
C. virginica. The walls retain the shape of the prisms and are iridescent.

38

FISH AND WILDLIFE SERVICE

t

Millimeters

"^.5

Figure 46. — Cross section of a piece of young shell of C.
virginica (mounted in bakelite and ground on a glass
wheel with carborundum, about 80 x). Periostracum
(top line), prismatic layer (middle), and calcite-ostracum
(lower).

form of thin, homogenous sheets of organic material
kept together like pages of a book. Tliis sub-
stance, discovered in 1S55 by Fremj^ is known as
conchiolin. The name is applied to the organic
material insoluble in water, alcohol, ether, cold
alkaline hydroxides, and dilute acids. In the
literature it appears also under the names of
conchin, periostracum, epidermis, and epicuticuhi.
Conchiolin is a scleroprotein, the structural for-
mula of which has not yet been determined. The
elementary analysis of conchiolin of 0. edulis
(Schlossberger, 1S56) is as follows; H, 6.5 percent;
C, 50.7 percent; N, 16.7 percent. Wetzel (1900)
found that conchiolin contains 0.75 percent of
sulfur and Halliburton (quoted from Haas, 1935)
assigned to it the following formula: C30, H4g, Ng,
0,1, which also appears in the third edition of
"Hackh's chemical dictionary" (Hackh, 1944).
Similarity of conchiolin to chitin - leads many
investigators to an error in ascribing cliitinous

' composition to structures which were found in-

; soluble in alkaline hydroxides and dilute acids.

i Thus, the presence of chitin was reported in the
shell and ligament of Anodonta, Mya, and Pecten

'• (Wester, 1910). The application of the Schulze's

test for chitin (intense violet coloration after
treatment for 24 hours in diaphanol [clilorodioxy-
acetic acid], followed by a solution of zinc cldoride
and iodine), does not confirm these findings (Lison,
1953).-'

To the naked eye and under the light micro-
scope the conchiolin appears as amorphous, viscous
and transparent material which hardens shortly
after being deposited. Using the electron micro-
scope technique, Gregiore, Duchateau, and Florkin
(1955) found that the conchiolin of gastropods and
bivalves consists of a fine network with many
meshes of irregular shape and variable dimensions.
This is, however, not the case in oyster shells.
Conchiolin of the genus Ostrea lacks meshes and
under the electron microscope is of uniform ap-
pearance (personal communication by Gregoire).

Cross sections of decalcified shells of C. vircjinica
show a distinct difference between the staining
properties of the conchiolin of the prismatic and
calcite-ostracum layers. On the cross sections of
shell shown in figure 49 the two parts can be
recognized by the typical foliated appearance of
the calcite-ostracum and the meslilike structure
of the prismatic layer. In the preparation stained
with Malloiy triple dye the organic matter of
the walls of the prisms are stained reddish-brown
while the foliae of the calcite-ostracum are bluish.
Differential staining indicates the dift'erence in
the chemical composition of the two parts.

The amount of conchiolin in the oyster shell was
studied by several investigators. As early as
1817 Brandes and Bucholz estimated that organic
material of the shell constitutes about 0.5 percent
of the total weight. Schlossberger (1S56) found
6.3 percent of organic matter in the prismatic
layer of the oyster but only from 0.8 to 2.2 percent
in the calcite-ostracum. According to Douville
(1936), the albuminoid content of the oyster shell
is 4.8 percent.

According to the determinations made by A.
Grijns for Kt)rringa (1951), the conciiiolin content
of the prismatic layer of (). edulis varied from 3.4
to 4.5 percent against the 0.5 to 0.6 percent in the
calcite-ostracum. The conchiolin content was
calculated from the percentage of X (by Kjeldahl
method) nmltiplied by 6.9. Tlie results of my
determinations of the wciglit of organic material

* Inasmuch as the same reaction is obtained with cellulose and tunicine,
additional tests should he made using Lupol solution and 1 to 2 per cent
sulphuric acid (Ii2S04). With this test chitin is colored hrown. while cellu-
lose and tunicine are blue.

MORPHOLOGY AND STRUCTURE OF SHELL

39

0 0.3

Millimeters

Figure 47. — Cross section of the shell of adult C. inrginica embedded in bak(-lite and polished on a glass wheel with carbo-
rundum. Two upper layers consist of clialky deposits.

after decalcification of the calcite-ostracum of
C. virginica shells from Long Island Sound and
Cape Cod waters are in agreement with those
given for 0. edulis. The content of conchiolin in
my samples varied from 0.3 to 1.1 percent with
the mode at 0.6 percent. For these analj'ses 23
pieces of shell were taken from 16 adult oysters
not damaged by boring sponge. The samples
varied in weight from 0.5 to 15 g.

Higher percentage of conchiolin in tlie prismatic
layer may be expected because this layer represents

the new growth of shell whicli lias not yet com-
pletely calcified.

The role played by conchiolin in the deposition
of calcium salts in the form of calcite or aragonite
presents a very interesting problem which has not
yet been solved. Recent electron microscope
studies of pearl oyster shells made by Gregoire
show tliat the organic material in which aragonite
crystals are laid (Gregoire, Duchateau, and
Florkin, 1950) is arranged as a series of bricklike
structures. No such arrangement has been de-

40

FISH AND WILDLIFE SERVICE

X

_L

Centimeters

Figure 48. — Left valve of 0. (Alectryonia) mcgodon cut along the principal axis of growth. Ilypo.stracum (dark .striated
layer) forms a pronounced platform for the attachment of the adductor muscle, and can be traced to its original position
in the young oyster (right). Chalky deposits are regularly arranged between the layers of calcite. Also see fig. 41.

scribed for calcite shells. Present knowledo;e of
the cliemistry of tlie org;anic constituents of tlie
sliell is inadequate. It seems reasonable to
assume that conchiolin like other proteins is not
a single chemical substance common to a large
number of firganisms, but that it differs specifi-
cally from animal to animal and may even vary
in the different parts of the same shell.

The analysis of amino acids obtained by hy-
drolysis f)f conchiolin prepared from decalcified
shells showed (Roche, Ranson, and Eysseric-
Lafon, 1951) that there is a difference in the shells
of the two species of European oysters, O. edulis
and f. angulata (table 4).

Table 4. — Amino acids from the conchiolin of two species
nf oysters

[In part.s of 100 parts of protein according to Roche, Ranson. and Eysseric-
Lafon (1951)1

Amino acids

CrassostTea
angulata

Ost
edu

rea
lis

Ar^inine

0.45

2 90

0. fiS

Lysine -

3.55
15. 70
0.51

4 30

15.70

Leucine.-

3.27
0.95

Vtiline

0. 9S

Methionine

1.77

1 fi*

Taking advantage of the fact that both calcite
and aragonite are present in the two distinct
layers of shell of the fan oyster (Pinna) and of the
pearl oyster (Findada) , the French investigators
(Roche, Ranson, and Eysseric-Jjafon, 1951) at-
tempted to determine whether there is a difference
in the chemical composition of the organic material
of the two layers of the shell of the same species.
They found that tyrosine and glycine occur in
higher concentrations in the prismatic layer than
in the nacreous part of shells. In the prismatic
layer of calcite portion the content of tyrosine
varies between 11.6 and 17.0 percent and that of
glycine between 25 and 36 percent. In the
nacreous part made of aragonite the concentration
of tyrosine was from 2.S to 6.0 percent and that
of glycine varied between 14.9 and 20.8 percent.
The significant differences in the contents of the
two amino acids in the two parts of the shell
may provide a clue for further studies of the role
of the organic component on the mineral form in
which the calcium carbonate is deposited by the
mantle.

MUSCLE ATTACHMENT

The place of attachment of the adductor muscle
or muscle scar is the most conspicuous area of the

MORPHOLOGY AND STRUCTURE OF SHELL

41

Millimeters

ro

Figure 49. — Cross section of shell of an adult C. virginica
after decalcification in weak acid, Mallory triple stain.
Conchiolin of the prismatic layer is reddish-brown; that
of calcite-ostracum is bluish.

oyster shell. In C. virginica, C. amjulata, and
many other species this area is highly pigmented;
in 0. edulis, C. gigas, pigmentation is either
absent or very light.

The muscle scar in C. virginica is located in the
postero ventral quadrant of the shell (figs. 15, 21,
33). To a certain extent the shape of the scar
reflects the shape of the shell, being almost round
in broad and round oysters and elongated in
narrow and long shells. The area of scar is
slightly concave on the side facing the hinge and
conve.x on the opposite, i.e., ventral side. Curved
growth line, parallel to the curvature of the
ventral edge of the valve, can be seen on the
surface. They are most pronounced in the ventral
part of the muscle impression. Size and shape of
the scar is variable and often irregular (fig. 50).
The outlines of the impressions shown in this

figure were obtained in the following manner:
the periphery of the impression was cu'cumscribed
with soft pencil; a piece of transparent Scotch
adhesive tape was pressed on the impression and
the outline was lifted and mounted on cross-
section paper; the area occupied by the impression
was measured by counting the number of squares.
Using this method, I obtained the replicas of
muscle impressions from 169 shells taken at
random from various oyster beds of the Atlantic
and Ciulf Coasts. The impressions are arbitrarily
arranged in four series (A-D) according to their
shape and size. The impression areas of round
and broad shells are shown in the two upper rows,
A and B; those of long and narrow shells are
arranged in the two lower rows, C and D.

It may be expected that the larger is the shell
the greater is the area of muscle impression.
The relationship, as can be seen in fig. 51, is
rectilinear although the scatter of plotted data
is considerable and the variability increases with
the increase in size. The ratio of nmscle impres-
sion area to sliell surface area varies from S to 32
with the peak of frequency distribution at 16 to
18 (fig. 52).

A small oval and unpigmented area on the

C3?e7 o c? cp

0

6 o

Centimeters

Figure 50. — Variations in .shape and size of muscle scars
on the shells of C. virginica. Rows A and B show the
types of scars normally found on broad and rounded
shells, the length of which is almost equal to or exceeds
the height. Rows C and D are the scars often found
on long and narrow shells in which the height exceeds
the length. Replicas of scars were made from shells
collected at random.

42

FISH AND WILDLIFE SERVICE

Table 5. — Chemical composition of oyster shells in percent
of shell weight
[From Hunter and Harrison, 1928]

20 0 300 40 0

SHELL AREA, cm^

Figure 51. — The relationship between the area of muscle
scar and the area of the shell of C. virginica.

dorsal half of each valve is the imprint of a
vestigial muscle in the mantle, discovered in 1867
by Quenstedt in the valves of the early Jurassic
oyster, Gnjphaea arcuata Lamark, and found by
Stenzel (1963) in C. virginica. In my collection
of living C. virginica the imprint is hardly visible
(figs. 15, 21, and 22). Slight adhesion of the
mantle to the valve indicates the location of this
area which Stenzel calls "imprint of Quenstedt's
muscle."

32

-

30

28

-

26

-

24

-

22

20

1

18

16

,

14

12

10

8

6

4

2

1

, 1

'

0

2

4

6 8

10

12

14

16

18

20

22

24

26

28

30

32

SHELL AREA-MUSCLE ATTACHMENT AREA

Figure 52. — Frequency distribution of the ratio of muscle
scar area vs. shell area in the shells of C. virginica of
Atlantic and Gulf States.

Constituents

Sample 1

Sample 2

Al

0.045
38.78

0.043
38.81
0.0025
0.09
0.189
0.009
0.073
0.580
0. 0009
0.0035

Ca

Cu

Fe

0.11

0.183

0.009

0.075

0.570

Mg

Mn... .

PiOs

SiOa

7.n

CI

0.0034
' 57. 19

COj

Fl

N

0.196

0.196

As

Organic matter 1

1.41

0.27

1 51

Water2

0.28

' Loss above 110° C. Ignited.

■ Loss to 100° C.

' Average for samples 1 and 2.

CHEMICAL COMPOSITION

The oyster shell consists primarily of calcium
carbonate, which composes more than 95 percent
of the total weight of the shell. The balance is
made up by magnesium carbonate, calcium sul-
fate, silica, salts of manganese, iron, aluminum,
traces of heavy metals, and organic matter.
Several analyses of oyster shell found in the
literature are incomplete, particularly with refer-
ence to trace elements. Analysis made for the
U.S. Bureau of Fisheries by tlie Bureau of
Chemistry of the Department of Agriculture and
published in 1928 (Hunter and Harrison, 1928) is
given in table 5.

Dead oyster shells buried in tlie mud of the
inshore waters of Texas and Louisiana are exten-
sively dredged by commercial concerns primarily
for the manufacture of chicken feed. Analysis of
these shells as they are received at the plant after
thorough washing in sea water is given in table 6.

The calcium carbonate content of these shells is
probably lower than in live oysters due to their
erosion and dissolution of lime in sea water.
The chloride content is affected by the retention of

Table 6. — Chemical composition of mud shells received al
the plant of Columbia-Southern Corporation at Corpus
Christi, Tex.

[Percent of constituents in samples dried at 110° C.)

Chemical

Percent

CaCOs..

93 88

S04as CaSOi

.MgCOj

0 88

SiOj

I 40

Alj03+Fei0i

0 3"'

CI as NaCl

0.27

NajO (other than NaCl)

0 46

Loss at 500° C

1 60

(.Analysis supplied by Columbia-Southern Corporation and copied with
their permission.)

MORPHOLOGY AND STRUCTURE OF SHELL

43

these salts in the shells after thorough washing
with sea water of greatly variable salinity. The
percent of silica, aluminum, and iron, which are
also higher than in the analyses of shells of Hve
oysters, is at least in part influenced by the
efficiency of plant operations in removing mud
from the surface of the shells.

Chemical composition of shells of 0. edulis is
not significantly different from that of C. virginica.
Table 8 gives the results obtained by European
scientists. The data quoted from various sources
are taken from Vinogradov (1937).

A nmch more detailed analysis of dead oyster
shells dredged from the bottom of Galveston Bay
8 miles east of San fjeon was made recently by the
Dow Chemical Company (Smith and Wright,
1962). The shells were scrubbed in tap water
with a nylon brush, rinsed in distilled water, dried
at 110° C, and ground in a porcelain mortar.
With the kind permission of the authors the
results are given in table 7. Additional 19 ele-
ments were sought but not found at the following
sensitivity limits:

10 p. p.m. — arsenic, barium.
1 p. p.m. — antimony, chromium, cobalt, ger-
manium, gold, lead, lithium, mercury,
molybdenum, nickel, vanadium, and
zirconium .
0.1 p. p.m. — beryllium, bismuth, cadmium,
silver, and tin.
The authors remark that traces of clay entrapped
within the shell may have influenced the findings
for titanium, manganese, copper, or zinc; and
that individual variations in silicon, iron, and
aluminum were due to contamination not remova-
ble by washing. It appears feasible that these
variations may have been caused by spicules of
boring sponges and algae infesting the shells.

Table 7. — Composition of C. virginica oyster shell dredged
from Galveston Bay, according to Smith and Wright
(1963)

Table S. — Chemical composition of shells of O. edulis (in
percent of ash residue)

Constituent

Concen-
tration

Constituent

Concen-
tration

Calcium (CaO) .-- ---

Percent
64.6
43.5
0.32
0.33
0.16
0.16
0.12
0.58

Organic Carbon as CH4--
Chlorine (CD

P.p.m.
400

340

Sodium (Na^O)

Aluminum (Al)._-

200

Magnesium (MgO)

Sulfur (SOil

Iron (Fe)

180

Phospliorus (P).

116

Silicon (SiO:)

Manganese (Mn)_

Fluorine (F)

110

64

Moistnrp (H^OI

Potassium (K)

30

Titanium (Til

12

Total of major con-

99. 8 %

Boron (B) - ..-

5

Copper (Cu)

Zinc (Zn)

Bromine (Br)

3

1

0.5

Constituent

Sample
1

Sample
2

Sample

Sample
4

CaCO,

98.60
1.21

97.65

96.54

97.00

Ca3(POi)!

MgCO]

0.312

0.52

trace

0. 9125
0.058

P2O5 . _

0.09

(Al Fe)203

0.03

Fe203

0. 0719

CaSO)

1.456

2.00

Si02

0.813

0.50

0.5-4.6

According to Creac'h (1957), all shells of 0.
edulis and C. angulata contain traces of phos-
jjhorus. The French biologist found that the
pliospliorus content is variable. Expressed as
P2O3, it varies in C. angulata from 0.075 to 0.114
percent. There is a significant difference in the
phospliorus content in various parts of the shell.
The amount of phosphorus per unit of volume nf
shell material is lower in the chalky deposits than
in the hard portion of the shells. Thus, in laying
a chalky deposit the moUusk utilizes from 2.4 to
2.6 times less phosphorus than is needed for
secreting the same volume of harder shell
substance.

Tlie presence of small quantities of strontium
in calcareous shells of mollusks is of particular
interest because of its apparent relation to arago-
nite. The marine organisms containing calcium
carbonate as aragonite have relatively higher
strontium content than those liaving calcite shells.
The relationship between the two elements is
expressed as strontium-calcium atom ratios
(Thompson and Chow, 1955; Trueman, 1944; and
Asari, 1950). In C. nrginica and C. gigas the
strontium-calcite ratio x 1,000 varies l)etween
1.25 and 1.29. Ostrea lurida from California has
a lower strontium content, the ratio being 1.01.
The percentages of Ca, Sr, COo, and organic matter
in the shells of three species of oyster and in Mya
arenaria, in wliich the content is the highest
among the bivalves, given by Thompson and
Chow (1955), are summarized in table 9. The

Table 0. — The percentage of calcium and strontium in the
shells of oysters and soft shell clam
(According to Thompson and Chow, 1965]

Species

Calcium

Strontium

Carlion
dioxide

Organic
matter

Atom
ratio
Sr/Ca
xl.OOO

0. turida _.

C, virginica _

C. gigas

M. arenaria

38.6

33. 7-37. 8

34. 6-36. 2
38. 6-38. 8

0.085
0. 92-0. 107
0. 097-0. 100
0.181-0.246

42.5
41.8^2.4
32. 6-42. 6
42. 2-42. 3

1.68
2. 16-2. 34
1.33-1.71
2. 22-2. 44

1.01
1.2.5-1.29
1,26-1.28
2. 16-2. 91

44

FISH AND WILDLIFE SERVICE

salinity and temperature of tlie water have ap-
parently no influence on Sr/Ca, which remains
fairly constant in calcareous shells. The possible
role of strontium in the mineralization and for-
mation of shell is discussed in chapter V.

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Journal of Microscopical Science, vol. 94, part 1,
pp. 57-70.
Pelseneer, Paul.

1920. A propos de la formation et de la composition
chimique de la coquille des moUusques. Annales
de la Societe Royale Zoologiquo et Malacologique
de Belgique, tome 51, pp. 70-74.
Quenstedt, F. A.

1867. Handbuch der Petrefakenkundc. Laupp,
Tubingen, 2d ed., p. 598.
Quilter, H. E.

1891. On tlie molluscan shell and periostracum .
Conchologist, vol. 1, pp. 5-8.
Ranson, Gilbert.

1939-41. Les huitres et le calcaire. Bulletin du
Museum National d'Histoire Naturelle, Paris, serie
2, tome 11, pp. 467-472; ibid, tome 12, pp. 426-
432; ibid, tome 13, pp. 49-66.

1943. La vie des huitres. Histoires Naturelles- 1 ,
Collection dirigee par Jean Rostand, (iallimard,
Paris, 261 pp.
deR£aumur, M.

1709. De la formation et de I'accroissement des
coquilles des animaux tant terrestres qu'aquatiques,

46

FISH AND WILDLIFE SERVICE

soit de mcr soit de riviere. Histoire de I'Acadcmie
Royale des Sciences avec les Memoires de Mathc-
matique et Physique, pp. 475-520. [Reprinted as
a separate: Academie Royale des Sciences Paris,
M6moircs, pp. 364-400.)

DERfiAUMUR, M.

1716. Eclaircisseniens de qiielques difficultcs sur la
formation et I'accroissement des coquilles. His-
toire de I'Academie Royale des Sciences avec les
memoires de niathematique et physique, pp. .384-
394. [Reprinted as a separate: Academie Royale
des Sciences Paris, Annee 1716, Memoires, pp.
30.3-311.]
Roche, Jean, Gilbert Ranson, and Marcelle Eys-
seric-Lafon.

1951. Sur la composition des seleroprotcines des
coquilles des MoUusques (conchiolines). Coniptes
Rendus des Seances de la Soeiete de Biologic et de
ses Filiales, tome 145, pp. 1474-1477.
Saville-Kent, W.

1893. The great barrier reef of Australia: its products
and potentialities. W. H. Allen and Co., Ltd.,
London, 387 pp.
ScHENCK, Hubert G.

1934. Literature on the shell structure of pelecypods.
Bulletin du Musce Royal d'Histoire Xaturelle de
Belgique, tome 10, No. 34, pp. 1-20.

SCHLOSSBERGER, J.

1856. Zur niiheren Kenntniss der Muschelschalen,
des Byssus und der Chitinfrage. Liebigs Annalen
der Chemie, Band 98, pp. 99-120.
Schmidt, W. J.

1931. f'ber die Prismenschicht der Schale von
Ostrea edulis L. Zeitschrift fur Morphologic und
Okologie der Tiere, Band 21, pp. 789-805.

SCHULZE, P.

1922. Uber Beziehungen zwischen pflanzlichen und
tierischen Skelettsubstanzen und ilber Chitinreak-
tioncn. Biologisches Zentralblatt, Band 42, pp.
388-394.
Smith, R. A., and E. R. Wright.

1962. Elemental composition of oyster shell. Texas
.Journal of Science, vol. 14, No. 2, pp. 222-224.

Sporn, E.

1926. Cber die Gesetzmassigkeiten im Baue der
Muschelgehatise. Wilhelm Roux' .\rchiv fur Ent-
wicklungsmechanik dor Organismen, Band 108, pp.
228-242.

Stenzel, Henry B.

1963. Aragonite and calcite as constituents of adult
oyster shells. Science, vol. 142, No. 3.589, pp.
232-233.

Oysters. In Treatise of invertebrate paleontology.
Part X — MoUusca, 6 (Bivalvia). [In press.)
Tanaka, Kojiro.

1937. On the chalky deposits in oysters. Fishery
Investigation, Imperial Fishery Experimental Sta-

tion, Tokyo, Supplementary Report No. 4, No. 46,
pp. 36-44. [English translation by the Atlantic
Biological Station, St. Andrews, Canada, 1953.)
Tanaka, Kojiro.

1943. On the effect of the concentration of salt on
the chalky deposits in oysters. Suisan Kenkyu-
shi (Journal of Fisheries), vol. 38, No. 12, pp.
222-224. [English translation by the Atlantic
Biological Station, St. Andrews, Canada, 1953.)

Thompson, D'Arcy W.

1942. On growth and form. 2d ed. Cambridge
University Press, Cambridge, England, 1116 pp.

Thompson, Thomas G., and Tsaihwa J. Chow. 1955.
The strontium-calcium atom ratio in carbonate-
secreting marine organisms. Deep-Sea Research,
vol. 3, suppl, pp. 20-39.

Trueman, E. R.

1944. Occurrence of strontium in molluscan shells.
Nature, vol. 1.53, No 3874, p. 142.

Vinogradov, A. P.

1937. The elementary chemical composition of
marine organisms. Composition of shells of
LamcUibranchiata. Translation in Memoirs, Sears
Foundation for Marine Research, No. 2, 1953, pp.
303-309.
Watabe, Norimitsu.

1954. Electron microscopic observations of the ara-
gonite crystals on the surface of cultured pearls.
I. Report of the Faculty of Fisheries, Prefectural
University of Mie, vol. 1, No. 3, pp. 449-454.
Wester, D. H.

1910. tlber die Verbreitung und Lokalisation des
Chitins im Tierreiche. Zoologische Jahrbucher,
.Abtcilung fiir Systematik, Geographic und Bio-
logic der Tiere, Band 28, pp. 531-558.
Wetzel, G.

1900. Die organischen Substanzen der Schaalen
von Mytilus und Pinna. Hoppe-Seyler's Zeit-
schrift fiir physiologischc Chemie, Band 29, pp.
386-410.
WoRSNOP, Edith, and James H. Orton.

1923. The cau.se of chambering in oysters and other
laniellibranchs. Nature, vol. Ill, No. 2775, pp.
14-15.

YONGE, C. M.

1952a. The monomyarian condition in the Lamelli-
branchia. Transactions of the Royal Society of
Edinburgh, vol. 62, part II, pp. 443-478.

1952b. Observations on Siliqua patula Dixon and on
evolution within the Solenidae. University of
California Publications in Zoology, vol. 55, pp. 421-
438.
Zander, Enoch.

1897. Vergleichende und kritische Untersuchungen
zum Verstandnisse der Jodreaktion des Chitins.
Pfltigers .\rchiv fur die gesammte Physiologic des
Mcnschen und der Thiere, Band 66, pp. 545-573.

MORPHOLOGY AND STRUCTURE OF SHELL

47

CHAPTER III
THE LIGAMENT

Page

Appearance and structure 48

Chemical composition 56

Elastic properties 59

Bibliograpliy R3

APPEARANCE AND STRUCTURE

The significance of the ligament in the pliylDgeny
and classification of bivalves was a favored
subject in inalacological studies of the past
centuiy. Lengthy theoretical speculations about
this structure are fcunul in the papers of Bower-
bank (1844), Jackson (1890, 1891), TuUberg
(1881), Ball (1889, 1895), Reis (1902), Bieder-
mann (1902), Stempell (1900), and others. A
review of the literature from the earlier years to
1929 is adequately presented by Haas (1935).
These investigations give little information, how-
ever, concerning the microscopic structure, origin,
chemical composition, and functif)n of the liga-
ment. The latter subjects receive attention in
the more recent works of Mitchell (1935) on tlie
ligament of Cardium corbi.s, in a series of detailed
studies by Trueman (1942, 1949, 1950a, 1950b,
1951, 1952, 1953a, 1953b) on the hgaments of Myfilus,
Pecfen, Nucula, Osfrea edulis, Tcllina fcuuis, and
the Semelidae, and in the paper of Owen,
Trueman, and Yonge (1953) on the ligament
in the bivalves.

The ligament of the Atlantic oyster is a narrow
band of dark, elastic material situated along the
edge of the hinge between the two valves. The
ligament does not extend deep into the shell, is
not visible from the outside, and is called internal
or ligamentum internum by Haas (1935) and
"alvincular" by Ball (1889). The latter term is
no longer used in nialacological literature.

The ligament performs a purely mechanical
function. Its elastic material, compressed when
the contractif)n of the adductor muscle closes the
valves, expands and pushes the valves apart when
the tension of (ho adductor is released. Tlie
extent to which tlie valves may gape de[)ends
largely on the shape and size of the beaks. In

48

the specimen shown in figure 17 the large, tri-
angular space beyond the hinge permits wide
excursions of the valves and their gaping may
consequently be very broad.

On the other hand, the narrow and crooked
beaks shown in figure 53 greatly restrict the
movement of the valves along the pivotal axis
regardless of the degree of relaxation of the nmscle.
Small pebbles, pieces of lu-oken shell, and other
foreign particles often found lodged between the
beaks may further limit the opening (jf the valves.
The possibility that such purely mechanical
ol)structions can impede the movement of the
valves should be kept in mind in evaluating the
results of physiological tests in which the degree
of shell opening is recorded.

The youngest part of the ligament is that which
touches the inside of the valves; the oldest
portion, which is usually dried, cracked, and
nonfunctional, faces tiic outside. When the

Centimeters

I'^iGURE 53. — Longitudinal section through tlic beak and
hgament of C. virginica. l.v. — loft valve; r.v. — right
valve; Ig.c.p. — functional, compressible part of the
ligament; Ig.n.f. — nonfunctional, old part of the liga-
ment.

FISHERY bulletin: VOLUME 6 4, CHAPTER III

valves are forcibly separated, the ligament breaks
approximately along the pivotal axis of the shell
(fig. 54, piv. ax.) and the two parts remain
attached to the respective valves.

The three parts of the ligament at the edge of
the valves differ in color, size, and shape. The
usually brownish central (inner) part called
resilium forms a bulging ridge marked by fine
striations visible to the naked eye or under a low
magnification. The resilium is attached to a
groove called resilifer or chondrophore (figs.
54, 2, 16). The dark olive anterior and posterior
portions of the ligament called by Olsson (1961)
tensilia are attached to the edges of the valves
(nymphae).

The resilium consists of tightly packed lamellae
arranged at about right angles to the longitudinal
axis of the ligament; they can be seen on the ex-
posed surface of the central part. These lamellae
are intersected by fine striations visible on the side
of the resilium after the removal of the adjacent
lateral part (fig. 55).

When, the valves are closed the resilium is com-
pressed because of its considerable thickness while
both lateral parts (tensilia) are slightly stretched.
It can be seen in a series of cross sections of the
hinge nuide at right angles to its pivotal axis (fig.
56) that the curved lines of the compressed resilium
(2) are deeply arched, while those in the lateral
parts arc almost straight. This observation
agrees with the description of the operation of the
ligament of 0. edulis by Trueman (1951). Since
the beaks of the oyster illustrated are asynunetri-
cal, the distance between the two valves is greater
at the anterior than at the posterior end (fig. 56,
3, and 1) and, conseciuently, the anterior portion

-PIV, AX.

Cent I meters

Figure 54. — Ligament of large C. virginica attached to
the right valve. View from the inside, piv. ax. —
pivotal axis. The resilium occupies the central position
and on both sides is flanked by tensilium. Slightly
magnified.

0

Millimeters

Figure .5.5. — Central portion of the ligament (resilium)
attached to the groove (bottom) of a valve. Note the
lamellar structure and fine striations visible on the right
side of the figure. C. virginica.

of the ugament stretches more than its posterior
part .

The ligament effectively seals the space between
the dorsal edges of the valves and forms an elastic,
watertight joint that prevents the entry of water
and organisms which otherwise could easily invade
the mantle cavity.

The spring-like action of the ligament is a func-
tion (if the elasticity of its component parts.
Examination of transverse and longitudinal sec-
tions of fresh ligament under low power discloses
its amazingly complex structure. A cross section
made witii a razor blade at a right angle to the
pivotal axis of the valves shows a series of well-
defined curved lines extending from tiie right to the
left valve, and a complex system of lamellae ar-
ranged perpendicularly to the curves. Both sys-
tems are clearly seen in unstained preparations
mounted in glycerin jelly or in balsam (fig. 57).
The pivotal axis of the ligament lies in the center
of the drawing, perpendicular to the plane of the
paper; tlie valves (not shown in the figure) are on
the right and left sides, and the newly deposited
portion of the ligament lies at the bottom of the
drawing. The most conspicuous arches extend
almost without interruption from one side to an-
other; the lighter ones can be traced only for short
distances over the cross-sectional area. The struc-
tiu-e of the resilimn resembles a leaf plate of an
old-fashioned automobile spring, suggesting that
the arches are the lines of stresses corresponding
to the deformation of the ligament under com-
pression. Within the mass of the ligainental ma-

THE LIGAMENT

49

Centimeters

Figure 56. — Three longitudinal sections through hinge and ligament of the shell of C. virginica. (1) posterior portion;
(2) central portion or resilium; (3) anterior portion, h. — beaks, Ig. — hganient, l.v. — left valve, r.v. — right valve.
Note the arched lines of the resilium (Ig.) in the central drawing (2). Slightly magnified.

terial they are the visual evidence of these stresses.
Since the "springs" (if the resilium do not consist
of separate structure parts joined together into a
complex unit, the comparison is only superficial.

The ligament is a nonliving structtu'e secreted
at a varying rate by the highly specialized epithel-
ium of the subligamental ridge of the mantle (see
p. 89). Structurally, the arches, visible at low
magnification, represent stages of growth; fimc-
tionally and in accentuated form, they reflect
compressional deformation in the operating
structure of the ligament.

Under a binocular microscope the lamellae of
the resilium, when separated with fine needles,
appear slightly bent and zigzagged. A small
piece of the resilium cut in the dorsoventral
plane and magnified about 250 times (fig. 58)
can be seen to consist of fibrillar material and
of dark bands of variable width composed of
tightly packed, oval, birefringent globules. Pres-
sure over the cover slip does not change the shaj)e
of the globules, which appear to be firmly em-
bedded in the ground substance. The globules
contain no acid-soluble material since they are
not afl'ected by strong hydrochloric acitl, nor are
they soluble in alcoliol or .xylol. Preparations
mounted in balsam j)resent the same ajipearauce
as nondehydrated sections mounted in glycerin
jelly. Besides the globules concentrated in the
dark bands within a delicately fibrillar grounii
substance, some of them are arranged in longi-
tudinal lines at riglit angles to the dai-Jc bands.
Some of the horizontal bands (upper part of

figure 58) are of mucli greater complexity than
the others; they consist of oval-shaped light areas
surrounded by globules. The two structural
elements, namely, the bands of fibrils and the
rows of globules, repeat themselves with regularity,
the successive layers varying oidy in width and
in the concentration and size of globules. The
fibrils intersect the arches either perpendicularly
or at about 45° (lower part of figure 58) and
probably exert additional elastic force under
compression.

The anterior and posterior parts of the ligament,
the tensilium of Olsson (19G1) or outer layer of
Trueman (1951), are made of tenacious material
which withstands considerable stretching without
breaking. This can be easily ascertained by
trying to tease or to pull apart the dissected
parts of the tensilium. In this respect the material
of the tensilium differs from that of the resilium,
which is weak under tension but strong untier
compression. Tiie color of the tensilium dilfers
from that of the resilium. In New P^ngland
oysters it is usually dark green on tlie surface,
wliile the resilium is liglit brown. Tiie tensilium is
made of tough lamellae which in a transverse section
appear as slender, transparent cylinders of slightly
yellowisli sul)stance (fig. 59). Both r.'silium and
tensilia jire secreted by highly specialized epi-
tlielial cells whieh imderly the ligament. The
thickness of eacii kunella corresponds to the width
of a ruffle at the edge of the secreting epithelium
(See chajiter \, p. ,s9). At low magnification
the nniterial of the tensilium appears to be non-

50

FISH AND WILDLIFE SERVICE

■'V■\\^lWli^^;'^■^■i^>:^|l;v;i

'^0i0iM^^ih.

'." ***' ' ""' * '"' y '^ '.. -■;"■■■■■■■■' --"v •■■■-- ^^ ■' —

"... ■ ' ■: ; . ^,^-.■■M V, ■

;.•■■

0

Millimeters

0.5

Figure 57. — Cros.s .section of the central portion of the ligament of C. virginica made perpendicular to the pivotal axis
of the valve. Arches (curved lines of dense material) extend from left to right: the valves are not shown in the
drawing.

fibrillar, but at higher magnification the fibrillar
structure becomes clearly visible. Two types of
fibrils can be distinguished on the photomicro-
graph of tensilium shown in figure 60. Heavy and
well-defined bundles of fibers originated along the
vertical jdane of the lamellae (up and down bundles
in fig. 60) and short and slender fibrils in places
at right angles to the large bundles (the lower
half of fig. 60). Large oval-shaped bodies on
the upper right and lower left part of the figure

are the accumulation of calcium carbonate crys-
tals. Single minute crystals are scattered over
the body of the lamella. The outer dark layer
is very thin, its color is due to densely packed
narrow iilirils. Large and small globules which are
conspicuous in tlie architecture of the resilium
are absent in the tensilium, and the structure of
the latter lacks the complex arrangement of
globules and fibrils found in the former.

Tlie complexity of the microscopic structure

THE LIGAMENT

51

.^r\

j«

if' y

ffl '!}''••> t.

Jm

ik

H

i\

Vi

0 0.1

Millimeters

I 't-s..

/'

"•Jl^f

Figure 58. — Longitudinal (dorsoventral) .section of the resilium of C. virginica. Two structural elements are seen:
Band of fibriUae extending in vertical direction in the plane of the picture, and horizontal bands of various thicknesses
consisting of numerous globules.

52

FISH AND WILDLIFE SERVICE

0 0

Millimeters

Figure 59. — Transverse section of tensilium showing lamellar structure and darkly pigmented surface, C. virginica-

Photomicrograph of unstained and nondecalcified preparation.

suggested that electron microscopy might reveal
some interesting details. Small pieces of the
resilium fixed in 1 percent osmic acid were em-
bedded in plastic and sectioned. Although the
material is ver\' hard, it was possible to obtain
sections from 0.3 to 0.5ai in thickness. The
electron micrograph (fig. 61) shows bands of
fibrils varying in diameter from 370 to 500 A.

A section made across the plane of the arches
(fig. 62) shows a membrane honeycombed by holes
about 500 A in diameter. Two interpretations
seem possible: (1) that the fibrils are tubular,
the light areas corresponding to the centers of the
tubes, or (2) that the empty circles represent
spaces between the fibrils. The first interpreta-
tion is more plausible because of the gradation

THE LIGAMENT

53

V* *

/n

-.'^i'

%m

#

Microns

30

Figure 60. — Large and small bundles of fibers of the tcnsiliuni of C. tirginica seen in unstained and nondecalcified
preparation of the material teased in glycerin. Photomicrograph.

from circular to elliptical liiiht areas as the plane
of section of the fibrils becomes tangential (see
fig. 62).

Sections made at risilit anoles to tlie fibrils (fig.
62) demonstrate a certain similarity to those of
the organic membranes of the aragonitic jjart of
the shells of mollusks and ])earls. According to
Gregoire, Duchateau, and Florkin (1950, 1955),

54

such organic membranes have a lace-like structure
consisting of meshes and holes of dift'erent size and
pattern. In tltese inxestigations by Belgian
biologists the material was first decalcified, and
the layers of organic substance then separated by
idtrasonic oscillation to obtain the ultrathin mem-
branes suitable for electron microscopy. The
films of the calcite-ostracum layer of the siiclls of

FISH AND WILDLIFE SERVICE

,4

<4

Figure 61. — Electron micrograph of the ligament of C. mrgniica sectioned parallel to the fibrils.

pelecypods whicli have no true nacre (0. edvli.'i,
0. tulipa, Yfildia, Acta, and others) were found to
consist "of heterogenous niateriul, tlie more repre-
sentative elements of which are amorphous, vitre-
ous phites, sometimes granuhxr and devoid of
visible (or unquestionable) pores." (1950, p.

30)." In the absence of ultrasonic equipment in
my laboratory this method could not be used at
Woods Hole, Mass. Comparison of figures pub-
lished by Gregoire and his associates with the
photograpii reproduced in figure 62 suggests that

5 Translation bv Paul S. GaUsofF.

THE LIGAMENT

55

0 0.5

Microns

Figure 62. — Electron micrograph of a .section of the ligament of C. virginica made acro.ss the fibrils.

the structure of the ligsiment of C. inrginica has
some similarity to that of the organic membranes
of the aragonite shells. Recently Stenzel (1962)
has found that the resilium of the Ostreidae con-
tains aragonite.

One of the sections of the ligament of C. viryinica
studied with the electron microscope shows a series
of bhick, oval-shaped bodies arranged along curved
lines and separated from one another by fibrils
(fig. 63). The black bodies probably correspond
to the small globules visible under the liglit micro-
scope. Their nature has not been determined.

The action of the ligament can be demonstrated
by a rather crude model consisting of two slightly
curved pieces of wood, representing the valves,
joined by a series of brass rods. The rods are
bent and arranged to correspond to the course of
the arches as the latter are seen in an enlarged
photograph of a transverse section of the ligament
(fig. 57). Thin rubber tubing interwoven between
the arches corresponds to the bundles of fibrils.
Since the diameter of rubber tubing used in the
construction of the model greatly exceeds the
comparable diameter of the fibrils, this portion of
the model is not in scale. Another departure

from actual conditions is the interweaving of the
rubber tubing between the arches, a method used
to simplify construction although no such arrange-
ment of fibrils was disclosed by microscopy. The
model is shown in fig. 64. If the sides of the
structure are pressed together, the arches curve
up and exert lateral pressure at the same time
that the increased rigidity of the rubber tubing
adds to the elastic force. One can easily feel this
pressure by touching the rubber tubing witli the
finger tips while bringing the "valves" together.

CHEMICAL COMPOSITION

The chemical composition of the ligament is
essentially the same as that of the organic matrix
of the shell (Mitchell, 1935: Trueman, 1949,
1951). The proteins forming the lateral (tensil-
ium) and the central (resilium) portions of the
ligament are not, however, identical. The differ-
ence can be demonstrated by staining reactions
and by various chemical tests. For instance, in
Tellina tenuis the lateral parts of the ligament are
stained red or yellow by Mallory triple stain,
while the inner part turns blue, a difl'erence
comparable to that between the staining reaction

56

FISH AND WILDLIFE SERVICE

Fir.uRE 63. — Electron micrograph of the ligament made near one of the arches parallel to the fibrils of C. virginica.
bodies probably correspond to the smallest globules seen in the light microscope.

Dark

of the conchiolin of the prismatic hiyer and that
of the calcite-ostracum discussed on p. 42. True-
man (1949) conchides that the two types of
conchiohn seem to correspond respectively to the
two components of the hgament. The tensihum
gives a positive reaction with the xanthoproteic,
Millon's, and Merker's reagents, whereas the
reaction of the resihum to these reagents is
negative. Brown (1949) points out that most

of the epithehal skeletal proteins of invertebrates
that have been examined seem to be coUagens
and that their physical properties depend upon
degree of hydration. The electron micrographs
of the ligament (figs. 61 and 63) do not, however,
show the axial periodicity of about 640 angstrom
(A.) which is the most common characteristic of
collagen fibrils (Gross, 1956). Other authors
describe fibrils of 270 A. period which participate

THE LIGAMENT

57

Figure 64. — Mechanical model of the ligament of C.
virginica. Arches are in scale and correspond to the
curves visible in a cross section of the ligament at a
magnification of about 100 X . Diameter of rubber tub-
ing representing fibrillae is not in scale.

in the formation of the mature 640 A. period
collagen (See pp. 512-513 of S. L. Palay [editor]
Frontiers in Cytology, 1958), as well as smaller
fibrils in the embryonic tissues. The latter
probably represent a very early stage in the
formation of collagen.

Collagen fibers can be tanned in vitro, that is,
they can be converted by various agents to a form
in which they swell less and develop greater
chemical resistance. The taiming of protein
structures by an orthoquinone occurs naturally
among many invertebrates and has been demon-
strated for the cuticles of a number of arthrojjods
(Dennell, 1947; Pryor, 1940; Pryor, Russell, and
Todd, 1946) and for the cliaetac of eartliwornis
(Dennell, 1949). There is also evidence that a
similar phenomenon takes place in the ligaments
of bivalves (Friza, 1932). In Anodorda, for in-

stance, the amber coloration of the lateral layer of
the ligament is considered to be the result of tan-
ning by an ortli(K|uinone. This conclusion is
based on the fact that even after boiling this layer
induces rapid oxidation of the mi.xture of dimethyl-
para]ihenylenediamine and a-naphthol (Nadi re-
agent), which is frequently employed to indicate
the presence of orthoquinones in the cuticles of
insects and crustaceans (Dennell, 1947). In the
ligament of 0. edulis the differentiation between
the two layers may be made visible by Mallory
triple stain. The lateral layer (tensilium) consists
of quinone tanned protein whereas the central
layer (resilium) is built of calcified proteins (True-
man, 1951).

Few chemical studies have been made on the
ligaments of oysters, but chemical analysis of the
two portions of the ligament of the related
pelecypod Tellina made by Trueman (1949) shows
the following difTerences summarized in table 10.

It is rather surprising to find that an elastic,
nonliving structure functioning through a con-
siderable period of time (according to Trueman,
several years in Tellina) is heavily calcified. Tlie
resilium of C. virginica contains a mucli larger
amount of calcium carbonate than the outer parts:
determinations made in my laboratory on the
ligaments of 5- and 6-year-old oysters dried at
55° C. show that the calcium carbonate content
of the resilium varied from 30 to 67 percent of the
total weight of the sample, while in the tensilium
the content of calcium carbonate was only from
5.3 to 8.5 percent.

It is apparent that f:nowledge of the chemistry
of conchiolins and other substances found in
molluscan shells and ligaments is incomplete and
that much remains to be discovered about the
composition and structure of these proteins which
play such an important role in tlie life of all
bivalves.

Table 10. — Results of chemical tests of the ligament of
Tellina tenuis, according to Trveman

Five ptTcent HCI

Saturated KOII (hot)

Xanthoproteic reaction...

Millon's reagent

Ritiret reaction

Ninitydrin

Morner's reagent

("hitosan test (Camphell)

Chitintest (Schulze)

Argentaffine

Outer
layer

Inner
layer

No effect
-\11 dissolves

+

-I-
+

Faint .

58

FISH AND WILDLIFE SERVICE

ELASTIC PROPERTIES

It has long: been known tluit the hganient per-
forms a mechanical function by automatically
pushino- the vah-es apart when the tension of the
adductor nniscle relaxes. In a live oyster, how-
ever, tlie gaping of the valves never attains the
potential maximum limited by the angle and length
of the beaks. This can t)e demonstrated by a
simple test: if the entire adductor muscle is
severed, the valves open to a much greater angle
than that maintained l\v a full}' narcotized oyster
with a completely relaxed muscle attaclied to the
shell. It follows from this observation thtit during
the entire life of the oyster the adductor muscle,
even at the periods of its greatest relaxation, exerts
a certain pulling force against the elastic tension
of the ligament.

In view of the voluminous literature dealing
with tlie structure and function of bivalve muscles
it is surprising to find how little attention has been
given to the study of the physical properties of
the ligament. The first attempt to determine the
pulling force of the muscle sufficient to counteract
the elasticity of the ligament was made in a rather
crude manner in 1865 by Vaillant who tried to
measure the elastic force of the ligament of
Tridacna shells. Trueman (1949) erroneously
gives credit for this pioneer work to Marceau
(1909), who only repeated the method used by
earlier investigators (Plateau, 1884).

After removing the soft body of Tridacna,
Vaillant set the empty shell on a table with the
flat valve uppermost and placed a glass graduate
on top of it. Water was poured into the graduate
until the valves closed. Then the volume of
water was read and its weight computed. The
weight of the water plus the weight of the glass con-
tainer and of the valve gave Vaillant a value which
he called the resistance of the ligament. For a
shell of Tridacna, apparently one of small size, he
gives the following figures: weight of water re-
quired to close the valves — 250 g. ; weight of the
vessel — 700 g. ; weight of the valve — 632 g. The
total force needed to overcome "the resistance" of
the ligament is, therefore, 1,582 g.

A similar method was used by -Plateau (1884),
the only differences being that weights were added
to a metal pan suspended from a lof)p encircling
the valves, as sliown in figure 65, and tliat the
shell was placed on a metal ring. The elastic
force exerted by the ligaments of several common
bivalves, as determined by Plateau, was found to

FiouRE 6.5. — Plateau's method of measuring elasticity of
the ligament.

be as follows: Oxtrea edulis — 333.8 g.; Venus
verrucosa — 500.0 g.; Mya arenan'a — 620.0 g.; and
Mytdus edulis — 1,051.8 g. In Marceau's paper
of 1909 the data taken from Plateau's work are
repeated without change or verification.

Trueman's investigation of the ligament of
Tellina (1942) marks a renewal of interest in the
study of the physical properties of the ligament.
In a later paper (1951) he finds that in very j'oung
0. edulis the outer layer of the ligament (ten-
silium according to our terminology) forms a
continuous band along the entire dorsal margin
of the hinge, but that in adults this outer layer
separates into the anterior and posterior portions,
leaving the inner layer (resilium) exposed at the
dorsal edge. The axis about which the valves of
the adult 0. edulis open (pivotal axis) is the same
in C. rirginica (figiu-e 54, piv. ax.). In the closed
shell of Osfrea and Crassosfrea the central part of
the ligament (the resilium) is under compression
and the two flanking portions (tensilium or outer
layer of Trueman) are under tension.

To measure the opening moment of thrust of a
iiinge ligament, Trueman (1951), uses the fol-
lowing method, sliown diagrammatically in figiu'e
66: soft parts of tlie body are removed and the
lower valve embedded in plasticine; a counter-
balanced beam is erected above the valve in such
a way that the weight placed on the pan at the
left end is applied at the center of the upper
valve. The distance from the left end of the
l)eam to the arm touching the centroid of the

THE LIGAMENT

59

////////////////

Figure 66. — Trueman's nipthod for measuring the mo-
ment of thrust of bivalve ligament. Redrawn from
Quarterly Journal of Microscopical Science, 19.51,
.series 3, vol. 92, part 2, p. 137.

valve is so adjusted that the weight at the point
of application to the centroid is twice that placed
on the pan. Weights are gradually added until
the valves just close so that the opening moment
M is exactly counterbalanced. The ratio M'
between the opening moment M and the surface
area of the valve A is determined by the formula:

M' = d-^ , where d is the straight line

A

distance from the point of weight application on

the shell to its pivotal axis; W is the weight

applied; and V is the weight of the upper valve.

There are two objections to this metliod. The
central point of the valve can be accurately
determined only for round, symmetrical shells;
for tlie irregularly curved shells of C. virgifnca,
C. anyulata, or C. (jigas, its position can only be
guessed. Another more serious objection refers to
the determination of the weight under which the
valves "just closed." Experimenting with C.
rirginica, I found that visual observation, even
with a magnifying glass, is not sufficient to deter-
mine when the valves are completely closed.
Freciuently a tiny slit between the valves cannot
be seen but becomes apparent on a magnified
kymograph record of shell movement. Trueman's
method with modifications was used by Hunter
and Grant (1962) to study the mechanical ciiarac-
teristics of the ligament of the surf clam, Spmda
solidissima. They found that the ligament of the
clam is about 3.5 times stronger (in terms of
opening moments) than that of Alya arenaria.
The mechanical differences, according to their
opinion, reflect the modes of life of the two clams.

The moment of thrust measured by Trueman's
method is of no particular significance to the
physiology of the oyster because it does not repre-
sent the pulling force which the adductor nuiscle
must exert to close tlie valves or to I^cep them
partially open. This force differs from Trueman's

moment of thrust because the site of the attachment
of the adductor muscle is located not in the center
but in the ventroposterior quadrant of the valves.
The following method overcomes these difficulties:
the body of the oyster is removed without injuring
the ligament; tlie gaping shell is placed with the
left valve resting on concave cement support (fig.
67) and immobilized by small lead wedges. Tite
right valve is connected to writing lever N of
kj-mograph K. A glass hypodermic sj-ringe of
10 ml. capacity, mounted on wooden frame G, is
placed so that its plunger F touches the valve
over the center of the muscle attachment area.
The flattened end of the plunger is cut off, and its
stem is sharpened to a point. A three-way stop-
cock L is attached by hard rubber tubing to the
upper end of the syringe; one of its arms is con-
nected to a hand pump D (automobile or bicycle
tire type) ; the other arm leads to an open mercury
manometer C. Two dry cell batteries E activate
the recording electro-magnet M which makes a
mark on the drum only when the key switch 5*
is pushed down. As the pump is worked the
pressure created in the system forces the plunger
down, gradually closing the shell. Each time the
mercury column rises 2 mm. the operator pushes
the signal key down. Pumping is continued after
the valves are closed until the horizontal line on
the drum record indicates that increase in pressure
produces no further change in the position of the
upper valve. The point corresponding to the
complete closure of the valves is easily determined
by placing a ruler against the horizontal portion
of the kymograph curve and noting the point at
which the line begins to curve down (fig. 68).
The number of signal marks from the beginning
of the recording to the end of the curved line
multiplied by two gives the height of the mercury
column in millimeters. The manometer must he
calibrated to correct for tlie error resulting from
slight irregularities in the diameter of the glass
tubing in its two arms.

To minimize friction between the walls of the
syringe and its piston, several lubricants were
tried until it was finally discovered that a minute
quantity of high-speed centrifuge oil permits free
movement of the piston under its own weight.
The weight of the piston in the operating position,
determined by placing the balance pan under the
point of the piston, was recorded at 17. Og.; weight
of the same piston taken out of the syringe was

60

FISH AND WILDLIFE SERVICE

Figure 67. — Apparatus used for determining the elastic force of the ligament. A — oy.ster shell; B — support; C — mercury
manometer mounted on wall; D — air pump; E — two dry cells to operate the signal magnet M ; F — plunger of hypo-
dermic syringe resting on right valve above muscle attachment area; G — stand upon which the syringe is mounted;
K — kymograph; L — three-way stopcock; M — signal magnet with writing pen; N — lever connected to upper valve of
the oyster A; S — key switch for signal magnet.

18.45g. Both syringe and piston were cleaned and
lubricated at the beginning of each series of ob-
servations and the weight of the piston in the
operating position checked frequently. Prior
and during the determination, which required
only a few minutes, the ligament was kept moist
by frequent applications of a few drops of sea
water.

To convert the manometer readings into force
in grams, the following simple computation was
made: since the cross-section area of the piston
in the syringe is 1.971 cm.^ and the specific gravity
of mercury is 13.95, the weight of the column of
mercury is equal to 1.971 x 13.95 x H wliere H
is the height f)f that column in centimeters.
Determinations of elastic force made by this
method are accurate within 5.3g. since readings
were taken at 2 mm. intervals and the weight
of a mercury cokunn of 1 cm. height is 26.71g.

With exposure to air the elasticity of the liga-
ment changes, gradually losing its resilience. As

THE LIGAMENT

733-851 O— 64 5

drying progresses greater force must be applied
to bring the valves together, and the ligament
becomes harder and more brittle until it finally
breaks along the pivotal axis. The rate of these
changes was ascertained in two tests with large
xVmerican oysters from Peconic Bay, N.Y. After
the shell was placed in the apparatus (fig. 67)
determinations were made at 15-minute intervals
between which the ligament was not moistened.
Room temperature varied slightly from 68° to
70° F., and relative humidity in the laboratory
was 46 percent. The results of testing which
continued for 5 hours and 5 minutes indicate that
under the conditions of the experiment no signifi-
cant change in the physical properties of the
ligament is noticeable during tiie first 90 minutes.
After that the hardness of the ligament increases
steadily as can be seen from the shape of the curve
in figure 69. The test repeated a second time
yielded similar results. It can, therefore, be
deduced that under the given experimental condi-

61

_______

162 ,^^ y^^""^

161 ^^

161 ^ ^^ _._^^^^^, ^^^>_^^^^^^.^^, ^H,H,^^

2 MM. HG

Figure 68. — Two kymograph records of the closing of oyster valves under pressure applied at the upper valve over the
muscle attachment area. Marks on the bottom lines refer to each 2 mm. increase in the height of the mercury column
in the manometer. Vertical lines indicate the point on the abscissa at which the final reading was made.

450

400

350

300-

250

200

100 200 300

MINUTES OF DRYING

Figure 69. — Effect of drying on elasticity of the ligament
of adult C virginica from Peconic Bay, New York.
(At temperature of 6.S° F.)

tions drying can not affect the values of readings
obtained within a few minutes after tlie removal
of the shells from water.

The question arises whether there are significant

V5

I

-4

differences in the elastic properties of the ligaments
of oysters living in different ecological environ-
ments. The problem was studied by obtaining
samples of oysters from the following localities:
Peconic Bay, N.Y. (nearly oceanic water of high
and stable salinity); upper part of Narragansett
Bay, R.I. (187oo to 247oo); Chesapeake Bay, Md.
(107oo to 16°/oo), both localities characterized
by considerable daily and seasonal fluctuations
in salinity of water; Apalachicola Bay and East
Bay, Fla., representing typical southern conditions
of warm water and great fluctuatic^ns in salinity.
Oysters from East Bay (near Pensacola, Fla.)
were taken from three different zones: A — inter-
tidal flat; B — bottom level; and C — below low water
level in the area of exceptionally strong tidal
currents. Each sample consisted of either 30
or 50 adult oysters of marketable size. After
arrival at Woods Hole, Mass., they were kept at
least 5 weeks in the harbor water (31%o to 32°/oo)
before they were tested. iVll experiments were
conducted during the winter when harbor water
temperature was about 4° C. and laboratory
air temperature about 21° C.

The results of the tests, expressed as the pulling
force in g. pei- cm.* of the muscle scar area necessary
to counteract the elasticity of the ligament, are
sunmiarized in the series of histograms shown in
figure 70. 1 1 is apparent that the elastic properties
of the ligament vary greatly within each group
but especially in the Peconic Bay and Apala-
chicola oysters. A comparison of tlie modes of
the elastic forces in ligaments of oysters from
different environments gives the following values

62

FISH AND WILDLIFE SERVICE

RHODE ISLAND

EAST BAY, FLA

160 MO iio ^ho 460 54o h a'o ito sio lio 4^ 6 eb ite 2Jo 320

RESISTHNCe OF LIGAMENT TO CLOSmG, GRJiMS/CU Of MUSCLE UREA

Figure 70. — Frequency distribution of the elastic property
of the ligaments in seven groups of adult C virginka.
The elastic property is expressed in the pulling force of
the adductor muscle (in g. per cm.2 of muscle area)
needed to counteract the action of the ligament.

expressed in g. per cm.^ of transverse section oi
muscle area arranged in diminishing order:

Peconic Bay (Fireplace oysters) 252g.

East Bay, Fla.— C, fast tide 178g.

Apalachicola Bay 128g.

Chesapeake Bay, Md 99g.

East Bay, Fla.— B, bottom 93g.

East Bay, Fla. — A, intertidal zone 91g.

Narragansett Bay 79. g

Whether the values observed do actually depend
on ecological conditions cannot be stated without
further investigation.

BIBLIOGRAPHY

Bevelander, Gerrit, and Paul Benzer.

1948. Calcification in marine molluscs. Biological
Bulletin, vol. 94, No. 3, pp. 176-183.

BlEDERMANN, W.

1902. LIntersuchungen tiberBau und Entstehung dcr
Molluskenschalen. Jenaische Zeitschrift fur
Naturwissenschaft, Neue Folge, Band 29, pp. 1-164.

1914. Physiologic der Stutz- und Skelettsubstanzen.
Hans Winterstein's Handbuch der Vergleichenden
Physiologic, Rand 3, 1 hiilfte, Teil 1, pp. 319-1,188.
Gustav Fischer, Jena.

BOWERBANK, J. S.

1844. On the structure of the shells of molluscous
and conchiferous animals. Transactions of the
Microscopical Society of London, vol. 1, pp. 123-
154.
Brown, C. H.

1949. Protein skeletal materials in the invertebrates.
Experimental Cell Research, Supplement 1, pp.
351-355.

1950a. A review of the methods available for the
determination of the types of forces stabilizing
structural proteins in animals. Quarterly Journal
of Microscopical Science, series 3, vol. 91, part 3,
pp. 331-339.

1950b. Quinone tanning in the animal kingdom.
Nature, vol. 165, No. 4190, p. 275.
Dall, William Healey.

1889. On the hinge of pelecypods and its develop-
ment, with an attempt tow'ard a better subdivision
of the group. American Journal of Science, series
3, vol. 38, pp. 445-462.

1895. Contributions to the tertiary fauna of Florida,
with especial reference to the IVIioccne silex-beds of
Tampa and the Pliocene beds of the Caloosahatchie
River. Part III. A new classification of the
Pelecypoda. Transactions of the Wagner Free
Institute of Science of Philadelphia, vol. 3, part 3,
pp. 479-570.
Dennell, R.

1947. The occurrence and significance of phenolic
hardening in the newly formed cuticle of Crustacea
Decapoda. Proceedings of the Royal Society of
London, series B, vol. 134, pp. 485-503.

1949. Earthworm chaetae. Nature, vol. 164, No.
4165, p. 370.

Friza, Franz.

1932. Zur Kenntnis des Conchiolins der Muschel-
sehalen. Biochemische Zeitschrift, Band 246, pp.
29-37.
Galtsopf, Paul S.

1955. Structure and function of the ligament of
Pelecypoda. [Abstract.] Biological Bulletin, vol.
109, No. 3, pp. 340-341.
GRfiGOiRE, Charles, Gh. Duchateau and M. Florkin.

1950. Structure, etudi^e au microscope ^lectronique,
de nacres decalcifies de Mollusques (Gast^ropodes,
LameUibranches et Ci^phalopode) . Archives In-
ternationales de Physiologic, vol. 58, pp. 117-120.

1955. La trame protidique des nacres et des perles.
Annales de I'lnstitut Oc^anographique, nouvelle
serie, tome 31, pp. 1-36.

Gross, Jerome.

1956. The behavoir of collagen units as a model in
morphogenesis. Journal of Biophysical and Bio-
chemical Cytology, vol. 2, No. 4, part 2, suppl., pp.
261-274.

Haas, F.

1935. Bivalvia. Teil 1 . Dr. H. G. Bronns Klassen

und Ordnungen des Tierreichs. Band 3: MoUusca;

Abteilung 3: Bivalvia. Akademische Verlagsgesell-

schaft, Leipzig, 984 pp.
Hunter, W. Russell, and Davio C. Grant.

1962. Mechanics of the ligament in the bivalve

Spisula solidissima in relation to mode of life.

Biological Bulletin, vol. 122, No. 3, pp. .369-379.
Jackson, Robert Tracy.

1890. Phylogeny of the Pelecypoda. The Aviculidae
and their allies. Memoirs of the Boston Society of
Natural History, vol. 4, No. S, pp. 277-400.

Jackson, Robert Tracy.

1891. The mechanical origin of structure in pele-

THE LIGAMEXT

63

cypods. American Naturalist, vol. 25, No. 289,
pp. 11-21.
Marceau, F.

1909. Recherches sur la morphologie, I'histologie et
la physiologie compardes des muscles adductcurs
des mollusques acdphales. Archives de Zoologie
Experimentale et Gfe^rale, serie 5, tome 2, fas-
cicule 6 (tome 42), pp. 295-469.
Mitchell, Harold D.

1935. The microscopic structure of the shell and
ligament of Cardimn (Cerastoderma) corbis Martyn.
Journal of Morphology, vol. 58, No. 1, pp. 211-220.
Olsson, Axel A.

1961. MoUusks of the tropical Eastern Pacific
particularly from the southern half of the Panamic-
Pacific f aunal province (Panama to Peru) . Panam-
ic-Pacific Pelecypoda. Paleontological Research
Institution, Ithaca, N.Y. Norton Printing Co.,
Ithaca, N.Y., 574 pp.
Owen, G., E. R. Trueman, and C. M. Yonge.

1953. The ligament in the Lamellibranchia. Nature,
vol. 171, No. 4341, pp. 73-75.
Palay, Sanford L. (editor).

1958. Frontiers in cytology. Yale University Press,
New Haven, Conn., 529 pp.
Plateau, Felix.

1884. Recherches sur la force absolue des muscles
des invertebros. I. Force absolue des muscles
adducteures des mollusques lamellibranches. Arch-
ives de Zoologie Expc^rimentale et G(5n6rale, s6rie
2, tome 2 (tome 12), pp. 145-170.
Pryor, M. G. M.

1940. On the hardening of the ootheca of Blalla
orienlalis. Proceedings of the Royal Society of
London, series B, vol. 128, pp. 378-393.
Pryor, M. G. M., P. B. Russell, and A. R. Todd.
1946. Protocatechuic acid, the sub.stance responsible
for the hardening of the cockroach ootheca. Bio-
chemical Journal, vol. 40, pp. 627-628.
Reis, Otto M.

1902. Das Ligament der Bivalven. (Morphologie
seines Ansatzfeldes, seine Wirkung, Abstammung
und Beziehungen zum Schalenwachstum). Jahre-
shefte des Vereins fiir vaterlandische Naturkunde,

Wurttemberg, Band 58, pp. 179-291. Stuttgart.
Carl Grlininger, K. Hofbuchdruckere: zu Guten-
berg (Klett -|- Hartmann).
Stempell, Walter.

1900. Ueber die Bildungsweise und das Wachstum
der Muschel- und Schneckcnschalen. Biologi-
sches Centralblatt, Band 20, pp. 698-703; ibiA.
pp. 731-741.
Stenzel, H. B.

1962. Aragonite in the resiltum of oysters. Science,
vol. 136, No. 3518, pp. 1121-1122.
Trueman. E. R.

1942. The structure and deposition of the shell of
Tellina tenuis. Journal of the Royal Micro-
scopical Society, series 3, vol. 62, pp. 69-92.

1949. The ligament of Tellina tenuis. Proceedings
of the Zoological Society of London, vol. 119, pp.
717-742.

1950a. Observations on the ligament of Mytilus
edulis. Quarterly Journal of Microscopical Science,
series 3, vol. 91. part 3, pp. 225-235.

1950b. Quinonc-tanning in the Mollusca. Nature,
vol. 165, No. 4193, pp. 397-398.

1951. The structure, development, and operation of
the hinge ligament of Ostrea edulis. Quarterly
Journal of Microscopical Science, series 3, vol. 92,
part 2, pp. 129-140.

1952. Observations on the ligament of Nucula.
Proceedings of the Malacological Society of London,
vol. 29, part 5, pp. 201-205.

1953a. The structure of the ligament of the Semo-
lidae. Proceedings of the Malacological Society of
London, vol. 30, pp. 30-36.

1953b. Observations on certain mechanical proper-
ties of the ligament of Pecten. Journal of Experi-
mental Biology, vol. 30, No. 4, pp. 453-467.

TuLLBERG, TycHO.

1881. Studien iiber den Bau und das Wachsthum
des Hummcrpanzers und der MoUuskenschalen.
Kongliga Svenska Vetenskaps-akademiens Hand-
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Vaillant, L£on.

1865. Recherches sur la famille des Tridacnid6s.
Annales des Sciences Naturelles: Zoology, serie 5,
tome 4, pp. 65-172.

64

FISH AND WILDLIFE SERVICE

CHAPTER IV
GENERAL ANATOMY

Page

IntrodiK'tory remarks - 65

Methods of study., 65

Organs underlying the shell 66

Organs underlying the mantle '0

The visceral mass 71

Nervous system "l

Anatomiea! peeuliarities "2

Bibliography "2

Before proceeding with the detailed description
of the structure and function of various organs
in tlie oyster, it appears desirable to present a
general anatomical picture of this niollusk and to
show the arrangement and topography of the
various systems of organs.

The anatomy of edible oysters is described in
several papers. Brooks (1905), Moore (1S9S),
Churchill (1920), and Galtsofi' (195S) give general
accounts of the anatomy of C. virginica. The
structure of the European oyster, 0. edulis, is
described by Orton (1937), Ranson (1943), and
Yonge (1960); a brief and partial description of
the anatomy of C. angitlata by Leenhardt (1926)
includes the histology of the species. The struc-
ture of the Bombay oyster, 0. cncullafa, is described
by Awati and Rai (1931); and a short anatomical
sketch of the Australian oyster, 0. comrnercialis,
is given by Roughley (1925).

The anatomical sketch of an adult C. virginica
given in this chapter describes the principal
organs of the oyster as they can be seen by dis-
secting the mollusk and e.xaniining the preparation
under a low-power microscope. Details that
can be observed by sectioning, staining, and
reconstruction are described in the chapters of
this book dealing with the respective organ
systems.

METHODS OF STUDY

Successful dissection of the oyster depends to a
considerable degree on the condition and sliape of
the specimen selected for study. It is convenient
to work with broad and large oysters measuring
about 4 to 5 inches in lieight and containing

FISHERY bulletin: VOLUME 64, CHAPTER IV

lean meats. In fat or in se.xually mature speci-
mens the large quantities of glycogen and of se.x
cells covering the organs make their anatomy
difficult to trace. Oysters most suitable for
anatomical study are those which have completed
spawning but have not yet accumulated much
glycogen. In New England waters such oysters
can be found in September and early October.

For dissection the oyster should be opened by
removing its flat (right) valve, a process facilitated
by fli-st narcotizing the oyster. Narcosis elimi-
nates the necessity of forcibly prying apart the
valves, a manipulation which in the hands of
an inexperienced person frequently results in
injury of the underlying tissues or, even more
often, to the hand of the operator by the sharp
edge of the shell. Another advantage of working
with a fully narcotized specimen is that the organs
and tissues remain fully expanded in their normal
position and are undistorted by contraction.

The best method of narcotizing is to use technical
magnesium sulfate (Epsom salt) as follows: The
oyster is thoroughly washed and scrubbed to
remove fouling organisms and then placed in a
suitable container, about S to 9 inches in diameter
and 3 inches deep, filled with sea water. During
the first 24 hours small quantities of Epsom salt
are gradually added until a concentration between
5 to 10 percent is reached, then the oyster is left
undisturbed for another 24 or even 48 hours at
room temperature. The magnesium sulfate should
be added very gradually because an excess of it
at the beginning of narcosis may cause the oyster
to close its valves and thus prolong the process.
Additional amounts of salt may be added because
oysters tolerate much higher concentrations of
magnesium sulfate and recover from it upon
being placed in running sea water. Completely
narcotized oysters do not respond to touch or
prick at the edge of the mantle.

With the narcotized oyster grasped in the liand,
right valve uppermost, a knife is inserted between

65

the mantle and the shell and carefully pushed
ahove the meat toward the adductor muscle,
its edge always at a sharp angle to the inner
surface of the valve. Actually it is preferahle
to move the oyster right and left, while gently
pressing it against the edge of the knife, rather
than to move the knife itself. After the attach-
ment of the adductor muscle to the sliell is severed,
the flat, right valve is lifted up until the ligament
breaks and the oyster is exposed in the cupped
valve which retains sea water. (In oyster bars
and restaurants raw oysters are usually served
on the right (flat) valve and the cupped left valve
is removed.) If it is necessary to open an un-
narcotized oyster, I prefer first to break the liga-
ment with a screwdriver, then to lift the valve
carefully and cut the adductor muscle. This
method reduces the chances of cutting the visceral
mass.

Dissection is mucli facilitated by allowing tlie
tissues to harden in 3 percent formalin for at least
1 day. For tracing the digestive tract and the
blood vessels I recommend, furthermore, tlie
in}ection of these systems with colored moulage
latex. For study of the digestive tract the follow-
ing method gives satisfactory results: the mouth
of the oyster is exposed by cutting out a small
triangular section of both valves and pushing up
the underlying tissues (the mantle cap). Blue
or red latex diluted with aliout 20 percent water
is injected into the mouth through a wide glass
pipette slightly flattened at the tip and supplied
with rubber bulb. During the injection the oyster
is held in a vertical position. Sometimes it is
difficult to fill the entire digestive tract with latex
injected through the mouth. An additional in-
jection can then be made through the anus with
a 2-ml. capacity hypodermic syringe, preferably
one of metal since latex rapidly adheres to glass
and causes the plunger to stick.

For injecting blood vessels through tlie ventricle
or auricles I prefer to use either latex diluted
with about .30 percent of water or vinyl resin
solution. Injection should be completed witliout
interruption in one operation, after which the
injected specimen is immediately placed in 5 per-
cent formalin in tapwater and left undistin-bed for
several hours or overnight to allow complete set-
ting of the latex or plastic. Preparations may be
indefinitely preserved in 3 to 5 percent formalin.

Various cavities and chambers of the oj'ster body
can be advantageously studied by making plaster

of paris casts. The valves of a live oyster are
forced apart by inserting an oyster knife at the
ventroposterior margin of the shell and gradually
rotating the knife until its edge is perpendicular
to the surface of the valves. The valves should
be opened very slowly to avoid tearing the ail-
ductor nuiscle. After a small wooden wedge is
inserted to prevent closing of the valves, freshly
made plaster of paris paste of the consistency of
heavy cream is injected into the cloaca and into
the opening of the promyal chamber. From time
to time the injected specimen is tapped gently
against the table to insure complete penetration
of the plaster into the smallest ramification of the
giU tubes. After the filling with plaster of paris is
completed, the wedge is removed and the valves
are pressed together. The preparation is left
undisturbed for 24 hours. After the plaster of
paris has hardened the shells and the soft parts
of the body are removed, the cast is dried for 24
hours at 56° C. and finally may be dipped in a hot
mixture of beeswax and turpentine to prevent
breaking of the finest ramification of the replicas.

ORGANS UNDERLYING THE SHELL

After the valve is removed, the body of the
oyster is seen to be covered with a soft membrane
called the mantle (figs. 71, 72). The mantle is a
bisymmetrical organ. Its left and right folds are
joined together at the dorsal edge where small
and slightly pigmented fold (not shown in the
figure) marks the position of the ligamental ridge,
a special organ which secretes the ligament. The
joint portion of the two lobes forms a cap which
covers the mouth and its associated structures
(fig. 71, m.). The remaining mantle edges are
free except for a point at the extreme ventral
margin (f.) where the two opposing lobes are
fused together to form a wide funnel-like channel,
the cloaca (fig. 72, cl.).

The edge of the mantle consists of three pro-
truding fringes, two of which, the outer and the
middle, are beset with highly sensitive tentacles
(t.). The tentacles and the edge of tlie mantle are
commonly pigmented.

Tlie parts of the mantle not attached to under-
lying organs enclose a large space filled with sea
water and known as the mantle cavity. Some-
times the open space under the mantle is referred
to as the shell cavity and the sea water retained in
it as tlie shell liquor. The space between the
mantle and the gills is often called the "gill cavity,"

66

FISH AND WILDLIFE SERVICE

_L

_L

0

Centimeters

Figure 71. — Organs of C. virginica seen after the removal of right valve, ad.m. — aflductor muscle; an. — anus: e.g. —
cerebral ganglion; f. — fusion of two mantle lobes and gills; g. — gills; h. — heart; l.ni. — left mantle; l.p. — labial palps;
m. — mouth; per. — pericardium; r. — rectum; r.m. — right mantle, sh. — shell; t. — tentacles. The right mantle con-
tracted and curled up after the removal of the right valve, exposing the gills. Portion of the mantle over the heart
region and the pericardial wall were removed. Drawn from live specimen.

an undesirable term because of possible confusion
with the inner spaces (cavities) of the-gills. The
correct terminology for the latter is water tubes
and gill chambers. The expression mantle cavity
seems to be more appropriate than tlie shell
cavity. As to the shell liquor, the term is well
establislied, especially in papers dealing with tiie
bacteriology of the oyster and, therefore, it should
be retained.

Under normal conditions the mantle underlies
atid adheres sliglitly to the shell, the secretion of
wliich is its principal function. As will be shown
later, this organ also participates in several other

functions; it controls the flow of water for respira-
tion and feeding; plays an important role in female
spawning; and receives and transmits sensory
stimuli.

In a living oyster the mantle cavity is always
full of sea water. As tlie shell is closed the surplus
water is ejected, but tiiat remaining in the free
spaces between the mantle lobes (fig. 73, m.c.)
keeps the enclosed organs constantly bathed in
water.

Various products of oyster metabolism accumu-
late in the shell liquor as well as considerable
quantities of mucus and blood cells discharged

GENERAL ANATOMY

67

Centimeters

Figure 72. — Oyster viewed from the right side. Portion of the right mantle and the wall of the epibranchial chamber
cut off to expose the gills and their water tubes, the cloaca, and the lower part of the gonad, ad.m.t. — adductor
muscle, translucent part; ad.m.w. — adductor muscle, white (opaque) part; an. — anus; bl.v.- — blood vessels of the
mantle; cl. — cloaca (the arrow indicates the direction of the outgoing current of water) ; cp.a. — circumpallial artery;
cp.n. — circumpallial nerve; ep.br. ch. — epibranchial chamber of the gills; f. — fusion of gills and mantle; g. — gills;
gd. — gonoducts; py.p. — pyloric process; q.m. — rudimentary Quenstedt's muscle; t. — tentacles; ur.v. — opening of the
urinogenital vestibule ; v.g. — visceral ganglion ; w.t. — water tubes of the gills. Drawn from a preserved specimen.

through the mantle and gills. The alkalinity of
the shell liquor retained in the mantle cavity
therefore decreases with time as the oyster remains
closed. Although liquor may become slightly
acid, excessive acidity is stopped by the buffering
action of calcium carbonate dissolved from the
shell.

The ability of the oyster to retain shell liquor
is a useful adaptation to life in the intertidal zone
permitting the animal to survive many days of

exposure to air. It is equally useful to those
oysters which live below tlie low water mark and
are never exposed to air. By closing their shells
tightly and by retaining some sea water they are
able to survive unfavorable conditions caused by
floods or by tlie temporary presence of toxic or
irritating substances in the water.

The color of the surface of the mantle facing tlie
shell is variable. Lean oysters devoid of glycogen
are usually of a dull grayish color whereas "fat"

68

FISH AND WILDLIFE SERVICE

JK.e.

m.c.

t

-U.5

Centimeters

rme.

Fir.vwE 73. — Transverse section of the dorsal part of an adult C. virginica a short distance below the labial palps. Drawn
semidiagranimatically from an enlarged photograph of stained section. Bl.s. — blood sinus; br.ef.v. — branchial
efferent vein; c.af.v. — common afferent vein; c.t. — connective tissue; di.d. — digestive diverticula; ep.br. ch. — epi-
branchial chamber; g. — gills; g.m. — gill muscles; g.r. — gill rod, gn. — gonad; in. — intestine; k. — kidney; l.af.v. — lateral
afferent vein; l.m. — left mantle; l.m.e. — edge of left mantle; m.c. — mantle cavity; pr.ch. — promyal chamber; r.af.v. —
right afferent vein; r.m. — right mantle; r.m.e. — edge of right mantle; st. — stomach; w.t. — water tube of the gills.

GENERAL ANATOMY

69

oysters are white and those full of spawn are
creamy-yellowish. The green color of oysters
from certain localities is attributed to the accumu-
lation of copper or to the absorption of blue-green
pigment from certain diatoms upon which they
feed. Mantle color is always a good indication of
the condition of the mollusk.

Several ramifying blood vessels can be easily
seen on the surface of the mantle (fig. 72, bl. v.).
A broad blood vessel along the periphery is the
circumpallial artery (fig. 72, cp. a.). A narrow
and darker line immediately adjacent is a circum-
pallial nerve (fig. 72, cp.n.). A small, slightly
pigmented depression in the dorsal end of the
mantle marks the position of a nonfunctional
Quenstedt's muscle (fig. 72) barely attached to
the valve.

The most conspicuous element of the oyster
anatomy visible after the removal of the valve is
the posterior adductor muscle. Tliis ovate organ
consists of a larger dorsal, translucent portion
(fig. 72, ad.m.t.) and a consistently smaller,
ventral opaque part (ad.m.w.).

A semitransparent oval membrane covers the
pericardium, the chamber in which the heart is
suspended (fig. 71, per.). On the left side of the
oyster the pericardial wall lies directly under the
valve, but on the right side the large and asym-
metrical promyal chamber (fig. 73, pr.ch.)
separates tlie pericardium from the mantle.

ORGANS UNDERLYING THE MANTLE

Directly under the free edge of the mantle
along the entire anteroventral side of the oyster
lie the gills (fig. 72, g.). They can be exposed by
cutting off the mantle along the line of its attach-
ment to the base of the gills, or by lifting the
mantle and pulling it up. If a piece of shell is
sawed ofl^ at the anterior edge of the valve the
corresponding portion of the mantle curls up and
exposes the gills underneath. For several days
the opposite fold of the mantle retains its normal
position with the tentacles (t.) spread over the
edge of tlie shell, while the curled mantle edge
under the cut secretes a vertical plate. Later
on the mantle fold of the intact side of the oyster
also curls up and by depositing new shell material
at the angle to the valve closes the gap. Cutting
off a portion of one valve proved to be a useful
procedure for observing the functions of tlie gills
and mantle.

The gills consist of two pairs of lamallae or

gill plates, one pair on each side (figs. 71, 72, g.).
At the anterodorsal margin thei.r free and gently
curved edges touch the lower tips of the labial
palps (fig. 71, l.p.) and their bases are joined to
the mantle. In the ventroposterior part of tlie
body tiie gills and the two lobes of the mantle
join to form a channel (fig. 71, f.) which marks
the entrance to the cloaca (fig. 72, cl.).

Tlie moutli (fig. 71, m.), a narrow horizontal
slot above the dorsal edges of the two posterior
labial palps, lies under the liood or cap formed by
the anterior fusion of the two mantle folds. It
can be seen by cutting off the mantle cap and
pressing down the upper (dorsal) edges of the
palps.

The cloaca (fig. 72, cl.), a large funnel-shaped
space between the ventral side of the adductor
muscle and the gills, is a continuation of tlie
epibranchial chamber (fig. 72, ep.br. ch.) which
extends along the gills. The latter can be ex-
posed by cutting along the wall of the cloaca,
starting from the mantle junction (fig. 71, f.) and
following the edge of the muscle. The epi-
branchial chamber extends along the base of the
gills. Wlien the dissected portions of the cloacal
wall are pulled apart, the following structures are
revealed: the rectum and anus (figs. 71, 72, r.,
an.), located on the ventroposterior border of the
adductor muscle; the blunt tip of the pyloric
process (fig. 72, py.p.) of the visceral mass, which
projects into the epibranchial chamber; the small
and almost invisible opening of the urinogenital
groove or vestibule (fig. 72, ur.v.), located on the
wall of the pyloric process; and the visceral
ganglion (fig. 72, g.), situated in a shallow de-
pression between the two divisions of the adductor
muscle and partially covered by the pyloric
process.

The heart (fig. 71, ii.), seen after removal of the
pericardial wall, consists of one ventricle and two
pigmented auricles. Two aortae (not shown in
the diagram) emerge from the tip of the ventricle,
and large venous sinuses (also not shown) empty
into tlie auricles. The slightly pigmented struc-
ture extending dorsally from tlie auricular side of
the pericardium along tlie base of tlie gills is the
organ of excretion (kidney) frequently called the
organ of Bojanus (fig. 7:5, k.). Inasmuch as there
is no doubt regarding the function of this tubular
thin- walled organ it seems preferable to call it the
kidney. Urine is collected in a large reservoir in
the lower (ventral) part of the kidney before

70

FISH AND WILDLIFE SERVICE

being discharged through the uriiKigenital vesti-
bule. On each side of the auricular part of the
pericardium there is a fine opening from which a
canal leads to the kidney. This renopericardial
opening and tlie canal are difficult to see witii the
unaided eye.

THE VISCERAL MASS

The \ery short esophagus enters tiie large and
somewliat twisted stomach, into which a wide sac
containing tlie crystalline style also opens. The
visceral nuiss (figs. 72, 7.'3) occupies the dorsal
half of tlie body, above the adductor nmscle. It
consists of esophagus, stomach, crystalline style sac
inside the pyloric process, and intestine embedded
in connective tissue. The stomach directly com-
municates with the digestive diverticula, a green-
ish mass of glandular tissue (fig. 73, St., di. d.),
which completely surrounds both stomach and
intestines. The intestine, after leaving the stom-
ach, makes a loop (fig. 73, in., and fig. 197, ch. X)
which ends in the rectum (fig. 71, r.) at the dorsal
edge of the adductor muscle. A snuiU rosette at
the tip of the slightly protruding rectum surrounds
the anus (fig. 72, an.), located in the area con-
tinuall}' swept by the current of water from the
cloaca (fig. 72, cl.).

Between the digestive diverticula and the
surface epithelium lie the gonads (fig. 73, gn.).
After spawning these layers disappear almost
completely, being represented only by a thin
germinal lining. The gonad is not visible to the
unaided eye at this stage. At the time of full
sexual development the layer of gonadal tissue in
large specimens may reach several millimeters in
thickness. Manj^ branching channels, the gono-
ducts (fig. 72, gd.), through which sex cells are dis-
charged, are clearlj' visible on the surface of a
sexually mature specimen. They all empty into
a common gonoduct leading into the urine >-
genital groove (fig. 72, ur. \-.) from whicli eggs or
sperm ai-e discharged into the epibranchial cham-
ber (ep. br. ch.). Secondary sex cliaracters are
absent.

The sex of the oyster can be recognized by
niicrosocopic examination of thegonad. Hermaph-
rodites among adult C. virginica are rare. Out of
many thousands of oysters examined in the course
of my studies I have found only one oyster with
tlie gonads containing both eggs and sperm. The
European 03'ster, (>. edulis, and tiie Olympia
oyster, (>. Iiirida, are hermaphroditic (A full

discussion of sex in the ovster is found in chapter
XIV.)

The position of the fully developed gonad in
relation to other organs of the visceral mass can
best be studied in a series of trans\'erse sections of
the dorsal half of the oyster. Figure 73 shows the
relative position of the organs as seen on the sec-
tion made just below the labial palps. The
gonad (gn.) is irregular in shape and located close
to the surface of the body. The digestive divertic-
ula (di. d.) occupy the larger part of the visceral
mass between the gonad and the digestive tract
itself, which at this level is represented by the
stomach (st.) and two cross sections of the in-
testines (in.). The rest of the visceral mass
consists of connective tissue (c.t.) containing
irregular blood sinuses (bl. s.), which may be full
of blood. The series of twisted tubules compris-
ing the kidney (k.) is located near the surface on
both sides of the body above the gills.

The large empty chambers between the gills
and the visceral mass directly communicate with
the water tubes (w.t.) of the gills (g.). The
epibranchial chamber (ep. br. ch.) on the left side
is much smaller than the corresponding chamber
on the right side. The latter, called by Nelson
(1938) promyal chamber (pr. ch.), extends to the
dorsal end of the oyster and opens to the outside
in the posterodorsal part of the body, independ-
ently of the cloaca. Water tubes (w.t.) inside the
gill plates open into these chambers. Soft and
delicate tissue of the gills is supported by the
framework of chitinous rods, the largest being
located at the base of the gills (g.). Two sets of
muscles (g.m.), one below and one above the
largest gill rods, control the mo\ements of the
plates. The five principal blood vessels of the
gills are located above the skeletal rods: the com-
mon afferent vein (c. af. v.) ; two branchial efferent
veins (br. ef. v.), one on each side; and two lateral
afferent viens (1. af. v.), also one on each side
of the oyster.

NERVOUS SYSTEM

The nervous system can be studied best by
reconstructions made from sectioned material
since only the principal ner\'es and ganglia can
be revealed by dissection. The \isceral ganglion
(fig. 72, v.g.) is located in a slight depression on
tlic anterior side of the adductor muscle, partially
hidden Ijy the tip of the pyloric process. It can
be observed by cutting the wall of the cloaca and

GENERAL ANATOMY

71

of the epibranchial chamber, then placing the
oyster on its posterior edge, lifting its ventral
side slightly toward tlie observer, and pulling the
dissected portions of tiie wall apart. The ganglion
and its nerves are then \'isible against, the back-
ground of the surrounding tissues. Some of the
individual nerves, namely, the posterior pallial
nerve (fig. 253, in ch. XIII, p.p.n.) and the lateral
pallial nerves (l.p.n.), emerge from the posterior
end of the ganglion and can be followed without
much difficulty until they begin to ramify. The
posterior pallial nerve follows the right side of the
adductor and sends a short branch to a sense
organ — a small unpigmented protulterance called
the pallial or abdomiiuil organ (p.o.). On the left
side of the oyster the pallial organ is much smaller
and is located much closer to the ganglion; in fact,
in many oysters only the right pallial organ is
present.

The anterior pallial nerve (fig. 253, in ch. XIII,
a.p.n.) and branchial nerve (br.n.) leave the
dorsal end of the ganglion and for some distance
follow the nerve trunk which leads to the cerebral
ganglia and is known as the cerebrovisceral con-
nective (c.v.con.). The cerebral ganglia (fig. 71,
e.g.) are embedded in connective tissue at the
bases of the labial palps. A very thin cerebral
commissure passes dorsally over the esophagus
and connects the cerebral ganglia in a typical loop
or ring. The circumpallial nerve (fig. 72, cp.n.)
follows the circumpallial artery and can easily be
seen at the edge of the e.xpanded mantle. The
other nerves emerging from the visceral and
cerebral ganglia can be more conveniently studied
on sectioned preparations and are described in
chapter XIII.

ANATOMICAL PECULIARITIES

In several respects the anatomy of the oyster is
simpler than that of other bivalves. The absence
of a foot results in the lack of pedal ganglia; only
the posterior adductor muscle is present, and
there are no specialized organs of siglit, although
the animal is sensitive to change of illumination.
On the other hand, the edge of the mantle is
fringed with highly sensitive tentacles abundantly
supplied with nerves leading to the ganglia. As
in other bivalves, the nervous S3^stem is not
centralized but is represented by widely separated
ganglia. The structure of the cerebrovisceral
connectives, of the circumpallial nerve, and other
large nerves resembles more the structure of

ganglia than that of the nerve, a condition which
undoubtedly results in a high degree of coordina-
tion among the various parts of the organism.

In addition to performing their principal func-
tions, several organs of the oyster also participate
in other activities. The gills, for instance, are
not ou\y the organ of respiration but collect and
sort food as well. The mantle is used extensively
in the control of the flow of water through the
body; the coordinated action of the adductor
muscle, gills, and gonad is necessary for the
effective discharge of eggs by the female oyster.
In other animals such functions are performed by
special organs, but in the evolution of the oysters
the high degree of coordination developed among
different parts of the body eliminates the need for
specialized structures, and new and complex
functions are successfully performed by synchro-
nizing the work of the existing parts.

BIBLIOGRAPHY

AwATi, P. R., and H. S. Rai.

1931. Oslrea cucidlala (the Bombay oyster). The
Indian Zoological Memoirs on Indian Animal
Types, III. Methodist Publishing House, Luck-
now, India, 107 pp.
Brooks, William K.

1905. The oyster. A popular summary of a scien-
tific study. 2d ed. The Johns Hopkins Press,
Baltimore, Md., 225 pp.
Churchill, E. P., Jr.

1920. The oyster and the oyster industry of the
Atlantic and Gulf Coasts. [U.S.] Bureau of Fish-
eries, Report of the Commissioner of Fisheries
for the fiscal year 1919, appendix 8 (Document
890), pp. 1-51.
Dahmen, Peter.

1923. Anatomie von Oslrea chilensis Philippi. Jen-
aische Zeitschrift fiir Xaturwissenschaft, Band 52,
pp. 575-626.
Galtsoff, Paul S.

1958. The oyster. Encyclopaedia Britannica, 1958
ed. vol. 16, pp. 1001-1004. Encyclopaedia
Britannica, Inc., Chicago, 111.
Leenhardt, Henry.

1926. Quelques etudes sur Grayphea angulata (Huitre
du Portugal). Annalos dc I'lnstitut Oceano-
graphique, nouvelle serie, tome 3, fascicule 1,
pp. 1-90.
MooRE, H. F.

1898. Oysters and methods of oyster-culture. U.S.
Commission of Fish and Fisheries, Part 23, Report
of the Commissioner for the year ending June 30,
1897, pp. 263-338.
Nelson, Thurlow C.

1938. The feeding mechanism of the oyster. 1.
On the pallium and the branchial chambers of
Ostrea virginica, 0. edulis and O. angidaia, with

72

FISH AND WILDLIFE SERVICE

comparisons with other species of the genus. Collection dirig^e par Jean Rostand. Gallimard,

Journal of Morphology, vol. 63, Xo. 1, pp. 1-61. Paris, 261 pp.

Orton, James H. Roughlby, T. C.

1937. Oyster biology and oyster-culture, being the 1925. The story of the oyster. Australian Museum

Buckland Lectures for 1935. Edward Arnold and Magazine, vol. 2, No. 5, pp. 163-168.

Co., London, 211 pp. Yonge, C. M.

R.\NSON, Gilbert. 1960. Oysters. Collins Clear-Type Press, London,

1943. La vie des huitres. Histoires Naturelles-1, 209 pp.

GENERAL ANATOMY 73

CHAPTER V
THE MANTLE

Page

Appearance 74

Anatomy _,_ 74

Rudimentary muscle of the mantle 79

Histology 79

Connective tissue 79

Muscles 83

Blood vessels 84

Epithelium, tentacles, and nerves 84

Periostracal groove and gland 86

Suhligamental ridge 89

Functions of the mantle 90

Formation and calcification of shell 91

Theories of calcification 94

Cytological identification of calcium 101

Sources of calcium _._ 103

Mineralogy of calcium carbonate in molluscan shells _ 103

Rate of calcification. - 104

Bibliography 107

The inner organs of all mollusks are covered
with a soft and fleshy fold of tissue called the man-
tle or pallium (Latin for cloak or coverlet). The
structure of the mantle is relatively simple: the
organ consists of a sheet of connective tissue con-
taining muscles, blood vessels, and nerves and is
covered on both sides by unicellular epithelium.
Many blood cells invade and wander throughout
the entire thickness of the mantle, infiltrating the
spaces (sinuses) in the connective tissue, and
crawling through the epithelium to aggregate on
the outer surface of the mantle.

Although tlie principal role of the mantle is the
formation of the shell and the secretion of the
ligament, the organ plays a major part in several
other functions. It receives sensory stimuli and
conveys them to the nervous system and assists
in the shedding and dispersal of eggs during
spawning (see ch. XIV). The mantle also par-
ticipates in respiration by providing direct ex-
change of gases between the surface tissues of the
oyster and the surrounding water. It stores re-
serve materials (glycogen and lipids), secretes large
quantities of mucus and, finally, aids in excretion
by discarding blood cells loaded with waste
products.

APPEARANCE

The appearance of the mantle reflects the condi-
tion of the 03-ster. At the time of sexual maturity

74

it is a creamy-yellowish color. In oysters which
have accumulated large amounts of glycogen with
the onset of the cold season the mantle is white
and thick. In oysters of poor quality or in those
which have not yet recovered after spawning, the
mantle is so transparent that the brown or green-
ish color of the underlying digestive organ is
clearly visible through the thin and watery tissue.
Oysters in this condition are particularly suitable
for the study of muscles, blood vessels, and nerves
which in good quality, "fat" oysters are covered
by a thick layer of reserve materials.

Pigment cells are concentrated along the free
edge of the mantle and in the tentacles in a band
varying in color from light brown to jet black.
Also, accunuilation of copper in the blood cells
may produce a distinct green coloration. Difi'er-
ent intensities of pigmentation are often found
in oysters of identical origin growing together,
and cannot be correlated with geographical loca-
tion or type of botton\.

ANATOMY

For a detailed study of the mantle the oyster
should be fully narcotized by Epsom salt (see
p. 65) or by refrigerating it overnight at a tempera-
ture of about 2° to 4° C. After the valves are
forced apart and the body dissected along the
median plane, the two halves of the oyster are
left attached to their respective valves and the
mantle is preserved in its natural position by hav-
ing a large quantity of fixing fluid poured over it.
Portions of the mantle required for study are cut
off, stained, delwdrated, cleared, and mounted.
In this way very satisfactory whole mounts can
be obtained.

The two lobes of the mantle are joined together
at tlie dorsoposterior margin, and form a cap or
hood which covers the mouth and the labial
palps (fig. 71). Along the anterior and ventral
sides of the body the lobes are free and follow the
curvature of the shell. When the oyster opens
its shell the mantles separate with the valves to

FISHERY bulletin: VOLUME 64, CHAPTER V

which they adhere, leaving a narrow opening
between the two lobes through which sea water
can enter the mantle cavity. The edge of the
mantle, may, however, occupy various positions:
it may extend parallel and beyond the edge of the
valves to leave a wide space between the two
opposing lobes, or it may bend inward almost
perpendicular to the shell surface (fig. 74) to
reduce or completely close the opening between the
two lobes and thereby limit the access of water
to the mantle cavity. The behavior of the
mantle edge as a regulatory mechanism controlling
the flow of water through the mollusk will be
discussed later (p. 185). In a closed oyster the
mantle edge is located about midway between
the distal margin of the gills and the edge of the
shell. Its position is marked by an impression
called the pallia] line, which is less pronounced in
the oystei's than in clams and some other bivalves.
At the ventroposterior end of the body the two
opposing lobes of the mantle join the gills to form

the delicate outside wall of the cloaca (figs. 72
and 75, cl., f.). On the left side of the body the
mantle is joined to the visceral mass; on the
right side it is separated from the visceral mass
by the promyal chamber. The fusion of the
mantle with the visceral mass and with the bases
of gill plates forms the wall of the epibranchial
chamber, which leads to the cloaca (fig. 75, d.).
The relative position of the epibranchial and
promyal chambers can be seen in the cross
section of the oyster made through the dorsal
part of the body (fig. 73, ep.br.ch.; pr.ch.).

An oblong slit between the two mantle lobes on
the dorsoposterior side of the body marks the
opening of the promyal chamber. The inside of
this chamber can be examined by completely
narcotizing the oyster and forcing its valves apart
as far as possible without tearing the adductor
muscle. Viewed from the posterior side the
promyal chamber in a relaxed oyster appears as
an oval cavity (fig. 75) to the left of the adductor

illimeters

FiouRE 74. — Cross sections of the valves, mantle, gills, and adjacent portion of the visceral mass of C. virginica. In both
diagrams the valves are open; the open pallial curtain (at left) permits free access of water to the mantle cavity; the
closed pallial curtain (at right) prevents water from entering the mantle cavity. The outer lobe adheres closely
to the valve and is not visible. Drawn from the photomicrographs of cross section of adult oyster. Bouin, hema-
to.\ylin-eosin.

THE MANTLE

75

--— — _2.>^

-^^

. ~~~; r-^

X

^s^^^^^

■ ■■ ■■;_:::i-ii-^^>-— -

"SS^ ^

^te^^gC^^^^ ^^'-l- - -

r.-^

1

^V^^^^^^j^

ir^---i-^.^ :-.■-: ■■■:■:■.•-,. -■

j::^<ii**aeixii^

^■^^^"^^Nis^^

' '"\^^i^2l?^2riiii5=OTvr

j|gjpr^;;r^^T^'^

' "" """""W^ N

^;;^^^'*i?_;3_^^_2^|]

1

i

r~s:j!s!Jig\i

jc y

1 1 1 1

r ®®oo/^

^^^^*^^J^

pr. ch."

"""■■^■(aj^ -^■^^"^

^^---..-rSSr

sss^^fesa

;>^^'*^^--ci.

-^m^

^^ad. m.

0

Centimeters

5

Figure 75. — Promyal chamber (at left) and cloaca (at right) viewed from the posterior side of a large oyster (C. virginica)
completely relaxed by narcosis. Note the fusion of the two opposing lobes of the mantle, and the adductor muscle
(in the middle). Drawn from life. Actual size. ad.m. — adductor muscle; cl. — cloaca; f. — fusion of mantle lobes
and gills; pr.ch. — promyal chamber; r. — rectum.

muscle. The large round openings of the water
tubes of the gills can be seen on the inner wall of
the chamber. The rectum extends along the
edge of the chamber, ending with a round anus
adhering to the side of the adductor muscle; the
opening of the cloaca lies to the right of the muscle.
The water tubes emptying into the cloaca and the
fusion of the mantle with the gill lamellae are
also clearly visible.

The most conspicuous components of the mantle
are the radial muscles, the blood vessels, and the
nerves (fig. 76). All these structures can be
identified in a piece of fresh tissue stretched over
a glass slide and examined under strong illumina-
tion with a low-power microscope. For more
detailed study, it is necessary to prepare whole
mounts or to section the preserved tissues.

The radial muscles extend from the place of
their attachment to the visceral mass to the edge
of the mantle. At about two-thirds of their
length from their base they begin a fanlike ex-
pansion toward the periphery before terminating
in the base of the tentacles. The majority of
the muscles are accompanied along their length
by nerves, blood vessels, and blood sinuses. Much
more slender than the radial muscles are the
concentric muscular bands wliich parallel the free
edge of the mantle (not shown in fig. 76) and are
more abundant at its thickened distal edge.

Because of its strongly developed musculature,
the mantle is highly contractile. It may stretch
a considerable distance beyond the edge of the
valve, or withdraw inside the shell, and even roll
up into a tube. Contraction of the radial muscles
will throw the inner surface of the mantle into

ridges which serve as temporary channels for
discarding mucus and foreign particles accumu-
lated on it. These movements may involve
either the entire surface of the mantle or only a
small portion of it, depending on the intensity
of stimulation received by the tentacles.

The wide circumpallial artery (fig. 76, cp.a.)
follows the entire periphery of the mantle. At
low magnification it is usually visible as a wide
tubular structure with many branching vessels
which communicate with the irregular spaces
(blood sinuses) within the connective tissue. A
large pulsating blood vessel, called the accessory
heart (ch. XI, fig. 236), is located in the ante-
roventral part in each lobe of the mantle. The
structure and the function of this vessel are dis-
cussed in chapter XI, p. 254.

Just outward from the circumpallial artery runs
the circumpallial nerve, which also extends along
the entire margin of the mantle. In whole mount
preparations seen under low power, the circum-
pallial nerve appears as a compact unbranching
band. Examination under higli power, however,
reveals a fine network of small nerves connecting
the circumpallial nerve with nerves and with the
visceral and cerebral ganglia. Since nerve fibers
on the surface of the mantle and in the tentacles
lead to the circumpallial nerve, stimuli received
by the neuroreceptors of these areas are trans-
mitted through the circumpallial nerve to the
radial nerves and reach either the visceral or the
cerebral ganglia.

The thick and muscular border of the mantle
is divided into three lobes (fig. 77) which have
been described in the literature as "folds" (Awati

76

FISH AND WILDLIFE SERVICE

Figure 76. — Whole
portion of connect
Safranin stain,
blood vessels; cp
cumpallial nerve;
tain (inner lobe) ;
or shell lobe; ti—
of middle lobe,
muscles are not
toxvlin.

Cenfimeters

mount of a piece of mantle. .Major
ive tissue was removed by maceration.
Magnified about 10 times, bl.v. —
a. — circumpallial artery; cp.n. — cir-
m.l. — middle lobe: p.c. — pallial cur-
r.m. — radial muscle; o.l. — outer lobe
-tentacles of inner lobe; t2 — tentacles
Radial nerves surrounded by radial
visible. Formalin 5 percent, hema-

and Rai, 1931), "reduplications" (Nelson, 1938;
Pelseneer, 1906), "lamellae" (Hopkins, 1933),
"lames" in French (Leenhardt, 1926), and
"Klappe", in German (Rawitz, 1888). The term
"reduplication" is misleading because the lobes
are not formed by the duplication of the mantle
tissue, being comparable rather to a fringe or
flounce at the margin of a soft material. To avoid
confusion the term marginal lobes is retained in
this text.

The mantle border of all the species of oysters
studied, namely, C. mrginica, C. angulala, C.

gigas, 0. edulis, and 0. lurida is divided into
three projecting lobes, the outer or shell lobe
(sh.L), the middle lobe (m.l.), and the inner lobe
or pallial curtain (p.c). Hopkins' statement
(1933, p. 483) that "The border of the mantle
(of C. gigas) divides into two lamellae, each
bearing a row of tentacles" is an obvious
inaccuracy of description.

The outer or shell lobe (sh.l.) is narrow and
devoid of tentacles. It lies in contact with the
margin of the shell and may be seen protruding
beyond the edge of the valve diu-ing periods of
rapid growth. The middle and the inner lobes
each bear a row of sensitive and highly contractile
tentacles.

The inner lobe or pallial curtain (fig. 77, p.c.)
is especially broad and turned inward. In de-
scribing this structure in scallops Pelseneer named
it the "velum" (1906). Although that term has
been used by several investigators (Awati and
Rai, 1931; Dakin, 1909b) Nelson (1938) pointed
out that the term "velum" is better known as the
swimming organ of the pelecypod larvae and
proposed to call the inner lobes of the mantle
the "pallial curtains". This term seems to be
appropriate, but is used in this book in the singular
since there appears to be no advantage in the
plural recommended by Nelson.

The inner lobe may be projected into the mantle
cavity (fig. 74). Depending on the degree of
contraction of various sets of muscles the inner
lobe assumes different angles in relation to the
mantle as a whole. In a fully relaxed mollusk
the lobe of each side extends outward in the general
plane of the mantle and shell. In a contracted
state the lobes on both sides project inward almost
at right angles to the surface of the mantle; in
this position the mantle borders touch and the
tentacles of the two sides interlock, effectively
sealing the entrance to the mantle cavity. This
function of the inner lobe was first described by
Rawitz in 1888 and was redescribed in 1933 by
Hopkins. As will be shown later (p. 304) the
pallial curtain also plays an important role during
the spawning of female oysters.

The deep furrow between the shell lobe and
the middle lobe is called the periostracal groove
(fig. 77, per.gr.), tlie name referring to the secre-
tion site of organic shell material by glandular
cells concentrated in the deepest portion of the
groove and collectively known as the periostracal
or conchiolin gland (^c.gl). During the shell-

THE MANTLE

733-851 O — 64-

77

Figure 77. — Transverse section of the edge of the mantle of adult ('. virginica. Bouin 3, heniatoxylin-eosin. The outer
or shell lobe at left faces the valve (not shown) and is bent as a result of fixation. The section passes between the
tentacles of the inner lobe (pallial curtain) ; only the tentacle of the middle lobe (m.l.) is seen. c.gl. — conchiolin (or
periostracal) gland; conch. — sheet of conchiolin spread over the shell lobe; cp.a. — circumpallial artery; cp.n. — circum-
pallial nerve; el.f. — elastic fibers; ep. — epithelium; l.m. — longitudinal muscles of tentacles; m.l. — middle lobe; ob.m. —
oblique muscles; p.c. — pallial curtain (inner lobe); per. gr. — periostracal groove; sh.l. — shell or outer lobe; tr.m. —
transverse muscles.

growing season, viscous yellowish material (fig.
77, conch.) accumulates in the groove and grad-
ually oozes out to the periphery of the outer
mantle lobe, where it solidifies into the perio-
stracum. The groove between the middle lobe
(m.l.) and the pallial curtain secretes mucus,
which is gradually moved by ciliary currents to
the outer margin of the mantle and there discarded.
It has already been noted that the edges of the
middle and the inner lobe each bear a row of
highly e.xtensible, tapering tentacles; however,
their arrangement and size in th-e two lobes are
different. Two types are clearly visible along the
edge of the middle lobe: numerous short and slen-
der tentacles, and less abundant long and stout

ones (fig. 76). The order of the tentacles follows
a certain pattern, namely, each long tentacle is
succeeded by a group of four to si.x small ones (t2).
The stout tentacles frequently occupy a position
slightly out of line with the small ones, being a
little nearer to the inner fold. The inner lobe
bears only the long and stout tentacles (ti).

There is great variation in the size of all the
tentacles and in their pigmentation. Since they
are highly sensitive to touch and other stimidi and
retract at the slightest disturbance, tlieir relative
size can be observed only wlien they are completely
relaxed. In fully narcotized adult oysters the
ratio between the niunbers of tentacles on the

78

FISH AND WILDLIFE SERVICE

inner and middle lobes was found to vary from
10:18 to 10:32.

It has not yet been definitely established
whether the two types of tentacles contain different
receptors and therefore respond to different
stimuli. According to Elsey (1935) the large
tentacles of C. gigas are more sensitive to hydro-
chloric acid than the small ones. Hopkins (1932)
does not specify which row of tentacles was under
(ihservation in his work on sensory stimulation
of C. virginica. In my experiments (see p. 293)
dbservations were made exclusively on the long
tentacles of the inner lobe.

A narrow and slightly pigmented cylindrical
structure along the dorsal edge of the mantle
(fig. 78) marks the position of the subligamental
ridge, the organ which secretes the ligament.
The ridge consists of a layer of specialized epi-
thelium underlined by connective tissue. Large
blood vessels are found close to the base of the
ridge. Microscopic structure of the ridge is given
on p. 83.

RUDIMENTARY MUSCLE OF THE
MANTLE

A small and sometimes hardly visible muscle is
located on the dorsal part of the mantle. Its
location is sometimes marked by light violet
pigmentation and by a shallow depression in the
corresponding part of the valve to which the
muscle adheres. The attacliment is weak, and in
the majority of oysters the muscle Separates from
the valve when the valve is lifted. Leenhardt
(1926) states, however, that in some 0. ednUs the
muscles were so strongl,y attached to the shell that
they could not be separated without rupturing the
mantle tissue. Examination of sections of the
mantle of C. virginica from the Woods Hole area
convinced me that muscle fibers do not extend
from one side to the other, but end in the con-
nective tissue of the mantle. Th& muscle is
apparently nonfunctional and morphologically is
not analogous to the anterior adductor of bivalves.
Leenhardt (1926) considers the rudimentary
muscle of the mantle as a vestige of the larval foot
retractor which disappears during metamorphosis.
Stenzel (1963) states that this musde is present in
all the Ostreidae and calls it Quenstedt's muscle in
honor of its discoverer (Quenstedt, 1867).

HISTOLOGY

The mantle consists of connective tissue which
envelops the muscles, blood vessels, and nerves

and is covered on both sides with the epithelium.
CONNECTIVE TISSUE

The most conspicuous structural element of the
connective tissue is the vesicular cell, characterized
by large globular or oval body and relatively small
nucleus without nucleoli. In zoological literature
these cells appear under a variety of names and
were even incorrectly considered as lacunae
(Leenhardt, 1926) and mucus cells (List, 1902).
Well-developed membranes outline cell boundaries
sharply; the protoplasm within forms a delicate
network of fine granules. In preparations dehy-
drated with alcohol the inside of the vesicular
cells appears almost empty, but in tissues treated
with osmic acid and in frozen sections stained
with Sudan II and other fat stains large globules
of lipids are seen to fill the inside of the cells (figs.
79 and 80). Less abundant are the smaller round
cells with more compact protoplasm. They often
occur near small arteries (fig. 81, r.c). The
fusiform cells (f.c.) with small bodies and oval
nuclei form long branching processes which anasto-
mose and touch each other.

Examination of frozen sections of connective
tissue treated with toluidine blue or other meta-
chromatic stains shows clearly the presence of a
cytoplasmic ground substance with a very fine
reticulum supporting various inclusions. After
the removal of glycogen this substance can be
stained very deeply with periodic acid fuchsin
(McMannus reagent )or with Hale stain which is
used to test for acid polysaccharides of the hyalu-
ronic acid type (Hale, 1946). The results of such
staining reactions have been interpreted in the
literature as indicating the presence of mucopoly-
saccharides or mucoproteins. Histological meth-
ods are not entirely dependable (Meyer, 1957),
but so far no chemical analyses of the connective
tissue of the mantle have been made. It is known,
however, that acid mucopolysaccharides are
among the components of the ground substances
in mammalian tissues. It is very likely tnat they
are also present in the connective tissue of the
oyster.

Elastic fibrils are scattered throughout the
connective tissue of the entire thickness of the
mantle but appear to be more abundant at the free
edge and in the layers underlying the surface
epithelium (fig. 77, el.f.). Muscle fibers are also
very abundant and will be discussed in detail later.

In some specimens the mantle may be thin and
transparent whereas in others it is thick and

THE MANTLE

79

0

Microns

200

Figure 78. — Longitudinal section of the subligamental ridge made at right angles to its dorsal surface. Bouin 3,
hematoxylin-eosin. bl.v. — large blood vessel; cl.m. — basal elastic membrane; ep. — epithelium; m. — muscle fibers;
pig.c. — pigment cells; po. — pockets between the epithelial cells; v.c. — vesic\ilar cells.

80

FISH AND WILDLIFE SERVICE

Microns

Figure 79. — Vesicular cells of connective tissue from the mantle of an adult C. virginica surrounding the blood sinus.
Blood cells crawl between the cells of connective tissue and penetrate into the sinus. Bouin 3, hematoxylin-eosin.

opaque. These changes in appearance usually
coincide with seasonal cycles in the glycogen con-
tent of the connective tissue and with the progres-
sive stages of gonad development.

The presence of glycogen can be easily demon-
strated by treating the tissue with Lugol solution
(1 percent iodine in 2 percent potassium iodide
in water). Specific reagents used for the identi-
fication of glycogen, such as Best's carmine and
Bensley's modification of Bauer-Feulgen reagent
(which stains glycogen granules red-\iolet), also
give good results.

In the live oyster glycogen can be seen as small
colloidal granules which ooze from the tissue under
slight pressure. In preser\ed and stained
material it appears in the form of granules or
rods (fig. 82). The total amount of glycogen in
tlie connective tissue may be so great that the
blood vessels and nerves of the mantle are com-
pletely hidden under it and cannot be traced by

0

30

Microns

Figure 80. — Vesicular cell of connective tissue
globules. Frozen section. Sudan IT.

THE MANTLE

with fat

81

r. c.

v.c.

Microns

Figure 81. — Cross section of a small artery of the mantle, bl.c. — blood cells; e.lf. — elastic fibrils; end. — endothelium;
f.c. — fusiform cells; r.c. — round cells; v.c. — vesicular ceils. Kahle, hemato.xylin-eosin.

0

Microns

30

Figure 82. — Two vesicular cells from the mantel of an adult C. virginica. Left — the cell contains f^lycogen stained
with Best's carmine; fat globules were dissolved in processing. Right — similar cell after fixation with Bouin :{; note
complete absence of glycogen and fat, both dissolved during fixation and dehydration.

dissection. Such nhuiulaiice of reserve material
led one of the earlier iinestigators (Creighton,
189G, 1(S99) to conclude that its storage in tlie
connective tissue of laniellibranclis is a special
adaptation compaiai)le to the storage of fat in
the connectixe tissues of vertebrates.

The (|uantity of glycogen stored in connectixe
tissue gTaduallj' decreases as the gonads of the

oyster increase in hulk. This was first reported
for (). ((htlis by Pekelharing (1901) and confirmed
by the more recent investigations of Bargeton
(1942). Evidence presented in the latter work
strongly suggests that tlie growing sex cells utilize
tlie glycogen stored in the vesicular cells sur-
rounding the gonad tulndes, but cytological details
of this process are still unknown and tlie ])roblein

82

FISH AND WILDLIFE SERVICE

has not yet been studied from a biochemical
point of view.

After the disappearance of their contained
glycogen the vesicular cells do not slirink or
collapse. A hypothesis was therefore advanced
(Semichon, 1932) that the glycogen granules are
supported by a framework of a special substance
which remains intact after the dissolution of
glycogen. It is claimed that this framework can
be revealed by staining with black anilin inks.
The evidence for the existence of such a special
substance is not, however, convincing. In cells
with a moderate content of glycogen the latter
can be seen in close contact with the protoplasmic
network typical for vesicular cells. Furthermore,
the walls of tlie vesicular cells are fairly rigid and
the cells retain their shape even wlieii they are
empty. The shrinkage of connective tissue fre-
quently caused by changes in osmotic pressure
when the salinity of the water smTounding the
oyster is suddenly increased is not associated
with the disappearance of glycogen.

The fat globules in vesicular cells vary greatly
in size and number, usually forming distinct
vacuoles that are easily dislodged. The relation-
ship between the fat and glj^cogen content of the
oyster and the role of lipids in the physiology of
laniellibranchs have not been studied.

Large oval cells containing a brown pigment are
scattered throughout the connective tissue of the
nuintle. The pigment is not soluble eitlier in
acids or fat solvents. Its chemical nature and
physiological significance are not known.

\Vandering blood cells are commonly seen in
the mantle. They crawl between the connective
tissue cells, aggregate in the vicinity of blood
vessels and blood sinuses (fig. 79), and are grad-
ually discarded through the sui-face of the mantle.
As a rule, the oyster continually loses a certain
amount of blood by diapedesis or bleeding. Any
excess of heavy metals accumulated by lilood
cells (see p. 390) is also discarded by this normal
process.

MUSCLES

The radial muscles consist of large, regularly
spaced bands of fibers which extend almost the
entire width of the mantle from the line of its
fusion with the visceral mass and with the ad-
ductor muscle to the free margin. For a study of
the anatomy of the muscular system the con-
nective tissue in which the bands are firmly
enclosed should be macerated in 1 percent potas-

sium hydroxide for about 24 hours. After being
washed in distilled water the loosened tissues are
removed with a small stiff brush and fine forceps.

The radial muscle bands are composed of
large bundles of unstriated fibers which begin to
branch toward the distal edge of the mantle about
one-third of the distance from that edge. At
this level the muscles appear fanlike and enter
into all three lobes, where they terminate.

The central part of a muscle band is usually
occupied by one or two radial nerves, although
muscles witliout a central nerve (figs. 83 and 84)
do occur.

The contraction of the radial muscles pulls the
entire mantle inside and throws its surface into
ridges. Such a general reaction usually precedes
the contraction of the adductor muscle and the
closing of the valves. The contraction may
occur spontaneously in response to some internal
stimulus or it may develop as a result of external
irritation produced by chemicals, mechanical and
electrical shock, or sudden change in illumination.
In response to a weak outside stimulus only a
small sector of the mantle contracts, making a
slight V-shaped indentation along its periphery.
This response may or may not be followed by
contraction of the adductor muscle. Strong
stimuli, as a rule, result in complete withdrawal
of the mantle, contraction of the adductor muscle,
and closing of the valves. Besides the large
radial bands there are many smaller bundles of
transverse fibers (fig. 77, tr.m.) extending diag-
onally across the thickness of the mantle, a well-
developed system of longitudinal muscles (l.m.),
and the oblique muscles (ob.m.) of the tentacles.

The longitudinal or concentric muscles foUow
the general outlines of the edge. They are more
abundant at the thickened distal edge of the
mantle but do not exhibit the definite pattern
of distribution apparent in the radial muscles.
The transverse muscle fibers are more numerous
in the pallial curtain (fig. 77, tr.m.) than in the
other parts of the mantle. They are so arranged
that the position of the curtain may be quickly
changed in response to external or internal stimuli.

All the muscle cells are of the smooth, non-
striated type with typical elongated nuclei. In
some bivalves the nmscle fibers of the mantle
appear to slmw a double obli((ue striation; tliis
was shown to be an optical effect created by a
series of fine fibrillae spiralling around tlie larger
fibers (Fol, 1S8S; Marceau, 1904). Muscle fibers

THE MANTLE

83

0

Microns

200

Figure 83. — Cross section of the radial muscle of the mantle of an adult C. virginica.

two nerves. Bouin .3, hematoxylin-eosin.

The muscle completely surrounds

witli true transverse striation, described in tlie
mantle of Pecten jacobaeus and P. opercular)':
(Dakin, 1909a), are not found in the oyster mantle.

BLOOD VESSELS

The principal blood vessels of the mantle
(fig. 232 in ch. XI) are the circumpallial artery
(cr.p.a.), which runs along its entire periphery and
sends out many branches; tlie common pallial
artery (co.p.a.); and a large pulsating vessel in
the anteroventral part of the mantle called the
accessory heart (fig. 236 in ch. XI). The latter
can be observed by dissecting the wall of the
epibranchial chamber and spreading the cut tis-
sues apart. The structure and function of these
vessels are discussed on page 2.53.

The small arteries and veins of the mantle can
be recognized easily bv their histolo2:ical charac-

teristics. The walls of the arteries have a thick,
elastic, muscular layer lined with endothelium
(fig. 81, end.). In the veins the elastic layer is
much less developed and the endothelium absent
(fig. 85). The sinuses (fig. 79) are irregularly
shaped spaces in the connective tissue. Since
they have no walls of their own tliey cannot con-
tract. The size of the opening or lumen may be
reduced by growth of the surrounding vesicular
cells and by accumulation of Ijlood cells.

EPITHELIUM, TENTACLES, AND NERVES

Both sides of the mantle are covered by cylin-
drical epitlielial cells set on an elastic basal mem-
brane (fig. 77). Large goblet cells which secrete
mucus and cells containing eosinophile granules
are abundant on both sides of the mantle. The
cells of the side facing the pallial cavity are long

84

FISH AND WILDLIFE SERVICE

0

Microns

200

Figure 84. Cross section of the radial mviscle of an adult C. virginica. The muscle is not accompanied by nerve. Bouin

3, hematoxylin-eosin.

and ciliated; those on the outside under tlie valves
bear no cilia and are much shorter, in places
almost cubical.

Tlie two sides of the mantle perform differei^t
functions. The inner side maintains ciliary cur-
rents, whicli in general move from the base of the
mantle to its edge and carry mucus and sediments
settled from the water; this material is passed to
the margin of the shell to be discharged. Tiie
epithelium of tlie outer side secretes the inner
layer of the shell, the so-called calcito-ostracum.

Although the ciliated epithelium of the edge of
the mantle contains the same kind and proportion
of cellular elements found in other parts of tlie
organ, the cilia at the b<irder of the mantle are
especially powerful. The tentacles themselves
consist of a core of connective tissue with associ-

ated blood vessels, elastic fibrils, and muscle
fibers which emerge from branches of the radial
muscles. On the outside the tentacles are covered
with a single layer of ciliated epithelium to which
black or brown pigment imparts a dark color.
Special sense organs are absent but the tentacles,
especially the long ones, are well supplied with
nerves branching out from the nerve which enters
tlie base of the tentacle and is itself connected
with the nervous system of the mantle (fig. 86).

The circumpallial nerve provides communica-
tion between the tentacles and the radial nerves.
The structure of this nerve resembles that of a
ganglion: numerous nerve cells of the types found
in visceral and other ganglia (see p. 28S) occupy
the peripliery of the nerve; its center consists of
nerve bundles with occasional small ganglion cells.

THE MANTLE

85

Figure 85. — Transverse section of a small vein of the
mantle. Note the ab.sence of endothelium and poorly
developed elastic layer. Bouin, hematoxylin-eosin.

Close nerve contact between the muscles and
other organs of the mantle is maintained through
a fine nerve network which can be made visible
by using the gold impregnation method (fig. 87).
I have had no success in revealing it with vital
stains.

PERIOSTRACAL GROOVE AND GLAND

The narrow space between the middle and the
outer lobes of the mantle edge, called the perio-
stracal groove (fig. 77, per.gr.), is lined with
ciliated epithelium which is replaced at the bot-
tom of the groove by glandular cells. The inner-
most part of the groove is called the periostracal
gland (fig. 88), although it would have been more
appropriate to refer to it not as a gland but as a
secretory epithelial surface (Maximow and Bloom,
1930). This surface is covered with a single
layer of glandular cells different in appearance
and structure from the epithelial cells of the
distal part of the groove. Unlike a true gland,
it does not form a compact body extending under
the surface of the groove and it has no duct. On
transverse sections of the mantle edge the gland
sometimes appears as a round structure sur-
rounded bv connective tissue. Examination of a

0 0.3

IVI i 1 1 i melers

Figure 86. — Innervation of the tentacles of the middle
lobe. Formalin 5 percent, gold impregnation. Whole
mount.

series of sections shows, however, that this appear-
ance is caused by the invaginations of the inner
surface of the lobe. The periostracal gland is
present in all lamellibranclis and was the object
of many histological studies (I^eenhardt, 1926;
List, 1902; Moynier de Villepoix, 1895; Rassbach,
1912; Rawitz, 1888).

There is a conspicuous difference in the appear-
ance of the cells along the two sides of the groove.
Those lining the outer lobe (fig. 88, left side) are
distended at the distal ends and taper toward the
base into slender rootlike processes which, accord-
ing to Rawitz (1888) who described tliem in tlie
oyster, penetrate the underlying connective tissue.
1 was not able to reveal such rootlets in my
material. None of these cells bear cilia, although
the distal part of the groove, not shown in figure
88, is lined with ciliated epithelium. Typical
goblet cells containing eosinophile granules,
anioebocytes, and round mucus cells are present
in the epithelial layer of botli sides of the groove.

86

FISH AND WILDLIFE SERVICE

Microns

30

Figure 87. — Small area of mantle showing nerve net. Formalin 5 percent, gold impregnation. Whole mount. Camera

lucida drawing.

At the very bottom of the groove the tall epithelial
cells are suddenly replaced by short cubical cells
(fig. 88, right side) which extend a short distance
along the inner side of the groove.

The material secreted by the periostracal gland
accumulates at the bottom of the groove and in
the majority of my preparations appears to
adhere to the cells of the outer side (left side of
figure 88). This, however, is the result of shrink-
age caused by dehydration during the processing
of sUdes. In preparations mounted in glycerin
the conchiolin can be seen in close contact with
the epithelium of both sides of the groove.

The function of the periostracal gland is to
supply large quantities of the material required
for new shell growth at the edge of the valves.

The organic matrix (conchiolin) and foliated
layers of calcite needed for increasing thickness of
the valves, on the other hand, are secreted by
the epithelium covering the entire outer surface
of the mantle and in close contact with the inner
surface of the valve. The epithelium consists of
nonciliated cells which are cylindrical near the
free margin of the mantle but become flattened
and almost cubical in more proximal areas. Both
conchiolin-secreting and calcium-secreting cells are
present in this epithelium but their cytological
differentiation by means of staining reactions or
by precipitation of calcium oxalate is not reliable.
Mucus cells and oval cells containing eosinophile
granules also occur throughout the entu'e surface
of the epithelial covering.

THE MANTLE

87

Microns

100

Figure 88. — Transverse section of the periostracal groove. The cells on the left side are distended with secretion; those
at the very bottom are short, almost cuboid. The black mass at the bottom of the groove is conchiolin. Bouin,
hematoxylin-eosin.

Owing to the presence of the conchioHn-secreting
cells, the entu-e outer surface of the mantle is
sticky and adheres closely to tlie inner surface of
the sliell. List (1902) advanced a theory, not
well supported by observation, that in the
Mytilidae the mantle adheres to the shell by
means of fibrillae which originate in the myoepi-
thelial cells and pass tlirough the epithelium. Such
an arrangement is not found in ('. virginica or in
C angulata, and according to Leenhardt (192())
does not exist in Mytilun.

The epithelial layer along both surfaces of tlie
mantle including its free edge contains alkaline
phosphatase, an enzyme involved in the calcifi-
cation of the shell. The presence of the enzyme
can be demonstrated by the Gomori method

(Gomori, 1939, 1943) based on the formation of
insoluble calcium phosphate as a result of phos-
phatase action on sodium glycero-phosphate and
calcium ions. Furtlier treatment with 5 percent
silver nitrate (or with cobalt nitrate) converts
tlie calcium phosphate to silver (or cobalt) phos-
phate which turns black after exposure to light.
Both reagents gave satisfactory results in demon-
strating the localizatiiin of the enzyme in the
epithelium of the mantle. Tlie strongest reaction,
judgetl by the opacity and width of the black
layer, was ftnmd to occur along the edges of the
mantle and in the area of the periostracal groove.
Even the tips of the tentacles contained noticeable
amounts of the enzyme (fig. 89). These obser-

88

FISH AXD WILDLIFE SERVICE

0 0.3

Millimeter

Figure 89. — Localization of alkaline phosphatase in the mantle of C. virginica (longitudinal section).

of a preparation treated by Gomori method.

Photomicrograph

vations are in full agreement with the results
obtained by Bevelander (1952).

SUBLIGAMENTAL RIDGE

A small ridge marking the dorsal edge of the
mantle along the fusion of its two lobes is known
as the subligamental ridge. Its length in the
anteroposterior du-ection corresponds exactly to
that of the ligament, which is secreted by the
epithelial cells of the ridge. The base of the
ridge is flattened and rests on basic elastic mem-
brane; the body of the ridge is semicjiindrical in
cross section, its surface slightly undulating, as
can be seen from the longitudinal section sh(')wn
in figure 7S.

The histological structure of the ridge has been
studied in MijMlu.y (List, 1902; Tullberg, ls,si),
in Anodonta (Moynier de Villepoi.x, 1S95;
Rassbach, 1912), and in the Portuguese oyster
Crassostrea (Gryphaea) angulata (Leenhardt,I926).
Leenhardt and Moynier de Villepoix call the

structure "bandelette paleale" (pallial strip) but
the term subligamental ridge seems to be more
descriptive.

In C. virginica the subligamental ridge is always
well developed and easily recognizable by its
shape and by its coloration, which is usually
darker than that of the adjacent part of the man-
tle. The epithelium (fig. 7S, ep.) covering the
ridge presents a most striking picture. It con-
sists of a layer of extremely tall and narrow cells
arranged in fanlike groups and set on a well-
developed basal elastic membrane. The length
of the cells varies from 50 to 200 n depending on
the position they occupy within the layer. The
cells are very thin, with granular protoplasm and
an oval-shaped nucleus. At the distal portion
of the ridge the boundaries of the cells become
indistinct and their protoplasm darker, presum-
ably due to the concentration there of the organic
material which thej' secrete. The free surface of

THE MANTLE

89

the epithelium is not attached to the ligament as
was described by Moynier de Villepoix (1895).

At regular intervals the row of epithelial cells
is interrupted by oval-shaped pockets which
appear to be empty, with the exception of oc-
casional amoebocytes and a few connective tissue
cells. The significance of these pockets is not
clear. The elastic membrane under the epi-
thelium, thicker here than in the other parts of
the mantle, includes many muscle fibers arranged
parallel to the length of the ridge (m.). Large
oval cells containing yellow-brownish granules
(pig. c.) are abundant. The ridge is well supplied
with blood through a large blood vessel (bl. v.),
around which the connective tissue consists of
tightly packed globular and spindle-shaped cells.
Directly under the basal membrane of the ridge,
however, the connective tissue of the mantle is
made up of large vesicular cells.

FUNCTIONS OF THE MANTLE

Ciliary currents along the inner surfaces of the
mantle form a definite pattern which may be
easily observed. If one valve of the oyster is
removed the corresponding mantle rolls up and
exposes the gills and the inner surface of the man-
tle on the opposite side. In such a preparation
the intact lobe of the mantle remains fully
stretched and the ciliary currents can be observed
by sprinkling the surface with small quantities of
carmine, colloidal carbon, powdered shell material,
carborundum, or other powders insoluble in sea
water. It is best to use very fine particles, such
as powdered mineral wiUemite and colloidal
carbon. Willemite phosphoresces a brilliant green
under ultraviolet light, which makes it possible
to locate even the tiniest particles not otherwise
recognizable. As a source of ultraviolet light I
used a small Mineralight lamp. Hard and heavy
particles of this mineral may stimulate the cilia
by their weight, but this difficulty is avoided by
using colloidal carbon.

As can be seen from the diagram in figure 90,
drawn from life, the general direction of the cur-
rents is from the base of the mantle to its periph-
ery, with the ciliary motion strongest in the
anterodorsal sector. In the large oyster (5 inches
in height) used for the drawing, this area extended
along the margin of the mantle from the level of
the labial palps approximately halfway down the
anterior side. The upper part of the mantle was
usually completely cleared 2 or 3 minutes after

Centimeters

Figure 90. — Discharge areas of the mantle. Note the
two large lumps of the discarded material at the edge of
the mantle which mark the boundaries of the principal
discharge area. Small arrows indicate the direction of
ciliary currents. The clear path along the periphery of
the mantle in the upper left side, indicated by short
arrows, marks the ciliary tract located over the circum-
pallial artery. Long arrow at right indicates the direc-
tion of exhalant current of the cloaca. Drawn from life.

it was sprinkled with powder, while in the same
specimen 5 to 10 minutes were required to clear
the lower (ventral) part. Although the ciliary
currents along the posterior side of the mantle
in the area adjacent to the cloaca are also directed
from the base toward the periphery, this area is
swept clear by an exhalant current from the gills
(fig. 90, long arrow) which is much stronger than
those produced by the mantle epithelium.

The currents along the anterodorsal part of the
mantle (upper left of the figure) adjacent to the
lal)ial palps are directed at an acute angle to its
free margin. There is also a well-defined tract of
ciliary movement about 1.5 mm. wide parallel to
the edge of the mantle. Upon reaching the level
of the lower corners of the labial palps this current

90

FISH AND WILDLIFE SERVICE

tums sharply about 90 degi'ees across the mantle edge.
This point marks the upper limit oi the area
tliroughout which the material settled on the
mantle is discarded ; in the oysters which received
powdered suspensions large lumps of particles
entangled in mucus are usually seen here. A
similar accumulation of matei'ial ready to be dis-
carded marks the anterior boundary of the dis-
charge area which may vary in dimension and
location in different oysters.

Along the ventral portion of the oyster body
the ciliary currents sweep across the numtle at
right angles to its edge and material is discarded
along the border. In this respect my observations
differ from those of Nelson (1938, p. 24), who
thinks that the current in this area is directed
along the free margin toward the mouth. Such
a current was not present in the large oysters
(C. virginica) I used in my experiments.

The existence of a special discharge area located
in the zone below the labial palps is biologically
significant. The so-called pseudofeces, or masses
of discarded food particles and of detritus
entangled in nmcus, which accumulate in this
area either slide over the free edge of tlie mantle
and drop off or are forcibly ejected by the snapping
of the valves. There is no doubt that the presence
of pseudofeces near the edge of the mantle stimu-
lates the strong ejection reaction which constitutes
one typical pattern of shell movement in tlie
oyster (see p. 169).

FORMATION AND CALCIFICATION OF
SHELL

Tlie principal function of tlie mantle is the
formation of the shell and its calcification. Tlie
great structural complexity and intricate pattern
of pigmentation found in some species are produced
by the mantle. The regulatory mechanisms re-
sponsible for this process are not known because
the morphogenesis of molluscan shells has never
been studied experimentally. From observations
on shell growth in some gastropods and lamelli-
branchs it is clear that the shape of the sliell as
well as the pattern of pigmentation result from
the position assumed by the edge of the mantle
during periods of shell secretion and from the
rate of deposition of calcium salts and pigments.

It can be easily observed in oysters, scallops,
and other bivalves in which tlie edges of the mantle
are not fused together that during periods of
growth the mantle extends a considerable distance

beyond the border of the shell. In some species it
even stretches far out and folds back over the outer
surface of the valve. In this way, for instance,
the mangrove oysters produce hooks or similar
structures by which they attach themselves to
branches of trees (fig. 5).

The differential rate of growth along the periph-
ery of the shell as well as the formation of spines,
nodes, ridges, and similar sculptural elements are
both caused by changes in the rate of deposition
of shell material. Two distinct phases may be
distinguished in the shell-forming process: (1) the
movements of the mantle which stretches and
folds itself in order to provide a matrix or mold
upon which the shell is formed, and (2) the
secretion and deposition of the shell material
itself. It is probable that the circumpallial nerve
plays a role in the first phase of the process by
controlling the muscular activity of the mantle.
Our present knowledge of the physiology and
biochemistry of shell secretion is inadequate to
propose an explanation of the morphogenetic
processes involved in shell formation. These
processes are not haphazard but follow a definite
and predetermined course. This is self-evident
from the fact that the final shape of the shell has
definite mathematical characteristics (see p. 24)
which can be attained only by orderly and
regulated deposition of organic framework and
mineral salts.

The first step in the formation of the oyster
shell is the secretion of conchiolin from the perios-
tracal gland. This process can be easily observed
by cutting off a small section of the edge of the
upper valve and exposing the intact valve and the
underlying mantle of the opposite side. Under a
low-power binocular microscope one can see a
clear, viscous, and sometimes stringy substance
oozing out of the periostracal groove. While
secretion is taking place the edge of the mantle
appears to be very active, expanding and retract-
ing as successive layers of conchiolin are laid
down. Figure 91 shows the position of the mantle
at the time of its retraction.

The newly deposited shell (n.sh.) extends out-
ward along the plane of the valve; the edge of the
mantle (mn.e.) rolls upvvard; its outer lobe
(o.mn.l.) is parallel to the plane of the valve,
while the middle and inner lobe (m.l.) face the
observer. The tentacles of the inner lobe extend
down; those of the middle lobe are slightly con-
tracted. The outer lobe underlies the sheet of

THE MANTLE

91

0.771 J].

p OS. a

i.i

r"-:v'.i^iS:-".-:^.t--^-.....f-*»-...

«>??;-\- -Ttf-

I ^2.5

k.

■f'rn

co/z. sA.

t%^;»^,,,s.-...-,.'i-t(S**v.r^<^l y nifi. £

0

Millimeters

Figure 91. — Small area of the mantle edge with the adjacent part of the newly secreted shell viewed from above. The
mantle was exposed by cutting off a piece of the opposite valve, and the oyster was placed in sea water under a
binocular microscope, con.sh. — conchiolin sheet; mn.e. — edge of the mantle; i.l. — inner lobe; n.sh. — new shell;
o.mn.l. — outer lobe of the mantle, contracted; p.os.g. — periostracal groove. Drawn from life. The position of
structures at the edge of the mantle in relation to one another: the new shell area (n.sh.) marked on three sides by a
brolien line is in the plane of the drawing, next to it is the outer lobe (o.mn.l.), then the conchiolin sheet (con.sh.),
the middle lobe (m.l.), and the inner lobe (i.l.) is at the top, nearest to the observer.

viscous conchiolin (conch. sh.) which oozes out
from the periostracal groove (p.os.g.) between the
outer and middle lobes. The distal edge of the
conchiolin sheet (end of stippled area) indicates
the previous maximal extension of the outer lobe
before the withdrawal of the mantle edge. The
entire group rests on the newly formed and already
solidified shell (n.sh.) .

During the secretion of conchiolin the edge of
the mantle frecjuentlj' extends out and then with-
draws to the position recorded in the drawing.
At the time of expansion the outer lobe temporarily
supports the semiliquid conchiolin and by moving
in and out spreads it over the shell. Because of
this action the pro.ximal part of the newly formed
valve receives a larger amount of conchiolin and

92

becomes thicker than the distal portion. When
secretion is interrupted, the conchiolin layers
become incorporated into the shell substance and
the concliiolin sheet as shown in figure 91 is no
longer visible.

The rate of secretion of the new shell varies at
different parts of the mantle edge. Quantitative
data are lacking, but observations made during
the periods of more rapid growth in (\ virginica,
(May to June and October to November in New
England waters) show that the area of newly
formed shell is always largest at the ventral side
of the valves near the principal axis of growth
(fig. 92).

The organic matrix of the shell can be produced
by the pallial epithelium at any place along the

FISH KSV> WILDLIFE SERVICE

Centimeters

Figure 92. — New shell growth formed during 1 year along
the periphery of the valve of an adult oyster from Long
Island Sound planted in the Oyster River, Chatham,
Mass. The newly formed shell is recognizable by zigzag
lines of the material: its width is greatest along the
ventral edge.

entire outer surface of the mantle and is not re-
stricted to the periostracal groove. Such secre-
tion, first observed in pearl oysters (B0ggild,
1930), can be experimentally demonstrated in C.
virginica. Oysters with one valve removed and
the edges of the mantle cut ofT above the peri-
ostracal groove secreted a new conchiolin layer
over the entire surface of the exposed mantle
within 5 days. Although the operated specimens
remained alive in the laboratory tanks at Woods
Hole over 3 weeks this conchiolin membrane
remained uncalcified. In another experiment
three adult oysters were removed from their shells
and kept alive in sea water for 3 weeks. They
formed rather thick coats of periostracum which
was very lightly calcified. The repair of holes
made in oyster shells by boring snails and sponges
also shows that conchiolin is secreted by the entire
surface of the mantle. The damaged area is rapidly

THE MANTLE

733-851 0—64 7

covered by a layer of organic material which
later becomes calcified.

Soon after being secreted, the conchiolin be-
comes calcified. Progressive stages of this process
can be observed on the growing edge of the shell,
or by inserting pieces of plastic or small glass cover
slips between the edge of the mantle and the valve
and removing them at regular intervals for in-
spection. The earliest stage of calcification is
recognized by the appearance of minute granules
of calcium salts, which become visible in polarized
light as brightly sparkling dots (fig. 93) . At this
early stage the distribution of the granules (cal-
cospherites) does not show any definite pattern
or arrangement. In a living oyster they can be
found entangled in strands of mucus left on the
conchioUn sheet by the back and forth movements
of the mantle edge. Within the next 24 to 48 hours
typical hexagonal crystals of calcite can be seen
(fig. 94, black crosses). They gradually increase
in size and present a picture of great brilliance
and beauty in polarized light (fig. 95) .

Distribution of calcospherites at the stage of
their transformation into small calcite crystals on
the surface of the newly secreted shell (fig. 96)
does not show any distinct orientation in relation
to the growth axis of the shell. Some of the cal-
cospherites are scattered over the entire field of
vision, while others are packed tightly between
the larger crystals (see large group of crystals at
the lower part of figure 96). Within the next 48
hours the calcite crystals increase in size (fig. 97) .
In the final stage of shell formation the calcite
crystals become arranged in a distinct pattern to
form the prismatic layer in which each unit is a
prism oriented with its long axis at about a 90°
angle to the edge of the shell (fig. 98). The form
of the individual prisms varies greatly, some of
them are even wedge-shaped and slightly curved.
This can be observed after boiling a piece of shell
in a strong sodium hydroxide solution to separate
the prisms (Schmidt, 1931).

Each calcite prism is surrounded by a capsule
of conchiolin. By dissolving the mineral in weak
hydrochloric acid it is possible to obtain intact
the organic meshwork of the conchiolin layer.
The walls of each capsule, as can be seen in figure
99, are very thin and slightly iridescent. Since in
the earliest stages of shell formation the conchiolin
sheet appears to be amorphous under the light
microscope, it is reasonable to assume that the
organic capsules of the calcite prisms are formed

93

Figure 93.— Small

30
Microns

granules
en

es (calcospherites) in conchiolin shortly after its secretion by the mantle. Black and w
ilargement of a Kodachrome photograph taken with polarized light.

■hite

by later deposition of conciiiolin, the secretion of
which continues during calcification. The details
of this prosess have not yet been described.

THEORIES OF CALCIFICATION

Studies of shell calcification fall into two major
categories. One type of work places the emphasis
on the identification of calcium-secreting cells or
organs; the other approaciies the problem from
the biochemical point of view. It has been

generally accepted that calcium carbonate, sepa-
rated from blood, is secreted as colloidal gel by
certain cells at the edge of the mantle and that
crystallization takes place outside the cells
(Crofts, 1929;Dakin, 1912;Kuyper, 1938) between
the conchiolin sheet and the mantle. Separation
of calcium is not, however, confiiied to the surface
cells of the mantle. The calcium-secreting cells
may be subepithelial, as in Patella (Davis and
Fleure, 1903). In the calcification of the epi-

94

FISH AND WILDLIFE SERVICE

\

1°

J L

Microns

200

Figure 94. — Calcite crj'stals of new o.vster shell about 24 to 36 hours after its formation. Black and white enlargement

of a Kodachrome photograph taken with polarized light.

phragm of Helix pomaha (Prenant, 1924, 1928),
the calcium is liberated by the leucocytes in the
connective tissue of the mantle. In the case of
pearl formation, Boutan (1923) has shown that
calcareous deposits are formed by amoeboid cells
which crawl through the mantle epithelium, while
the latter secretes the concentric layers of the
organic matrix (conchiolin).

De Waele (1929) approaciied tlie calcification
problem from the phj-siochemical point of view.

Working wnth Anodonta cygnea he has shown that
tiie e.xtrapallial fluid between the mantle and the
shell is chemically identical with blood. Ex-
posure of this fluid to air causes the formation of a
precipitate, which consists of a suspension of
calcium spherules in protein solution. He there-
fore assumed the existence in the pallial fluid of a
hypothetical compound consisting of protein,
carbon dioxide, and calcium carbonate. The
escape of carbon dioxide would then cause the

THE MANTLE

95

Microns

200

Figure 95. — Early stage of the formation of prismatic layer. Photographed with polarized light.

precipitation of calcium carbonate. Dotterweich
and Elssner (1935) found, however, that calcium
carbonate crystals are formed in the extrapallial
fluid of Anodonta only in an atmospiiere containinjj;
less than 1.5 percent carbon dioxide. In Helix,
regeneration of the shell will take place in an
atmosphere containing up to 15 percent of carbon
dioxide, according to Manigault (1933). Al-
though the latter accepted De Waele's theory, his
own results seem to prove its inadequacy; and
Robertson (1941) remarks that De Waele's hypo-
thetical protein compound is without a real
chemical basis. Furthermore there are other

discrepancies in De Waele's results which invali-
date his theory. The calcospherites and the
protein precipitated from blood and from extra-
pallial tluid contained 50 percent organic matter,
whereas the new shell contained only 4 percent of
it. To reconcile these facts it would be necessary
to assume that a great proportion of the organic
matter in the new shell must be reabsorbed. The
entire process as outlined by De Waelc appears to
be higlily improbable.

Steinhardt (1946) assumed that calcification of
the oyster shell is associated with tlie formation
of citrate, probably the tricalcium-citrate

96

FISH AND WILDLIFE SERVICE

A

o

^:iD

k — ^

V

rr~

* I 1 I I

Microns

Figure 96. — Photomicrograph of a piece of new shell of Crassostrea virginica taken 28 hours after the beginning of calcifi-
cation. Small calcite crystals are randomly distributed, and calcospherites scattered over the entire field of view
are in places densely packed between the larger crystals.

(C6H507)2Ca3 + 4H20. The observation that citric
acid is formed in connection with carbo-
hydrate metabolism, and that citrate is quaUta-
tively precipitated from a solution which also
contains phosphate and calcium ions in a suitable
concentration (Kuyper, 1938, 1945a, 1945b),
forms the basis of his conclusion. The citrate in
the precipitate is found not as calcium citrate but
in a somewhat more comple.x form in which cal-
cium is combined with both phosphoric and citric
acids. This is verified by the results of the
analyses shown in table 11, in which the oyster
shell was presumably 0. eduiis. It is rather
difficult to arrive at a definite conclusion regarding
the role of citric acid in the calcification of oyster
shells, but Steinhardt's observations establish the
presence of calcium phosphate in the oyster shell,
which was supposed to consist primarily v( car-
bonates; and an abundance of calcium phosphate
in the mantle was demonstrated by Biederniann
(1914).

During recent years (Bevelander, 1952; Bevel-
ander and Benzer, 1948; Bevelander and Martin,
1949; Hirata, 1953; Jodrey, 1953) considerable
advances in the study of the processes of calcifica-

THE MANTLE

Table 11. — Analyses to calcified materials according to

Steinhardt

[All figures are in percentages]

Material

Citric acid

Phosphorus

Calcium

Concretions from crayfish stomach

1.56

0.15

0. 013-0. 024

0.017

9.0
0.154
0.007
0.019

25.6
33.3

White coral

35.2

32.8

tion have been made. It had been generally
assumed that the small granules appearing on the
surface of the conchiolin consisted of calcium
carbonate, but Bevelander and Benzer found that
the}' are made of calcium phosphate. It is not at
all clear how the calcium phosphate of the granules
is converted into calcium carbonate, which is the
final product of calcification in the oyster shell.
It is doubtful tliat the conversion is accomplished
by threct reaction between the calcium phosphate
and tlie carbonate, because such a process would
require very higli concentrations of carbonate.
Tiie explanation proposed by Bevelander and
Benzer implies that calcium phosphate may be
dissolved by the action of organic ions which in
some manner bind calcium. Phosphatase may

97

rS^'

0

Microns

60

Figure 97. — Calcite crystals deposited on a piece of conchiolin. Photomicrograph taken 38 hours after the secretion of

conchiolin has started.

contribute to this process by transferring phos-
phate to some substrate and removing the phos-
phate ions. This tentative e.xphination suggests
a number of biochemical studies that should he
made to obtain a better understanding of the
process of calcification.

An important factor in the process of shell
calcification is the enzyme phospiiatase, which is
generally present in the ossifyiiig cartilages of
young animals and in other tissues and organs in

which calcium is deposited. The action of tlie
enzyme consists of liydrolysis of he.xosemonoplios-
phoric ester and glycerophosphoric ester and
consequent liberation of inorganic phosphate.
Tlie role of phosphatase in tlie shell formation of
niollusks was established by Manigault (1939),
who found a direct correlation between ph,os-
phatase activity in the digestive diverticula,
numtle, and blood and precipitation of calcium in
the shell. He concluded tluit phosphatase is

98

FISH AND WILDLIFE SERVICE

Figure 98. — Prismatic layer of the new growth of shell at the edge of the mantle. Four to five days old. Photomicrograph

of an unstained whole mount.

probably a transfer agent involved in tiie mobiliza-
tion of calcium. The localization of tliis enzyme
along the border of the mantle and in the surface
epithelium of the oyster, shown by tlie Gomori
technique (fig. 80), confirms tlie opinion of
Manigault and of Bevelander that tlie phosphatase
plays an active role in the calcification of oyster
shells.

During the last decade consideraT)le advance
was made in studies of the metabolic aspects of
shell formation. Hammen and Wilbur (1959)
paid particular attention to carbon dio.xide con-
version to shell carbonate and to the secretion of
conchiolin matri.x in whicli the calcium carboiuite
crystals are deposited by Mie oyster (C. inrgiriica).
Tlie work of Jodrey and Wilbur (1955) on high
activity of the enzyme oxalocetic decarboliylase
in the mantle tissue of this species suggested that
tlie deposition of carbonate may be related to
decarboxylation reactions of tlie mantle. Experi-

mental work conducted by Hammen and Wilbur
at the Duke University Marine Laboratory at
Beaufort, N.C., corroborated this hypothesis.
Living oysters and isolated shells were placed for
12 hours in sea water containing 240 microcuries
of NaHC'^Oa per liter. The radioactivity of the
shell surface was determined near the posterior
margin of the right valve and corresponding cor-
rection was made for self absorption on the surface.
By incubating pieces of oyster tissues in NaHC'^Os
it was found that C" is incorporated into organic
acids of the mantle. More than 90 percent of the
radioactivity occurs in succinic and smaller
amounts in fumaric and malic acids. The initial
step in the process is the fixation of carbon dioxide
by propionic acid resulting in the formation of
succinic acid. Botii acids were found in relatively
iiigii concentrations in the shell forming tissues
of the oyster. The fact that in these experiments
labeled amino acids were found in the radioactive

THE MANTLE

99

Figure 99. — Photomicrograph of organic meshworlc of prismatic Layer of shell after decalcification in weak hydrochloric
acid. Note that the outlines of the capsules retain tlie shape of the mineral prisms.

conchiolin of tlie shell indicate that carbon dio.xide
fixation also contributes to the syntheses of the
organic matrix of the shell.

Calcium enters the mantle directly from sea

water, as was demonstrated by Jodrey (1953)
usino; mantle-shell preparation and radioactive
Ca*^, and can he taken up through other parts of
the moUusk and transported to the mantle. The

100

FISH AND WILDLIFF SERVICE

enzyme carbonic anhydrase which is present in
\arious mollusks may be expected to accelerate
deposition of calcium carbonate, and the rate of
deposition is retarded by carbonic anhydrase
inhibitors.

Complex metabolic cycles involved in shell for-
mation have been reviewed by Wilbur (1960),
and probable relations of carbon dioxide to shell
conchiolin and carbonate deposition are shown by
him in a summary diagram (fig. C, p. 25 of Willnu-'s
paper) .

CYTOLOGICAL IDENTIFICATION OF CALCIUM

Several methods for the identification and local-
ization of calcium salts in the oyster tissues are
available, but none are completely reliable.
Goniori (1939) suggests that soluble calcium could
be demonstrated by treating the frozen sections
with ammonium oxalate, the insoluble octahedral
crystals of calcium oxalate being easily recognized.
The use of a fixative consisting of formalin and
ammonium oxalate was also proposed (Rahl,
cjuoted from Gomori). Both methods tried in
my laboratory on sections of oyster mantle gave
unsatisfactory results. The diflRculty is the dis-
lodging of calcium-bearing granules and mucus
during sectioning, since the granules are easily
carried out by the knife's edge from their original
location inside the cells to the outside of the epithe-
lium. This difficulty can be avoided to a certain
extent by double embedding the tissue in colloidin-
paraffin.

Indirect methods of Ca''"''' identification are
based on the use of heavy metals (silver, cobalt,
copper, and iron). Because almost all insoluble
calcium compounds in the tissues are either
phosphate or carbonate, any procedure which
would demonstrate the presence of these anions
may be considered specific for calcium. When the
sections are immersed in a solution of one of the
heavy metals the corresponding metallic salt is
formed at the sites of phosphate or carbonate.
The reduction may be effected by exposing to light
if silver nitrate is used, or by immersing in appro-
priate reducing reagents (ammonium sulfide,
acidified potassium ferricyanide). Identification
by staining of calcium is based on the formation of
insoluble lacs with several hydroxyanthraquinine
dyes (alizarin sulfonic acid, purpurin, anthrapur-
purin). Calcium deposited in the process of shell
formation may, however, contain substances which
interfere with the lac-forming reaction of alizarin.
Also, tlie dye frequently fails to stain old deposits

and its color is affected by the presence of iron.
Although these complications limit the usefulness
of alizarin as a reagent for the determination of
calcium, I found that a 1 percent water solution of
alizarin S (sodium alizarin sulphonate) is probably
the best histochemical reagent for identification of
calcium in the oyster mantle. It readily reacts
with new deposits of calcium carbonate or calcium
phosphate and forms compounds resistant to both
acids and alkalies.

To study the cytology of calcium secretion,
the deposition of conchiolin and its calcification
was stimulated by cutting oft" small pieces of shell
along the posterior margin of the oyster. Labora-
tory experience shows that such injury made
during the warm season is rapidly repaired.
Small pieces of the mantle border with the adhering
and partly calcified conchiolin were excised and
3 days later preserved in neutral formalin or
absolute ethyl alcohol. Sectioned tissues were
stained with alizarin S and other reagents for
demonstration of calcium. The preparations
showed a large number of alizarin stained globules
or granules, about 1 .5 /i or less in diameter adhering
to the surface of the mantle. Identical granules
were found inside the goblet cells of the epithelial
layer along both sides of the mantle (fig. 100).

The results of the staining and other histo-

IVlicrons

Figure 100. — Calcium containing grannies discliarged by
the epitlielial cells at the cilgc of the mantle of C.
virginica. Neutral formalin 3 percent, alizarin.

THE MANTLE

101

0 0.3

Millimeters

Figure 101. — Photograph of crystals of a mixture of calcite and gypsum formed in the mantle cavity of C. virginica.

chemical reactions show that the secretion of
calcium is not confined to special sites but takes
place over the entire edge and outer surface of
the mantle. The intensive coloration of the
granules by alizarin suggests that they contain
a considerable amount of calcium, probably
bound in organic compounds of the globules.
Amoebocytes present in the material secreted
by the mantle also may be involved in the mobili-
zation of calcium during the formation or repair
of shells.

Sometimes the mineral crystals formed by the

mantle are not incorporated in the conchiolin but
accumulate in the pallial cavity and are eventually
ejected. On several occasions fairly large quanti-
ties of a wliite powdered material were found in
front of the discliarge areas of oysters which were
ke])t iti glass trays in running sea water in the
laboratory. The material consisted of crystals
(fig. 101) wliich, according to the X-ray analysis
kindly performed by Marie Lindberg of the
Geochemistry and Petrology Branch of the
Geological Survey of the U.S. Department of the
Interior, were found to consist of a mixture of

102

FISH AND WILDLIFE SERVICE

calcite and gypsum (hydrous calcium sulfate),
with the latter present only as a minor constituent.
The oysters appeared to be normal in every respect
and showed good growth of shells. The presence
of gypsum is of interest since it is not a normal
constituent of oyster shell. What particular
disturbance in tlie calcium metabolism produced
its formation is unknown.

SOURCES OF CALCIUM

It has been suggested (Pelseneer, 1920; Galtsoff,
1938) that lamellibranchs may remove calcium
directly from sea water. Pelseneer (1920) cites
an example of a young Anodonta cy(jnea wliicii in
2 months removed all the calcium from 5 1. of
water in wliicli it was kept. Definite proof of the
direct absorption of calcium by the oyster mantle
is given by tlie experiments with ('. i'ir(jinica
(Jodrey, 1953) in which radioactive Ca^° was used.
Calcium turnover was also studied by Hirata
(1953) in mantle-shell preparations made by cut-
ting off tiie adductor muscle and the visceral
organs, and leaving the intact mantles spread over
their respective valves. Tlie mantle remained
alive for several days and deposited tlie shell
material, although at a lower rate tlian does the
intact oyster. Jodrey placed a mantle prepara-
tion in 500 ml. of aerated sea water with a Ca"
activity of 5.8 microcuries. At least part of the
calcium of the newly formed sliell substance came
directly from the sea water, and the deposition of
calcite took place in tissue isolated from the circu-
latory and digestive systems. The experiments also
demonstrated tliat the greater portion of calcium
in the mantle appears to be inert. Only 2.5 per-
cent of the total calcium content was renewed
every 24 minutes, the turnover being 0.6 mg. of
calcium per minute per gram of mantle. In
addition to entering the mantle directly calcium
can be taken up by other organs of the oyster and
transported to the mantle (Wilbur, 1960).

MINERALOGY OF CALCIUM CARBONATE
IN MOLLUSCAN SHELLS

Calcium carbonate is known to occur in 12
mineral forms (Prenant, 1924), but only three of
these have been found in aninuils. In tlie shells
of mollusks, calcium carbonate usually occurs as
calcite and ai'agonite. There are many species in
which both minerals occur together although in
different parts of the shell. Prenant (1928), who
contributed much to the study of calcification,
found that besides calcite and aragonite the animal

tissue may contain small spheres (sphaerolithes) or
tiny needles of the mineral called "vaterite", after
tlie mineralogist Vater who discovered it. Vater-
ite was reported to be present in the connective
tissue of certain gastropod mollusks, cestodes, and
trematodes, and in the fat tissue of insects (Dip-
tera). Its presence in the tissues of the oyster
has not been reported.

The various forms of calcium carbonate secreted
by aninud tissue can be identified l)y their crystal-
lograpliic properties, birefringence, density, and
chemical reaction. 8ome of these distinctive
cliaracteristics are summarized in table 12, taken
from Prenant (1924).

Impurities always present in material secreted
by living forms can sometimes make the mineral-
ogical identification of calcium carbonate doubtful.
Calcite and aragonite can be distinguished by
means of tlie polarizing microscope. Calcite
crystals examined uiuler crossed nicols give a
brilliant jjicture of various colors, and a distinct
Ijlack cross appears when the optical axis is aligned
parallel to tlie axis of the microscope (fig. 94.)
In tlie case of aragonite, hyperbolic arched lines
appear instead of the black crosses. Exact
identification of minerals can of course be made by
X-rays, but this method is rarely available to the
biologist.

Among various chemical identification methods
tiie Meigen color reaction can be most easily
em])loyed (B0ggild, 1930, p. 238). In a weak
solution of cobalt nitrate aragonite becomes violet,
the intensity of coloration increasing as tlie solu-
tion is warmed. Calcite, however, remains pale
blue even in a heated solution.

The conditions under which a mollusk secretes
calcium carbonate in a specific mineralogical form
are not at present understood. It is reasonable
to presume that the organic matrix of the shell is
someliow involved in this process. Roche, Ran-
son, and Eysseric-Lafon (1951) found that in the
shells of mollusks consisting both of calcite and
aragonite the conchiolin associated with tiie calcite
of the prismatic layer had higher concentrations
of glycine and tyrosine than were present in the
nacre of the same shell consisting of aragonite
(see cii. II, p. 41). The causal relationship be-
tween the mineralogical forms of carbonate and
amino acids of its conchiolin lias not been
demonstrated.

A iiypothesis that carbonic anliydrase, an
enzyme present in tiie tissues of the mantle, plays

THE MANTLE

103

Table 12. — Distinctive properties of principal mineral forms of calcium carbonate found in invertebrates '

Name

Chemical
composition

Optical System

Birefringence

Index of
refraction

Density

Meigen
reaction

Calcite

CaCO]

Rhoraboedric, uniaxial .

Strong (0 172)

1.658-1.486

1.686-1.530

About 1.55

Near 1.5

About 1.5

2.714
2.95

2. 5-2. 65
2. 25-2. 45

1.777

Negative.

Positive.

Do

Aragonite

CaCOj

CaC03

Monoclinic, biaxial

Slightly weaker, (0.156)...
Weak

V'aterite _.

Sphaerolithes, optically negative

CaCOj

Hydrated carbonate

CaC03 6HjO

Prisms or Monoclinic tablets

Near 0 085

I From the data published by Prenant, 1927.

an important role in the formation of calcium
deposits in molluscan shells has been advanced by
Stolkowski (1951). According to this theory the
enzyme exerts its effect by orienting the calcium
carbonate molecules in the aragonite crystal
lattice. The action of carbonic anhydrase in this
admittedly very complex process is not, liowever,
satisfactorily explained and should be more
thoroughly investigated before its role in the
formation of aragonite or calcite in mollusk shells
is definitely established. In its present state the
hypothesis fails to explain the existence of shells
in which both aragonite and calcite are present.
Recently Stenzel (1963) reported that in the shells
of C. virginica aragonite covers the areas of attacli-
ment of the adductor muscle, tlie inipiiiit of
Quenstedt's muscle, and is found in the ligament.

Another explanation of the formation of the
less stable aragonite instead of calcite suggests
that strontium and magnesium carbonates in-
fluence the formation of aragonite in shell. Some
support to this idea is found in the fact that in
vitro the crystallization of aragonite is facilitated
by strontium and lead salts. This observation
made by Prenant (1924) apparently influenced
Trueman's (1942) hypothesis that strontium,
magnesium, and probably other salts found in
living mollusks influence the crystallization of
aragonite.

That there may be some correlation between
the predominance of the particular mineralogical
form of calcium carbonate and the temperature
of the surrounding water has recently been
suggested by some geologists. Through quanti-
tative X-ray analysis of shells they have demon-
strated that in certain polyclad worms (Serpulidae)
and in some gastropods and pelecypods {Mytilus,
Vohella, Pinctada, Anomia, and others) the con-
centration of aragonite in shells increases with
increasing temperatm'es (Epstein and Lowenstam,
1953; Lowenstam, 1954). In Mytihts, for in-
stance, only the sliells of warm water species are
composed entirely of aragonite, whereas those

from colder waters contain varying amounts of
both calcite and aragonite. This interesting
ecological observation does not, however, provide
a clue to the nature of the biochemical processes
which control the predominance of one or another
crystallization system.

RATE OF CALCIFICATION

The calcification rate of the left valve of C.
virginica is significantly higher than that of the
right one, as can be readily seen by examining
newly formed shells. The calcareous material
deposited by the left mantle is thicker and
heavier than that deposited during the same time
by the right mantle (Galtsofl', 1955). I made
the following observations on shell growth rate of
adult C. virginica. After the new growth of shell
along the valve edge was carefully removed the
oysters were placed in tanks abundantly supplied
with running sea water. About 2 months later
the areas of newly deposited shells on each valve
were measured with a planiineter, carefully re-
moved from the shell, rinsed in distilled water,
dried in air, and weighed. The results are
summarized in table 13. In every case the
amount of calcified material deposited over a
unit of area was considerably greater on the left

T.^BLE 13. — Areas of new yroivth and rate of deposition of
shell material by C. virginica in mg. per day per C7n.'
during April to June 1934, ^Voods Hole, Mass.

Oysters

Area
of new
shell

Weight
per cm.s

Depo-
sition

per cm.!

pay day

Days
under
obser-
vation

Ratio

weight
of left to

weight
of right

valve

Tive-year-Dld, Narragansett Bay
Left valve

Cm.'
6.80
5.16

7.1

7.7

6.1

8.8

3.68
4.20

6.83
7.35

Mg.
156.0
59.3

123.0
19 9

74.2
26.6

163.8
52.0

71.2
33.0

Mg.
2.8
1.1

1.8
0.3

1.09
0.37

2.98
0.95

1.3
0.6

A'o.
55

68

68

55

55

2.6

Right valve

.\dult, Narragansett Bay

6.2

.\dult. Narragansett Bay
Left valve

2.9

Right valve

Two-year-old, New Hampshire
Left valve.

3.2

Right valve

\'ery old. New Hampshire

2.2

Right valve

104

FISH AND WILDLIFE SERVICE

\alvc (lower) than on the right one (upper),
the difference varying from 2.2 to 6.2 times.

The rate of deposition of calcified material by
the surface of the mantle may also be studied by
inserting between the mantle and shell small
pieces of plastic or other nontoxic material of
known area and weight. Results obtained with
this method vary greatly. Observations made
on 16 adult oysters at Woods Hole during the
period of August 9 to 20, 1953, show that in 15
oysters the daily rate of shell deposition per square
centimeter varied from 0.4 to 2.1 mg. One
oyster deposited 14.2 mg. in 2 days or 7.1 mg. per
day. The amounts of shell material deposited
by 20 Narragansett Bay oysters kept in laboratory
tanks for 68 days during the period of April to
June varied from 0.1 to 0.79 mg. of shell substance
per day cm.^ In some of these oysters the
presence of the plastic material induced patholog-
ical conditions which resulted in the formation
of leathery capsules similar to the blisters fre-
quently found on the inside of shells near the
adductor muscle. The formation of such blisters
was accompanied by deposition of calcite greatly
in excess of the rate of calcification under normal
conditions.

Seasonal variation in rate of shell deposition
over the inner surface of the valves was also
studied, using 20 adult oysters for each set of
determinations. Observations were continuous
from June 1954 until the end of February 1956.
To avoid possible injury to the mantle while in-
troducing pieces of plastic, the oysters were fully
narcotized in magnesium sulfate solution and
insertions made when the mantle was completely
relaxed and did not respond to touch. Thin sheets
of plastic were cut into rectangular pieces 0.5 cm.'
in area and weighed before inserting them under
the mantle, theu- weight varying from 5.5 to 6.0
mg. Some of the pieces introduced were ejected
by the oysters, but losses were minimized when the
insertion was made under full narcosis. The
treated oyster was then marked and placed on its
left valve in a large tray supplied with running
sea water. The temperature of the water was
recorded twice a day. Each set of 20 oysters was
kept in water as long as the seasonal rise or fall of
water temperature did not exceed 2.5° C.

To obtain measurable quantities of shell de-
posits the pieces of plastic were left inside the
oysters for a longer time in winter and in August,
after the completion of spawning, than during the

rest of the year. The number of days the oysters
with inserted pieces were left undisturbed varied
as follows: from 10 to 16 days in April to July;
from 25 to 30 days in August; from 13 to 18 days
in vSeptember to November; for 30 days in De-
cember; and 70 days in January to March. Ob-
servations were continued for 14 months. No
shell was formed in January to March except in a
few oysters in which the mantle was injured during
insertion. These samples were not included in
the data plotted in fig. 102. Laboratory observa-
tions showed that shell opening and feeding of the
oysters at Woods Hole are as a rule temporarily
reduced after the discharge of sex products which
takes place late in July and early in August.
Unequal time intervals in observing shell deposi-
tion do not affect the validity of the results since
the rates of shell formation shown in figure 102
are expressed as weights of shell deposited per
cm.- in 1 day.

At the end of each period the oysters were re-
moved, the pieces of plastic recovered, rinsed in
distilled water, dried at 55° C, and weighed. The
results sunmiarized in figure 102 are shown as medians
(Md.) of the rate of shell deposition per cm.^ per
day, and as lower (Qi) and upper (Q3) quartiles.

The curves show two periods of accelerated shell
growth in Woods Hole water, one in May to June
and another in October, and no shell growth dur-
ing winter from December to the end of April
when the temperature of the water varied between
1° and 2° C. These observations are in agreement
with many field data and with the experiences of
practical oyster growers of the North Atlantic
states, who found that oysters grow more rapidly
in the spring and in the autumn and cease to grow
when the water temperature drops to about 5° C.
The relatively low rate of shell deposition during
the summer is attributable to the inhibitory effect
of fully developed gonads. Observations fre-
quently made in the Woods Hole laboratory show
that shell growth in the winter will begin within
24 hours after the transfer of oysters from the
harbor to much warmer sea water in the labora-
tory.

Under normal conditions no shell is deposited
in winter. In several instances, however, large
amounts of shell material were secreted over an
area of the mantle which was apparently injured
by the insertion of plastic. One of these cases is
shown in figure 103. In this oyster a heavy pocket
of shell material was deposited on the valve over

THE MANTLE

105

25

220
a.

15

UJ

10

< 5

UJ

2.0'

<
o

UJ
Q.

O

u

1.5

1.0

CO

o
a- 0 5

MdL MEDIAN

Q^ LOWER OUARTILE

Qj UPPER OUARTILE

i i

VI VII

MONTHS

Figure 102. — Seasonal changes in the rate of deposition of shell material over the inner surfaces of left (lower) valv'es.

the area occupied by a piece of plastic, and a shell
ridge was formed along the edge of the mantle,
which was withdrawn a considerable distance back
from its normal position. It can be deduced from
these observations that injury to the mantle stim-
ulates the shell secretion and that deposition may

take place even at low temperatures when normal
shell growth is inhibited. This would indicate
that the cnzynmtic system involved in shell dep-
osition is always present and may become active
in res]3onse to pathological conditions in spite of
the inhibitory effect of winter temperatures.

106

FISH AND WILDLIFE SERVICE

0 3

Centimeters

Figure 103. — Abnormal deposition of shell material along
the edge of the mantle (black line) and over the piece of
plastic quadrangle, which was completely encapsulated
in a pocket of newly secreted shell. Mantle is shown by
stippled area. Winter observation at Woods Hole.

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1932. Sensory stimulation of the oyster, Ostrea vir-
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Jodrey, Louise H.

1953. Studies on shell formation. Ill, Measure-
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ment of Science, Washington, D.C., Publication
No. 64.

110

FISH AND WILDLIFE SERVICE

CHAPTER VI

THE LABIAL PALPS

Page
111
111
114
118

Anatomy.--

Histology "

Direction of ciliary currents

Reaction to stimuli

1 1Q

Bibliography

ANATOMY

The four soft flaps which lie at. the anterodorsal
side of the body under the mantle hood are the
labial palps. Their trianoular members are at-
tached by their broad bases to tiie visceral mass
and have slightly curved margins which extend
ventrally to the point where they touch the free
edges of the gills (fig. 104).

The two pairs of palps, one on each side, are
joined together into a single unit which serves
primarily for final sorting of food particles and for
the delivery of food to the mouth. Each pair
consists of one external and one internal palp
(r.i.p. ; r.o.p.) . The two external palps join together
above the mouth (m.) where they form the upper
lip (u.l.) ; the two internal palps are united below
the mouth into a lower lip (1.1.). As a result of
this arrangement the mouth is an irregularly
shaped, narrow, curved slit. Both lips are arciied ;
the lower one is shorter, and its edge is thicker than
that of the upper lip.

At the central junction of the two internal palps
there is a median gutter which leads to the center
of the lower lip. The two lateral gutters (l.g.)
formed on each side where the external palp
meets its opposing internal member are the prin-
cipal paths by which the food is conveyed to the
corners of the mouth (fig. 104). The surface is
smooth along the outer part of the external palps
and on the inner palps along the n\edian plane,
where the palps meet; along the lateral gutters,
both have a striated appearance due to numerous
ridges and grooves which empty into the corre-
sponding gutter. This arrangement is significant
for an effective sorting of food.
HISTOLOGY
Each labial palp consists of a layer of connective
tissue covered on both sides by columnar ciliated

FISHERY bulletin: VOLUME 64, CHAPTER VI

epithelium set on a basement membrane. These
are made of large vesicular cells of the type found
in the mantle (fig. 105, c.c). Within the body
of a palp there are many blood vessels and blood
spaces or sinuses. These spaces are splits or
interstices between the connective tissue cells and
have no lining or wall. The longitudinal and
transverse muscle fibers are numerous l;)ut not as
well developed as they are in the mantle.

The smooth sides of the palps are covered mth
small epithehal cells, about 5 to 6m long, with
minute cilia not exceeding .3m in length. Awati
and Rai (1931) maintain that in O. cucullata only
some of the epithelial cells of this surface are
ciliated. They suggest that tlie ciliated cells have
sensory function but present no evidence to
support this view. The absence of cilia on some
of the cells of this layer may be due to then-
destruction during the processing of tissue.

In C. virginica the epitlielium of the smooth
surface of the palp consists of almost cubical cells
with relatively large nuclei and small cilia (fig. 105,
c.ep.). Cell boundaries are indistinct, the cells
tliemselves are crowded and compressed, and there
is a very thin and transparent cuticle on the
periphery. In the subepithelial layer large eosino-
philic cells (e.c.) and mucous cells (m.c.) are very
abundant. The mucous cells are frequently pear-
shaped, but their appearance varies, depending
on the amount of secretion they contain. Then-
length is between 25 and 30 m- Wherever the
secretion of mucus is least, the outer surface of the
palp is slightly ruffled, as shown on the right side
of figure 106,' representing a longitudinal sectu)n
of the palp viewed at low magnification.

The inner or ridged surfaces of the palps present
a dift'erent picture. The entire surface is folded
into deep ridges and grooves. In figure 106 the
I'idges are about 0.3 mm. high along the central
axis. The ciliated cells of the ridged surface are
slender, cvlindrical, and tightly packed, with
small, round nuclei. They form a layer varying
from 40 to 60 m in thickness. The difference m

111

0

Millimeters

Figure 104. — Labial palps of C virginica. The mantle hood is cut off at the midrlle, and the sides are pulled aside to
expose the lips and the mouth. Drawn from life. h. — hood; I.g. — lateral gutter; 1.1. — lower lip; m. — mouth;
r.i.p. — right inner palp; r.o.p. — right outer palp; u.l. — upper lip.

the shape and size of the cells of tlie two sides
of the palp is clearly seen in this figure.

Thin and transparent cuticle covers the epithe-
lium of the ridged surfaces. The cilia are robust,
ranging in length from 8 ^ in the grooves to 20 n
on the tips of the ridges, with the longest cilia
found near the free edge of the palps. Mucous
cells are present but are less abundant than on
the smooth side.

The ridges themselves are set at an acute angle

to the surface of the palp and recline toward its
free edge. There is a noticeable difference in the
epithelium of the two sides of the same ridge.
Tlie cells lining the sides which face the mouth
(lower sides of ridges in figure 106) have longer
cilia, and tlie entire epithelium is slightly ruffled.
On the <ipposite sides the ciliated cells are uniform
and have smaller cilia. This difference appears
on a tangential section of the palp shown in
figure 107.

112

FISH AND WILDLIFE SERVICE

-^?g^::}^

Microns

30

Figure 105. — Cross section of the smooth side of labial palp of C. virginica. c.ep. — cubical ciliated epithelium; e.c. —
large eosinophilic cell; m.c. — mucous cell; m. — muscle fibers; c.c. — vesicular cells of connective tissue. Note the
the infiltration of subepithelial layer by mucus and invasion of blood cells. Bouin, hematoxylin-eosin.

The epithelium rests on a thin basal membrane
consisting of connective tissue fibers and muscles.
The core of the ridge is made of delicate connective
tissue with occasional large vesicular cells con-
taining eosinophilic granules. The description of
microscopical structure of the palps given al)ove
is in agreement with the observations made on
various bivalves by previous workers (Leenhardt,
1926; List, 1902; Siebert, 1913; Thiele, 1S86; and
Wallengren, 1905a, 1905b).

The palps are well supplied with blood delivered

through the anterior aorta, pallial artery, and
short tentacular artery. The velar artery
branches off from the short tentacular artery
and runs the entire length of the palp, giv-
ing off numerous ramifications. According to
vSchwaneke's observation (1913) on Anodonta,
tlie blood from the palp is retiu'ned to the heart
by the way of the mantle.

The palps are innervated by the nerve emerging
from the cerebral ganglion nd entering the
anterior end of the junction between the paired

THE LABIAL PALPS

113

Microns

250

FiGfRE 106. — Longitudinal section of a portion of the
right external palp. The right (smooth) side is only
slightly ruffled. The left side is folded and forms deep
ridges and grooves. Bouin, hematoxylin-eosin. a.s. —
anterior slope of the ridge; g. — grooves; l.m. — longi-
tudinal muscles; p.s. — posterior .slope of the ridge; r. —
ridge covered with ciliated epithelium; t.m. — transverse
muscles.

lobes. A nerve net was described in the palps
of Anodonta (Matthews, 1928). I was not able
to demonstrate its presence in the oyster palps.

DIRECTION OF CILIARY CURRENTS

The most conspicous features of the palps are
the abundance of mucous cells on their smooth
sides and the powerful ciliated layer covering the
ridges of the inner sides. The idea that labial
palps of bivalves represent a sorting apparatus
was clearly stated by Coupin (1893), who observed
that in Mytiluji, Cardium, and Pholas the more
voluminous particles are discarded by the palps
while the finest ones are delivered to the mouth.
He concluded that the principal function of the

Microns

200

Figure 107. — Tangential section of the inner surface of
labial ])alp of C. virginica made through two and a half
ridges. The ruffled sides of the ridges are directed
anteriorly toward the mouth. Bouin, hematoxylin-
eosin.

palps of Pholas is to prevent the bulky material
from reaching the digestive tract.

The sorting of food is accomplished by a complex
system of ciliary ciu'rents along the surfaces of
the palps. With reference to C. virginica, Kellogg
(1915) states that the smooth sides of the two
internal palps facing each other direct the food
particles backward to the tips of the palps. This
statement is only partially correct.

In describing the feeding habits of fresh-water
mussels Allen (1914) noticed that the ridges of
the inner surfaces of the palps normally overlap
one another in a reclining position. This is also
the usual position of the ridges in C. virginica
(figs. 106 and 108). On the anterior slope of each
ridge (fig. 106, a.s.) the currents are directed
toward the free end of the palp; the currents of
the posterior slope (p.s.) lead toward the mouth.
Thus the final direction of the movement of a
particle along the surface of the palp depends on
the position of the ridges. Allen thinks that, as
long as no adverse stinmli are received, the par-
ticles which lie between the palps pass on forward

114

FISH AND WILDLIFE SERVICE

Ml llimeters

Figure 108. — Longitudinal section of the palps along the
lateral groove at the place of entrance to the mouth.
l.g. — lateral groove; l.m. — longitudinal muscles; m. —
mouth; r. — ridges of the inner sides of palps. Bouin,
hematoxyli n-eosin.

from one ridge to another and eventually reach
the mouth. In case of hritation the reflex erec-
tion of the ridges brings uppermost the cilia
leading backward, and the particles on the surface
of the palp are pushed from summit to summit
to the edge of the palps and discarded. It is not
clear from Allen's description whether his expla-
nation is based on observations or on an assumption
that the ridges are capable of changing their
position in response to stimulation.

During observations of the feeding of an oyster
spat less than 3 mm. long and attached to a glass
slide, Nelson (192.3) found that the filaments of
the palps narcotized in magnesium sulfate solu-
tion lose tlieir power of erection with the result
tliat large masses of material passing over the
filaments accumulated near the mouth and blocked
it. This effect may have been caused by the in-
hibition of ciliary motion as well as by the sup-
pression of muscular contraction.

The "filaments" of the palps of the spat ob-
served by Nelson undoubtedly correspond to the
ridges of the inner surfaces of the palps of adult
oysters. The theory of sorting out of the material
advanced by Allen and accepted by Nelson implies
that the change in the direction of movement of
the particles toward the mouth or away from it is
controlled by the erection of the ridges. With a
microscope I examined the palps of oysters intact

THE LABIAL PALPS

except for the removal of one valve and was not
able to observe changes in the position of the
ridges. Similarly, no erection of ridges was
noticed on the excised palps which were kept in sea
water and examined with high power. The ciliary
Clu-rents remained, as a rule, undistm'bed for
many hours.

Tonge (1926) states that in 0. ediilis "there are
no muscles within the folds such as could cause it
to contract downwards." This statement cannot
be confirmed by my observations on C. virginica.
In the palps of the American osyter the longi-
tudinal muscles are clearly visible (figs. 106 and
108, l.m.). They extend along both sides of a
ridge from its base to the very tip. The contrac-
tion of muscle fibers on one side of the ridge pre-
sumably may change the position of the ridge and
make it stand at right angles to the sm-face of the
palp. There is no evidence that this actually
happens.

Churchill and Lewis (1924) arrived at the con-
clusion that in fresh-water mussels the cUia cover-
ing the upper portions of the ridge beat forward
(toward the mouth) while in the deeper part of
the groove between the ridges they beat in the
opposite direction. They also make a generaliza-
tion that "the palps appear to be a mechanism for
reducing the c}uantity of material to an amount
that can be handled by the mouth." According
to these authors the two sides of the palps perform
distinctly different functions. In fresh-water
mussels the ciliary currents along the outer
(smooth) surfaces remove the particles from the
mantle chamber, while on the ridged surfaces the
currents are directed toward the mouth.

The currents along the labial palps of C.
rirginica are, however, more complex and some-
what different from those described for fresh-water
mussels. To observe these currents I removed
one valve, dissected the mantle hood, and pushed
apart its sides to expose the entire surface of the
palps. I placed the oyster in sea water in a
sliallow tray and observed it with a low-power
microscope under strong illumination directed at
an angle of about 20 degrees to the surface.
Suspensions of colloidal carbon or finely powdered
willemite in sea water were used to flood the sur-
face, and the distribution of the brilliantly fluores-
cent willemite particles was observed under
ultraviolet light. A series of pencil diagrams
outlining the positions of the palps were prepared
in advance and were used to mark the direction

115

m.sh.

m.b.

0

Millimeters

10

Figure 109. — Ciliary currents along the outer surface of
the right external palp of C. virginica. Dark streaks
(dotted areas) represent the distribution of mucus
strands with black particles entangled in them 2 or 3
minutes after the surface was flooded with suspension
of colloidal carbon. Large arrows indicate the direction
of principal currents; small arrows show the secondary
currents directed toward the free edge of the palp. The
material entangled in a slimy ball (m.b.) is discarded at
the corner of the palp. m.sh. — mucus sheet. Drawn
from life.

of principal currents as they were observed im-
mediately upon the addition of the suspended
material. Figures 109 through 112 summarize
the results of many observations on several large
oysters.

The palps are usuaJly totally covered with a sheet of
mucus, but the secretion of the mucus is greatest
on the outer surfaces of the e.xternal palps. Num-
erous mucous glands under the surface epithelium
(fig. 105) are easily stinuilated by the slightest
irritation and begin to discharge large amounts of
mucus as soon as their surface is flooded with water

Mil limeters

10

Figure 110.— Principal ciliary currents along the smooth
surfaces of internal palps, C. virginica. Small arrows
indicate the weak countercurrent directed toward the
mouth at the bottom of the central gutter. Drawn
from life.

containing suspended particles. Figure 109 shows
the two systems of currents, both directed away
from the mouth. A strong current along the base
and an ecjually powerful current along the free
edge run toward the lower free corner of the palp.
Weaker currents (small arrows) are directed across
the surface from the base of the palp to its free
edge where the particles cleared from the area are
picked up by the strong ciliary current at the
j)eriphery and are carried to the corner of the palp
touching the edge of the gill (m.). Here a strong
eddy is formed, probably as a result of combined
downward currents of the palp and of the ciliary
epithelium of the gills. The particles discarded
from the palps rotate in this area until a fairly
large ball measuring about 0.5 cm. is formed and

116

FISH AND WILDLIFE SERVICE

Millimeters

Figure 1 11. — System of currents along the ridged surfaces
of the palps, C. virginica. Currents along the edges of
the palps and at the bottom of lateral gutter are shown
in heavy long arrows. The current and countercurrent
along the ridges are shown by short arrows. Long
narrow arrows denote the movements of particles across
the palp toward its free edge. Drawn from life.

the mass is dropped to the surface of the mantle
and discarded through the principal discharge
area (fig. 90). Tlie currents along the smooth
surfaces of the two internal palps (fig. 110) are
directed primarily away from the mouth and
toward the free edges along the entire length of
the palps. There is a much weaker counterciuTent
along the central gutter. The material brought
by this current reaches the lower lip and may be
puslied into the mouth (fig. 11.3). Some of the
particles wliich reach the rounded rim of the lower
lip drop back to the surface of the palp and are
carried away by the principal currents.

The system of currents on the ridged surfaces
of the palps is very complex (fig. 111). There are

Mill i meters

10

Figure 112. — The paths of two particles across the ridged
surface of a palp (small arrows), C. virginica. Small
particle at the lower part of the diagram fell between
the two ridges into the furrow and was carried away by
the current along the edge. A slightly large particle
(upper half of drawing) settled on the top of a ridge and
was kicked from one crest to another until it reached
the bottom current of the lateral gutter and was pushed
toward the mouth. Drawn from life.

two major and opposing cmTents along the lateral
groove. The bottom cm'rent runs toward the
mouth; at a slightly higher level (heavy long
arrows) the currents run in the opposite direction.
Equally strong currents directed away from the
mouth run along the free edge of the palps.

There are at least three major currents along
the inner sm'faces of the palps: (a) from the base
across the palp to the edge, (b) in an opposite di-
rection from the edge to the base (fig. Ill, short
heavy arrows), and (c) slightly weaker currents
from the central part of the palps at about 45
degrees to the edge (long slender arrows).

A particle moving along the ridged surface of
the palp may follow a very irregular path. Figure
112 diagrams the route of two particles which both
were pushed away from the surface. One particle
(upper part of fig. 112) settled on the edge of a
ridge and was pushed by the uppermost cilia from
the top of one ridge to the others until it reached

THE LABIAL PALPS

117

Millimeters

Figure 113. — Tangential section across the mouth and
palps. Semidiagrammatic. e.g. — central gutter; l.g. —
lateral gutter; 1.1. — lower lip; l.i.p. — left inner palp;
1.0. p. — left outer palp; m. — mouth; mn. — mantle; r.i.p. —
right inner palp; r.o.p. — right outer palp; u.l. — upper
lip. Formalin and hematoxylin.

the base and was carried toward the mouth.
Another particle (lower part of fig. 112) fell into
the furrow; several times it was kicked over two or
three ridges, but it always fell to the bottom of a
furrow and finally was carried toward the edge of
the palp and discarded (heavy arrow). Fre-
quently the medium-sized particles entangled in
mucus are caught in an eddy between the ridges
and begin to rotate in a turbulent current. Addi-
tional particles are entangled, and as the ball
grows it is kicked out and caught by the current
along the edge. There was no evidence of the
reversal of ciliary motion and the pattern of
currents described above was maintained through-
out the periods of observations.

An effective sorting mechanism, which prevents
all but the smallest particles from reaching the
mouth, is provided by the system of ciliary move-
ment. The main supply of food is delivered by the
lateral gutters (fig. 113), which lead directly into

the corners of the mouth. Only a small amount
of food reaches the mouth via the central gutter
where it must be pushed over the edge of the lower
lip. Observations of the ciliary motion of the
palps give one the impression that the number
of food particles delivered to the mouth constitutes
only a small fraction of the total quantity which
reaches the palps. It appears that the principal
function of the palps is to eliminate a large part of
food gathered in feeding and allow only a very
small portion of it to reach the digestive tract.
The system of currents and countercurrents ob-
served in the palps of C virginica is much more
complex than that described by Yonge (1926) for
the palps of O. edulis.

Mucus plays an important role in the sorting of
food. The discharge of mucus is directly propor-
tional to the amount of particles which settle on
the surface of the palps. The sheet of mucus is
pulled over the ridges of the palps by the action
of the more powerful cilia at the tops of the ridges.
The smaller cilia of the grooves may not be in
direct contact with the mucus sheet above them
and beat in a different direction. Only the small-
est particles, free of mucus, are moved directly by
the cilia.

A study of the ciliary currents of the palps
suggests that the food-sorting mechanism of the
oyster is designed for continuous feeding in water
containing a low concentration of minute food
organisms. Under such conditions only a limited
amount of mucus is used for sorting out the suit-
able forms. The question of whether the oyster
is capable of selecting its food on some other basis
than size has not yet been adequately studied.

REACTION TO STIMULI

The palps respond both to mechanical stimula-
tion and to the chemical stimulation of weak acids,
solutions of mineral salts, etc. If a weak mechani-
cal stimulus is applied to the edge of the palp, the
affected portion contracts, making a shallow in-
dentation which is proportional in size to the in-
tensity of the stimulus. ^Strong mechanical
stimulus or application of such irritating solu-
tions as weak hydrochloric acid, ethyl alcohol,
adrenaline, etc., provoke general contraction of
the palps and gradual curling up of their edges.
The external palps bend to the outside, while the
internal palps curl in the opposite direction and
seal the access to the central gutter (fig. 114).
Autonomous responses of the labial palps of Ano-

118

FISH AND WILDLIFE SERVICE

0

Mil limeters

FioDRE 114..— Labial palps. Left— normal position. Right— position taken after mechanical stimulation. Drawn

from life. Slightly enlarged.

donta were studied by Cobb (1918), who found
that curling can be evoked by a jet of warm water
(54° C), a beam of sunhght, faradic current, and
by a number of chemicals. His conclusion that
chemical stimulation is more effective than are
the mechanical stimuli in provoking a response
could not be verified. While it is easy to deter-
mine the effectiveness of various concentrations of
chemicals or to register the response to mechanical
stmiuli, a direct comparison between the intensi-
ties of chemical vs. mechanical stimulation is im-
possible without having a common standard for
reference.

BIBLIOGRAPHY

Allen, William Ray.

1914. The food and feeding habilts of freshwater
mussels. Biological Bulletin, vol. 27, No. 3, pp.
127-141.
AwATi, P. R., and H. S. Rai.

1931. Ostrea cucuUata (the Bombay oyster). The
Indian Zoological Memoirs on Indian Animal Types,
III, Methodist Publishing House, Lucknow, India,
107 pp.
Churchill, E. P., Jr., and Sara I. Lewis.

1924. Food and feeding in fresh-water mussels.
Bulletin of the U.S. Bureau of Fisheries, vol. 39,
for 1923-1924, pp. 439-471. (Document 963.)

Cobb, P. H.

1918. Autonomous responses of the labial palps of
Anodonta. Proceedings of the National Academy
of Sciences of the United States of America, vol.
4, pp. 234-235.
CoupiN, H.

1893. Sur I'elimination des matieres gtrangeres chez
les Acephales et, en particular, chez les Pholades.
Comptes Rendus Hebdomadaires des Seances de
r Academic des Sciences, tome 117, pp. 373-376.
Kellog, Jame.s L.

1915. Ciliary mechanisms of lamellibranchs with
descriptions of anatomy. Journal of Morphology,
vol. 26, No. 4, pp. 625-701.
Leenhardt, He.nry.

1926. Quelques etudes sur Gryphea angulata.
(Hultre du Portugal). Annales de I'lnstitut
Occanographique, nouvelle sdrie, tome 3, fascicule
1, pp. 1-90.
List, Theodor.

1902. Die Mytiliden. Fauna und Flora des Golfes
von Neapel und der AngrenzendenMeeres-Absehnitte
herausgegeben von der Zoologischen Station zu
Neapel. 27 Monographic. R. Friedliinder und
Sohn, Berlin, 312 pp.
Matthews, Samuel A.

1928. The palps of lamellibranchs as autonomous
organs. Journal of Experimental Zoology, vol.
51, No. 3, pp. 209-258.
Nelson, Thurlow C.

1923. The mechanism of feeding in the oyster.
Proceedings of the Society for E.xperimental Biology
and Medicine, vol. 21, No. 3, pp. 166-168.

THE LABIAL PALPS

119

SCHWANECKE, H.

1913. Des Blutgefasssystem von Anodonta cellensis
Schrot. Zeitschrift fiir wissenschaft lithe Zoologie,
Band 107, pp. 1-77.

SlEBERT, WiLHELM.

1913. Das Korperepithel von Anodonta cellensis.

Zeitschrift ftjr wissenschaftliche Zoologie, Band

106, pp. 449-526.
Thiele, Johannes.

1886. Die Mundlappen der Lamellibranchiaten.

Zeitschrift ftir wissenschaftliche Zoologie, Band

44, pp. 239-272.

Wallengren, Hans.

1905a. Zur Biologie der Miischcln. I. Die Wasser-
stronuingen. Lunds Universitets Arsskrift, Ny
Foljd, Afdelningen 2, Band 1, No. 2, pp. 1-64.

1905b. Zur Biologie der Muscheln. II. Die
Nahrungsaufnahme. Lunds Universitets Arsskrift,
Ny Foljd, Afdelningen 2, Band 1, No. 3, pp. 1-59.
Yonge, C. M.

1926. Structure and physiology of the organs of
feeding and digestion in Ostrea edulis. Journal of
the Marine Biological Association of the United
Kingdom, vol. 14, No. 2, pp. 295-386.

120

FISH AND WILDLIFE SERVICE

CHAPTER VII
THE GILLS

Page

Anatomy of the gills --- 121

Promyal chamber --- - 123

Gill lamella - 125

Skeleton.. 126

The filaments -- 126

Ostla -- 127

Ciliary tracts..-. 128

Terminal groove --- 129

The muscles of the gills.. 130

Ciliated cells 132

Fine structure of the cilia... - 132

Mechanical properties of the cilium 135

Metachronal rhythm. ._ 136

Frequency of beat .- 137

Effect of temperature 138

Composition of sea water and ciliary motion.. 139

Effects of chemicals on ciliary motion 139

Metallic ions... 139

Hydrogen ions 140

Various drugs _. HO

Inhibition of ciliary movement by antiserum 141

Effect of pressure on ciliary motion 142

Ciliary currents of tlie gills 142

Mechanical work of the laterial cilia.. 143

Carmine cone method.. 144

Effect of temperature .._ 145

Hydrostatic pressure inside the gills 146

Spontaneous inhibition of ciliary motion 146

Bibliography 147

The gills of the oyster and other bivalves per-
form several important functions. They play a
major part in respiration to which the mantle
contributes a minor share. They maintain a
steady current, filter the water, and collect food
particles which are sorted and separated from
detritus and other materials in suspension. Tliey
serve for the dispersal of sex cells at the time of
spawning, and are used for the incubation of
fertilized eggs in tlie larviparous species. The
effectiveness of these functions is dependent on
coordinated performance of the gill apparatus and
on the contractions of the adductor muscle.

ANATOMY OF THE GILLS

Within the class of bivahes the structure of
the gills varies in an increasingly complex series
of modifications. The simplest of these is one
pair of plumlike single gills or ctenidia with two
rows of flattened filaments on each gill. This
primitive type, present in tlie order Protol^rancliia

(fig. 115, A), is found in Ahicula, Yoldia, Leda,
Solenomya, and others. More complex structure
(fig. 115, B) occurs in the ark shells (Arcidae),
scallops (Pectinidae), oysters (Ostreidae), sea
mussels (Mytilidae), and other families of Fili-
branchia. This type is characterized by long and
slender filaments kept in place by patches of
interlocking cilia. In some of the bivalves of
this gi'oup, including edible oysters, the gill
lamellae are plaited into vertical folds and the
reflected plates of the gills are completely united
with the mantle and visceral mass. These
were formerly designated as a separate order of
Pseudolamellibranchia.

The highest degi-ee of complexity is found in
the gills of fresh-water mussels (Unionidae),
cockles (Cardiidae), clams (Veneridae), and many
other mollusks of the order Eulamellibranchia.
In these bivalves the lamellae are joined by bars
of connective tissue, the filaments ai-e firmly
connected by vascular junctions, and the entire
gill has the appearance of a perforated, leaflike
organ (fig. 115, C).

In the order Septibranchia {Poromya, Cux-
jiidaria) the gills are degenerate. They are
modified into perforated, muscular partitions
between the two pallial chambers, and the gill
filaments are greatly reduced (fig. 115, D).

The oyster gills consist of four folds (demi-
branchs or plates) of tissue suspended from the
visceral mass. Two folds on each side of the
body arise from a common ridge or gill axis,
which is composed of connective tissue and
muscles (fig. 73, g.in.). In a cross section made
at right angles to the axis of the gill, each demi-
branch is V-shaped, with the two branches of the V
forming its ascending and descending lamellae.
The descending lamella arises from the gill axis;
its opposite number is the ascending lamella.
The two outer ascending lamellae, one on each
side of the body, are fused with the mantle.
The two inner demibranchs are joined together at

FISHERY bulletin: VOLUME 64, CHAPTER VII

121

A

B

D

Figure 115. — Diagram of the four types of bivalve gills according to A. Lang (1900). A — Protobranchia type {Nucula,
Yoldia, Leda, and others); B — Filibranchia type (Pectinidae, Ostrcidae, Mytilidae, and others); C — Eulamellibran-
chia type (Unionidae, Cardiidae, Veneridae, and others); D — Septibranchia type (Poromya, Cuspidaria).

the central axis of the gills under the common
afferent vein (fig. 73, c. af. v.). The section
across all four demibranchs resembles two W
letters joined together at the center.

Along the entire length of the gill runs the
interlamellar space, which in places is divided
by the interlamellar partitions or septae into a
series of vertical compartments called the water
tubes. The septae end close to the junction of the
demibranchs and the visceral mass, so that the
upper portion of the interlamellar space forms a

continuous channel along the horizontal axis of
each demibranch. This condition is found only
at the upper (dorsal) part of the body at the level
of the stomach and above it (fig. 116, A). At
approximately the level of the heart the entire gill
structure has only three points of attachment, in
the center at the visceral mass and at each side
where the gills fuse with the mantle (fig. 116, B.).
The entire chamber system of the gills can be
visualized as four passages which at the level of the
heart merge into two epibranchial chambers. The

ep.ch

add.m

ep.ch.

Centimeters

A

B

C

Figure 116. — Transverse sections of 1-year-old C. virginica (diagram drawn from sectioned and stained preparations).
A — Section at the level of the stomach, s.; four epibranchial chambers at the base of the gills, ep.ch., and proniyal
chamber, pr.ch., at right. B — Section near the level of the heart, two epibranchial chambers, ep.ch., are separated
by the pyloric process, p. C — Section at the level of the adductor muscle below the heart, common epibranchial
chamber, ep.br.ch. Bouin, hematoxylin-eosin.

122

FISH AND WILDLIFE SERVICE

latter in turn lead to a common epibranchial
chamber which, tliruugh the cone-shaped cloaca,
opens to the outside.

There are no partitions, Aalves, or any other
featui-es for regulating the flow of water inside the
chamber system. The current is maintained
solely by the beating of the ciliary epithelium of
the gills and of the lining of the chambers. Below
the level of the heart the fusion of the gills with
the median axis is lost and the two chandjers,
separated here by the pyloric process of the vis-
ceral nuiss (fig. 72, py.p.), merge together to form
a common! epibranchial cb.amber (figs. 72 and 116,
ep.br.ch.). This leads to a wide cone-shaped
exhalant chamber or cloaca (fig. 72, cl.) which can
be examined by forcing apart the posterior end of
the vahes and focusing a beam of light on its
inner sm-face. The water tubes appear as large,
round holes (fig. 72, w.t.).

PROMYAL CHAMBER

In oysters of the Ostrea type all the water col-
lected by the gills is discharged through a single
opening of the cloaca. In Cra^aostrea, however,
the exhalant system is modified by the presence of
an asymmetrical space on the riglit side of the
body called the promyal chamber (fig. 116,
pr.cii.). This irregularly shaped pocket between
the mantle and the visceral mass extends in a
dorsoposterior direction from the level of the
pericardium to a wide outside aperture which may
be seen by forcing the valves apart and examining
the space between them. Openings of the water
tubes similar to those found on the inner wall of
the cloaca are visible inside the chamber (fig. 75)

The promyal chamber was first observed by
Kellogg (1892), who suggested that the water
from the gills may be discharged througli it.
StalTord (1913) showed the chamber in one of the
illustrations of his book but did not refer to it in
the text. The full anatomical significance of the
promyal chamber in C virginica was described by
Nelson (19.3S) and by Elsey (1935) for C. gigas.

The position occupied by the promyal chamber
makes it apparent that water from the dorsoan-
terior part of the right demibranch is discharged
through this chamber and does not pass tlu'ough
the cloaca. After releasing carmine suspension
near the gills of an actively feeding oyster {C.
virginica), one can observe some of the red dis-
colored water being expelled through the promyal
chamber while the principal stream is passing

THE GILLS

through the cloaca. In spawning males the sperm
shed from the right gonoduct is frequently
discharged through the promyal chamber.

In assessing the relative importance of the
promyal chamber in the movement of water
through the gills. Nelson (1938) states that out of
36 water tubes in the right demibranch of C.
virginica the first 14, ct)mprising more than half
the length of the demibranch, are in free C(3mmu-
nication with the promyal chamber. The remain-
ing 22 water tubes discharge water into the nar-
rowed portion of the epibranchial chamber beneath
the adductor muscle or into the cloaca. In the
absence of measurements of the amount of water
discharged through the promyal chamber, it is
impossible to state what percentage of the total
output is discluirged through the chamber. From
anatomical evidence it may be concluded that the
greatest part of the water used for ventilation of
the gills leaves through the cloaca and only a
minor portion through the promyal chamber.

Relative dimensions of the different parts of the
exhalant system of the gills can be demonstrated
clearly by casts made with plaster of Paris or with
latex. Before being injected, the oyster should
be completely narcotized. The material used for
injection is then forced through both the cloaca
and the promyal chamber while the oyster is
gently and frequently tapped to permit good
penetration to the very ends of the water tubes.
When all the passages are fidl the valves are
pressed together and tied with a string. The ma-
terial is permitted to set for 24 hours before the
shells are opened and the soft parts of the oyster
removed.

The various parts of the water exhalant system
of C. virginica are shown in figure 117, which rep-
resents the cast of the inner gill chambers viewed
from three different angles. The promyal cham-
ber (fig. 117, left, pm. ch.) occupies an irregular
area on the right side of the visceral mass and
ends in an aperture almost twice as large as the
opening of the cloaca (cl.). The relation between
the promyal chamber and the two demibranchs of
the I'ight side is shown in tlie central drawing (fig.
117) of the cast viewed from the left side. The
right and left demibranchs are separated by a
septum (s.) in the upper portion of the posterior
side of the gills (fig. 117, right). Most of the
iimer spaces of the gills are in direct connection
with the cloaca; less than one-half of the right
demibranch empties into the promyal chamber.

123

Figure 117. — Water exhalant system of the gills of C. virginica. Plaster of Paris cast of a large specimen. Left —
view from the right side. Center — view at sharp angle from the left. Right — view from the posterior side of the
gills, cl. — cloaca; l.d. — left demibranch; o.o. — outside opening (aperture) of the promyal chamber; pm. ch. —
promyal chamber; r.d. — right demibranch; s. — septum separating the right and left demibranchs.

The relative size of the promyal chamber is
variable. In some specimens it extends over
three-fifths of the length of the gills and forms a
spacious pocket (fig. 118). The exhalant system
of C. gigas (fig. 119) is similar to that of C. vir-
ginica. In the specimens I examined, the promyal
chamber extends approximately one-half the
length of the giU, and the funnel-shaped cloaca
was much wider than in C. virginica. In 0.
edulis the promyal chamber is absent and the
entire system of water tubes, epibranchial cham-
bers, and cloaca is synnnetrical (fig. 120). In
comparison with C. virginica and C. gigas, the
cloaca of 0. edulis is much broader and longer,
which is probably the result of the almost circular
shape of the body. The water tubes are shorter
than in Crassostrea.

The promyal chamber also has been described
for the Australian rock oyster, Ostrea (Crassostrea)
cucvllata, Born (C. comercialis I. and R.), and in
Ostrea (Crassostrea) Jrons L. from the mangrove
roots in Florida (Nelson, 1938).

Oysters of the Crassostrea type inhabit and
thrive in the brackish and nmddy coastal waters,
which are less suitable for the mollusks of the
Ostrea tj^pe. It has been implied by some investi-
gators (Elsey, 1935; Nelson, 1938) that the toler-
ance to muddy waters is due to a superior cleaning
mechanism which is somehow associated witli the
presence of the promyal chamber and prevents

Figure 118. — Cast of the water exhalant system of a very
large C. virginica. Note the extent of the promyal
chamber along the gill axis and the impression made by
the distended pallial arteries at the lower end of the
right demibranch.

124

FISH AND WILDLIFE SERVICE

Centimelers

Figure 119. — Cast of the water exhalant system of the
gill of C. gigas viewed from the left side. cl. — cloaca;
l.d. — left deniibranch; pm. eh. — promyal chamber;
r.d. — right demibranch.

the accumulation of sediment in the paihal cavity.
No evidence has 3-et been presented to corroborate
this view. The physiological significance of the
chamber is not known beyond the fact that it pro-
vides an additional outlet for the discharge of
water through the gills. Elsey (1935) remarks
that in C. gicjas the promyal chamber allows a free
passage of water when the gonads distend before
spawning and encroach into the branchial space,
partially closing the water tubes. Sections of the
visceral mass of C. virginica with fully developed
gonads do not show any "encroachments" into
branchial space. Tlie latter remains unob-
structed, and the water tubes fully open. At
present it is difficult to see what structural and
physiological advantages are gained by the pres-
ence of a promyal chamber in Crassostrea oysters.

GILL LAMELLA

Each lamella of a gill consists of a great number
of tubular filaments arranged at right angles to the
axis of the gill. At the edge of the plate the fila-
ments are reflected on themselves and continue

upward along the plane of the ascending lamella.
The filaments forming the gill-lamella do not lie
smoothly on one plane; they are arranged in a
series of transverse folds or plicae that give the
surface of the gill a plaited appearance noticeable
to the naked-eye. A transverse section (fig. 121)
shows the arrangement of filaments in alternating
grooves and ridges. The number of filaments on
a single fold of an adult C. virginica is not constant.
In my preparations it varied from 10 to 16 per
fold.

There are three types of filaments that can be
distinguished by their position, shape, and dimen-
sions. The larger, or principal filaments (fig. 121,
p.f.), are located at the bottom of the groove
between the plicae. In cross section they have a
triangular shape with two bulky chitinous rods
forming two sides of this triangle. The rods are
fused at the apex but are separated at the base,
which contains a narrow blood vessel. The two
transitional filaments (t.f.), one on each side of
the principal one, are smaller and diff'er in shape
from the ordinary filaments (o.f.), which form the
rest of the plica. Sometimes the difll'erence is
insignificant. In general the ordinary filaments
seen on cross section are elongated, club-shaped
units.

Throughout their length the filaments of each
plica are joined at the bases by regularly spaced
interfilamentar junctions (if.j.), which consist of
narrow bands of vascular connective tissue. The
free portions of the filaments surround the oval-
shaped openings, called ostia or fenestrae (o.),
through which the water enters the inside passages
of the gill. Numerous muscle fibers follow the
interfilamentar junction and extend along both
sides of the plica to its distal part (if.m.). At
intervals, the two lamellae of each demibranch
are connected by the partitions (interlamellar
septa) made of connective tissue (il.s.) which run
across the plate from one lamella to the otiier.
These partitions are more numerous toward the
distal (free) edge of the demibranch and diminish
toward the base of the gill, where they are found
only at about every sixth plica. The interlamellar
junctions contain numerous muscle fibers, blood
vessels, and nerves. The muscle fibers are
arranged in three systems: the longitudinal mus-
cles (l.m.) seen in cross section extend vertically
from the proximal to tlie distal end of the lamellae;
the transverse muscles (tr.m.) go from one side of
the lamellae to the other; and the tangential

THE GILLS

73S-851 0—64-

125

Figure 120. — Inner cast of the water exhalant system of the gills of 0. edulis. Right-
Left — viewed from left side.

k-icwed from posterior side;

interlamellar muscles (il.m.) underlie the surface
epithelium of the junction between the ascending
and descending lamellae. At both ends of the
junction near the location of the principal fila-
ments the muscles branch off and form well-
pronounced bands underlying the chitinous rods.
It can be deduced from the pattern of distribution
of the interlamellar muscles that their contrac-
tions bring the plicae of the two sides of the
demibranch together, constrict the blood vessels,
and reduce the diameter of the water tubes.

SKELETON

A framework of chitinous rods forms a scaffold-
ing which supports the entire gill structure. The
skeleton can be isolated from the tissues by brief
treatment with a weak solution of sodium hy-
droxide, which does not affect the chitin. Struc-
tural elements cleared by this method are shown
in figure 122. The skeleton of each filament
resembles a ladder with the horizontal rungs
slightly curved on one side and joined to the
vertical elements by knobs of chitin. On both
sides the supporting ladderlike unit of each
filament is joined by cross pieces to tlie next
units, forming a continuous framework. The
vertical and horizontal bars surround the open-

ings (ostia) between the filaments (o.). Each
skeleton unit supports two adjacent filaments.
The vertical bars correspond to the walls of the
two adjacent filaments, while the horizontal
members (the rungs of the ladder) are embedded
in the tissue of the interfilamentar junctions.
Two purposes are accomplished by such a pattern.
The gill acquires rigidity and at the same time
provides strong support for the delicate, sievelike
membrane through which water passes into the
water tubes. Heavy rods support the principal
filaments (fig. 123). At the base of the gills the
skeleton rods form massive V-shaped arches
embedded in fibrous connective tissue (fig. 124).

THE FILAMENTS

The structural unit of the gill is a tubular
filament of ciliated epithelium supported by
skeleton rods. The central part of tlie filament
is occupied by a space which is periodically filled
with blood as the gill plates expand and contract.
Connective tissue underlies the proximal part of
the filament which consists of nonciliated, almost
cuboid cells, tightly packed in a two-cell layer.
The distal part of the filament is covered with
ciliated cells (fig. 125). Bulky nmcous cells
occur at irregular intervals and discharge their

126

FISH AND WILDLIFE SERVICE

Millimeters

FiGTRE 121. — Transverse section of the demibranch of C. viriginica. Kahle fixation, hematoxylin-eosin. ch.r. — chitinous
rods; g. — groove; if.j. — interfilamentar junction; il.m. — interlamellar muscles; il.s. — interlamellar septum; l.m. —
longitudinal muscles of the interlamellar septum; o. — ostium; o.f. — ordinary filament; p.f. — principal filament;
pi. — plica; t.f. — transitional filament; tr.m. — transverse muscle of the interlamellar septum; w.t. — water tube.

content to tlio surface of the gills. The secreted
mucus spreads over tlie gill plates and entangles
the particles which settle on them. At the distal
edge of the gill the filaments are fused together
to form a terminal groove along wliich food is
conveyed toward tlie mouth. The principal
filaments differ from the others by their larger
size and nontubular appearance. At tlie base of
each filament there is a blood vessel located in a
space between tlie chitinous rods. The epithelial
cells are slightly larger than those of the ordinary
filaments and have longer cilia (fig. 126).

OSTIA

Ostia or fenestrae, the oval-shaped open spaces
between two adjoining filaments (figs. 121 and 125,
o.), are framed by two vertical and two horizontal

skeleton bars covered witli epithelium. Their
configurations and dimensions vary, depending
on the degree of contraction of the filamental
musculature and the distention of blood spaces.
In an actively feeding oyster the contraction and
expansion of the ostia regulate the passage of water
through tlie gills. This can be observed on the
surface of a gill exposed by cutting off a piece of
shell. The dimensions of the ostia are somewhat
correlated to the size of the oyster ova which pass
through the gills during spawning (see ch. XIV,
p. 303). In a viviparous 0. lurida the ostia are
large, varying from 90 by 45 /u in contracted
state, to ISO by 60 n when fully expanded. The
ova of this species average 90 m in diameter. In
C. gigas and C. virginica, which have smaller eggs,

THE GILLS

127

200

Microns

Figure 122. — Side view (A) and front view (B) of a
skeleton of ordinary gill filaments of C. virginica. Soft
tissues removed by sodium hydroxide. Unstained
preparation, whole mount.

the ostia are approximately only one-third the
dimensions of those in 0. lurida.

CILIARY TRACTS

The surface of the filament is covered by
several different ciliary tracts. Cells of uniform
size on the outer surface bear the frontal cilia,
which are relatively small and beat parallel to the
surface of the filament (fig. 125, fr.c). They are
flanked on each side by a single laterofrontal cell
(If.c.) of larger size with a blade-shaped cilium,
which according to Atkins (1938) occurs in the
family Ostreidae and is somewhat different from
the laterofrontal cells of other bivalves. In
fixed and stained preparations this wide and
curved cilium is frequently frayed. The shape
of the cilium and the presence of the basal granules,
typical for normal ciliated cells, both indicate
that the laterofrontal cilium is formed by the
fusion of ordinary filaments, a view which is con-
firmed by studies made with the electron micro-
scope (see p. 132). The laterofrontal cells of C.
virginica are fairly large and cone-shaped; their
relatively small nuclei are located at the narrow,
proximal end of the cell; and the protoplasm is
devoid of granules and deeply stained with
hematoxylin. The laterofrontal cilia of gills
preserved in a relaxed state extend toward their
opposing numbers on the adjacent filament and
touch their tips. In a contracted gill they are
bent and almost undistinguishable from the
cila of the frontal cells. The length of the latero-
frontal cila on sectioned and stained preparation
varies from 11 to 15 m. Accurate measurements,
however, are difficult because of the bending of
the cilia wliich do not remain fully extended even
in completely narcotized cells. In O. edulis,
according to Atkins (1938), tlie length of the
laterofrontal cilia varies from 14 to 25 ^i. The

0

Millimeters

0.5

Figure 123. — Chitinous rod of the principal filament of C. virginica. Soft tissues removed by sodium hydroxide. Un-
stained preparation, whole mount.

128

FISH AND WILDLIFE SERVICE

f.c.t

ch.a.

0

_L

Microns

200

Figure 124. — Longitudinal section near the base of the
gill of C. rnrginica. Chitinous arches, ch.a., are em-
bedded in fibrous connective tissue, f.c.t. Osmio acid
fixation, iron hemato.xylin.

frontal and laterofrontal cilia of tiie principal
filaments have the same structure as those of the
ordinary filaments, difTering only in their <>reater
size. A short distance below the cell surface each
cilium terminates in a basal body, a tiny granule
from which a pair of rootlets extends deeper into
the protoplasm and becomes undistinguishable
near the nucleus (figs. 12(1 and 127).

^vlj^

Microns

Figure 125. — Transverse section through ordinary fila-
ment of C. virginica. Vertical chitinous rods (stippled
areas) and blood space are at the center, fr.c. — frontal
cilia; If.c. — laterofrontal cilia; I.e. — lateral cilia; o. —
ostium. Kahle fixation; hematoxylin-eosin.

Besides the frontal and laterofrontal cilia,
Atkins (1938) distinguishes in 0. edulis the "fine
frontal" and "paralaterofrontal" cilia, which run
on both sides of the central portion of the frontal
tract (fig. 127, f.f.c, para l.f.c). He states (1938,
p. 367) that: "Subsidiary laterofrontal cilia are
present in Ostreidae, but are very difficult to
distinguish even in the living gill." I was unable
to identify these cells in sectioned preparations or
in the living gill filaments of C. virginica examined
under high power. The frontal cilia of this
species appeared to be of uniform length along
the entire cross section of the tract (fig. 125).

Beneath the laterofrontal cilia of the filaments
there is a group of six large cells, four of them
broad and two narrow, which bear large, stout
cilia about 17 to 18 m in length. These are the
lateral cilia (fig. 125 and 127, I.e.), which bend
forward slightly toward the outer surface of the
filament and touch the ciha of the opposite group.

TERMINAL GROOVE

The free edge of a demibranch formed by the
concrescence of the ascending and descending
lamellae is a shallow trough called the terminal

THE GILLS

129

Microns

Figure 126. — Transverse section of the principal filament of the gill of C. virginica drawn from the same preparation as
in figure 125. Note the well-developed muscle fibers, m., under the large skeleton bars, ch.r.. c.t. — connective
tissue; bl.v. — blood vessel; bl.s. — blood space; fr.c. — frontal cilia; If.c. — laterofrontal cilia; I.e. — lateral cilia; m. —
muscle.

groove. This depression at the border of the
gills extends their entire length. The epithelial
lining of the terminal groove consists of columnar
ciliated cells with large cilia and numerous mucous
and eosinopliihc cells. The epithelium rests on
a basal membrane. Transverse muscle fibers
extend between the two sides of the groove.
During feeding the grooves are open, the condition
which is shown in figure 128. Their contraction
brings the edges together and closes the groove.
In this way the oyster discards some of the
material which was collected by the surface of
the gill. The rejected particles entangled in
mucus are dropped to the inner surface of the
mantle and are discharged. The direction of the
ciliary beat along the four terminal grooves is
always toward the labial palps and the moutli.

THE MUSCLES OF THE GILLS

The gills of an actively feeding oyster contract
and expand at frequent, although irregular, inter-
vals. This behavior is difficult to notice in an

intact oyster, but it can be observed in an oyste^"
in which much of the valve has been cut off
without injuring the adductor muscle and the
gills. The mantle at the exposed area rolls up
and leaves tiie gills in full view, and if carefully
performed, the operation has no visible ill effect
on the function of tlie gill.

The most conspicous movements which can be
seen with the naked eye are the muscular con-
tractions at the bases of the gills and the corres-
ponding changes in the position occupied by the
demibranchs. These four structures may stand
apart like stiff leaves of a wide open album or
tiiey remain parallel, touching one another like
the pages of a closed book. There is also a lateral
movement of the filaments which brings them
together or pushes them apart. This movement
frequently occurs independently of the contrac-
tions of tlie demibranchs and nnvy be limited to
a small portion of the plica. Both types of move-
ments afl'ect the opening of the ostia, which are
widely stretched when either the four demi-

130

FISH AND WILDLIFE SERVICE

Microns

20

Figure 127. — Cross section of the filament of the gill of
0. eduUs, according to Atkins, 1938. an.l.fc. — anterior
laterofrontal cilia; c.f.c. — central frontal cilia; f.f.c. —
fine frontal cilia; para l.fc. — paralaterof rental cilia;
m.g. — mucous gland; 1. — lateral cilia; t.m. — transverse
muscle fiber. Chitinous rods are shown as black areas
under the epithelium.

branchs or only a group of filaments stand apart
and are constricted when the latter are drawn
together.

Changes in the position of the demibranchs de-
pend on two distinct systems of muscles located at
the gill axis above and below the skeletal arches.
In general the muscle fibers follow the configura-
tion of the arches. The larger bands located in-
side the arches are the flexor muscles, which are
attached to the inner sides of the two arms of an
arch (fig. 129, f.). Their contraction brings the
two adjacent demibranchs together. The smaller
bands at the base of the arch (ex.) are the extensor
muscles, which cause the demibranchs to stand
apart. The action of the two bunds shown in the

Millimeters

0.5

Figure 128. — Terminal groove at the edge of a demi-
branch of C. virginica. Longitudinal section of the
demibranch. Bouin, hematoxylin-eosin.

figure is antagonistic. The extensor bands are
smaller, probably because the elasticity of the
chitinous arches pushes the demibranchs apart and
this springlike action means that less force is re-
quired of the extensor muscles than of the flexor
bands.

Other muscle bands of the gills, although less
conspicuous than the flexors and extensors of the
arches are, nevertheless, of great importance in
regulating the transport of water through the com-
plex gill apparatus and in facilitating the exchange
of blood inside the gill filament. Water tubes of
the gill can be constricted by the contraction of
the muscles underlying the epithelium of the inter-
lamellar septa and extending from one lamella to
another (fig. 121, il.m.), while the contraction of
the transverse muscles of the interlamellar septa
compresses the blood vessels. The contraction of
the longitudinal muscles of the septa (fig. 121,
l.m.) results in the withdrawal and shortening of
the entire demibranch. This reaction occurs spon-
taneously but can also be induced by stimulation.
The contraction of the interfilamental muscles
(if.m.) brings together the vertical rods of the gill
skeleton, causes the curving of the crossbars, and
constricts the blood space of the filament, forcing
blood into the pallial veins.

Contractions afi^ecting only part of the gill cause
the blood to oscillate inside the gills. Because of
the open nature of the lamellibranch circulatory
system the direct return of blood from the gills to
the auricles cannot be accomplished by the pump-
ing action of the heart. Contractions involving
the entire gill apparatus are needed to complete
the renewal of blood.

THE GILLS

131

0

Microns

200

Figure 129. — Longitudinal section through the base of
a demibranch of C. virginica. Kahle, Mallory triple
stain, ex. — e.xtensor muscles; f. — flexor muscles. Pieces
of the skeleton arch are shown in black.

CILIATED CELLS

The structure and functioti of vibratile elements
of tlie cells have been the object of numerous inves-
tigations beyond the scope of this book. The
reader is, therefore, referred to comprehensive re-
views of the problem of ciliary motion made by
Gray (1928) and more recently by Atkins (1938)
and Brown (1950). Several theories based on
studies of the structure and action of cilia fail to
give a satisfactory e.xplanation of ciliary motion,
which at present still remains a biological mystery.

Cilia examined in transmitted light or viewed on
a dark background in reflected light appear to be
optically homogenous. In polarized light they
are birefringent (Schmidt, 1937). Observations
with the light microscope disclose the presence of
an axial filament (axoneme) surrounded by a thin
sheet of cytoplasm (^\enyon, 1926). As a rule,
the cilia emerge from tiny basal granules
near the cell surface and penetrate through the
cuticle, which under the light microscope appears
as a thin homogenous membrane. Studies of
the role and origin of basal bodies in various
ciliated cells have resulted in a great number
of speculations. Experiments by Peter (1899)
showed that in small fragments of a crushed
protozoan the cilia continued to beat as long
as they were in organic connection with the
adjacent pieces of cytoplasm. He deduced from
tiiis observation that the ciliary mechanism is
located near the surface of the cell. Similar results
were obtained with lateral cells stripped away

from the filaments of Mytilus gills. The cilia
that were removed from the basal granules re-
mained motionless while those connected with
them continued to beat (Gray, 1928). The
microdissection technique in more recent years
supports these findings. It was demonstrated
that in the ciliated cells of the gills of Anodonta
the motion of the cilia ceases when the cell is cut
transversely in the immediate region of the basal
granules. Transverse cuts made at any level
within tiie proximal two-thirds of the cell had no
efi'ect on ciliary motion, but if the cut was made
across the zone occupied by the fibrillae or rootlets
in the distal third of the cell, the coordination of
the ciliary motion was destroyed although the
cilia continued to beat. These observations
seem to support the validity of the theory, ad-
vanced hidependently by Henneguy (1897) and
Lenhossek (1898), that the basal granule, homol-
ogous and sometimes identical with the centro-
some of the mitotic figure, is the center which
controls the activity of the cilium.

FINE STRUCTURE OF THE CILIA

With the advance of electron microscopy con-
siderable progress has been made in the study of
the fine structure of cilia. It has been discovered
that throughout the plant and animal kingdoms,
regardless of the position of the organism on the
evolutionary level and irrespective of the organs
studied, cilia have a common structural pattern.
The cilia of the gill epithelium of the oyster are
no exception to this rule. Thin sections of the
frontal and lateral cells of the filaments fixed in
buffered osmic acid and examined under the elec-
tron microscope show a structure which is undis-
tinguishable from that of the cilia of vertebrates,
protozoa, or the tails of spermatozoa. The cilium
consists of a protoplasmic matrix in which are
embedded 11 filaments; 2 single filaments are at
the center and 9 double ones are arranged in a
ring on the periphery. The central pair is con-
nected to the peripheral ring by radial trabeculae
or spokes. Short pieces of dense ntaterial join
the outer filaments to the membrane (fig. 130),
wliicli binds more osmium and is, therefore, darker
tlian their interior, making the cilia appear tubular
(Fawcett, 1958). The two central filaments are
o\al shaped in cross section. The plane in which
these filaments are oriented is similar for all the
cilia of the cell and is thought to be perpendicular
to the direction of the ciliary beat (Fawcett, 1958).

132

FISH AND WILDLIFE SERVICE

^

.^-»f

**

^ <.MJtK

0

Micron

0.5

Figure 130. — Cross section of the group of frontal cilia of the gill of C. virginica. Microvilli of the cell surface are seen
at the bottom. Electron micrograph. Buffered osmic acid 1 percent.

The orientation is apparent in the electron micro-
graph (fig. 1.31) of a longitudinal section of the
distal part of the lateral cell of the filament of
C. virgmica and on transverse sections of the
frontal cilia (fig. 130). Because the latter cilia
are curved in the direction of the beat, they were
cut transversely and appear in the micrograph a
short distance above tlie cell surface. Tlieir oval-
shaped axial filaments are oriented parallel to the
surface of the cell, i.e., in the du'ection of ciliary
beat. The membranelike laterofrontal cilia con-
sist of several individual cilia embedded in a
protoplasmic membrane, but each element retains
the typical structure of a single cilium (fig. 131).
The basal corpuscles of cilia are arranged in
rows (fig. 132), and the central part of each is
siu'rounded by denser cortex, giving the appear-
ance of an empty central cavity. In the longi-
tudinal section (figs. 131 and 132) they are
elongated with a pair of rootlets arising from
each proximal end. Rootlets of the cilia of the
clam, EUiptio complaiiatus, have a periodic stria-

tion of about 750 A. Similar periodicity appears
in electron micrographs of oyster cilia made in
the course of my studies, but the pictiu'e is not
as clear as that published by Porter and Fawcett
(see DeRobertis, Nowinski, and Saez, 19.54, p.
382).

The distribution of rootlets follows a precise
pattern. Each rootlet of a pair turns at an acute
angle and crosses over the rootlet of the adjacent
corpuscle. The rootlets may be followed fiu-ther
down the cytoplasm toward the nucleus (not
shown in the micrograph) ; some of them cross
the second rootlet emerging from the other side
of the same corpuscle as can be seen at the center
and left side of figure 132. The crossed rootlets
are in close contact with each other, but it is
not clear whether or not they are fused. Ap-
parently direct communication between the basal
corpuscles is lacking.

Tlie cjuestion of whether tiie rootlets are simply
the anchoring structures of the cilia or play an
active part in its movement remains unanswered.

THE GILLS

133

I*

i,

* ." >■*

^f

0~ 0.5 '

J.' ^ Micron

Figure 131. — Longitudinal section of the distal portion of laterofrontal cell of the gill of C. virginica. Since the plane
of section passes at the middle of the cilium only single axial and two peripheral filaments can be seen. The basal cor-
puscle and the beginning of rootlets are at the lower part of the micrograph. Electron micrograph. Buffered osmic
acid 1 percent.

There is the possibility that they may represent a
coordinating mechanism of the cihary epithehum.
The fact that the rootlets of the two adjacent
corpuscles cross each other is in favor of this
view, which was advanced by Grave and Schmitt
(1925) on the basis of their observation of the
cilia of fresh-water mussels made with the light
microscope. Exploration with the electron micro-
scope gives additional support to their hypotliesis
which, however, requires further corroboration.

The free surface of the ciliated cell appears as
a thin homogenous layer, devoid of visible struc-
ture, when examined in the light microscope. In
reality this layer consists of fingerlike processes
called microvilli (figs. 130 and 132), which are
found in various tissues; they are considered a
device to increase the surface of the cell. Their
number has been estimated as high as 3,000 per
single cell of intestinal mucosa, and there is no
doubt that numerous fingerlike processes greatly
increase the surface area of the eill and facilitate

the exchange of gases and ions. In figure 132
the layer of microvilli, about 0.5 /j in thickness,
rests upon the plasma membrane of the cells.
The cytoplasm under the membrane contains
numerous mitochondria.

The complex ultrastructure of the ciliated cell
of the oyster gill is shown diagrammatically in
figure 133, which represents a reconstruction of
the principal features seen on electron micro-
graphs. The diagram is based on a large number
of micrographs and summarizes our present
knowledge of the dimensions and arrangement of
the various parts which comprise the ciliated
apparatus of the oyster gill.

Although the mechanism of ciliar_v motion is
not known, studies of tlie ultrastructure of the
cilia suggest that tlie molecular organization
of both cilia and myofibrillae of the muscle cells
are homologous and that the mechanism of their
contraction is similar. This conclusion gains
further support from bioclieniical studies which

134

FISH AND WILDLIFE SERVICE

Micron

Figure 132. — Section perpendicular to the surface of frontal cilia of the filament of the gills of C. virginica. The curved
frontal cilia are cross sectioned. Note the row of basal corpuscles with rootlets; the sharp line, parallel to the surface
of the cell corresponding to plasma membrane; and the microvilli above it. Electron micrograph. Osmium fixa-
tion. Buffered osmic acid 1 percent.

show that both the contraction of the muscles
and the movement of bactei'ial ciha is stimulated
by adenosinetriphosphate (DeRobertis, Nowin-
ski, and Saez, 1954, pp. 389).

MECHANICAL PROPERTIES OF THE
CILIUM

Most of the observations on tlie structure and
and movements of lamellibranch cilia were made
on the gills of Mytilus. There is no reason to
tliink, however, that the cilia of the oyster oill
are fundamentally different from those of the
mussel.

The gifl cilium is a flexible and elastic rod wliich
can be deformed by mechanical pressure applied
with a microdissection needle. Tiie deformity is
repaired rapidly wlien the pressure is removed.
Gray (1928) interprets these observations on
Mytilus cilia, as an evidence of transverse elas-
ticitv of the cilium.

Tlie movement of the cilium consists of two
distinct phases, the forward effective stroke and
the much slower recovery stroke which brings
the cilium to its initial position. The velocity of
the eft'ective stroke is considered to be five times
that of tiie recovery stroke (Kraft, 1890). Tlie
eft'ective stroke begins with the curving at the
tip and extends down to the base, bending the
entire cilium into an arch of 180°; throughout this
period the cilium behaves as a rigid rod mounted
to the cell by its end. During the recovery
stroke the cilium straightens from tlie base to
the tip and moves backward as a limp thread.
Botii t!ie effective and the recovery strokes take
place in tlie same plane, which remains constant
(Gray, 1922a; Carter, 1924).

The movement of a cilium results from con-
traction of its filaments. It is not clear, however,
wliether all 11 filaments are eciually involved in
the effective and recovery strokes. Furtliermore,
it appears probable, although definite proof is

THE GILLS

135

Figure 133. — Diagrammatic reconstruction of the distal
portion of the ciliated cells of the gill epithelium of C.
virginica. ax.f — axial filament; b.pl. — basal plate and
basal corpuscle; c. — cilium; c\v. — plasma membrane;
mit. — mitochondria; r. — rootlet. Cross section of the
cilium shown at upper left corner.

wanting, that the pair of a.xial fihinients gives the
ciHum the necessary rigidity but does not partici-
pate in tlie movement.

METACHRONAL RHYTHM

Automatism is a general characteristic of ciUary
motion. This typical property of ciliated epi-
thelium, common to all animals which have
ciliated cells, is a fundamental characteristic of
the ciliary motion of lamellibranch gills. As
Gray (1928, p. 4) stated: "There can be little
doubt that all cilia are fundamentally automatic
in their movement and that the power possessed
by organisms to inhibit the locomotion of their
cilia is of extraneous nature."

In any ciliated surface there is some sort of
coordinating mechanism that manifests itself in
the metachronal rhythm of the beat. The term
metachronal rhythm or metachronism denotes the
regular sequence of ciliary motion in which any
cilium in a given series is slightly out of phase

with the ciliiun behind and in front of it. Since
the cilia in one row of the epithelium beat at the
same rate but are in different phase, their com-
bined movement gives the optical appearance of a
wave passing over a wheat field on a windy day.
The beating of the lateral cilia along the isolated
filament of an oyster gill is an excellent object in
which to observe the metachronal wave. In the
drawing of an exposed surface of the giUs of a live
oyster examined under a compound microscope
(fig. 134) the metachronal waves along the two
rows of the lateral cilia move in opposite direc-
tions. The effective stroke of the lateral cilia in
this case is at right angles to the direction of the
metachronal wave (i.e., perpendicular to the plane
of the drawing). The crest of the wave includes
the cilia that are ready to begin their effective
stroke; in the troughs are the cilia that are about
to start the recovery stroke.

The direction of the metachronic wave is not
disturbed by the temporary cessations caused by
such extraneous agents as narcotics or cold.
Upon recovery the metachronic wave proceeds in
the same direction as when the motion was
artificially stopped. In the ciliated epithelium of
the roof of a frog's mouth the metachronic wave
is not disturbed even if a piece of epithelium is
cut off and then placed back after rotating it 180°
(Briicke, 1917). Transplantation of the gill
epithelium of an oyster was tried in the Bureau's
shellfish laboratory without success. Copious
discharge of mucus, continuous bleeding of the
wound area, and the cm'ling up of the filaments
interfered with the implantation of the excised
pieces. In all my experiments the host animals
discarded the implants in a short time.

The fact that small pieces of ciliated surface

Figure 134. — Two tracts of the lateral cilia of C. virginica
along the two filaments on both sides of the ostia.
Small black particles suspended in water are drawn into
the ostia while the large ones are discarded by tlie
recovery strokes of the lateral cilia. Drawn from life.

136

FISH AND WILDLIFE SERVICE

or even single ciliated cells removed from the or-
ganism contiime to beat for a long time leads to
the conclusion that in the majority of cases the
ciliary motion is independent of nervous control
of the organism. This is, however, not a general
rule since the ciliary motion on small fragments
of the hps of the snail, Physa, removed with the
attached nerve, soon ceases unless the nerve is
stimulated (INIerton, 1923b). Numerous investi-
gations give support to the concept that in many
invertebrates and vertebrates the nervous system
is an effective agent in the control of coordinated
activity of cihary tracts (Babak, 191.3; Carter,
1927; GothHn, 1920; Lucas, 1935; McDonald,
Leisure, and Lenneman, 1927; Seo, 1931).

Bipolar cells and nervelike fibers immediately
below the ciliated epithelium of the gills of fresh-
water mussels, Lampsilis and Quadrula, described
by Grave and Schmitt (1925), were supposed by
these authors to serve as conduction paths for
stimuli which they claim regulate and coordinate
ciliary movements of the gills of these mollusks.
According to their point of view, the ciliated cells
of the bivalve gills have a dual control. They
may be perfectly autonomous and continue to
beat in the complete absence of neural connections;
on the other hand, the automatic beat of the cilia
may be regulated through supplementary nervous
connections in conformity with the state of the
organism as a whole. These authors assume that
ciliated tissues of fresh-water mussels are botli
autonomous and under the control of the nervous
system.

Intracellular fibrillae of the gills of Mya, Lamp-
silis, and Quadrula were considered by Grave and
Schmitt (1925) to be the conductive paths for
coordinating and regulating ciliary movement. A
complex system of interconnecting rootlets of the
ciliated cells of oyster gills described above
(fig. 132) gives additional support to this view.
Grave and Schmitt (1925) described also the
nervelike apparatus of bipolar cells and fibers.
Reinvestigation of the tissues of fresh-water mus-
sels by Bhatia (1926) did not support these
findings. No such structures were found in my
preparations of the gills of C. virginica, or, ac-
cording to Lucas (1931), in the gills of Mytilus
edulis. Their existence in the gills of fresh-water
mussels seems to be doubtful.

FREQUENCY OF BEAT

The rate of ciliarj- beat can be observed easily
on lateral cilia because of their relativelv large

size and well-pronounced metachronic wave. Ob-
servations must be made on small excised pieces
of gill since the position of the lateral cilia on the
sides of the filaments makes it impossible to
watch their activity on an intact demibranch.
In my preparations the filament or a group of
them was separated by using fine needles, and
kept in a micro-aquarium filled with sea water.
The temperature was controlled by circulating
cold or warm water in the outside jacket of the
microacjuarium.

The frequency of beat was determined by using
a stroboscope of the type manufactured by R.C.A.
and sold under the name "Strobotac". The
reddish flickering light given off by this instrument
is sufficient to observe cilia under a magnification
of about 250 X. Readings are made directly
on the panel of the instrument by rotating the
knobs controlling the frequencies. The instru-
ment must be adjusted to the zero point and
frequently checked.

Gradual decline in the frequency of beat on the
excised filament becomes apparent after several
hours; the disturbance of the metachronism in
the preparations kept for more than 24 hours is
a sign of pathological conditions. Such prepa-
rations should be discarded.

The frequency of beat varies greatly in different
oysters of the same age, origin, and environment.
For instance, among the 12 large adult specimens
from New England waters tested in August 1956,
the range of variation at room temperature of
22° to 23° C. was from 16 to 27 beats per second.
All the specimens were in excellent condition and
appeared normal in every respect.

In addition, there are sometimes wide variations
in the frequencies of ciliary beat in the adjacent
filaments of the excised gills. In studies of the
effect of temperature and other environmental
factors on the rate of beat, therefore, all the read-
ings must be made over the same portion of the
ciliary tract. This is sometimes diflacult because
of the mobility of the excised pieces and copious
secretion of mucus which interferes with the
observations.

In the data summarized in table 14 the beat
frequencies were recorded in a selected locus of the
tract of lateral cilia kept at nearly constant tem-
perature. The filaments were taken from the 14
different oysters listed in the first column of the
table. Observations lasted from 10 to 30 minutes.
The maximum range of variation recorded during

THE GILLS

137

each test was from 16.6 to 20.5 beats per second.
The greatest difference ])etween the individual
oysters was recorded in two lipe males; one had the
median frequency of 15.5 per second (at 23.3° C.)
while in the other the ciha beat at the rate of 24.8
per second (at 25.1° C). In the majority of the
oj^sters the median rate of ciha beat varied between
18 and 22 per second.

Table 14. — Frequency of heat of lateral cilia of 14 adult

C. virginica recorded at nearly constant temperatures

[Readings were made at intervals of 1 or 2 minutes]

Oyster

Spawned out, sex un

determined

Two-year-old

Ripe male

Spawned out male.-.
Spawned out male. . .

Ripe male

Spawned out female.
Spawned out female
Spawned out female.

Ripe female

Ripe female

Spawned out female.
Spawned out female
Spawned out female

Beats per second

Dura-
tion

Temper-
ature

range

Max.

Min.

Me-
dian

Min-

nies

" C.

No.

No.

No.

21

23. 8-23. 8

20.7

18.3

19.6

10

24. 1-24. 4

17.3

15.7

16.3

15

23, 3-23. 3

15.3

16.0

15.5

10

23. 2-23. 6

21,5

20.5

21.2

10

24. 0-24. 2

21,3

19.3

20.0

12

25. 1-25. 2

25,2

23.5

24.8

30

25. 2-25. 4

23.7

21.1

22.1

15

24.2-24.3

20.5

16.6

18.5

10

25.1-25.1

20.6

17,3

19.0

10

24. 5-24. 5

18.8.

15,6

17.3

20

26. 5-26. 5

18.0

15,5

17.3

10

23,0-23.1

21.5

19,3

20.7

10

23.0-23.1

19.6.

18.8

19 3

30

22. 4-23. 2

20.7

19.7

20.6

Record-
ings

20
10
15
10
10
10
15
15
10
1(1
20
10
10
15

EFFECT OF TEMPERATURE

In evaluating- the biolooical sionificance of the
experimental data of the effect of temperature on
beat frequencies, one should remember that the
pieces of isolated tissue used were in an abnormal
situation. They were deprived of blood supply,
separated from close association with other struc-
tural elements of the gill, and subjected to in-
creased concentrations of metabolites. It is con-
ceivable that under normal conditions the lateral
cilia of an intact gill may react somewhat
differently.

Stroboscope observations fully confirm the fact
that temperature controls the ciliary beat. This
effect was observed in a series of determinations
made during the summer using small pieces of
filaments taken from .39 adult New England
oysters kept in water at various temperatures.
At the start of each series of readings 10 minutes
were allowed for adjustment to the desired tem-
peratm-e which was kept constant within plus or
minus 1 ° C. Ten stroboscope readings were made
at 1-minute intervals and repeated at higher or
lower temperature. Xo more than three different
temperature levels were used on one preparation.
Careful precautions were taken to prevent the
movement of the e.xcised filaments in the micro-

aquarium so that all the readings would be made
on exactly the same locus of the ciliary tract.
This was necessary because of the considerable
differences in the rate of beating which occasionally
occur along the adjacent filaments.

The results, summarized in talile 15, show the
maximum median frequency' of 27.7 beats per
second at temperatures of 25° to 27° C. The
ciliary activity became irregular at about 35° C,
and the movement ceased at 37° to 38° C.
Wliether these limits are applicable to oysters
from warm southern waters is not known, since
all the obser\'ations were made only on the New
England oysters. Between 35° and 37° C. the
motion was so irregular that its frequency could
not be recorded with certainty. Irregular beating
at the rate of about two beats per second was ob-
served in some specimens during short exposure
til tlie temperature of 45.6° C. Judging by the
median values of the beat frequencies, the opti-
mum temperature is between 23° and 27° C. (see
fourth column, table 15). The ciliary activity
tleclines rapiiUy below 21° C. and ceases com-
pletely at 5° to 7° C.

Individual variations in the frequency of beat
among oysters of a single population suggest dif-
ferences in their physiological states and different
reqiurements for food and water for respiration.
As a rule, spawned-out females remained inactive
for some time in late August ami early September.
During this period the gonads containing un-
spawned sex cells were reabsorbed and tissues be-
came watery because of the reduction in solids
content. The adductor muscles remained con-
tracted, and the shells were closed for unusuallj'
long periods, lasting from 3 to 4 days, or opened

T.^BLE 1.5. — Frequencies of heal of lateral cilia of the gills of
adult C virginica at different temperatures

[Stroboscope readings made on excised filaments kept in sea water]

Temperature

Frequency of beats per second

Prepara-
tions
used

Oysters

range

Minimum

Maximum

Median

used

" C.
36-37 .,

Number
Irregular

Number
Irregular
24.2
27.7
23.8
27.7
33.3
33.3
27.7
20.3
16.7
11.6
11.9
8.9
2.6
1.9

Number
Irregular

22."5'
23.8
23.3
27.7
2.5.6
20.8
17.2
13.7
10.5
10.2
5.6
2.1
1.8

Number
2
1
5
1
3

7
13
3
6
6
7
8
8
5
5

Number

1

33-35

31-33

17,3
23,8
19,3
20,8
21,3
16,6
16.7
13.3
10.0
3.6
2.2
1.6
1.5

0

29-31

1

27-29

o

25-27

3

23-25_

21-23

3

6

19-21

2

17-19

1.5-17

2

13-15

11-13

9-11

3
3
2

7-9

5-7,

5

138

FISH AXD WILDLIFE SERVICE

only for a short time. Even when the valves
opened, the gills produced a weak and unsteady
current interrupted by frequent cessations of ciliary
motion.

The effect of temperature on ciliary activity can
be seen more clearly in the experiments in which
only a single gill filament was used. The results
are shown in figure 135 in which the median fre-
quencies of the beat are plotted against the tem-
perature. As in previous observations 10 readings
were made at each temperature step and the entire
experiment was completed in about 2K hours.
The frequency of beat rapidly increased between
10° and 25° C. The slowing down of ciliary mo-
tion below 10° C. was gradual until all movements
ceased at about 6° C. The curve shown in figure
135 has four distinct slopes that indicate the dif-
ferences in the response of the lateral cilia to tem-
perature changes: a) a very slow increase between
6° and 11° C; b) a more rapid acceleration
between 11° and 15° C; c) a gradual increase be-
tween 15° and 25° to 26° C; and d) a decline as
the temperature rises toward the 30° C. mark.

COMPOSITION OF SEA WATER AND
CILIARY MOTION

Ciliary motion may be affected by changes in
the chemical composition of sea water and by
various drugs. Ionic balance t)f the outside me-
dium is one of the principal conditions for con-
tinuous ciliary activity of the gill. The most
important ions are sodium, potassium, calcium,
and magnesium; the increase in concentration of
one without a corresponding compensation in the
concentration of another or the withdrawal of one
of the ions may completely disrupt tlie ciliary
motion.

EFFECTS OF CHEMICALS ON CILIARY
MOTION

METALLIC IONS

The most favored object for study of the effect
of ions on ciliary motion of bivalve gills lias been
the frontal cilia of the excised pieces of Mytilus
gills (Lillie, 1906; Gray, 1922b). Only occa-
sionally were tlie lateral cilia used in these obser-
vations.

The monovalent metallic ions are important in
the stability of the ciliated colls and maintenance
of ciliary motion. By using a series of samples of
artificially varied sea water it can be shown ex-
perimentally that the replacement of sodium by

03

30

\

\
\

/

25

/

6 20
en

•/
/•

UJ

^ 15

/

y>

/

<

UJ

/

00

10

*r

5

^

1 1

1

10 15 20

TEMPERATURE,

Figure 135. — Effect of temperature on the median fre-
quencies of beat in number per second of the lateral
cilia of C virginica. Readings were made with Strobotac
on a single filament of the gill kept in sea water in a
microaquarium. Temperature was changed by circu-
lating warm or cold water in the jacket of the micro-
aquarium. Duration of the experiment lYi hours.

other monovalent cations rapidly affects the rate
of ciliary beat. The effect is the greatest with
lithium and smallest with potassium. In the
order of their effectiveness the ions can be placed
as follows: Li< Na< NH3< K. There is, how-
ever, a marked difference between the effects
produced by sodium and potassium. The frontal
cilia beat more rapidly in solutions containing
greater concentrations of potassium and are less
affected by changes in the concentration of sodium.
The laterofrontal cilia of Mytilus are affected by
potassium in a manner not observed in other cilia.
The first reaction to the increased concentration
of this ion is an increase in the rate of beating.
With further addition of potassium the recovery
stroke becomes incomplete and the cilia vibrate
very rapidly with greatly reduced amplitude and
impaired efficiency.

Magnesium inhibits the beat of the lateral cilia
of the excised pieces when the concentration of this
metal in the surrounding water exceeds its con-
centration in the blood. Potassium antagonizes
the action of magnesium while sodium produces no
such effect.

Tlie difference between the effects of magnesium
and potassium is also apparent in tlie way these

THE GILLS

139

ions act on the stability of the intercellular matrix.
Under normal conditions magnesium is essential
for the maintenance of stability. If the gill
preparation is placed in a medium containing
sodium and magnesium, the cells remain stable;
these deteriorate rapidly in a mixture of magnesium
and potassium. It is probable that the potassium
ion drives away magnesium from certain areas
inside the cell and sodium ions do not (Gray,
1922b). In the absence of calcium the rate of
ciliary beat is gradually decreased and eventually
ceases (Gray, 1924), but the increase of calcium
in the surrounding water produces no marked
effect on ciliary motion.

As long as the normal equilibrium of tlie cations
sodium, potassium, calcium, and magnesium is
maintained in the surrounding medium, the ciliated
cells (of Mytilus) are insensitive to changes in
the concentration of anions (CI", NO3", Br , I ,
acetate, and SO4 ).

It may be assumed that the results of observa-
tions on Mytilus gills are applicable to the oyster
and that changes in the ionic equilibria in sea
water may have a similar effect on the efficiency
of the ciliated mechanism of oysters.

HYDROGEN IONS

The effect of variations in the concentration of
hydrogen ions on the rate of ciliarv' motion in
bivalve gills is greater than that caused by changes
in the concentrations of any other ions. This
lias been demonstrated on tlie gills of Anodonta,
Mytilus, Mya, and Ostrea (Chase and Glaser,
1930; Gray, 1928; Haywood, 1925; Nomura, 1934;
Yonge, 1925). The greatest effect is produced
by those acids which, like carbonic acid, penetrate
the cell surface most rapidly. Nomura (1934)
found the following order of efficiency of acids in
arresting the ciliary motion of Pecten: H2C03>
CH3COOH>H3P04'>HCl. Ciliary activity ceases
in 1 minute at pH 3.8 wlien IICl has been added,
but with CH3COOH or IL.COs the stoppage
would occur in the same time at the much higher
pH of 5.5. A decrease in the pH values of sea
water from 8.1 to 6.1 reduces the ciliary motion of
the gills of C. viniinica to about 37 percent of their
normal rate. In tliese observations by Galtsoft'
and Whipple (1931) the pH of sea water was
changed by bubbling carbon dioxide, and measure-
ments were made of tlie rate of flow of water
produced by the lateral cilia. Ciliarv motion
stops completely over the entire gill surface of

the oyster when the pH of water is reduced to
5.3 to 5.6. Minimum pH in which the cilia can
function depends on the concentration with which
they are normally at equilibrium. This was
demonstrated clearly by Yonge (1925) on the
cilia of Mya. Thus the average pH inside the
style sac of this clam is 4.45 and the cilia of the
sac stop functioning below pH 3.5 to 4.0, while
the gill cilia normally surrounded by sea water
of about pH 7.2 come to a standstill at pH 5.2 to
5.8.

VARIOUS DRUGS

Tiie eft'ects of various drugs on ciliaiy motion
of the gill epitlielium of Anndonta, Pecten, Mytihis,
and Ostrea have been observed by various in-
vestigators.

The reaction to any effective drug usually
takes place in four consecutive stages: (1) re-
tardation of the frequency of beat, (2) disappear-
ance of metaciironism along the ciliarv tract
and its perseverance within the individual cells
(unicellular metachronism), (3) synchronous beat-
ing of the cilia of a single cell (disappearance of
unicellular metachronism), and (4) cessation of
beat.

The degree of depression depends on the con-
centration of the drug used and the duration of
its action. Cessation of beat in the gills of
Anndonta was observed in the following compounds
(Bethell, 1956): 0.5 percent chloral hytlrate (in
4 to 5 minutes) ; 1 percent novocaine (9 minutes) ;
1.5 percent pilocarpine hydrochloride (in 10
minutes). In 1 to 1.5 percent vera trine sulfate
the metachronal wave slows until movement
ceases. Caffeine (2 percent solution) accelerates
the ciliary motion for 3 minutes and in 6 minutes
completely depresses it. The effect of adrenaline
on the gills of C. (jieias was studied by Nomura
(1937). The rate of ciliary motion was observed
on excised oblong pieces of the gill that were
placed in a graduated, narrow glass tubing.
They crawled along the glass surface of the
tubing, and their advance during 1 minute was
recorded. The crawling velocity in various con-
centrations of adrenaline also was recorded,
and the degree of depression of ciliaiy motion
was expressed in percentage of the velocity
attained in natural sea water. The results show
that the ciliary movement is depressed in pro-
portion to the concentrations, which varied from
10-'° to 10-=^.

Observations made in the Bureau's shellfish

140

FISH AND WILDLIFE SERVICE

laboratory at Woods Hole using adult virginica
showed that 5 ml. of 1 percent solution of chloral
hydrate applied to the mantle cavity of an oyster
kept in a 4 1. tank of slowly changing water de-
pressed the beating of the lateral cilia by 50 to
87 percent. Twenty-five minutes after the re-
moval of the drug the effect disappeared and
normal (i.e., preceding) rate of ciliary motion was
reestablished. Application of 1 ml. of 0.1 per-
cent chloral hydrate to the mantle and gills had
no visible effect, but 4 ml. of the same concentra-
tion injected in the vicinity of the gills increased
the ciliary motion by 15 percent. The effect
lasted only a few minutes.

In the above e.xperiments the duration of the
drug action was brief since the oysters were kept
in running sea water. Different results were ob-
tained when the test oysters were left in stagnant
water. No appreciable effect was noticed in
0.015 percent solution of chloral hj^drate, a slight
decrease (about 12 percent) was recorded in 0.019
percent, and the ciliary action stopped in 0.03 per-
cent solution.

Slight depression of cihary motion (from 11 to
13 percent) was obtained by a single 1 ml. dose of
nembutal solution (concentration 0.02 g. per 1.)
injected directly into the mantle caWty. No
decrease in ciliary motion appeared in the control
tests in which 1 ml. of sea water was injected.
Ciliary activity in all these tests was measured
by determining the velocity of the cloacal current.

Introduction of 3 ml. of digitalin (1:500) into
the pallial cavity results in an immediate, 90 per-
cent depression of ciliary activity. Figure 136
represents part of the record obtained by using
the electric drop counting method described in
chapter IX, p. 190. The effect is dissipated in
about 2 minutes.

A solution of pilocarpine of 1:10,000 in sea water
applied directly to the excised pieces of C. virginica
gills has no effect on lateral cilia. In the test
made in the Woods Hole laboratory the frequency
of beat in natural sea water varied in this experi-
ment from 10.5 to 11.4 per second, and from 10.3
to 10.9 per second after addition of the drug. Tlie
concentration of 0.5 percent slowed down the
frequency by approximately 40 percent (6.5 to
6.8 per second). All observations were made at
23.5° C. Atropine sulfate solution of 1:1,000 had
only a slight effect on the frequency of beat of the
lateral cilia, reducing it by about 17 percent at
22.3° C

THE GILLS

733-851 0 — 64 10

_KJvlLJu^_J^_^_^Ju^LJ^_^_^_^_^_rouu^JvAJvAAAJlJu^JLJ^_I^JL
J 1 1

time jntervol - I second

Figure 136. — Kymograph record of the effect of digitalin
(1:500) on the rate of ciliary activity of the gill of C.
virginica. Electric drop counting method. First and
third line indicate time intervals of 1 second; dotted
line marks the 2 minute interruption in recording. Second
and fourth lines show the contacts made by each drop
of water discharged through the cloaca. Two ml. of
digitalin solution were injected into the pallial cavity in
5 seconds, which are indicated at the top by the straight
line which interrupts the beat recording. A signal key
was depressed for 6 seconds (upper line) when the
digitalin was being added.

The effects of acetylcholine and eserine are of
particular interest because of their importance to
the functioning of the neuromuscular mechanism.
Eserine inhibits the action of choline esterase,
the enzyme which hydrolizes acetylcholine and
prevents its accumulation. The latter would
cause an excessive neuromuscular activity.
Nomura and Kagawa (1950) found that at concen-
trations higher than 10"" both acetylcholine
chloride and eserine inhibit ciliary movement of
the gills of C. gigas. These investigators deduced
from their observations and from the experiments
of Nomura (1937) that acetylcholine and adrena-
line, while inhibiting ciliary motion in tlie oyster,
have the opposite effect on the lieart of this
mollusk.

INHIBITION OF CILIARY MOVEMENT
BY ANTISERUM

Antiserum produced in rabbits by the injection
of minced gill tissue of Anodonta inhibits ciliary
motion of the gills of this species. This observa-
tion of GaUi-Valerio (1916) was confirmed by
Makino (1934) for C. gigas, Meretrix, and other
bivalves.

The problem was furtlier studied by Tomita
(1954, 1955), who improved the technique of
preparation of the antisera by eliminating the
preservatives (merthiolate and phenol) which are
known to depress the ciliary motion in the con-
centrations commonly used for this purpose.

141

The antigens were prepared by Tomita in the
following manner. The gills of C. gigas, Anadara
inflata, and Pecten yessoensis were minced in 0.85
percent saline and homogenized in a blendor. The
protein content of the homogenate was estimated
from the determination of nitrogen made by
microKjeldahl method, and the preparation was
diluted with saline to give the final protein content
of 1 mg. per ml. Merthiolate in the concentration
of 1:10,000 was added as a preservative. On
alternate days a quantity of antigens containing
2.5, 5.0, and 7.5 mg. of protein per kg. of body
weight were injected into healthy rabbits. After

2 weeks the animals were bled and the antisera
were placed aseptically in sterile ampules without
any preservatives and stored in a refrigerator.

Small pieces of gill tissues, 3 to 4 mm. long and

3 mm. wide were cut from the free margin of the
middle demibranch and placed in sea water in a
glass tubing about 12 mm. in diameter. The rela-
tive speed of crawling estimated by Nomura's
method (1937) was taken as a measure of ciliary
activity in normal sea water (100 percent effi-
ciency) and in various dilutions of the antiserum.
Complete stoppage of crawling was recorded in tlie
dilution 1:40 after 32 minutes. Considerable de-
pression of ciliary motion was noticed in the dilu-
tion 1:320 after 77 minutes of exposure. It is
regrettable that no observations were made on the
ciliary motion of an intact gill or that the frecjuency
of ciliary beat in the excised pieces was not meas-
ured by a stroboscope or by any other technique
more reliable than the "crawling" method.

The antisera of the two other species of bivalves
(Anadara and Pecten) have an inhibitory effect on
the gills of Ostrea. The inhibition was, however,
less pronounced than that caused by the anti-
Ostrea serum. The anti-muscle serum tested on
the gills of all three species was less effective than
the anti-gill serum. The author deduced from
these observations that both "tissue-specificity"
and "species-specificity" are involved in the in-
hibitory effect of the antisera.

EFFECT OF PRESSURE ON CILIARY
MOTION

Observations of the effects of increased hydro-
static pressure on ciliary motion were made by
Pease and Kitciiing (1939) using the gills of
Mytilus edulifi. Part of an excised gill plate was
placed inside the glass chamber of a pressure bomb
designed by Marsland, and the surrounding sea

water was saturated with veratrine, which accord-
ing to Gray (1928) considerably prolongs the
activity of the cilia.

Under normal pressure the rate of beating,
measured stroboscopically, was about 9 to 10 times
per second, considerably slower than the normal
rate of 15 to 17 per second that one expects at the
temperatures of 21° to 24° C. at which the tests
were conducted. Apparently the use of veratrine
was unnecessary because the duration of the ex-
periments did not exceed 90 minutes and some of
them were completed within 8 to 16 minutes.
The tests show that a sudden increase in the hydro-
static pressure by 1,000 pounds per square inch or
more immediately increases the frequency of beat
of the lateral cilia. Decompression results in a
reduction in frequency below the normal level and
slow recovery. Pressure in excess of 5,000 pounds
per square incii decreases the frequency and causes
permanent injury. The authors claim that the
change in temperature due to compression or de-
compression is too small to account for the ob-
served effects, because, on theoretical grounds, it
may be expected that the temperature increases by
0.6° C. when the water is compressed adiabatically
to 5,000 pounds. The actual temperature in
chamber of the pressure bomb was not observed.

It would be of interest to repeat these experi-
ments using pieces of gill epithelium kept in normal
sea water not poisoned by veratrine.

CILIARY CURRENTS OF THE GILLS

The ciliary currents at the surface of the gills
of an intact organ can be observed by dropping
small particles (carmine, carborundum, colloidal
carbon, and willemite) on the surface of the demi-
branchs and following under the binocular micro-
scope their movement and direction. The most
important contributions to the studies of this
subject were made by Wallengren (1905a), Orton
(1912), Kellogg (1900, 1915), Yonge (1926), and
Atkins (1937, 1938).

There are five major tracts on the surface of the
gill fif C. virginica (fig. 137). The frontal cilia beat
parallel to the surface of the demibranch from the
base toward its free margin. This current, main-
tained along all ordinary filaments (or.f.), carries
the particles settled on the surface of the gill to the
terminal groove (tr.g.). This is lined with ciliated
cells that beat parallel to the edge of the gill and
push the particles entangled in mucus toward the
mouth. Between the plicae the current caused

142

FISH AND WILDLIFE SERVICE

Figure 137. — Diagram of the system of ciliary currents
on the surface of the demibranch of C. virginica. The
four plicae are shown slightly pulled apart to indicate
the principal (wide) filaments at the bottom of the
grooves. Open ostia, o., are shown only on the left
plicae; the mouth is toward the left; b. — base of the
gills; or.f. — ordinary filaments; o. — ostia; pr.f. — prin-
cipal filament; tr.g. — terminal groove.

by the frontal cilia of the principal filaments
(pr.f.) runs in the opposite direction, i.e., from
the free edge of the gill toward the base. Particles
carried by this current enter the track along the
base of the gills (b.), which rmis parallel to the
du-ection of the current in the terminal groove and
carries food particles toward the mouth. The
lateral cilia (not shown in the diagram) beat at
right angles to the surface of the gill and create a
cm'rent that forces water inside the water tubes
and into the epibranchial chamber.

Small single particles fall into the grooves and
eventually are carried by the principal filaments
toward tlie mouth while the larger particles or a
mass of small ones entangled in mucus are pushed
by the frontal cilia toward the free edge of the
demibranch and may be dropped from the gill
before entering the terminal groove. Frequently
a group of particles is passed from the edge of one
demibranch to the surface of the underlying one
before it is discarded. The complex system of
ciliary currents in the gill constitutes an efficient
selective mechanism for the sorting of food.
Final selection is made along the surface of the

labial palps, which reject a large portion of the
material brought in by the gills (see p. 115)

The ciliary tracts of the gills of 0. edulis de-
scribed by Atkins (1937), in general resemble
those observed on the gill of C. m.rginica (fig. 138).
In the three species of oysters C. virginica, 0.
edidis, and C. angulata, the ciliation is essentially
the same.

MECHANICAL WORK OF THE LATERAL
CILIA

The lateral cilia function principally as movers
of water. They force water tlu-ough the ostia into
the water tubes of the gills and maintain inside
the gill a current that passes through the branchial
chambers to the outside. The hydrostatic pres-

l-f.C.

pv-o-l-f.e.

i.f.

etc.

^^;^

■;i^<: ^\f/<

1

20

Microns

Figure 138. — Frontal view of a living filament of O. edulis.
c.f.c. — coarse frontal cilia; f.f.c. — fine frontal cilia; If.c. —
laterofrontal cilia; pro. If.c. — subsidiary laterofrontal
cilia. From Atkins, 1937, figure 1.

THE GILLS

143

sure inside the gill chamber is maintained solely
l)y tliese lateral cilia, which form a pumping
nieclianism with their synchronized beating over
the entire gill surface.

Local disturbance in the coordination of ciliary
motion caused by the change in the ratio between
the effective and recovery strokes or by the clianges
in the phase of beat results in a drop of pres-
sure and decrease in the current velocity. In the
absence of valves or any other regulatory devices,
the synchronous beat of the lateral cilia over the
entire surface of the gills is an essential condition
for the effective functioning of tlie gill.

One can see under the microscope tliat slight
mechanical disturbances, such as tapping of the
dish in whicli the gill fragments are kept, dis-
organize the metachronal wave of the lateral cilia
and affect the frequency of their beat. The gill
may be compared to a folded tubular sieve, witii
the meshes of the sieve corresponding to the ostia
surrounded by the lateral cilia. The contraction
of the gill muscles brings tlie filaments together,
constricts the ostia, and reduces the spaces be-
tween the filaments. In this way the passage of
water through the gill may be restricted.

CARMINE CONE METHOD

The efficiency of tlie lateral cilia can be measured
with a simple device kno\\ii as the carmine cone
method (Galtsoff, 1926). The method is based
on measurements of the velocity of the gill's
current in a liorizontal glass tubing introduced into
the cloaca. Tlie valves of tlie oyster are gently
forced apart until tliey are wide enough to allow
the insertion of soft rubber tubing into the cloaca.
A wooden wedge is placed between the valves to
keep them from closing. The insertion of rubber
tubing of a suitable diameter is made by gently
rotating it counterclockwise until tlie rubber is
slightly pressed against the outside wall of the
cloaca. The tubing is then secured in its position
by packing the space around it with cotton. A
cotton plug is inserted into the opening of the
proniyal chamber and is covered with plastic clay.
The entire operation can be performed within 2
or 3 minutes and is greatly facilitated by narcotiz-
ing the oyster in an S to 10 percent solution of
magnesium sulfate in sea water.

The oyster with rubber tubing in the cloaca is
tlien placed in a shallow wliite enamel tray filled
with sea water and gently tilted back and tnrtli
to remove anv air bubbles that may have remained

under the valves. A small balloon pipette is intro-
duced into the rubber tubing to suck out the air
bubbles that may be trapped in the epibranchial
chamber. The presence of the cloacal current is
checked by placing a drop of fine carmine suspen-
sion against tlie end of the tubing. The suspension
may be added to the gills as well, and in a few
seconds a fine carmine cone appears in the cloacal
current.

The end of the rubber tubing is now connected
to one arm of an inverted T tube which has a
slightly curved glass funnel sealed inside the other
arm. This arm is joined to a horizontal glass
tubing of known diameter, not less than 15 cm.
long and graduated in 0.5 cm. (fig. 139). A
thistle funnel filled with fine carmine suspension
is attached so the vertical arm of the inverted T
tube, and the tube and the funnel are held by two
clamps mounted on a heavy stand (not shown in
the diagram). The carmine suspension must be
released by a pinchcock without disturbing the
rubber tubing inserted in the cloaca, and the
amount released must be very small in order to
avoid back pressure of water in the gills. Because
of the frictional resistance of water moving inside a
circular tube, the highest current velocity is at
the center of the cross sectional area of the hori-
zontal tube. A minute quantity of carmine sus-
pension or of a solution of nontoxic dye in sea
water released from the funnel forms a sharply
defined cone inside the tube, the tip of which moves
from zero to 10 or 15 cm. mark; the time of its

Centimetelefs

Figure 139. — Diagram of the carmine cone method for
the study of the efficiency of the lateral cilia of the
oyster gill. In order to indicate the position of rubber
tubing inside the cloaca, the right valve is not shown;
the tank in which the oyster is kept is omitted from the
diagram. The funnel with carmine suspension is
perpendicular to the plane of the drawing.

144

FISH AND WILDLIFE SERVICE

passage is recorded by using- a stop watcli gradu-
ated to one-tenth of a second.

Glass tubing of sufficiently wide diameter should
be used to avoid turbulent flow. For large C.
virginica tubing of 5 to 6 mm. in diameter was
satisfactory.

The efficiency of the lateral cilia can be ex-
pressed either in terms of the velocity of the cloacal
current or by computing the mechanical work
they perform. The fact that a distinct cone forms
at the center of the tube through which the current
is running indicates that we are dealing with a
viscous flow for which the velocity can be ex-
pressed by the Poisseul's formula:

S--

D-Ap

In this formula S is the velocity at the axis of the
tube in cm. /sec; D is the diameter; and / the
length of the tube in cm.; A p is pressure drop
between the two marks along the tube in dynes/
cm.'; and n is viscosity of sea water in poises
(C.G.S. unit).

The mean velocity (Sm) of the current of the
entire cross sectional area of the tube is one-half
the velocity at the axis. The rate of discliarge,
V, in cc. per second is computed by using the
following formula:

V-

ttD'O.BS

The rate of mechanical work 11' (in ergs per
second) can be determined from the formula:
W=2irlixS^. For a detailed discussion of the
mechanical activity of oyster gills, the reader is
referred to the original publication of Galtsoff
(192Sb).

The cone method is simple and requires no
elaborate eciuipment. It can be used in any field
or temporary laboratory and is particularly use-
ful for rapid toxicity tests in tracing the phys-
iologically active components of various pol-
lutants. The method has, however, several
limitations that should be kept in mind. First,
the volume of water passing through the cloaca
does not represent the total amount transported
by the gills because a certain portion of the water
is discharged tln-ough the promyal chamber.
Second, the tests should be completed ui 1 day because
the prolonged presence of tubing inside the cloaca
and of the wedge between tlie valves may produce
pathological conditions. Because of the sensi-

tivity of the cilia to mechanical disturbance great
care should be exercised to avoid jarring, shaking,
and vibrating when preparing a test. The oj'sters
usually recover within 12 hours after being placed
in running sea water and show no ill eft'ects of the
narcosis and handling.

EFFECT OF TEMPERATURE

The cone method proved satisfactory in a study
of the effect of temperature on the efficiency of the
lateral cilia. Tlie results of many tests performed
in the Woods Hole laboratory show considerable
variability in the velocity of the cloacal current
of oysters of the same size and origin. At a given
temperature and under identical conditions the
lateral cilia of some oysters work faster than those
of others. Consequently, no definite rate of work
maintained by the gill epitiielium at a specified
temperature might be considered as typical or
normal for an oyster of a stated size and type.

An example of the effect of temperature on cur-
rent velocity produced by the lateral cilia of oysters
of identical size transporting water at different
rates is shown in figure 140. In both experiments
the water was agitated by an electric stu'rer and
its temperature was changed by using heating or
cooling units placed at the end of the tank far-
thest from the oyster. Not less than 15 minutes
for adjustment was allowed at each temperatui-e
step. Readings were made starting at 20° C. and
decreasing to the extreme low temperature at
which no cm-rent was produced. Then the water

Figure 140. — Effect of temperature on the velocity of
the cloacal current produced by slow (lower curve) and
fast (upper curve) adult oyster, C. viiginica, of about 4
inches in height. Each dot represents the mean
velocity of the current of 10 consecutive readings made
at intervals of 2 to 3 minutes. Carmine cone method.

THE GILLS

145

was warmed to the extreme high and cooled again
to 20° C. for the hist observation. Each circle
represents a mean of 10 consecutive readings made
at intervals of 2 to 3 minutes. The lower curve
represents the activity of an oyster in whicli slow
ciliary motion started only at 11.3° C. The upper
curve is typical for an oj^ster which maintains a
rapid transport of water. In both curves the
maximum activity occurred at 20° to 25° C.
Rapid acceleration in the rate of current took
place between 10° (or 11.3°) and 15° C. Essen-
tially the relationship between the temperature
and current velocity is similar to tiie effect of
temperature on the frequency of beat of lateral
cilia shown in figure 135, although the slope of the
latter curve is steeper than in the two curves
shown in figure 140. Within the range of the
temperature used in these tests, the action of tlie
cilia was completely reversible.

The increased rate of activity induced by
temperature may be expressed by temperature
coefficients determined at 10° intervals. Tliese
values, calculated from a large number of obser-
vations with the cone method and given in table
16, show considerable difference in Qio based on
the determinations of current velocity and on the
rate of work.

T.\BLE 16. — Temperature of coefficients {Qio), of the rale of
ciliary activity of lateral cells of C. virginica

Temperature range

Temperature

coefflclent

based on

velocity of

current

Temperature

coefficient

based on

rate of work

performed by

the cilia

5-10 ...

°C.

6.0
2.5
1.8
1.5
1.3

4.8

10-20

4.4

15-25 ---

2.4

20-30 -

1.8

25-35

1.2

The current velocity is not a true measure of
the work performed by cilia because the viscosity
of the water changes with temperature. In the
formula W=2ttIiiS'^ the work required to maintain
a current at a constant speed is proportional to
viscosity, ;u. Since at a lovverte mperature the
viscosity of sea water is greater than at higher
temperatures, more energy is required to propel
cold water. As the work needed to pr(jduce
current of a given velocity is proportional to the
square of the velocity at the axis of the current,
it is apparent that the decrease in the frictional
resistance due to lesser viscosity of water is not
sufficient to compensate for the additional energy

required for maintaining faster current. Tem-
perature coefficients computed on the basis of the
rate of work performed are, therefore, more
significant than the Qio based on the velocity of
current.

HYDROSTATIC PRESSURE INSIDE
THE GILLS

The velocity of the cloacal current is propor-
tional to tlie difference in hydrostatic pressure
inside the gill chambers and at the opening of the
cloaca. The pressure can be measured by in-
troducing an L-shaped glass tubing into the free
end of the rubber tubing inserted into the cloaca
and recording the difference between the level of
sea water in the tube and the level in the container
in which the oyster is kept. Correction should
be made for the position of the meniscus in the
tube due to surface tension. Using this simple
device I found that in an actively feeding adult
C vir(jinica the pressure inside the epibranchial
chamber may be as high as 7 to 8 mm. of sea-
water column. If the temperature and salinity
of water are known, the pressure may be calculated
in grams per unit area.

SPONTANEOUS INHIBITION OF
CILIARY MOTION

When tiie bivalve mollusks close their sliells
and cut off their access to outside water, they enter
a state of suspended animation during wiiich their
normal functions are greatly slowed down or
completely cease. This state of diminished ac-
tivity observed in Anodonta and Sphaenum
{Cydas) was regarded by Gartkiewicz (1926) as
sleep. Through the transparent shell of Sphacr-
i)i)7i he was able to see that the ciliary motion of
the gills and the beating of the heart were at a
complete standstill when the shells were closed.
This observation corrected the erroneous opinion
of earlier investigators (Wallengren, 1905a, 1905b)
that ciliary activity persists when the valves are
closed.

The cessation of ciliary motion after tiie closing
of shells was attributed to the accumulation of
carbon dioxide and the decrease of pH. A pH
of less than 6.0 probably does not occur in the
body fluids of bivalves after they close tlieir
valves because of the buffering action of carbonates
of the shell substance.

In the gills of C. lirginica ciliary motion ceases
shortly after the closing of the valves and is re-

146

FISH AND WILDLIFE SERVICE

newed after they open. It is probable that in
these cases the depression of cihary activity is due
primarily to the accumulation of metabolites
There exists, however, another type of inhibition
of ciliary motion that is not associated with
changes in the outside environment. It can be
observed on gills exposed by the removal of a
portion of the valve. The oyster is placed in a
suitable container supplied with slowly running-
sea water, and the gills are strongly illuminated
and examined under a dissecting microscope.

The time required for a small inert particle
(carmine, or powered oyster shell) to be moved
along the terminal groove between the two selected
points in the microscope's field of view is recorded
with a stopwatcli. Copious discharge of mucus
that impedes the transport of particles along the
groove was avoided by adding only minute cjuan ti-
tles of material in suspension. Readings were
repeated every minute, and the degree of expansion
of the gill lamellae and ostia were recorded.
The observations lasted from 10 to 30 minutes.
Ciliary motion over the terminal groove of the
gill frequently slowed down as the adductor con-
tracted, but previous rhythm was resumed within
a few seconds after relaxation of the muscle.
The most spectacular were the instances of com-
plete cessations of ciliary motion over the surface
of the entire giU following strong contraction of
the adductor muscle and complete closure of the
valves. Since a portion of the shell was removed
the surface of the giU remained m contact with
fresh sea water and the cessation of ciliary activity
could not be attributed to the accumulation of
carbon dioxide or other metabolites.

The association of the inhibition of ciliary
motion with the contracted state of the adductor
muscle is showii in table 17, which contains ex-
cerpts of the records of observations made on
two male and two female adult oysters. Tem-
porary depression and sometime stoppage of ciliary
motion were often observed after occasional
contractions of the gill muscles. In these cases
the inhibitory impulses seem to be less pronounced
than in the case of the contraction of the adductor
muscles. Electric shock applied from tlie DuBois
inductorium direct to the gill epithelium or to
the edge of the mantle liad no effect on ciliary
beat of the frontal and terminal cilia. Only in
the case of a sliock sufl^ciently strong to cause
contraction of the adductor muscle was there a
cessation of ciliary activity.

T.\BLE 17. — Association of the velocity of ciliary current
along the terminal groove of the external right demibranch
and the state of contraction of the adductor muscle

[Excerpts of the protocols of the four experiinenis with adult and sexually
mature C. virginica made in .\ugust at Woods Hole, Mass!.]

Sex

Time '

Tem-
pera-
ture

.\dductor

Time needed to

move a particle

over a distance

of 1 cm.

Min.

Max.

Female

7:42- 7:44 a.m.

7:45- 7:47 a.m.

7:48- 8:10 a.m.
10:20 10:22 a.m.
10:24^-10:27 a.m.

10:30-10:35 a.m.
4:58- 5:08 p.m.
5:25- 5:33 p.m.
3:21- 3:28 p.m.
3:31- 3:41 p.m.

'C.
21.6
21.6
21.6
21.0
22.0

22.0
21.5
22. 0
22.8
22.8

Relaxed

Contracted
Relaxed

Secmds
13.6
22.0
12.0
22.0
26.6

22.3
20 6

Secovds
13.8
22.0

Male

Relaxed..

Partially con-
tracted.
Rela.xed

23.0
29.2

22 6

Female

oo o

Male.

Contracted *

Relaxed.

no movement
14 4 ( 16 6

Contracted

no movement

1

I Readings made every minute within time shown in this column.
: Complete contraction. Ciliary motion stopped along the entire terminal
groove and on the surface of the gill.

Extu'pation of the visceral ganglion or its burn-
ing witli an electric needle had no effect on ciliary
motion of the gill, indicating that inhibition does
not originate in the ganglion. The frequent
comcidence of the cessation of ciliary motion with
the contraction of the adductor muscle and the
subsequent resumption of ciliary activity after its
relaxation suggests the possibility of a neuroid
transmission of the inhibitory impulse which may
originate during muscular activity and spread
over the ciliated surface of the gill.

Since the problem of the impulses causing
inhibition of ciliary motion has not been studied
sufficiently, it is impossible at this time to present
a reasonable explanation of this puzzlmg phenome-
non.

The transport of water by the gills during feed-
ing and respiration is discussed in chapter IX since
this function is controlled jointly by the mantle and
adductor muscle.

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THE GILLS

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THE GILLS

151

CHAPTER VIII
THE ADDUCTOR MUSCLE

Page

Anatomy — '■'-

M icroscopic structure ^^^^

White muscle fibers '■''■'

Dark muscle fibers IM

Attachment to shell 'tiO

Chemical composition of the adductor muscle 161

Inorganic salts 162

Organic components 162

Glycogen. 162

Proteins 164

Physiology of the adductor muscle 164

Chemical changes during muscular activity 167

Normal shell movements... 168

Method of recording 16S

Five major types of shell movement. 169

Type A --- 176

Type B... .--. -- 171

Type C 171

TypeD 172

Type E 172

Duration of periods of opening and closing 172

Effect of temperature.. 174

Effect oflight and darkness 17.i

Effect of tide 17S

Power of adductor muscle -- 17S

Tests made in air and in water 177

Cycles of shell movements 1^"

Bibliography l^^l

ANATOMY

The adductor muscle of tlie oyster is a massive
organ that controls the opening and closing of the
valves. It occupies a slightly asymmetrical posi-
tion at the ventroposterior part of the body and
is surrounded by the following internal organs:
tlie visceral mass, pericardium, epibranchial cham-
ber of the gills, and cloaca (fig. 72). Tlie rectum
adheres to the posterior side of tlie muscle. Tiie
protrusion of the visceral mass, containing the
crystalline style sac and the lowermost part of the
gonad, covers the anterior side of the muscle. A
wedge-shaped visceral ganglion located inside the
epibrancliial chamber rests in a slight depression
on the side of the muscle under the visceral pro-
trusion. The ganglion can be exposed by cutting
through the wall of tlie epibranchial chamber and
lifting the tip of the visceral mass.

The adductor muscle of the monomyarian mol-
lusks, i.e., those which have only one muscle (sucJi
as edible oysters, pearl oysters, scallops, and

Spondylus), corresponds to the posterior adductor
of other bivalves. The anterior adductor, present
in larvae, disappears during metamorphosis shortly
after the attachment of the larva.

Shortly after the metamorphosis of the larva
the posterior adductor muscle develops into the
most conspicuous and the heaviest organ of the
oyster. In valves of C. vinjinica and in some
other species of edible oysters the muscle scar
where the adductor is attached to the shell is
darkly pigmented. The shape and dimensions of
tliis area are variable (see p. 30 ch. II).

The weight of tlie nniscle of C. virginica ac-
counts for 20 to 40 percent of the total weight of
the tissues. After spawning, when other parts of
the body are watery and poor in solids, the rela-
tive weight of the adductor increases. Exam])les
of this condition, usually encountered after the
discharge of a large number of sex cells and be-
fore tlie accumidation of the reserve materials
(glycogen) in the connective tissue, are given in
table 18. It may be deduced from these data
that the weight of the adductor muscle is not
affected by the changes in the chemical composi-
tion which take place in otlier organs. For fur-
ther discussion of this problem the reader is
referred to chapter XVII of this book.

The adductor is cttmprised of two distinct parts.
Al)out two-tliirds of the total bulk of the muscle is
translucent, oval-sliaped, and slightly concave at

T\BLE \H.— Relative weiqht of the adilurtor mtmrle of six
adult C. virginica {/, to ~) inches in height) during the
spawning season {August) in Woods Hole, Ma.'is. (fresh
basis), 1951

Oyster

Ripe male

Ripe male

Ripe female

Kipe female

Partially spawned female

Spawned out, sex undetermined

Weight

Meat

Grams
17.8
15.0
18.7
6,5
6.5
5.2

Adductor
muscle

GraTus
3.5
3.7
4.4
2.1
2.3
2.2

.\dductor

muscle

(total

weight)

Percent
19.7
21. K
23.5
32.3
35.3
42.3

152

FISHERY bulletin: VOLUME 64, CHAPTER VIII

0

Millimeters

0.5

Millimeters

0.5

B

Figure 141. — Cross sections of the two portions of the adductor muscle of C. virginica. A — white or opaque part. B —
translucent part. The muscle bands of the white part are more compact and are surrounded by tougher connective
tissue than thase of the translucent part (right). Bouin, with formalin hemato.xylin-cosin.,

the dorsal side adjacent to the pericardiutii. This
portion is frequently called the vitreous or dark
part. The remainder is crescent-shaped and an
opaque milky-wdite. The fibers of this part are
tougher than those of the translucent portion;
the difference shows clearly when the nuiscle is
being cut or teased.

The fibers of the adductor muscle form dense
bands surrounded by connective tissue. In a
cross section examined under a low-power micro-
scope (fig. 141) the bands appear as separate units
packed more or less parallel to one another. This
arrangement is less pronounced in the translucent
part (fig. 141, right). The tissue that surrounds
the muscle bands is better developed in the opaque
section. A layer of connective tissue separates
these two major parts of the adductor.

Connective tissue provides a framework for the
muscle. Individual fibers do not run the full
muscle distance between the two valves; they are
anchored at one or both ends in the sheets of
tissue which surround the bands. A very thin
membrane, called endomysium, invests each mus-
cle cell; the sheathing around the bauds of cells is

epimysium ; the septa which radiate fi"om the latter
form perimj'sium.

The cross-sectional areas and the weight of the
two portions vary in different specimens. It was
reported by Hopkins (1930) that the ratio of
weight of the translucent to the white part of the
muscles of oysters growing near Beaufort, N.C.,
depends on ecological conditions. In the oysters
found at the upper limit of their vertical distri-
bution near the high-water level the ratio was 1.26,
while in the oysters taken at a level 2.5 feet lower,
where they were submerged during about three-
quarters of the time, the ratio was 2.51.

The entire adductor muscle is well supplied with
blood; wandering leucocytes are usually seen
between the fibers and in the connective tissue.
Both parts of the adductor muscle are abundantly
supplied with nerves. The innervation of the
muscle is discussed in Chapter XII.

MICROSCOPIC STRUCTURE

The muscle fibers of the two parts of the adduc-
tor differ in both size and structure. The white
muscles are smooth and wide, while the dark

THE ADDUCTOR MUSCLE

153

(translucent) fibers are thinner and have a peculiar
striation which has been described as oblique,
double-oblique, helicoidal, and spiral. Some in-
vestigators (Kellogg, 1S92; Orton, 19.35; Hopkins,
1936) and authors ot biology textbooks (Borradaile
and Potts, 1961) refer to the translucent part as
consisting of striated muscles.

Both types of fibers appear under the light
microscope as k)ng cylindrical cells, slightly thick-
ened in the middle and tapering toward the ends.
An oval-shaped nucleus with one or several
nucleoli is near tlie surface, outside the contracting
elements which make up the bulk of the cell.
Clear homogenous cytoplasm (sarcoplasm) which
can be seen under high magnification forms a very
thin surface layer of the cell and around the
nucleus. The major part of the cell is made of
slender fibrils that differ in their orientation in the
two types of muscle cells.

Tlie principal structural elements appear in
unstained, isolated fibers examined with phase
contrast oil immersion lens under high magnifica-
tion. Whole mounts can be made after pieces of
muscle are macerated in 20 percent nitric acid
and then placed in glycerol. Treatment witli
nitric acid apparently does not affect the visible
structure of the fibers. Preparations should be
made from fibers which have been taken from
both a fully relaxed and a completely contracted
adductor. The desirable state of relaxation is
obtained by narcotizing the oyster in 5 to 10
percent magnesium sulfate solution for 48 hours;
treating the mantle with a strong solution of
hydrocliloric acid causes long-lasting contraction.
In opening the oyster, care sliould be exeicised
not to damage the visceral ganglion, since injury
to this nerve center may cause relaxation of the
adductor.

WHITE MUSCLE FIBERS

White muscle fibers isolated from a completely
relaxed adductor of a full}' narcotized ('. rir<ivuca
are from 2 to 3 mm. long and about 10 ;u in diam-
eter. Tiie fibers are too short to stretch from one
valve to the other and, with the exception of
those attached to the shell, end in connective
tissue. Occasionally tliey bifurcate but do not
anastomose. The body of the fiber consists of
many fibrils of variable length and a diameter of
only a fraction of a micron. The fibrils are ori-
ented parallel to the long axis of the cell and those
close to the surface appear to be darker. The
arrangement of the fibrils changes somewhat, de-

pending on the state of contraction. Figure 142,
A-D, represents four camera lucitla drawings made
of a white auiscle fiber; (A) the fiber is in a com-
pletely relaxed state, (B) it is strongly contracted,
(C) it is partially contracted, and (D) a noncon-
tracted fiber is folded by the contraction of the
surroimding fibers. All drawings were made from
glycerin-mounted preparations examined with
phase contrast lens. The difference between the
relaxed and contracted fiber is primarily in the
thickness of the fiber, which in B is about three
times greater than in A. In both cases the orienta-
tion of fibrils is the same. In a partially con-
tracted and slightly twisted fiber, C, some of the
fibrils are at an angle to the long axis of tlie cell
while others retain their original orientation. The
fiber D, found in the same preparation with C, is
folded but not contracted. Its surface layer of
transparent cytoplasm was wider than in the
others and the fibrils followed the zig-zag outlines
of the fiber. Although the sample was isolated
from a contracted adductor, only a few fibers
were found in highly contracted state B. The
fiber A was separated from a completely relaxed
nuiscle.

DARK MUSCLE FIBERS

The fibers vi the dark (translucent) part of tlie
adductor are from 1 to 2 nun. long and in a relaxed
stale are about .5^ in diameter. When isolated in
teased preparations, the fibers have a tendency to
twist and coil. The connective tissue around them
is less tenacious than in the white muscle, and the
fibers can be separated easily by fine needles. As
early as 1869 Schwalbe showed that the fast ad-
ductor nmscle of Ostna is composed of fibers whicii
exhibit a clearly defined diamond lattice pattern.
Marceau (1909) maintained that double obliquely
striated muscles are widely distril)Uted in the fast
parts of the shell closing muscles of bivalves, and
Anthony (1918) advanced a theory tliat oblique
striations are a stage in the evolutionary develop-
ment of transverse striation. The fact that true
cro.'is striation occurs in the muscles of Pedeii,
Lima, Teredo, Spondylu.s, and other bival\-es leads
to a widely accepted belief that the dark portion
of the adductor muscle, also described by some
autliors as yellow, grey, or tinted (Kawaguti and
Ikemoto, 1959), consists of cross striated fibers and
tliat quick mo\einents of these aninuils are brouglit
about by their contraction.

From their study of the translucent fibers of the
adductor of C. angidata, Hanson and Lowy (1961)

154

FISH AND WILDLIFE SERVICE

B

Microns

Figure 142. — Small pieces of four white fibers of the adductor of C. v'rginica seen under phase contrast lens. Whole
mounts of a preparation teased after treatment with nitric acid. Glycerol. A — completely relaxed fiber from nar-
cotized oyster; B — strongly contracted fiber; C — slightly contracted fiber; D — folded but not contracted fiber.
Figures B and D are from one preparation of a highly contracted adductor.

concluded that the fibers of that part of the muscle
differ from true cross striated muscles in that the
bands (A and I) lie at about a 10-degree angle to the
fiber axis and are arranged helically around tlie
outer part of the fiber; this produces the double

oblique striation visible in the light microscope.
Hanson and f^owy's observations were based on
electron microscopy, and tlie bands they refer to
as A and I are not visible under the light micro-
scope.

THE ADDUCTOR MUSCLE

155

N

0

Microns

30

A

«•- 1«i,:

0

Microns

10

B

FiGiRE 143. — Small picco of dark muscle fiber from tho contracted adductor muscle of C. virginicn. A — Whole mount
in glycerol after nitric acid treatment. B — Small portion of the same negative magnified. Round glol)ules are
artifacts. Phase contrast lens.

156

FISH AND WILDLIFE SERVICE

Examination of relaxed fibers of C. virginica
with phase contrast lenses shows the existence of a
distinct diamond lattice pattern shown in figm-e
143. In the relaxed dark fibers this doul)le oblique
striation is absent and the fibrils are oriented
parallel to the axis of the cell. My observations
confirm the description made by Hanson and
Lowy (1957), who found that in helical configura-
tion of myofibrils of the "yellow" part of the ad-
ductors of oysters and Ensis ensis the angles be-
tween the lielix and the axis of the fiber increased
as the muscle relaxed. The so-called diamond
lattice pattern of striation is not a permanent
feature of the translucent fiber. It becomes visible
in a contracted muscle and is usually confined to
the cut ends of the fiber. This observation made
by Bowden (1958) for Ostrea edulis and C. angulata
is in accordance with my observations on C
virginica.

Considerable advance in the understanding of
fine structure of bivalve muscle cells was made l)y
Philpott, Kahlbrock, and Szent-Gyorgyi (1960),
in the work on C. virginica, Mya arenaria, Mer-

cenaria mercenaria, and Spixiila solidissima. Simi-
lar studies of C. angulata were nuide by Hanson
and Lowy (1961).

With respect to the ultrastructure of the fibers
of the adductor muscles of these species, the re-
sults of the two investigations are in agreement
although they present dift'erent theories of the so-
called catch mechanism of the adductor, which is
discussed later. In both parts of the muscle the
fibrils consist of two types of filamentous struc-
tures that can be clearly seen on the electron
micrograph of the transverse section of the fibril
(fig. 144). The thick filaments fonn the largest
part of the fibril; their diameter varies from 250
to 1,500 A. The thin filaments which occupy the
space around the thick ones are about 50 A. in
diameter. The thick filaments have the 145 A.
periodicity associated with paramyosin. The
authors surmise that actomyosin is localized in
the thin filaments. Hanson and Lowy (1961), in
confirming the presence of two kinds of filaments
in the fibrils of C. angvl.ata, assume that the
thinner filaments contain mainly actin. Accord-

'^k.*^^

Microns

Figure 144. — Electron micrograph of a small portion of a muscle fiber of the translucent part of the adductor of C.

virginica. Courtesy of Philpott and Szent-Gyorgyi.

THE ADDUCTOR MUSCLE
7133-851 O — 64 11

157

ing to their interpretation of the electron micro-
graphs which accompany their paper, both types
of fihiments are rehitively short in comparison
with the length of the fiber; they lie parallel to the
fiber axis and are grouped with separate arrays
which alternate with each other and appear to be
cross-linked by means of transverse projections
which belong to the thick filaments. The exist-
ence of the projections does not seem to be firmly
established, and their connections with the two
types of filaments require corroboration. It is
obvious from the electron micrographs published
by Philpott, Kahlbrock, and Szent-Gyorgyi,
(1960) that filaments are randomly distributed
throughout the cross-sectioned area of the fibril.

In tfie relaxed state the muscle cells are stretched
and on longitudinal sections of either part of the
adductor appear to be arranged in parallel lines
separated in places by connective tissue (fig. 145).

A contracted adductor muscle is strikingly
different in appearance from one which is relaxed.
Most of the muscle fibers are folded and the
entire organ has a herringbone appearance (fig.
\AQ). The uniform thickness of the folded fibers
indicates that their actual length is not shortened
by the contraction; the fibers are compressed to
occupy a shorter distance between the valves.
Folding implies the existence of a force that acts
parallel to the longitudinal axis of the fibers.
The question arises as to tlie nature of the force
that produces this effect. 1 n an attempt to answer

200

Microns

Figure 145. — Longitudinal section tlirougli a completoly
relaxod translucent part of the adductor muscle. Bouin,
hematoxvlin-eosin.

Microns

Figure 146. — Longitudinal section of the white jiart of the
adductor muscle of C. virginica preserved in Bouin with
formalin solution. Muscle is in a highly contracted
state. Hematoxylin-eosin. Camera lucida drawing.

this question I examined a series of sections of
muscles preserved at various degrees of contrac-
tion. Oysters were stimulated to close their
valves and were preserved in that state by using
a strong and rapidly acting fixative applied through
an opening cut in a portion of the shell. In such
preparations contracted muscle fibers were found
only in the area near the attachment to the valves.
In the two photomicrogi'aphs (fig. 147) the con-
tracted fibers, nearest to the valve (left side),
are short, thick, and deeply stained with eosin.
The fibers to the right in the same preparation
are narrow and fcilded.

In a partially closed oyster the contracted fibers
may be scattered between the folded fibers
throughout the entire cross-sectional area. This,
condition, shown in figure 148, is drawn from
preparations preserved in osmic acid and stained
witii iron hematoxylin. The contracted fibers
appear as isolated dark bodies scattered through-
out the moderately folded fibers. It may be
deduced from the histological picture that only a
small number of muscle fibers are in a contracted
state. In order to explain the folding of the
noncontracted portion of the adductor it is neces-
sary to assume tliat a rigidity develops in the
contracted fibers in two places— near their contact

158

FISH AND WILDLIFE SERVICE

r-^jTSIf^;

Microns

Figure 147. — Two photomicrographs of a longitudinal section of the translucent part of the adductor muscle near the
valve (the left side of the photograph) . The muscle was preserved in a contracted state in Bouin with formalin solu-
tion. Note the thick, short contracted fibers on the left and the beginning of folding at the right edge of it. Con-
tracted fibers are deeply stained with eosin. The photomicrograph on the right shows folded fibers a short distance
away from the area of the same section shown at left.

with the folded fibers and at theh' anchorage in
the connective tissue. Under this condition the
contracted portions will bring the valves together
and conapress the noncontracted fibers into folds.
This gives the oyster a considerable degree of
flexibility in controlling the degree of opening of
the valves.

Observations by Bandmann and Reichel (1955)
on Pinna nobilis deal with similar conditions. In
the smooth muscle of this mollusk plastic length-
ening is combined with an orientation of the fiber
structure without any changes of its elastic prop-
erties. The reverse process (disorientation) takes
place during contraction, which is accompanied by
an increase in dynamic stiffness. The authors
attribute plastic and contractile length alterations
to two different mechanisms: change in orienta-
tion and change in molecular shape within the
contractile elements.

No observations have l)een made in tlie living
oyster of the contractions of small bundles of
fibers that run parallel to the surface of the valves.

0

0.3

Millimeters

Figure 148. — Longitudinal section of partially contracted
translucent portion of the adductor. Contracted fibers
appear as black spindles. Osmium fixation. Iron
hematoxylin.

THE ADDUCTOR MUSCLE

159

These fibers, which are at right angles to the
main fibers extending from one valve to the other,
are found near the attachment of the adductor
to the valves (fig. 149). Their position suggests
that they act as braces by bringing together and
tiglitening the principal bundles.

250

Figure 149. — Longitudinal section of a piece of partially
relaxed muscle near the attachment to the valve (right
side). Note band of muscles at right angle to the main
fibers. Kahle, hematoxylin-eosin.

ATTACHMENT TO SHELL

Tlie adductor muscle of C. virginica is fastened
.so strongly to the sliell that when tlie valves are
forced apart the nmscle lireaks in the middle
instead of tearing from the shell. The adhesion
sometimes withstands a pulling force of 10 kg.
(22 pounds). On the other hand, the connection
between the muscle and the shell can be weakened
or completely destroyed by applying lieat to the
shell o\"er tlie area of the muscle scar. This con-
nection is smooth and glossy.

Brilck (1914) found that in the shells of AnodonUi

and Cyclas the muscles are fastened by means of
a specialized layer of cells which he called holding
or adhesive epithelium ("haft epitlielium").

Hubendick (19.58) used both electron and light
microscopy to demonstrate the presence of ad-
hesive epithelium in the areas of attachment of
the muscles of the fresh-water snail Acroloxus
lacustris (Maxwell). The surface of the cells
has a dense brush border of minute microvilli
which are transversed by very thin cytoplasmic
fibrils originating in the base of the cell. The
epithelial cells are fastened to the underlying
connective tissue by the evaginations which
extend into the base of the cells. Since the
muscles used by Hubendick were fixed in osmic
acid, which resulted in their detachment from
the shell, the electron micrographs published in
his paper do not show the actual connection
between the microvilli and shell material. The
shell surface over the area of the attachment has,
however, small depressions into which fit the tops
of the microvilli. It is, therefore, likely that in
Acroloxus the adhesion of the muscle is accom-
plished in this manner.

The holding epithelium of C. virginica can be
seen on transverse sections of decalcified shell and
muscle preparations. Individual cell boundaries
are indistinct, but the position of each cell is
clearly marked by a large round nucleus (fig. 150).
Fine strands resembling those described by
Hubendick originate in the base of the cells and
terminate at their surfaces. They are not visible
at low power but can be seen under oil immersion.
The holding epitheliiun of the oyster is a modifi-
cation of the surface epithelium of tlie mantle;
the transition from one type to another can be
seen in the areas adjacent to the muscle attach-
ment (fig. 151). The holding epithelium of C
virginica secretes an organic film of about 2 yu
in thickness that consists of adhesive material by
whicii the muscle fibers are attached to the shell.
The chemical nature of this film was not deter-
mined, but staining properties suggested the
presence of collagen. Since it is known that un-
der proper conditions collagen is digested by
coUagenase, I made a series of experiments at
Woods Hole to determine the effect of this en-
zyme on the attachment of muscles. Small
amounts of phosphate buffer solution (pH 8.4)
containing 1 mg. of collagenase per ml. were
injected into adductor muscles through holes

160

FISH AND WILDLIFE SERVICE

0

Microns

60

0

Figure 150. — Longitudinal section of tlie adductor muscle
of C. t'lrginicn where the muscle fibers are attached to the
shell. Note the holding epithelium and the cement layer
which in the upper part of tlie illustration is separated
from the epithelium and turned over. The decalcified
shell is out of the field of view. The cement film is
partially detached at the upper half of the preparation
and twisted exposing its surface facing the shell; the
width of the upper part of the film corresponds to the
thickness of the section. Kahle. Hematoxylin-eosin.

drilled in the valves. In another set of experi-
ments the muscles of oysters with the shells
attached to them were immersed in the solution
of collagenase and were kept at a temperattire of
24° to 25° C. for 24 to 48 hours. Solutions of
trypsin and phosphate buffer alone, without
collagenase, were used for control experiments.
In all cases the muscles treated with collagenase
became detached within 36 hours. In the con-
trols they remained attached to the shells (fig.
152).

Millimeters

0.3

Figure 1.51. — Cross section of the visceral mass of C. vir-
ginica near the adductor muscle. Notice the gradual
change of typical mantle epithelium (left side) into
tiolding epithelium covering the adductor muscle. The
shell is not shown. Kahle, hematoxylin-eosin.

CHEMICAL COMPOSITION OF THE
ADDUCTOR MUSCLE

The chemistry of the adductor muscle of oysters
has received less attention than that of the mus-
cles of clams, scallops, and sea mussels. Probably
the differences in the chemical composition of the
muscles of various marine lamellibranchs are not
of fundamental nature, although the proportion
of various components may vary greatly between
the species and even within moUusks of the same
species living in difi'erent environments. Older
reviews dealing with the comparative physiology
of the adductor muscle make no distinction be-

THE ADDUCTOR MUSCLE

161

tween the various groups of mollusks and combine
the data under the general and nonscientific
designation of "shellfish" (Katz, 1896; Riesser,
1936). Gross analysis of the adductor muscle
of the oyster {0. imbricata) CGrimpe and Hofl^-
mann in: Tabulae Biologicae, 1926) shows the
following composition: water 66.58 percent; pro-
tein 11.38 percent; fat 4.8 percent; and ash 1.1
percent.

INORGANIC SALTS

Studies of the content of the metallic salts in
the body of oysters and other bivalves were
made by many investigators interested in the
problem of osmotic regulation in marine inverte-
brates. Observations on European oysters, pre-
sumably 0. edulis, made by Krogh (1938) are of
particular significance. He found that in the
oysters living in waters of high salinity (35°/oo)
in France the concentrations of chlorine, sodium,
and potassium expressed on the basis of tissue
water, were as follows: chlorine 256 mM/kg.**;
sodium 265 mivi/kg.; potassium 46 niM/kg. The
next day the oysters were placed in water of
lowered salinity (25°/oo) in Limfjord, Denmark,
and individual samples were taken at intervals of
1 to 2 days. The results, though somewhat
irregular owing to individual variations, showed
a decrease in chlorine (221 to 138 niM/kg.) and in
sodium (258 to 139 niM/kg.). The potassium
increased from 46 to 98 mivi/kg.

The mean values for the concentrations of
some elements in the adductor muscle of the
Australian oyster, Crassosfrea (Saxosfrea) com-
mercialis, were found to be as follows (Humphrey,
1946):

Percent Mg.

Potassium 3S1. 7 ± IS. !l

Sodium 327.9 ±13.0

Calcium 45. 76± 3.28

Magnesium 70. 93 ± 3. 03

Chlorine 733.4 ±17.3

" Values f^iven in millimoles per kilogram of water.

In this case sodium and potassium were present
in almost equal amounts (Na:K = 0.9S) while in 0.
edulis potassium was present in much smaller
concentrations and the Na:K ratio varied from
1.6 to 5.8. The concentrations of calcium and
magnesium in the whole adductor muscle of C.
commercialis were found to be 1.1 and 1.5 x 10^ m,
respectively. Both elements are uniformly dis-
tributed between the two parts of the muscle
(Humphrey, 1949).

ORGANIC COMPONENTS

Glycogen

Bivalve mollusks accumulate considerable quan-
tities of glycogen in their tissues, including tlie
muscles. This reserve material is deposited
primarily in the connective tissue of the body
parenchym and in tlie mantle and in smaller
quantities is found in the gills and adductor
muscles. Analyses made in the Bureau's shellfish
laboratory show that on a percentage basis the
adductor muscle stores smaller (juaiitities of
glycogen than do the gills or visceral mass (table
19).

Table 19. — Solids, water, and glycogen content of the
adductor muscles, gills, and remainder of the bodies of
15 C. virginica of good quality from the vicinity of Charles
Island, Long Island Sound

[Average percentages of fresh substance, 1934. 1935]

Item

Mantle

Body

Gills

Adductor
muscle

November 30:

Water

73.24

26.76

7.96

74.0

26,0

3.96

80.20
19.80
4.68

88.62
11.48
1.53

78.67

Total solids

21.43

Olvcogen

1.69

January 3:

Water -.-

78.60
21.40
3.37

79.04

Total solids

20.96

Glycogen

1.40

Samples consisted exclusively of adult Long Island Sound oysters of good
commercial quality and high content of solids; they were analysed within
a few hours after removal from the bottom.

In the Japanese species, 0. circumpicta, the
percentage of glycogen in the two parts of the

Figure 152. — EfToct of collagenase on tho attacliment of the muscle of C. virginica. Upper row: control — trypsin injected
tliroiigh the hole in the riglit valve (on left) has no effect on the attachment of the muscle. Lower row: part of the
adductor is detached from the right valve after an injection of collagenase. The detached part is seen on the left
valve (riglit side). Twenty-four hours after injection, 24° to 25° C. Left vahcs of each oyster are on right.

162

FISH AND WILDLIFE SERVICE

•^

I I I I I I

0 ^ . 5

Centimeters

THE ADDUCTOR MUSCLE

163

adductor has been calculated as follows (Ko-
ba3^ishi, 1929):

Translucent portion —

October 1.12 percent.

November 1.0 percent.

White portion —

October 1.43 percent.

November 1.29 percent.

The figures are not essentially different from those
for C. vir<iln.ica. The questions of how much of
the glycogen in the adductor muscle is part of the
muscular mechanism and how much of it is stored
have not been answered with certainty.
Proteins

According to the data quoted from Tabulae
Biologicae (1926), the fresh adductor muscle of
0. imbricata contains 11.38 percent protein and
4.8 percent fat. No published data are available
for the protein content of the muscle of C. vir(jinica..
It may be assumed that in this species the protein
content is not essentially different from that
usually found in plain muscles in which it forms
from 14 to 18 percent (Evans, 1926).

The contractile mechanism of the adductor
nuiscle of bivalves has the same structural
elements as are found in vertebrate muscles:
myosin (Florkin and Duchateau, 1942), actin,
and adenosinetriphosphate (ATP). The actin and
myosin extracted from muscles of 0. edulis, Mij-
tilus edulis, and Pinna nohilis (Lajtha, 1948) have
solubility relationships similar to those of the
corresponding- substances of rabbit muscle (Szent-
Gyorgyi, 1951). The myosin is soluble in distilled
water, insoluble in dilute potassium chloride solu-
tion (0.002-0. OS m), and again soluble in 0.1 m
potassium clJoride and higher. It is also soluble
in the 0.1 m and stronger solutions of chloride and
magnesium chloride. Myosin and actin can be
precipitated at isoelectric points of 5.2 and 4.7.
They both show double refraction whicli dis-
appears in dilution or at higher concentration
(0.4 M potassium chloride for myosin). Actin has
a higher doulile refraction than myosin. It also
lias the peculiar property of undergoing reversible
change from the globular to the fibrous state and
vice versa, depending on the pH and ionic con-
centration of the medium.

Besides actin and myosin the adductor muscle
contains another protein called paramyosin, which
differs in solubility and X-ray diffraction from
nwosin (DeRobertis, Nowinski, and Saez, 1954).
Paramyosin was first detected in the adductor

164

muscle of the clam {Merce.naria iyenus) mer-
cenaria) by usmg electron stains (Hall, Jakus,
and Schmitt, 1945). Preparations of muscle
fibrillae treated with phosphotungstic acid reveal
a periodic structure of alternate bands that show
affinity for the stain. The distance between the
bands averages 145 A. At the same time there
is a larger period of 720 A. which is repeated
every five spaces of the smaller period (145 X 5).
It was concluded by Hall, Jakus, and Schmitt
(1945) that the fibrillae of this type consist of
paramyosin. Its content in various bivalves
varies but is cjuite high in Mytilus edulis in which,
according to Lajtha (1948), it exceeds the content
of myosin. Paramyosin of the adductor muscle
of C. rirginica was separated from actomyosin by
precipitation with three volumes of ethanol at
room temperature (Philpott, Kahlbrock, and
Szent-Gyorgyi, 1960) and resuspension of the
precipitate in 0.6 M potassium chloride at pll 7.4,
which was then dialyzed against the same solution.
By such treatment the paramyosin passed into
solution and the actomyosin remained precipitated.
The yield of paramyosin extracted in percent of
total protein was 22 percent in the opaque part,
16 percent in the translucent part. On tlie basis
of biochemical studies the authors suggest that
paramy(isin is localized in the thick filaments,
while the thin filaments consist of actomyosin.

Paramyosin is not found in vertebrate muscles
but is the principal protein in many invertebrates
' (Engstrom and Finean, 1958). Although its par-
ticular role in muscular contraction has not been
determined with certainty, it appears probable
that this protein is responsible for the maintenance
of the tonus of the adductor muscles.

PHYSIOLOGY OF THE ADDUCTOR
MUSCLE

The zoologists of the middle 19th century were
aware of the difference in the function of the two
parts of the adductors of bivalves. They re-
garded the white part as a bunch of elastic bands
which counteracted the pulling force of the valve
ligament and the translucent part as an ordinary
nniscle wliich brought the valves together (Bronn,
1862, p. 359). Goutance (1878) and Jhering
(quoted from Marceau, 1904a) and later Jolyet
and Sellier (1899) maintained that the translucent
part of the adductor muscle of Pecten maximus
Consists of striated anastomosing fibers whose ex-
clusive function is to close the valves; they

FISH AND WILDLIFE SERVICE

observed that the white part of tlie adductor con-
tracts very sh>wly and can remain in a contracted
state for a hmg time. Marceau (1904a, 1904b)
confu-med these results by a series of experiments.
He cut off either white or translucent portions and
found that in 0. ednlis the rapid closing of the
valves is accomplished by the contraction of the
translucent part of the muscle wliile the elasticity
and tonus of the white part counteract the pullino-
force of the ligament. Useful reviews of many in-
vestigations dealing with the muscle physiology
of bivalves and other invertebrates are found in
the papers of Ritchie (1928), Jordan (19.38),
Evans (1926), and others.

It is a well-established fact that the two parts of
the adductor muscle contract at different speeds.
In scallops the isolated striated (translucent) por-
tion contracts in about 100 microseconds in sec);
its rela.xation time is about 0.1 second (sec.)
(Bayliss, Boyland, and Ritchie, 1930). In the
slow part of the adductor the contraction time
varies from 500 m sec. to 2.5 sec. and the relaxation
time is from 10 to 45 sec. The contraction of the
adductor muscle of oysters is always several times
faster than its relaxation, the ratio var^ving
according to the type of muscular reaction. Mar-
ceau (1909) published a number of tracings of the
spontaneous movements of the valves of 0. edulis
in which only the white (slow) part of the muscle
was left. The time of relaxation was from 15
minutes to 1 hour.

In many bivalves the adductor muscle can re-
main contracted, keeping the valves closed
tightly, for a long time. This behavior varies,
however, in different species. For instance, com-
mon scallops of the American and Eiu-opean
coastal waters, Astro-peden inadians and Chlamys
opercularis, close their valves for only a short
time. Soon after being taken out of water the_y
gape, lose shell licjuor, and perish. My observa-
tions on pearl oysters of the Hawaiian Islands
and Panama {Pinctada (jaltmff, P. mazatlanica) ,
.show that shortly after being taken out of water
their shells gape and the muscle fails to contract.
These species cannot be transported over long
distances unless they are kept in frequently re-
newed water all the time. On the other hand, the
bivalves in which the adductor muscle remains
contracted for a long time can survive long ex-
posure and can be shipped alive over great
distances.

Oysters living within the tidal range on flats
thrive in this situation because they can keep
their valves closed during the time of exposure.
It is obvious that this ability provides a great
survival value for those sedentary animals that
can withdraw within their heavy shells to avoid
desiccation and remain protected against un-
favorable conditions or attacks of predators.

The abOity of bivalve muscles to keep the shells
closed is frequentl}' called a "catch" or locking
mechanism. The idea originated from observa-
tions made by Uexkiill (1912) on the scallop; if a
piece of wood is pushed between the valves the
adductor contracts with such force that the edges
of the shells may be splintered. The wooden
wedge is held as firmly as if it were in a vise and
can be removed only by twisting and pulling.
The valves, however, remain motionless, and the
muscle that holds them in their position shows
no elasticity. The muscular fibers seem to be
frozen solid. The shell cannot be opened, but if
the valves are pressed on both sides they may be
brought nearer together and remain fixed in their
new position. This ability Uexkiill called "Sper-
rung", which in English means "locking." Bay-
liss (1924) interpreted Uexkiill's expression using
the word "catch," probably influenced by Griitz-
ner's (1904) suggestion that the muscle fibers of
the bivalve adductor must somehow be "hooked
up" by a mechanical arrangement similar to a
ratchet consisting of two pieces with teeth facing
each other. In his proposal the upper piece
could be pushed only in one direction, shortening
the total length of the model, and the upper
teeth could not move back unless the two pieces
were separated from each other by the depth of
the teeth. There is nothing in the struct ui-e of the
muscle fiber which even remotely suggests the
existence of such a mechanism. The expression
"catch mechanism" imphes some mechanical de-
vice and is, therefore, misleading. It has been
used, however, for such a long time that the
literary meaning of the words has been lost and
the term simply refers to the continuous state of
contraction of the closing muscle of bivalves.

Several theories have been proposed to explain
the locking or catch mechanism of the adductor
muscles. Some investigators assumed that the
muscle twitch (i.e., the contraction in response
to single brief stimulus) is common to all muscles
and the difference between the behavior of the
adductors of bivalves and of the muscles of other

THE ADDUCTOR MUSCLE

165

types is due to the differences in time scale and
the condition of stimulation. It was claimed,
(Ritchie, 192S, p. 86), althoug:h not proved, that
tonus of the adductor muscle is maintained by
tetanic contraction. Another view (Winton,
19.30), which is more in harmony with the bio-
chemical data, explained the locking mechanism
as a result of physical changes during contraction,
particularly the alteration in viscosity of muscle
proteins. Experiments with byssus retractor of
Mytilus showed that after stimulation by dii-ect
current the viscosity of the muscle was raised
and remained high for about 2 hours. Xo such
effect was obtained if alternating current was
used. These observations suggest that viscosity
changes are involved in the contractions of the
adductor of bivalves.

The difference between the white and the
translucent parts of the adductor muscle may be
primarily of a quantitative character. This
suggestion was made by Shukow (1936), who
found that in Anodonta and Unio the two parts
of locking muscles actively participate in single,
spontaneous contractions and in the maintenance
of tonus. Shukow's observations indicate the
inadequacy of the theory that makes the main-
tenance of the tonus the exclusive function of
tlie white fibers.

Studies of the electric phenomena in the smooth
adductor muscles of lamellibranchs {Alytilus, AIo-
(lioliis, and smooth part of C'hiamys) lead Lowy
1953, 1955) to conclude that the hypothesis of
"catch mechanism" is unnecessary because, ac-
cording to his observations, the tonus in tlie intact
muscles of these mollusks is due to a shifting
pattern of tetanic stimuli controlled by the
nervous system, bringing it in line with the tonus
in otlier muscles. Since action potentials were
observed in muscles which were isolated from the
ganglia, Lowy suggested that they may be of
myogenic natiu^e. The question of whether the
tonic activity of lamellibranch muscles is neuro-
genic or m3'ogenic remains open. Lowy makes an
interesting statement that "lamellibrancli muscles
maintain a certain level of tension all the time
due to the activity of a peripheral automatic
system, which works by successive activation of
limited areas." ^ This conforms with the histo-
logical observations described above which show
that in an intact adductor muscle of the
oyster preserved in a contracted state only certain

' — Underscoring is mine. P. .S. G.

muscle bands are in a true contracted state whOe
others are folded. Lowy concludes that further
studies are needed before it is decided whether
lamellibranch muscles are directly innervated by
excitatory and inhibitory nerves or are acted on
indirectly via a peripheral ganglionic plexus. The
existence of inhibitory axons in Fecten was demon-
strated by Benson, Hays, and Lewis, (1942), who
found that the relaxation of the adductor of the
scallop was considerably accelerated by stimu-
lating certain nerve bands going to the msucle.
This is in accord with the evidence presented by
Barnes (1955) for tlie adductor muscles of Ano-
donta. His work implies that the adductor of
Anodonta is innervated by three types of nerves:
one group of motor fibers supplies the striated
muscles and produces phasic contractions which
may summate and produce tetanus; another group
of activating fibers supplies the unstriated nmscles
and produces increased tonus; the third group
consists of inliibitory fibers which decrease the
tonus. Barnes points out that the nervous mech-
anism controlling the adductor activity in Mytilus
may be the same as in Anodonta. Mytilus is
capable of both phasic and tonic contractions,
but there is no obvious differentiation of the
muscle into two parts. It nmst be accepted,
therefore, either that all nmscle fibers are capable
of exhibiting both types of contraction or that the
two types of fibers are present but completely
interspersed.

Electrical activities associated with the contrac-
tion of the adductor muscle of the oyster have
not been studied enough to warrant an evaluation
of their role in the locking mechanism of these
mollusks. An attempt to solve the paradox of
the catch muscle mechanism was made recently by
Johnson, Kahn, and Szent-Gydrgyi (1959) and is
based on the study of the property of paramyosin.
The solubility of this protein was found to be
critically dependent upon the pH and ionic
strength of the medium. Similar dependence was
shown in the glycerinated fibers of the anterior
byssus retractor of AI. edulw. The fibers were
stretched, and the tension thus developed was
measm-ed. To reduce the efl'ect of actomyosin,
10"" M Salygi-an and 10"^ m pyrophosphate were
added to the medium. Stiffness of the fibers was
measured at various values of pH. Below pH
6.5 and at low ionic strength of 0.07 m potassium
chloride the fibers were relatively stiff. This is
a range in which paramyosin crystallizes out of

166

FISH AND WILDLIFE SERVICE

solution. At higher pH values the fibers were
relatively plastic. The authors think that parallel
with the actoniyosin system which produces initial
tension of the adductor there is a second, or
paramyosin system, capable of maintaining the
tension developed by the first one by crystallization
of the paramyosin component caused by pH
shift within the muscle. The theory was tested
by Hayashi, Rosenbluth, and Lamont, (1959) on
the nmscle extracts of Mercenaria (Venus) mer-
cenaria and Spisula solidissima. The results of
these experiments tend to support the hypothesis
that crystallization of paramyosin elTectively
freezes the adductor nmscle at any state of
contraction.

In two papers dealing with the fine structure of
the small fibers of the oyster {C. angulata) and
other bivalves, Hanson and Lowy (1959, 1961)
have proposed two possible explanations of the
mechanism by which the closing muscles of mol-
lusks maintain tension "very economically,"
i.e., without using much energy. According to
their view, based on examination of electron
micrographs of muscle, the thick filaments of the
fibril (see p. 157 and fig. 144) are discontinuous
and do not contract; they slide as the nmscle
shortens the relative portions of the thick and thin
filaments. The tension is maintained by cross
links between the two types of filaments. Accord-
ing to their view the alternative hypothesis, which
supposes that tension is maintained by change
in the physical state of the protein within a
paramyosin system, is diflRcult to reconcile with
their observations. The sliding or so-called
interdigitatory model of the contractile structm-e
is based primarily on the studies of striated muscle
(Huxley, 1960), and the extension of the theory to
nonstriated muscles of bivalves is very attractive.
It is impossible, however, to state at present which
of the two theories interprets correctly the catch
mechanism. Further experimental studies are
needed to solve the puzzle which for a century
has baffled the biologist.

In spite of the substantial advance of bio-
chemical investigations, the problem of the lock-
ing mechanism recjuires further study. So far
no evidence has been presented to show that the
shift in the pH needed for the crystallization of
paramyosin actually takes place in the whole
living muscle of a bivalve. It seems that the
solution to the locking paradox should consider
the problem in its entirety, by taking into account

all the biochemical and biophysical processes
which accompany the prolonged tonus of the
adductor muscle.

Chemical changes during muscular activity

Chemical changes occurring during the contrac-
tion and relaxation of the nmscle are extraordinarily
complex. The reader interested in this problem
should consult the textbooks of general physiology
(Scheer, 1948), biochemistry (Needham, 1932;
Baldwin, 1957), and particularly the comprehen-
sive reviews of more recent works given by Szent-
Gyorgyi (1951) and Weber (1958). Most of the
work on the chemistry of muscular contraction
has been performed on vertebrate muscles. In
general the results were found to apply to the
muscles of scallops {Pecten, Astropecten, Chlamys),
sea mussels (Mytilus), edible oysters {Ostrea,
Crassostrea), and Anodonta.

A complex chain of events is involved in mus-
cular contractions. I will consider only the high
points. Glycogen appears to be the principal,
if not the only source of energy in this process.
Its content in the adductor muscles of bivalves
varies from less than 1 to about 3 percent. The
immediate source of energy for muscular con-
traction is not derived, however, from the break-
down of glycogen. Considerable quantities of
phosphate are released by the organic compounds
called phosphagens. These substances contain
(Weber, 1958, p. 5) an energy-rich phosphate
bond and, therefore, are the "stores of immediately
available energy." Creatine phosphate, identified
as a phosphagen of vertebrate muscle, does not
(iccur in mollusks; its place is taken by arginine
phosphate. Phosphagen decreases during con-
traction and is formed again during rest. After
prolonged contractions the tissues of the fatigued
muscle become acidic due to the accumulation of
lactic acid. Glaister and Kerly (1936) found that
iodoacetate, which inhibits the formation of lactic
acid in Mytilus nmscle does not materially inter-
fere with muscular contraction.

The key substance involved in the energy trans-
formatiDU in the muscles is, however, adenosine-
triphosphate (ATP); the presence of ATP is a
prerequisite to contraction. According to Szent-
Gyorgyi's theory ATP has a great affinity to
myosin and is strongly linked to it. Excitation
of the muscle implies the formation of actomyosin
(from actin -\- nayosin), a process which does not
take place in the absence of ATP (Szent-Gyorgyi,

THE ADDUCTOR MUSCLE

167

1951). Dephosphorylization of ATP to adenosine-
diphosphate (ADP) is believed to be the most
important reaction closely connected to the libera-
tion of energy in the contracting muscle. The
function of ATP, according to Weber (1958) is
twofold: "it acts as a contracting substance if it
is split and as a relaxing and plasticizing substance
if it is present without being split." The ATP
used in contraction is restored "almost as rapidly
as it is broken down by transphosphorylation of
phosphagens."

Since the phosphorylization of ATP is the main
stage in the energy-prox-iding reaction in the
muscle, it is of interest to know the splitting
capacity of this compound in the adductor muscles.
Investigation of this problem by Lajtha (1948)
showed that the phosphatase activity is much
lower in bivah'e muscles {Mytilun and Pinna)
than in rabbit muscle. Lajtha suggests that this
is correlated with the slow working of the adductor
muscle, which does not require the quick energy
changes needed in the more rapidly functioning
muscles of vertebrates and insects.

Chemical changes in the adductor muscle of the
oyster {C. commercialis) were studied by Hum-
phrey (1944, 1946, 1949, 1950), who demonstrated
the presence of arginine pliosphate and of several
phosphorylated breakdown compounds of glyco-
gen. The glycogen can be synthesized in both
parts of the muscle from glucose- 1 -phosphate, but
synthesis is more readily effected in the translucent
portion.

In the glycolysis of the oyster muscle the glyco-
gen breaks down in the presence of added potas-
sium, magnesium, and DPN (diphosphopyridine-
nucleotide) and yields a mixture of pyruvic and
lactic acids (Humphrey, 1949). The glycolytic
ability of the adductor muscle of the oyster is
several hundred times less powerful than that of
rabbit muscle.

Studies of the glycolysis in extracts of the
adductor muscle of C. commercialis (Humphrey,
1944) disclosed three essential facts: (1) phospliate,
potassium, magnesium or manganese, and DPN
are the essential parts of the system resulting in
the production of acid; (2) lactic and p.\Tuvic acids
are produced simultaneously; and (3) acid produc-
tion is inhibited by fluoride and iodoacetate. The
glycolysis in oysters and other invertebrates still
is not well understood, particularly witli respect
to the metabolism of pyruvate by oj-ster muscles.

The ATP present in the adductor muscle has a

definite relationship to glycolysis. The amount of
ATP in the muscle decreases when oysters are left
out of water. From this observation Humphrey
advances the hypothesis that the breakdown of
glycogen provides the enei'gy for the muscle to
resist the pull of the ligament. He thinks that the
regeneration of ATP proceeds through glycolj^sis,
wiiicii continues under both aerobic and anaerobic
conditions. Both conclusions require further cor-
roboration.

NORMAL SHELL MOVEMENTS

Studies of shell movements can give valuable
information regarding the physiological state of
the oyster and its reactions to the changes of
environment. Tlie only type of motion that can
be performed by an adult oyster consists of two
distinct components: the contractions of the
adductor muscle that bring the opposing valves
together and may completely seal off the soft parts
of the oyster, and tlie springlike action of the
ligament that pushes the valves apart during the
periods of relaxation. The purely mechanical
action of tlie ligament is counteracted by the
tonus of the muscle, which retains a certain degree
of elasticity even in the state of maximum stretch-
ing. If the muscle is cut off at the maximum
gaping, the valves are pushed farther apart by the
elastic force of the ligament.

METHOD OF RECORDING

Oysters selected for long-term observation (sev-
eral weeks or months) should be free of boring
algae and animals. The surface of the shell is
scrubbed with a metal brush, washed, and dried.
The left valve is embedded in a rapidly setting
mixtm-e of cement, sand, and unslacked lime in
proportion 1:2:1. Care should be exercised to
keep the edges of the valves free of cement mixture
and to wipe out and wash with sea water all excess
material. Mounted oysters are left in the air at
room temperature for 12 to 24 hours.

A small metal loop cut from a paper clip may be
used to attach strmgs which lead to a recording
lever. The two arms of the U-clip are bent
horizontally, and the loop is placed on the clean,
dry surface of the right valve and sealed in a
vertical position by a few drops of iiot colophonium
cement. For recording the up and down move-
ments of a valve iieart and muscle levers available
at scientific supply houses can be used. Adequate
levers can be made of strips of appropriate length
cut from a sheet of plastic and mounted on pivots

168

FISH AND WILDLIFE SERVICE

of a small glass rod inserted in a hole drilled in the
supporting arm. It is convenient to have at hand
levers of various lengths so that records of shell
movements of sevei'al oj-sters can be made
simultaneously on one kymograph drum. Unless
there are some special reasons for not changing the
sea water during the observations, the oj'sters are
placed in running sea water, and the temperature
of the water is recorded on a thermograph and its
salinity checked at regular intervals.

The records reproduced in this book were
obtained by using a slow-motion kjanograph.
The uppermost position of the writing pen always
corresponds in these tracings to the position of a
completely closed right valve; the lower position
of the line marks the various degrees of opening of
the shell. The magnitude of the up and down
excursions of the writing pen depends on the ratio
between the two arms of the lever, the distance
between the hinge ligament and the place of the
attachment of the string, and the height of the
oyster. The magnification of shell movements
recorded in the Bureau's shellfish laboratorj' at
Woods Hole varied from three to seven times the
actual excursions of the valves. A baseline
representing the position of the writing pen when
the shell is completely closed (not shown in the
records reproduced here) may be obtained by
rotating the drum rapidly before beginning
observations.

Under ordinaiy circumstances the opening and
closing movements of the shell are so small that
the corresponding up and down tracings on kymo-
graph paper are relatively short and are not dis-
torted by the actual movement of the lever, which
on wider tracings describes an arc on the side of
the rotating cylinder. In case of wide gaping
produced by experimental stretching of the muscle
the distortion becomes serious since the writing
point at the bottom moves ahead of the time
marker and draws a gentle slope instead of a steep
curve. To avoid possible misinterpretation the
true position of the writing lever at the time of
maximum stretching and its return to the top as
the muscle contracts are shown on the records by
dotted lines.

For long-term observations the speed of the
kjonograj)!! drum is adjusted to slow movement of
about 1 inch per hour, ^^^len studies are made
of the reactions of oysters to various stimuli the
speed of the rotation should be increased to about
three-eighths of an inch (1 cm.) per minute. With

the fast- and slow-motion kymograph used in
the Bureau's shellfish laboratory, the latter speed
corresponded to one complete revolution of the
drum per hour. With this technique several
thousands of records of shell movements of oysters
were obtained under a great variety of conditions
using both normal and diseased oysters. Speci-
mens used in the tests were taken from New
England waters, Chesapeake Bay, South Carolina,
the west coast of Florida, Mississippi, Louisiana,
and Texas. A relatively small number of records
were made of shell movements of C. gigas and
0. lurida of the Pacific Coast. Many records
were obtained while oysters were subjected to
various types of poisons (chlorine, phenol, black
liciuor and red liquor of pulp mill wastes, crude oil,
thiocyanates, etc.) or while they were given
various concentrations of carbohydrates and
suspensions of pure culture of Escherichia coli.

For a study of shell movements under normal
conditions the oysters were kept in running sea
water delivered at 10 times, at least, faster than
the rate at which it was transported through the
gills. Under this condition one can be certain
that the products of metabolism were removed and
the oj'Sters were not deprived of food.

Shell movements play an essential part in the
respiration, feeding, and rejection of silt, mucus,
and excreta that otherwise may accumulate in
the pallial cavity of the oyster. Material settled
on the gills and mantle is rejected by rapid and
powerful snapping of the valves. In addition to
this rejection reaction there are smaller and
slower changes in the tonus level of the adductor
which may be interpreted as adjustments to a
steady flow of water through the gills. It is not
surprising that shell movements of oysters show
great variations both in the rate and type of
contraction. Analysis of the records made under
known conditions in the laboratory indicates
that in spite of this variability the movements of
individual oysters can be grouped into five major
types characterized bj^ their responses to various
conditions.

FIVE MAJOR TYPES OT SHELL
MOVEMENTS

In comparing the records of shell movements it
is necessary to know the followang essential points:
the highest and lowest level reached by the
writing pen during the periods of closing and
opening of the valves, the frequency at which

THE ADDUCTOR MUSCLE

169

the contractions occur, and the speed of rotation
of the drum. PubHshed reports frequently fail
to mention these significant details. Another
feature of importance is the general level cor-
responding to the tonus of the muscle to which
the valve returns after each brief closing. Under
normal conditions the adductor muscle is never
completely relaxed. The distance to which the
valves are pushed apart by the hinge ligament is,
therefore, indicative ot the degree of rela.xation.
During my years of study, more than 2,000
tracings of shell movements of oysters were
obtained under a great variety of conditions. It
was possible to group them into five principal
types which for the sake of brevity are designated
by the first five letters of the alphabet.

Type A

The three curves shown in figure 15.3 (,A-1,
A-2, and A-3) indicate normal behavior of the
oyster. The differences in the appearance of the
curves are due primarily to differing speeds of
drum rotation. Curve A-2 is a continuation of
curves A-1 with the drum movement reduced
from 15.3 cm. to 3.6 cm. per hour. The extreme
right portion of curve A-2 indicates the summa-
tion of sevei'al stmiuli that caused brief closing of

the valves. The curve A-3 is a variation of type
A-1 and is essentially similar to ciu"ves A-1 and
A-2. The writing lever in curve A-3 was set in
such a way that the magnification of the vertical
excursions was only one-third of that used in
curves A-1 and A-2. The contractions were,
however, more frequent. Several downward
excursions of the pen indicate brief attempts to
widen the opening of the valves, but the general
tonus level of the adductor remained fairly
constant.

Type A shell movement, shown in figure 153,
represents movements of an undistiu'bed oyster
that maintains a steady current of water for the
ventilation of the gills and for the collection of
food. The general level of opening of the valves
is fairly constant (curves A-1 and A-2). Relaxa-
tion of the muscle immediately after rapid con-
traction is slow, and the resulting curve slopes
down gently (see right parts of curves A-1 and
A-2) . Sudden snapping of the valves is associated
with the discard of rejected food, mucus, detritus,
and other pai tides that accumulate on the inner
surface of the pallium. This rejection reaction is
an important feature of oyster behavior for it is
the principal method of keeping the pallial cavity
free from the accumulation of foreign matter.

A-1

A-2

^^Av_J,^

A-3

Figure 15.3. — Shell movcment.s of normally feeding oyster. Type A. Vertical magnification of curve .A.-3 is about one-
third of that in A-1 and A-2. In each curve the uppermost point corresponds to the position of the lever when the
shells are completely closed. Time interval: A-1, 5 min.; A-2, 30 min.; .\-3, 1 hour.

170

FISH AND WILDLIFE SERVICE

Numerous minor contractions (fie;. 153, A-2) that
occur between the rejection reactions only slightly
reduce the opening between the valves and are
more difficult to interpret because they are not
accompanied by the discharge of any material.
Possibly they represent the fine adjustments made
by the oyster in maintaining a steady flow of water
through the gills. On the other hand, it is also
possible that they are responses to minor physical
disturbances such as vibrations of laboratory
floors and slight changes in illumination. None of
the existing laboratories in the United States
have the shockproof floors and walls that would
assure complete elimniation of the outside dis-
turbances caused by street traffic and footsteps
within the building.

Type B

Type B shell movement is characterized by the
increased frequency and well-pronounced perio-
dicity of contractions and corresponds to the state
of increased excitability (fig. 154). Curve B-1
was observed in oysters which were exposed to a
rapid rise of temperature from 13° to 25.6° C.
B-2 represents the behavior of oysters aftected by

the metabolites accumulated in stagnant and
unaerated water. The uniform and rapid con-
traction shown in B-2 stopped mimediately when
the water was changed.

Cm've B-3 represents a similar activity recorded
on a rapidly moving drum. The relaxation peri-
ods are much shorter, but the level of the muscle
tonus remains constant. Shell movements of this
type were frequently observed in oysters which
were left after spawning in water containing large
quantities of oyster eggs and sperm. Normal
movements of the type A-1 were resumed as soon
as the water was changed.

TypeC

The cm've of type C shell movements (fig. 155)
illustrates periods preceding or following changes
in the degi-ee of opening and closing of the valves.
Both periods are characterized by a series of minor
contractions and relaxations until the final tonus
level is reached. The type shown in C-2 (left
part of the curve) is a typical "staircase" or
"Treppe" reaction of the adductor muscle, which
contracts in several distinct stages. This reaction
is the response to an irritating substance added

B-2^

B-3

^'WA/V\N^VvAA^/VY^^^^^^^

Figure 154. — Shell movements of type B are typical for the state of increased excitability frequently caused by the
accumulation of metabolites in sea water or rapid rise of temperature. Vertical magnification in B-.3 is about one-
fourth of that ii B-1 and B-2; uppermost points correspond to closed shells. B-1 temperature increased from 1.3° C.
at the start to 25.6° C. at the end. B-2, B-3 increased muscular activity due to the accumulation of metabolites.
Time interval: B-1, 1 hour; B-2, 1 hour; B-3, 1 minute.

THE ADDUCTOR MUSCLE

171

C-l

i^jj^^r..-^ ^jVUiloULXu^

C-2

J_JU-

FiGCEE 155. — Shell movements of type C, preceding the closing of valves (left side) and following their opening (right
side). Note staircase movement in C-2. Time interval: C-l and C-2, 1 hour each.

to the water. A "staircase" in reverse direction
sometimes tai^es place dming the opening of the
valves (C-2, right half). This behavior was pro-
voked by small doses of oyster sperm, vitamins,
and sugars injected between the valves into the
pallia! cavity. The "reversed staircase" may be
interpreted as a testing reaction of the oyster,
which adjusts the opening of the valves to a
needed rate of ventilation.

TypeD

Shell movement of type D (fig. 156) was ob-
served in oysters affected by various poisons which
caused increased excitability of the adductor
muscle (D-1). In case of a prolonged action of
poison the periods of greater excitability (D-2 and
D-3) are interrupted by gradually increasing du-
rations of periodical closure (D-4 and D~5). This
type of shell movement is a symptom of a highly
advanced pathological condition resulting from
poisoning, disease, or exposure to adverse physical
conditions. It is typical for dying mollusks (D-5).

Type E

The E type of shell movement associated with
the spawning of the female oyster is cliaracterized
by great regularity, rapidity, and rhythmic up
and down strokes (fig. 157). At the beginning of
the reaction the time needed to reach the maxi-
mum relaxation level is very brief, almost ef^ual
to the time of the contraction (see: E-2). Dur-
ing the relaxation phase (downward stroke) there
is a brief period of slowing down in the decrease
of muscular tension. On the curve this period is

represented by a small plateau. This moment
coincides with the passage of eggs tlarough the
gills into the pallial cavity. The eggs in the pallial
cavity are dispersed into tlie surrounding water
by rapid contractions of the adductor.

Shell movements that take place during the
spawning of a female do not occur at any other
time and cannot be induced by drugs. They cease
with the cessation of spawning. The factor that
induces female spawning (temperature or chemical
stimulation by sperm) has no efTect on tlie type of
shell movement of a male and is ineffective on non-
spawning females. It is probable that this type of
muscular activity is associated with the discharge
of eggs from the gonads.

DURATION OF PERIODS OF OPENING
AND CLOSING

The length of time the shells remain open or
closed and the conditions that affect this behaxior
are of importance to oyster biology. Obviously
the normal functions of the organism, such as
respiration, feeding, and elimination of waste pro-
ducts, can be performed only when tlie valves of
tlie mollusk are open. It does not follow, however,
that tlie opening of the shell indicates that the
mollusk is feeding or is ventilating its gills. Under
certain conditions water may be sliut off from the
pallial caxity by the pallial curtain or by the ces-
sation of ciliary motion wiiile the valves remain
open. However, in the majority of laboratory
observations of tlie behavior of oysters in unadul-
terated sea water tlie opening of the valves coin-

172

FISH AND WILDLIFE SERVICE

D-l

•^^AiKUMii

D-2

D-3

\liaj„ua^,..u^^

D-4

D-5

Figure 156. — Shell movements of type D are observed in the oysters poisoned by toxic substances or weakened by
adverse environment. D-5 shell movements of a dying oyster. Vertical ex'cursions of the writing pen are mag-
nified three times in all tracings. Uppermost level of the curves corresponds to closed shells. Time interval:
D-l, D-2, D-3, D-4, D-5, 1 hour each.

E-l

E-2

Figure 157. — Shell movements of a spawning female. Note the frequencies of up and down movements, brevity of
the relaxation periods and slowing down at the middle of the downward strokes; this brief period coincides with
the penetration of eggs through the gills. Time interval: E-l and E-2, 1 minute each.

THE ADDUCTOR MUSCLE
733-851 O— 64 12

173

cided with the maintenance of a steady cloaca!
current.

The determination of the number of hours the
oyster remains open under average normal condi-
tions is of significance in studies of reactions of
the mollusk to changes in its envh'onment. Cer-
tain industrial wastes discharged in sufficient
concentrations into natural waters reduce the
time the oysters stay open. It was found, for
instance, that the red lirjuor which is the waste
product of pulp mills using acid tligestion of wood
and the black licjuor of pulp mills which employ
a sulfate process exert this effect on the Olympia
oyster (Hopkins, 1931) mid on C. rirginica (Galtsoff,
Chipman, Engle, and Calderwood, 1947). Any
condition that forces oysters to remain closed for
an abnormally long time deprives them from
taking in food and eventually may harm them.

The percentage of time during each 24-hour
period that the oysters are open can be used as an
index of normal behavior, provided the shell move-
ments of the mollusk do not indicate pathological
conditions of the type shown in curves D-1 to
D-5. Failure to recognize the significance of this
type of shell movement while recording the time
the oyster remains open may lead to serious mis-
understandings and errors. Unfortunately there
are many published data in which the "time open"
was recorded without observing the character of
shell movements.

The length of time C. rirginica remains open is
also influenced by temperature and by the state
of the oyster itself. Since the shell movement is
influenced by several external and internal factors,
it is not surprising that there is a gi-eat discrepancy
in the estimates of the average duration of "open
shells" reported by various investigators.

In the Bureau's shellfisli laboratory at Woods
Hole from June 15 to October 15, 1926, 132 daily
records of 34 oysters observed gave an average of
17 hours 7 minutes for open shells. The tempera-
ture of the water during tiiis period ranged from
13° to 22° C, but daily fluctuations of temperature
were insignificant, never exceeding 1.5° C (Galt-
soff, 1928). Records of the three oysters kept by
Nelson under observation for 21 days in New
Jersey water indicated that the shells remained
open on tlie average of 20 hours per day at tem-
peratures varying between 22° and 25° C. (Nelson,
1921). For oysters kept in running sea water at a
Beaufort, N.C., laboratory, average time open in
October to November varied between 10 and 14

hours (Hopkins, 1931). The temperature of water
was not recorded. Two hundred and one daily
records of 49 York River (Virginia) oysters kept
under observation in the laboratory at Yorktown
showed that the periods of opening varied from 19.2
to 24.0 hours a day (Galtsoft", Chipman, Engle,
and Calderwood, 1947). Within the temperature
range of 17.0° to 28.0° C. Long Island Sound oys-
ters were found to remain open for an average
period of 22.5 hours. The latter data are based
on 64 records of 18 oysters (Loosanoff and No-
niejko, 1946). 0. lurida of the Pacific Coast
remained open for an average of 20 hours a day
at the temperature range of 5° to 17° C. (Hopkins,
1931).

A sample of oysters always includes several
individuals tliat may remain closed for 24 hours
or longer. One or two of them will reduce unduly
the average figure based on a small number of
observations. Furthermore, under identical con-
ditions of the normal environment (i.e., not
affected by pollution, dredging, or other disturb-
ances) an oyster may keep its shell open or closed
for varying periods of time depending on the
requirements of the organism for food and oxygen.
I found that immediately after spawning the
female oysters have a tendency to keep their shells
closed for several days. On the other hand,
oysters left overnight out of water open almost
immediately upon being returned to sea water.
It is reasonable to assume that they accumulated
an oxygen debt during the period of closure.
In view of these observations the differences in
the duration of periods of opening or closing
described for oysters of different localities have no
particular significance. Tlie average value may
be useful, however, in determining the adverse
effects of the changes in the population of oysters
in a given locality and in making a comparison
between the behavior of these individuals in clean
and polluted waters.

EFFECT OF TEMPERATURE

Temperature as such has no direct influence on
the diu-ation of shell opening. There was no
significant difl'erence in the length of time the
Woods Hole oysters remained open when kept at
temperatures varying from 15° to 30° C. (Galtsoff,
1928). It is rapid change in temperature, often
occurring in those laboratories where sea water is
subject to wide diurnal fluctuations, that has a
pronounced effect on shell movements. O. lurida,

174

FISH AND WILDLIFE SERVICE

Figure 158. — The average percentage of time open of two
specimens of 0. lurida at each hour of the day observed
over the '29-day period (solid circles). Average tem-
perature readings for each liour during the same period
(open circles). From Hopkins (1931), fig. 4, p. 6.

for instance, has a tendency to close with the fall-
ing of temperatm-e and open with a rise of tempera-
ture (Hopkins, 1931). The sensitivity of this
oyster to temperature changes was reported to be
greater at the lower range. At 4° to 6° C. the
oysters remained closed a relatively high per-
centage of the time; at 6° to 8° C. they were open
only about 6 hours, while at the maximum of
about 15° C. they remained open over 23 hours
per day. In both cases the diurnal curve of shell
activity was parallel to the curve of temperature
fluctuation observed by Hopkins (1931) although
the percentage of time open in warmer water was
consistently higher (fig. 158). It would be of
interest to repeat these observations and compare
them with controls kept at a constant temperature
since Hopkins' temporary laboratory near Olym-
pia, Wash, lacked adequate ecjuipment for regula-
tion of temperature. He concluded that "change
of temperatui-e is more important in affecting the
length of time Olympia oysters remain open than
the degree of temperature itself." The results
of his observations on (\ virginica at Beaufort,
N.C., bear close resemblance to those described
above but, unfortunately, they were not accom-
panied by thermograph records and so are
not entirely convincing. His conclusions need
clarification.

EFFECT OF LIGHT AND DARKNESS

Periods of light and darkness have no apparent
effect on the closing or opening of the valves.

Analysis of 103 daily records of shell movements
of oysters kept in the Bm'eau's Woods Hole lab-
oratory in running sea water at neai'ly constant
temperature (daily fluctuations ±0.5° C.) and
constant salinity sliows that of the total number
of 831 hours of inactivity (shell closures), 266 hours
or 32 percent occurred during the 8-hour period of
darkness and the balance of 565 hours, or two-
thirds of the total, took place during the remaining
two thirds of daylight (Galtsoff, 1928). During
the summer, from June to August inclusive, the
Long Island Sound oysters kept their shells open
for 94.4 percent of the total time during daylight
and 93.8 percent during the hours of darkness
(Loosanoff and Nomejko, 1946). These observa-
tions repudiate Nelson's conclusion that the
periods of inactivity (or closings) occur dm'ing
darkness (Nelson, 1921, 1923c).

EFFECT OF TIDE

There is no evidence that the opening and
closing of oyster valves is related to the stages of
tide. The idea that oysters living below the
low-water mark are relativel^y inactive during the
outgoing tide and that the times of cessation and
commencement of feeding are correlated to stages
of tlie tide, was several times expressed by Nelson
(1922, 1923a, 1923c, 1938) and without verification
was accepted by Orton in his article in Encyclo-
pedia Britannica (Orton, 1929). Loosanoff and
Nomejko (1946) analyzed the kymograph tracings
of shell movements of oysters kept under vu-tually
natural conditions on a platform installed on a
small oyster bed on the bottom of Milford Harbor
in Long Island Sound. They found that the shells
remained open on an average of 93.4 percent of
the time during the flood periods and 95.2 percent
of the time during the ebb periods. The tidal
changes in Long Island Sound are not accompanied
by the excessive changes in the temperature,
salinity, pH, and turbidity of water which fre-
f}uently take place in the tidal streams of the
southern Atlantic states and may influence the
shell movements of oysters.

POWER OF THE ADDUCTOR MUSCLE

Anyone who attempts to open a live oyster by
inserting and twisting a knife between the two
valves becomes aware of the considerable resistance
exerted by the mollusk. As a rule the valves of
healthy 03-sters just taken out of sea water are
difficult to pry apart. The power of the adductor
muscle, which is solely responsible for keeping

THE ADDUCTOR MUSCLE

175

the valves tightly closed, varies greatly in oysters

of the same size and environment. Prolonged

exposure to air so weakens the adductor that

oysters left out of water for several days can be

easily opened.

In attempts to measure the power of the

adductor of various bivalves Plateau (1S84),

Marceau (1905a, 1905b), and Tamura (1929, 1931)

drilled holes near the edge of the shells and inserted

rods or hooks to wliich they attached weights.

The opposite valve was immobilized. Assuming

that the adductor muscle is an elastic body, the

amount of work (W) done by the adductor

against the loaded weight (G) was calculated by

ac
using a simple formula W=— jG where ac is a

ad

distance in centimeters from the ligament to the

attachment of the weights; ad is the distance in

centimeters from the ligament to the center of the

adductor muscle ; and G is the weight in grams

applied to the valve. Under a known pulling

force the shell movements were traced on a

kymograph and a record was made of the time

and load under which the muscle fibers were torn

off. Continuous irritation of the adductor by the

foreign body (hook or rod) inside the sliell near

the mantle makes this technique objectionable.
Furthermore, the end point of tlie experiment,
the tearing off of the muscle, is of no biological
significance compared to a determination of the
tensile force of the muscle fibers.

The method used in the Bureau's shellfish
lalxiratory eliminates these objections. Tlie left
valve of tlie oyster is mounted on a heavj^ cement
block, using a very strong mixture of portland
cement and sand to which a small amount of
plaster of paris is added (fig. 159). The base is
bolted to the frame D which may be placed in the
a(|uarium tank B supplied with running sea water.
A galvanized iron screw (a) about 1 inch in length
is inserted into the valve at the center of the
attachment of the adductor muscle. Its tip
should not penetrate the valve. Enough portland
cement or other highly adhesive mixture is applied
to the shell surface around the screw to make a
cone of about 1 incli in diameter; the top of the
screw (a) should protrude above the cement. A
metal stirrup (E) consisting of a pair of iron bars
(b) with pronged arms at the lower end and a
hook (d) mounted at the upper end connect the
valve and the pan (e) of the laboratory balance

Figure 159. — Method of determining the resistance of the adductor muscle of C. virginica to a pulling force. A — cement
base, bolted to wooden frame D and placed in tank B; a — galvanized iron screw; b — bars of the stirrup E; c — adjust-
ing nut; d — hook for connecting the stirrup to the balance; e — left pan of the balance: F — seawater intake; H —
overflow; K — kymograph; L — writing lever; M — signal magnet and pen; R — Telechron timer; T — transformer.

176

FISH AND WILDLIFE SERVICE

placed on frame D. The length of the hook is
adjusted by turning the nut (c). The two pans
of the balance are placed in a zero position, and
the desired weight is put on the right pan. The
right valve of the oj'ster is connected to the
writing lever (L) of the kymograph (K). The
writing pen (M) is attached to a signal magnet
which is activated by an electric timer (R) and
transformer (T). The timer is made by mounting
a plastic disc on the axis of a Telechron motor
making one revolution every hour. A short piece
of copper wire at the periphery of the disc, indi-
cated in figure 159 by the arrows, completes the
circuit every 30 minutes (at the vertical position
of the arrow). The weight of the balance is
sufficient to keep the platform from floating when
it is placed under water. Sea water is supplied
through the intake (F); the overflow (H) controls
the water level. This setup was successfully used
in a large number of tests made both in the air
and under water.

Occasionally the bond between the cement cup
and the surface of the shell was insufficient for a
pull of 8 to 10 kg. and had to be adjusted by using
a stronger mixture and slightly roughening tlie
surface of the valve. In the majority of cases the
connection between the valve and the cement cap
remained intact even when the pulling force of
about 10 kg. was applied and occasionally the
muscle itself was torn in the middle.

The purpose of the test was twofold: to study
the behavior of the adductor under variable
pulling force and to determine the time required
to cause the loss of tonus by the muscles that were
being stretched by weights varying from 2 to 10
kg. directly over the muscle scar.

New England oysters kept in the harbor near
the laboratory were used in all the tests. The
oysters were about 5 inches in height and ap-
peared to be in good condition with the shells
undamaged by boring sponge.

TESTS MADE IN AIR AND IN WATER

Adult oysters exposed to air at room tempera-
ture are able to withstand the pulling force of
several kilograms for several days. Under the
weight of 7 to 8 kg. the adductor muscle opened
immediately (fig. 160). A force of 8 kg. (2,185.8
g./cm.^ of cross-sectional area of the adductor)
caused immediate stretching of the adductor to
about one-third of the maximum gaping distance,
which was attained within 6.5 hom's. During the

5 hours following the initial stretching there was
no shell movement but the adductor retained its
tonus level; the response to pricking (several
small upward strokes on the record) M^as very
slight. Final stretching to 1.5 cm. gaping dis-
tance of the valves was relatively rapid. At this
stage the adductor lost the tonus and failed to
respond to stimulation. Upon removal of the
weights the muscle regained its elasticity and
contracted (right side of fig. 160). For several
hours an oyster weighing only 18.2 g., exclusive
of shell, was capable of maintaining a constant
tonus level against the pull of 8 kg.

In all tests in which the pulling force of 10 kg.
per oyster was applied (from 2.5 to 3.0 kg./cm.^
of the muscle area) the muscle stretched im-
mediately and the gape of the valves reached the
maximum width of 15 to 18 mm. The muscle
failed to respond to pricking or to the application
of 0.1 A" hydrocholoric acid but retained a certain
degree of elasticity and was able to counteract
the pulling force of the ligament. As soon as the
muscle was cut off the valves opened several
millimeters beyond their former position.

Individual variations in the time recjuired for a
muscle to reach maximum stretching are consider-
able. The time needed to produce tonus loss is
inversely related to the weight applied to the
valves. The pulling force of 0.5 kg. (131 to 136
g./cm.^) applied for 15 days had no effect on the
opening of the oyster shell in the air (at room
temperature of 15° to 18° C). At the pulHng
force of about 500 g./cm.- the loss of tonus and
failure to respond to stimulus developed in 300

8 Kg
applied

Figure 160. — Record of shell movement of C. virginica
kept in air under the pulling force of 8 kg. (2185.8 g./cm.^
of cross-sectional area of the adductor muscle). Arrows
indicate time when the weight was applied (upper left)
and removed (lower right). Temperature 18° to 23° C.
Total weight of oyster meat 18.2 g.; of shell 166 g.
Maximum gap (right end of the curve) 1.5 cm. The
distortion of the lowermost position of the lever with
reference to the horizontal axis is marked by the heavy
arrow. Time interval: 0.5 hour.

THE ADDUCTOR MUSCLE

177

hours. To avoid desiccation the oyster in this
experiment was surrounded by a small moist
chamber. With the increase in weight the time
of complete loss of tonus rapidly decreases
(fig. 161).

Muscles which were kept for several hours under
a pulling force of about 1.7 kg./cm.^ of cross-
sectional area suffered a temporary injury which
resulted in abnornuxl shell movements after the
return of the oysters to sea water (fig. 162). The
two tracings reproduced in this figure are almost
identical, although in the case of oyster A a pulling
force of 6 kg. was used while S kg. were applied to
oyster B. In both instances the pulling force per
unit of muscle scar area was the same, 1,676 in A
and 1,675 g./cm.' in B. After a few days in
running sea water both oysters completely re-
covered and their shell movements became normal.

In oysters kept in sea water the relationship
between the weight applied to the valves and the
time needed to attain tonus loss is less regular
and individual differences are mucli greater tluui
for oysters left in the air. Witli a pulling force of

Figure 162. — Shell movements of C. virginica in sea water
after the removal of weight of 6 kg. or 1,676 g./cm.^ of
muscle area (upper line) and 8 kg. or 1,675 g./cm.^ of
muscle area (lower line) applied to the valves. Weights
were removed after complete loss of tonus was attained
in 43.52 and 52 hours exposure in air at temperature
23° to 24° C. Water temperature 13.5° C. Time
interval: A and B, 0.5 hour each.

about 1.5 kg./cm.^ of muscle area some of the
oysters showed tonus loss in less than half an hour
wliile others remained closed for many liours.
The relationship between the increasing pulhng
force and the time required to devek)p loss of
tonus is shown in figure 163.

Changes in the character of sliell movements of

\'o rs 2 0

K I LOG R6MS / CM^

Figure 161. — Time in hours reriuired to obtain loss of
tonus of the intact adductor muscle kept under constant
pull in kg. /cm. 2 of the cross-sectional area of the adductor.
Experiments with C. virginica kept in air at tempera-
tures between 18° and 24° C.

10 15 2,0

KILOGRAM S / CM^

Figure 163. — Time in hours required to obtain complete
loss of tonus in the adductor muscle of C. virginica kept
in water under a constant pulling force expressed in
kg. /cm. 2 of the cross-sectional area of the adchictor
mu.scle. Temperature 13.9° to 18.0° C. At the pulUng
force of 0.59 kg. complete tonus loss was obtained in 274
hours (11.5 days).

178

FISH AND WILDLIFE SERVICE

an oyster kept under the continuous pull of the
relatively light weight of 2 kg. (606 g./cm.^ of
cross section of muscle area) are shown in figure
164. The five lines represent excerpts of about
7.5 hours duration from a continuous recording
made at a temperature of 13.9° to 14.1° C. and
salinity of 31.3 °/oo- In line A the movements are
normal. Their amplitude is increased after the
application of a pulling force of 2 kg.; at the same
time the frequency of contraction decreases
(hne B). This condition continues until the
67th hour (line D, middle part) when the muscle
begins to stretch and the number of contractions
greatly increases. At the 71st hour (end of line
D) the muscle does not respond to stinmlation.
After removal of the weight (line E) shell move-
ments are restored. The frequency of contrac-
tions during the recovery period is greater than
under normal conditions. Within the next 48
hours normal shell movements of the type shown
in line A are resumed.

Similar experiments in tlie air at higher tempera-
tures varying from 18.5° to 24.0° C gave slightly
different results shown in figure 165. The pulling

Figure 164. — Shell movements of C. virginica in sea
water under continuous pull of 2 kg. (606 g./cm.^ of
cross-sectional area of the adductor muscle). Tempera-
ture 13.9° to 14.1° C. Salinity 31.3 °/oo- A— normal
shell movements before the application of weight.
R — immediately after the application of weight.
C — after 32 hours; note increased gaping. D — after
64 hours; wide gaping, complete loss of tonus and lack
of response to stimulation. Maximum valve opening
1.5 cm. E — increased muscular activity during the

recovery period following the removal of the weight.
Time interval: A, B, C, D, and E, 0.5 hour each.

WEIGHT OFF

Fir.uRE 165. — Excerpts of the continuous records of shell
movements of C. vinjinica in air under the pulling force
of 2 kg. (500 of the cross-sectional area of the adductor) .
A — line ends at the y6th hour after the application of
force; B— at I90th hour; C— at 274th hour when the
muscle failed to respond to stimulation. Widest gap of
valves 1.5 cm. Temperature 1S.5° to 24° C. Time
interval: A, B, C, 0.4 hour each.

force of 2 kg. per oyster applied in this case
was equivalent to 590 g./cm.^ of the cross-sectional
area of the muscle. Loss of tonus was attained in
this case after 274 hours (line C) when the gap
between the valves reached the maximum of 1.5
cm. Pathological condition of the muscle was
apparent after 96 hours (line A) and becam e pro-
nounced at 190 hours (line B). After removal of
the weights the oyster was left in running sea water
but failed to recover and died in 2 days.

A lighter weight (315.5 g./cm.'^ of muscle area)
appUed to an adult oyster kept in running sea
water at temperatures ranging from 13.9° to 18.0°
C. produced very slow changes in the normal shell
movements (fig. 166). The upper line of figure
166 represents normal movements recorded imme-
diately after the application of the weight. A
noticeable increase in the amplitude of contractions
began on the 3rd day and continued tlirough the
11th and 12th days. During the 13th and 14th
days the amplitude of up and down strokes was
greatly reduced; loss of tonus and failure to re-
spond to stimulation developed by the 18th day.
Tlie last line sliows tlie typical staircase contrac-
tion following the removal of the weight, indicating
that the muscle retained some of its elasticity. At
the maxinmm ampHtude of the contractions (9th
and nth days) the oyster periodically lifted the
weight of 1 kg. to the lieight of about 1 cm. Ten
days after the end of tlie test the oyster recovered
completely and its shell movements became
normal.

THE ADDUCTOR MUSCLE

179

- waiQht ramovQd

Figure 166. — Shell movements of C. rirginica in running
sea water under a continuous pull of 1 kg. (312.5 g./cm.^
of the cross-sectional area of the adductor muscle).
Temperature 13.0° to l.S.O° C. Time interval: 0.5 hour.

If imich greater weight (4 kg. per oyster or 1 ,150
g./cin.^ of muscle area) is applied shell movements
become abnormal at the very beginning of the test.
This is demonstrated in the records of two C'otuit
(Mass.) oysters (C rirginica) and one f. gigas
shown in figure 167.

The stretching of the adductor muscle by a pull-
ing force not exceeding 4 kg. per oyster did not
interfere with their feeding; a strong current was
maintained by the gills, and the feces were formed
and discharged in a normal way. However, the
secretion of mucus by the mantle and gills was
greatly increased. Vast quantities of slimy mate-
rial accumulated at the mantle edge and were dis-
carded as pseudofeces.

The resistance of the adductor nmscle to a pull-
ing force exceeds by many times the force required
to overcome the elasticity of the ligament and
close the shell. This additional force is apparently
needed to keep the valves hermetically sealed.
Tlie ability to keep the valves tightly closed has
definite survival value. Mollusks possessing it
are able to protect themselves against desiccation
when exposed to air, or against adverse conditions
caused by the presence of toxic substances in the
water. Powerful muscular mechanism also helps

Figure 167. — Shell movements of two Cotuit oysters, C.
virginica (lines A and B), and C. gigas (line C) in sea
water under a continuous pull of 4 kg. or about 1,150
g./cm.2 of cross-sectional area of the adductor muscle.
Temperature 14.5° to 16.5° C. Salinity 32.0 to 32.3 °/oo.
The exact time of tonus loss is shown by the broken line
and arrow. Time interval: A, B, and C, 0.5 hour each.

them to resist attacks of starfishes, crabs, and other
enemies that attempt to pry open their valves.

CYCLES OF SHELL MOVEMENTS

There is no indication of any periodicity in
muscular activity in the kymograph records of
shell movements of oysters that were kept in
running sea water in the laboratory or kept out-
side on a suitable platform submerged from a pier
(LoosanofT and Xomejko, 1946). Brown and his
associates (Brown, 1954; Brown, Bennett, Webb,
and Kalph, 1956) claim, however, that C. virginica
possesses a persistent lunar cycle of activity with
the maxima occuring at about 12.5 hour intervals.
Oysters used for obtaining tracings of shell move-
ments were kept for a fortnight or longer in about
4 or 5 1. of sea water which was not changed but
was adjusted by occasional addition of distilled
water to compensate for evaporation. The mean
daily cycles were calculated for 15-day periods
by obtaining the average value of opening for
each hour of tlie day and appljang to the data a
very complicated method of adjustment. The
main conclusions reached by the authors were
that: (1) oysters and qualiogs display "statistical
rhythms of opening of shell while the overt
rhythms are not apparent from kymograph
records", (2) short periods of opening tend to
occur about 6:00 a.m. and more or less prolonged
periods of openings happen through much of the
remainder of the day. The observations and
their mathematical treatment are of interest from
a theoretical point of view, but the ecological

180

FISH AND WILDLIFE SERVICE

significance of the times of maxima and minima
of activities in the daily cycle of the oyster are
difficult to imagine at the present time.

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Physiologie, band 80, pp. 214-222.
Lajtha, Abel.

1948. The muscle proteins of invertebrates. Pub-
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vol. 21, fascicolo 3, pp. 226-231.

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FISH AND WILDLIFE SERVICE

Leenhardt, H.

1923. Sur la presence d'un muscle pedieux chez Ics
Ostr^idfe. Bulletin de la Society Zoologique de
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LoosANOFF, Victor L., and Charles A. Nomejko.

1946. Feeding of oysters in relation to tidal stages
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Bulletin, vol. 90, No. 3, pp. 244-264.

LOWY, J.

1953. Contraction and relaxation in the adductor
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1955. The lamellibranch muscle. Contractile mech-
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1904a. Sur les fonctions respectives des dux parties
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1904b. Sur le mecanisme de la contraction des
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1921. Report of the Department of Biology of the
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1936. Vergleichende Muskelphysiologie. Ergebnisse
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THE ADDUCTOR MUSCLE

183

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184

FISH AND WILDLIFE SERVICE

CHAPTER IX
TRANSPORT OF WATER BY THE GILLS AND RESPIRATION

Page

Transport of water __ 185

Determination of tlie rate of water transport 186

Direct metliods 186

Constant level tanks 186

Current indicators 190

Indirect methods 193

Use of turbid water 193

Use of radioactive plankton 194

Control of the rate of water transport through the gills 194

Steady state 195

Reduction of water transport 196

Dissolved organic substances and rate of water transport 197

Respiration __ 200

Methods of study 201

Microdetermination of oxygen 204

Oxygen uptake _ 205

Oxygen uptake under continuous pull of the adductor muscle 209

Environmental effects 211

Seasonal changes in the rate of oxygen uptake 211

Effect of change in salinity and pH 211

Respiratory quotient, R. Q 212

Respiration In other species of oysters 212

Utilization of oxygen 214

Bibliography. ___ 215

All bivalves maintain a steady flow of water
through their gills for feeding, respiration, and the
removal of products of metabolism. In the litera-
ture on oyster physiology this process is described
under such names as pumping, filtration, ventila-
tion, respiratory current, and feeding. When
"feeding" is used in reference to the transport of
water by the gills, tiie term is misleading since
feeding imphes the acceptance of food by the
organism and involves sorting and rejection of
particles removed from the water by filtration.
The terms ventilation and transport of water
appear to be suitable expressions for denoting the
processes by which the oyster mainfains a flow
of water in a comple.x system of water tubes and
through the epibranchial chambers. Both terms
are used in this text interchangeably, depending on
whether the emphasis is on collection of food or on
respiration.

Maintenance of a steady stream of water and
respiration are the two principal functions of the
gin. Of lesser importance are the excretion
(through diapedesis) of certain products of
metabolism, tlie absorption of substances dissolved
in water, and the ingestion of particles settled on

FISHERY bulletin: VOLUME 64, CHAPTER IX

the gills by the leucocytes present on their surface.
Water transport and oxygen uptake are inter-
dependent functions that may be considered as
two phases of a single process. It is convenient,
however, to discuss them separately, keeping in
mind tliat both activities occur simultaneouslj'.

TRANSPORT OF WATER

The preceding chapter described how a steady
rate of water current inside the demibranches is
produced by synchronized beats along the thou-
sands of tracts of lateral cilia. Temporary cessa-
tion of ciliary motion along some of the tracts or a
disturbance in their rhythm of beating will result
in a drop of hydrostatic pressure inside the water
tubes and cause a leak of water through the ostia.
This disturbance may slow down or stop the
cloacal current.

The adductor muscle, the edge of the mantle, the
gill muscles, and the ostia all pla.v a part in the
regulation of the flow of water produced b}' ciliary
activity. It is self-evident tliat, within the limits
determined by the capacity of the gills' chambers,
the volume of water transported depends on shell
movements and the width at which the two valves
are kept apart. When the shell closes the current
stops. A similar efl^ect may be produced by the
edges of the mantle independently of the move-
ments of the valves. Sometimes the pallium
assumes a vertical position and the tentacles along
tlie edges of the opposing mantles interlock while
the valves remain wide open. Under this condi-
tion no water penetrates through the curtain wliicli
seals the entire pallial cavity. This behavior
occurs in the presence of low concentrations of
to.xic substances and during the spawning of the
female oyster (ch. XIV, p. 304). It is, therefore,
obvious that shell movements should not be con-
sidered as indications of the feeding of the oyster,
a mistake which frequently is found in papers
describing the "feeding" of oysters.

The rate of water transport may also be reduced

185

by contractions of gill muscles bringing together
the filaments of the plicae, followed by constriction
of the ostia. The contraction of the ostia some-
times takes place independently of the contraction
of gill muscles.

DETERMINATION OF THE RATE OF WATER
TRANSPORT

The rate at which water is transported through
the gills can be studied by both direct and indirect
methods. In direct methods the volume of water
discharged through the gills in a given time is
collected and measured, or is calculated from ob-
servations of the velocity of the cloacal current.
The indirect methods are based on determinations
of the rate of removal of particles suspended in
water. The powders used for the latter purpose
include kaolin, natural silt, calcium carbonate,
colloidal carbon, isolated chloroplasts, various
plankton microorganisms such as Euglena, (_'hlo-
rella, and Nitzschia, and cultures of unicellular
algae that have been made radioactive.

Direct methods

Tliere are two groups of direct methods. First,
the water disciiarged by the gills may be inter-
cepted, its volume measured, and the sample re-
tained for analysis. In the second method tlie
velocity of the current is determined by measuring
the angle of deflection of a light paper cone or
glass plate placed in front of the current. The
rate of transport of water masses may be computed
from the cross-sectional area of the column of
moving water and the angular deflection of the
plate. Both methods have certain advantages
and disadvantages.

The methods which make it possible to collect
and analj'ze water after it has passed through the
gills are particularly suitable for studies of feeding
and metabolism. The slight disadvantage is that
the mollusk must be partially enclosed in rubber
or plastic. In the second group of methods the
mollusk is not in direct contact with any foreign
material and is kept under normal conditions.
The drawback is, however, that the discharged
water can only be collected in very small quantities
directly from the stem of the current inside the
cloaca. Choice of method must be governed, of
course, by the purpose of the observations.

Constant level tanks. — A very simple device con-
sisting of two connecting vessels in which tiie level
of sea water is kept constant was designed by
Galtsoff (1926, 1928) and has been adapted and

modified by many investigators making long-term
observations on the feeding of the oyster. The
apparatus, shown in figure 168, consists of two
rectangular vessels, one large and one small, con-
nected by a semicircular trough or by glass tubing
of wide diameter inserted in the partition between
the two vessels. The large vessel should be just
big enough to accommodate an oyster mounted
on a stand, the inlet tube for sea water, and a
thermometer. The volume of the tank is made
as small as possible in order to permit rapid ex-
change of water. Convenient dimensions for
observations on adidt Crassostrea vinjrmca and ('.
gi'jas, are 10 by 6 by 4 inches for the large vessel,
and 2 inches by 1}^ by 2 inches for the small one.
The tanks are made of transparent plastocel,
lucite, or similar nontoxic material about }4-inch
thick. The edges are sandpapered and cemented
together by a solution of plastic in acetone, cello-
solve, or by undiluted methylene chloride, and
the small vessel is cemented to the side of the
large one. After the cement is dry the tank
should be washed carefully and rinsed in sea water.
A semicircular trough made of thin plastic bent in
hot water to the proper shape is mounted on the
wall between the two vessels. The drain pipe is
glass tubing, 6 to 8 mm. in diameter, inserted
through the bottom of the small vessel; its upper
end is slightly widened and adjusted to the exact
level of water in the large vessel, which is con-
trofled by a wide cut in the opposite wall of the
vessel (left side in figure 168). The level of water
is adjusted by moving the vertical overflow tube
up and down (right side of fig. 168) until excess
water running through the large vessel spills
tln-ough the overflow and nothing enters the
small vessel. Levels must be set very accu-

Frc.uRE 168. — Constant level tanks designed by Galtsoff
to measure the volume of water transported by the gills
of the oyster. Dimensions of the large vessel are in by
6 by 4 inches.

186

FISH AND WILDLIFE SERVICE

rately. When the correct height of the drain pipe
is reached a few drops of water added to the
contents of the small vessel will be discharged
immediately. Setting the level of water in the
small vessel too low may lead to serious error as
the water will be forced through the gills by
gravity. Since readings may be grossly influenced
by poorly adjusted levels, repeated checking of
the overflow must be performed at regular and
frecjuent intervals.

The rate of supply of sea water into the large
vessel should e.xceed the expected maximum rate
of water transport by the oyster, which rarely
exceeds 30 1. per hour. After equilibrium in the
water levels in the two communicating vessels has
been established , the tank is ready for use. Before
the oyster is placed in the tank the moUusk is
wrapped in thin rubber dam or in plastic sheeting
cut in the shape of an apron. This technique,
suggested first by Moore (1910), was adapted for
oyster research by Nelson (1936, 1938). A tri-
angular piece of sheeting cut to fit the shape and
size of the oyster is spread on a table. After the
valves are thoroughly scrubbed, washed, and
dried, the oyster is placed on its left valve on the
sheeting. The anterior half of the oyster is left
free; only the posterior half is wrapped in the
apron (fig. 168). The sheeting is attached to the
shell with melted colophonium cement, starting
with the lower (left) valve. Enough slack should
be left at the edges of the shell and at the hinge
side to allow free movement of the valve; small
cotton balls are inserted in both places to prevent
leakage of water. The two sides of the apron are
then brought together to make a sleeve of the
required diameter to fit the interconnecting trough
of the tank and are joined using hot colophonium
cement. A wide test tube is inserted in the apron
and the apron sleeve is sealed by pressing the two
sides of the apron against the glass while applying
the cement. Before the oyster is put in the tank
the apron should be tested against leakage by
gently blowing air into the sleeve.

A small metal loop cut from the end of a paper
clip and cemented to the Ihxt valve is used to at-
tach a string leading to the kymograph lever,
which records the shell movements. The water
levels in the tanks are checked again; the oyster
is placed on a stand inside the tank and if necessary
immobilized by plastic clay; the sleeve of the apron
is drawn over the trough (or tubing) and is secured
by a piece of string; the writing lever of the kymo-

graph is connected to the valve. The last ciieck
for a possible leakage of water is made after the
oyster opens its vah'es and begins to transport
water. The escape of water through the seams
of the apron can be detected by adding carmine
suspension and watching the currents. In a
correctly adjusted set all water transported by
the gills enters tiie small vessel through the sleeve
and can be collected, measured, and analyzed.

The transport of water can be recorded con-
tinuously by using various automatic devices, such
as electric drop counters and dumping vessels.
The latter turn over after the water has reached a
certain predetermined level. Any of tliese devices
can be connected to tlie kymograph lever which
makes a mark on a rotating drum. The shell
movements and the temperature of water are
recorded sinmltaneously on the same sheet. The
arrangement of various parts of an apparatus for
recording the oyster activity and for collecting
samples of water before and after its passage
tlu'ough the gills is shown diagrammatically in
figure 169. The oyster in this diagran: has been
placed in a constant level tank A, whicli is provided
with two connecting vessels B and C A valve
l)etween A and B may be lowered to disconnect
tank A, and cap L is used to close the connection
between vessels B and C. Vessel B is slightly
larger than C and is used for taking samples of
water for plankton study or for gas analysis. The
water in this vessel is protected from direct contact
with air by the paraffined float H. The sample is
taken through the drain tul)e X. The small vessel
C has an overflow Y, which controls the levels of
water in all three vessels. From the overflow Y
water is delivered through tube Z to a dumping
vessel E, which is mounted on a horizontal axis.
A diagonal wall divides the vessel into two parts,
of wliich only one (facing the reader) is being filled
with water. Float F activates the system of
levers and releases the catch L which holds the
\essel in an upright position. The vessel over-
turns but snaps back into the position shown in
the diagram. Proper adjustment of the vessel is
obtained by attaching small weights (not shown
in the diagram) to its bottom. The vessel with
its levers is mounted on a solid frame D set on a
heavy concrete platform. The water discharged
at each dump activates the springboard G, which
is connected to a writing lever N. Shell move-
ments are recorded by tlie lever M; the electric
time sional S connected to a timer T records time

TRANSPORT OF WATER BY THE GILLS AND RESPIRATION

187

Centimeters

50

Figure 169. — Diagram of a setup for simultaneous recording of the rate of water transport and shell movements of the
oyster. A — vessel with oyster; B and C — small connecting vessels; D — frame of the dumping vessel; E — dumping
vessel; F — float; G — springboard to record each overturn of the dumping vessel; H — paraffined float; K — slow motion
kymograph; L — cap to close the connection between the two vessel; M — lever recording shell movement; N — lever
recording the dumping of water; O — overflow; P — sea-water supply; Q — constant level siphon; R — barrels; S — signal
magnet; T — electric time recorder; X, Y, W — tubes for taking samples of water; Z — overflow tube leading to dumping
vessel. Temperature recorder is not shown.

iiiterviils. The supply of sea water is delivered to
the barrels R set on top of tlie stand, and the
ii\ erflow siphon Q keeps the water in the containers
at a coitstant level and insures a uniform rate of
deli\ery of water to the experimental tank A.

The size and shape of the dumping vessel run
he modified to suit the purpose of the experiment
and to facilitate its operation. The capacity of

the dumping vessels made in my laboratory at
Woods Hole varied from 55 ml. to 226 ml. The
methods described above were successfully used in
a number of investigations (GaltsofI, Prytherch,
Smith, and Koehring, 1935; Chipnum and Galtsoff,
1949a, 1949b).

All dumping vessels require frequent adjust-
ments and become unreliable if used continuously

188

FISH AND WILDLIFE SERVICE

for several days. For long-term observations
it is more practical to use a water v/heel made with
two plastic disks mounted on a horizontal glass
rod about )2-inch apart. The space between the
disks is divided by radial partitions into a series
of triangular compartments. The wheel is placed
under the overflow tubing of the small vessel
which receives water discharged by the oyster.
The compartments are arranged in such a way
that when they are full of water the wheel turns
slightly and the next empty compartment moves
into position under the pipe. The wheel is kept
half submerged in sea water to prevent spinning.
Under this condition tlie rotation proceeds in
smooth steps; when the wheel makes one com-
plete turn tlie little bar attached to its side touches
a string which moves tlie writing lever and makes
a vertical stroke on a slow moving drum of the
kymograph. The construction of the wheel (F)
and the arrangement of different parts of the
set in which it was used are shown in figure 170.
The wheel is calibrated by measuring the volume
of sea water needed to make it turn one complete
revolution. The test is repeated at least 10 times,

and the average value is taken as the true capacity
of the wheel. Wheels of different dimensions
may be used. In my e.xperiments I used wheels
of about 50- and 100-ml. capacities; the readings
were accurate within ±2.5 percent.

The setup shown in figure 170 is specifically
designed for studying the effects of various con-
taminants that may be added at a known rate to
the water supplied to tank E. Sea water from the
laboratory supply pipe C is delivered to three
5-gal. carboys from which it runs into two temper-
ing jars with electric heaters (group B) and
vessel C. The water then passes into mixing
chamber D to which the solution to be tested may
be added from the two flasks 0 and N, which
contain known concentrations of chemicals or a
desired dilution of a culture of microorganisms.
Test solutions in flasks O and N may be added
directly to the gills (as shown in the diagram,
flask N) or may be delivered to the mixing cham-
ber D. If the solution is to go into the mi-xing
chamber the siphon from flasks N or O is turned
around 180° so that the tip of the delivery pipe is
at the right end of the mixing chamber D. This

Figure 170. — Setup for automatic recording of the amount of water transported by the gills of the oyster. A — series of
5-gal. containers from which sea water is delivered to the tank with the oyster; B — two containers with electric heaters
and thermostatic control (not shown in the diagram); C — constant level jar from which water is delivered to mixing
chamber D; E — tank with oyster wrapped in apron; F — water w^heel; H — container in which the water wheel is
partially submerged; K — kymograph; L — writing lever activated by the turning of the water wheel; M — writing
lever recording shell movement; \ and O — flasks containing solutions or suspensions which may be added either to
the mixing chamber or directly to the gills of the oyster.

TRANSPORT OF WATER BY THE GILLS AND RESPIRATION
733-S51 0—64 13

189

arrangement was used by Galtsoff and Arcisz
(1954) in their work on the effects of known con-
centrations of Escherischia coli on oysters.

For observations intended to last several days
or weeks it is convenient to use a slow-motion
kymograph, rotating at the speed of about 1 inch
per hour. A time marker of 1-hour or K-hour
intervals can be made by mounting a Incite disk
on a Telechron type motor making one revolu-
tion per hour. A piece of copper wire attached
to the periphery of the disk acts as a contact
which slides over the two poles of an electric
circuit and activates a small signal magnet. Using
these methods it was possible to record the ventila-
tion of the gills for 26 consecutive days with only
occasional brief interruptions for cleaning the
tank and for the removal of feces accmnulated
inside the apron. An excerpt of such a record
is reproduced in figure 171.

0 2

Centimeters

Figure 172. — Electric drop counter.

Shell movement

rm n

Open

Closed

Discharge of water- each stroke corresponds to 250ml

iiiii Ill iiiiiiiiiiiiiiiHiiiiiiiiiiiiiiiiiiiimiiiiiiiiiiiiiMi

Figure 171. — Typical record of the shell movements
(upper line) and rate of transport of water by the gills
(second line) of an adult C. virginica kept in running
sea water at the fishery laboratory in Woods Hole at
the temperature of 21° to 22° C, Each vertical stroke
corresponds to the discharge of a dumping vessel of
250-ml. capacity. Time interval, 1 hour.

An electric drop counter (fig. 172) may be used
instead of a dumping vessel or water wheel if the
volume of water passed through the gills is small,
as for instance in juvenile American oysters or in
Ostrea lurida. The drop counter is made of a
short section of glass tubing with two platinum
wires sealed opposite each other. Each time a
drop of water falls between the wires the contact
is completed and electric current from a trans-
former or a battery activates the signal magnet
and makes a mark on the kymograpli drum. The
counter works satisfactorily in water of liigli
salinity but is not suitable for brackish water.
The number of drops per unit of time is counted
from the kj-mograph record shown in figure 173.

190

Current indicators. — The relative velocity of gill
current can be studied by recording on a kymo-
graph drum the deflections of a shallow cone
placed in front of the cloacal opening (Hopkins,
1933). The cone C (fig. 174) about 5 cm. in
diameter at its open end is made of lightweight
paper waterproofed by dipping in a dilute solution
of gum damar in xylene. The cone is mounted
on the lower end of vertical rod F^, which rotates
on horizontal a.xis A and moves the horizontal
lever F which indicates the cm'rent. The entire
system is very light, since lever and rod are made
of straw, and it is accurately balanced by con-
necting the vertical rod F' by a hair to a simple

I I I I I I I I I I I I I I I I I I I I I I I I

— \ \ — \ — I — \ — I I I I I

(— I — \ — I — I — I — I — \ — \ — I — \ — I — h

I I I

I I I

I I I I I I I I I I I I I I I I I I I I I I I

Figure 173. — Four parts of a record of the rate of water
transport by the gills of a 2-year-old C. virginica ob-
tained with an electric drop counter. Upper lines —
each stroke corresponds to one drop of water discharged
by the oyster; lower lines — time intervals of 1 sec.
Temperature of water 22.2° C.

FISH AND WILDLIFE SERVICE

i

F^

Figure 174. — Diagram of apparatus designed by Hopliins (1933, p. 474) to record relative rate of cloaeal current and
shell movements of oyster. S — lever for recording shell movements; s — fixed bar indicating on liymograph paper
closed position of valves; F — lever recording cloaeal current; f — fixed bar indicating zero position of F when no
current is present; F' — vertical rod bearing cone C against which the current strilces and turns it on axis A; balance
B; adjustable weight — W.

lever B with an adjustable weight W. The cone
and current recording lever are fixed to a single
stand and so placed that the cone is directly in
the path of the cloaeal current. The lever S
records the movements of the upper valve of an
oyster immobilized in cement. The two fLxed
bars s and f shown at left are set in such a way
that they record continuously on the kymograph
paper the closed position of the shell (s) and the
zero position of current flow (f). The actual
amount of water transported by the gill cannot be
measm'ed by this method, but the relative values
corresponding to the rate of discharge are com-
puted by measuring with a planimeter the area of
the record enclosed between the lines made by the
excursions of the lever dm-ing a known time. A
sample of the record obtained by this method is
shown in figure 175.

Records of changes in the velocity of the cloaeal
current obtained in this manner can not be ac-
curately calibrated. In my experience a cone
placed in front of the cloaeal current frequently
fails to come back to the zero position and the
entire delicate system easily gets out of adjust-
ment. Another weakness of the method is the
uncertainty of the correct position of the cone in
relation to the diameter of the stream; it is im-
possible to know whether the entire width of the
column of mo\-ing water strikes the cone surface.

A method based on a similar principle was
developed by Mironov (1948) in the course of
studies of water filtration by Black Sea mussels.
The mollusk (M, figure 176) is placed on a hori-
zontal platform (P) mounted on the side of an
aquarium about 5 to 6 cm. below the surface of the
water. A cover slip (R) freely suspended by two

Figure 175. — Relative rate of water transport by the gill of C. gigas obtained with Hopliins' method at 13.7° C. Repro-
duced in part from Hoplcins' paper, 1933. Portion of Ivymograph record shows: relative strength of cloaeal current
F; zero position of lever corresponding to absence of current f ; and 5-minute interval on record, two vertical lines T.

TRANSPORT OF WATER BY THE GILLS AND RESPIRATION

191

Figure 176. — Mironov's method of recording the rate of
water filtration by sea mussel. A — horizontal arm for
the suspension of cover slip R; M — mussel; P — platform ;
S — scale divided in angles. From Mironov, 1948.

glass strings from a horizontal arm (A) of a rod is
lowered to such a position that the center of the
cover slip is exactly in front of the exhalant siphon
of the mussel and is perpendicular to the axis of
the cloacal current. A thin glass indicator is
cemented by canada balsam to the cover slip; its
distal end moves along the scale (S) which is
divided in angles. The deflection of the indicator
is read every 5 minutes. The apparatus is cali-
brated by recording the deflection of the glass
plate caused by a current of known velocity. For
this purpose Mironov used the difference in water
levels in the two ac^uaria A and B shown in figure
177. Water from A runs through an inverted
U-tube into tank B. A small inverted T-tul)e (T)
with one end sealed is inserted into the left arm of
the siphon (U). The recording assembly of the

type shown in figm'e 177 is placed in front of the
cover slip, and the angles of deflection correspond-
ing to the various level differences are read: milli-
meters on vertical scales L and Li placed in each
tank. The velocity of the current is computed by
using the for nulla V = -v'2gH where H is the differ-
ence of the heights of water columns in the two
connecting tanks and g is acceleration due to
gi'avity. The volume is calculated by multiplying
the current velocity V by the cross-sectional area
of the opening of the siphon (2.4 mm.^ in Mironov's
experiments). The average rate of water trans-
port was found to be 15 ml./min., or 0.9 l./hr.
Mironov's observations, made within 24 hom-s,
showed great variations in the rate of water
transport which undoubtedly indicated that the
experimental animals were disturbed and had not
reached the steady state.

Lack of automatic registration of the rate of
discharge is the obvious deficiency of the method,
which could be improved by incorporating a me-

FiouRB 1 77. — Mironov's method of calibrating the angle of
deflection of a glass plate by the currents of different
velocities. A, B— two aquaria tanks; U — inverted tub-
ing acting as a siphon; T— tubing connecting the siphon
and the tank A; the horizontal arm of the tubing is
placed in front of the swinging glass indicator; L, L,—
scales mounted on side walls of tanks to read the differ-
ences in water level.

192

FISH AND WILDLIFE SERVICE

chanical or optical device for continuous recording
of changes in the position of the indicator.

Both Hopkins' and Mironov's methods are of
hmited value because the mollusks can not be
kept in running sea water which would disturb the
recording devices. This greatly restricts the
application of their technique.

Indirect methods

All indirect methods are based on measurements
of the rate of removal of particles suspended in
water. Since the volume of water is kept con-
stant, the observations must be completed in a
relatively short time in order to avoid the effect of
metabolites. This condition seriously limits the
usefulness of the methods.

Use of turbid water. — Viallanes (1892) was the
first to determine the relative rate of removal of
suspended particles by bivalves. He selected a
nimiber of small 0. edulis (18 months old),
C. angulata of the same age, and M. edulis of "an
average size" and placed them in separate crystal-
lizing dishes in a tank with running sea water.
Dishes of the same dimensions, but without mol-
lusks, served as controls. After several days the
sediment that accumulated on the bottom of the
dishes was collected, dried, and weighed. He
subtracted the quantity of material precipitated
mechanically in the controls from the total
quantity found in the dishes with mollusks, and
assumed that the remainder was proportional to
the volumes of water filtered by them. For each
liter of water filtered by 0. edulis, the C. arKjulata
filtered 5 1. and the mussel 3 1. Dry clay was
added to the experimental tanks in the proportion
of 0.0546 g./l. In 24 hours the mussel precipi-
tated 1.768 g. of clay, C. angulata 1.075 g. and
0. edulis 0.199 g. Essentiallj^ the same crude
method was employed 34 years later by Ranson
(1926). He did not record the temperature
during the obvervations and made no attempt to
observe the shell movements of the mollusks or
to note whether the valves remained open all the
time during the experiment. In later years the
ability of water-filtering bivalves to clear turbid
water has been studied by more elaborate methods.
The amount of material remaining in suspension
has been computed from turbidity observations
made by means of a nephelometer (Mironov, 1948)
or with an electrophotometer used as a turbidim-
eter (Lund, 1957). A great variety of suspen-
sions are used in this type of experiment — India
ink, colloidal graphite, carmine powder, powdered

eggs, ground diatoms, fuller's clay, milk, calcium
carbonate, dried mud and others. Mironov
(1948) reports that the best material for this pur-
pose is nephelinic grey clay; after washing it
gives a very stable suspension in which the pre-
cipitation of particles is so slow that it has vir-
tually no effect on experimental results.

In experimental studies of the rate of water
propulsion by the California mussel, Mytilus
calif ornianus, Fox, Sverdrup, and Cunningham
(1937) used a suspension of calcium carbonate
(CaCOa). The water was stirred continuously
to keep in suspension the calcium not removed
by the mussels. At frequent intervals analyses
were made of the calcium carbonate remaining
in suspension and from these data the rate of
water propulsion was computed. The rate of
precipitation of calcium in the control tanks
suggested that in the absence of mussels the
amount of calcium suspended in water can be
expressed as an exponential function of time and
that the amount precipitated in a unit of time is
proportional to the total amount which remains
in suspension. In the mathematical treatment
of the data Fox and his collaborators applied the
following exponential equation:

P=

nm

=^»'^-(lf +

a'\t=Poe-'"

where p is millgrams of suspended calcium per
liter; Po is the amount of suspended calcium in the
closed sj^stem at the beginning of the experiment;
n the number of mussels in the vessel; m the
volume of water (in liters) transported by one
mussel in a unit of time; M the total volume of
water in the vessel; t the time; e the Napierian
base equal to 2.71828; and "a" and "b" the
logarithmic decremental constants determined
experimentally.

Under the specified conditions of the experiment
in a closed system and using the above assump-
tions, the volume of water transported by one
mussel was calculated by the following equation:

=M

b—a

The values obtained for medium sized mussels from
95 to 130 mm. long varied between 2.2 and 2.9 l./hr.
Several drawbacks are common to all methods
based on turbidity determinations. The inollusks
are kept in a closed system and are subject to ab-
normal conditions caused by high content of sus-

TRANSPORT OF WATER BY THE GILLS AND RESPIRATION

193

pended material and accumulation of metabolites.
The methods are not suitable for continuous ob-
servations since they should be completed in the
relatively short time before tmbidity of the water
is changed because of the aggi-egation and floccula-
tion of suspended particles. Finally, computa-
tion by turbidity observations of the volume of
water filtered by mollusks is based on the assump-
tion that mechanical precipitation, due to gravity,
remains constant. This, however, is not the case.
J0rgensen and Goldberg (1953) found that C. vir-
ginica removes graphite particles from 5 to 10
times faster from a 4-hom" old suspension than
from a fresh one. This effect is explained by the
difference in the size of the particles which in the
aged suspension are about 2 to 3 /j in diameter;
in the fresh one they are less than 2 ;u. By adding
small amounts of carmine suspension to the gill of
the oyster it is easy to notice that the filtering
efficiency of the gills of C. virginica and C. gigas
is not high and that many small particles appear
in the cloacal cui'rent. J0rgensen (1943) assumed
that all particles are removed as the water is
filtered through the gills, and computed the rate
of water transport m by using the following
formula:

m-

(log conco— log cone,) M
log e ■ t

where m is the volume of water (in liters) trans-
ported in 1 hour; M is the volume of suspension in
liters; conCo and conct are the concentrations of
cells or particles at the beginning of the observa-
tions and after t hours ; and e is the Napierian base
(2.71828). The formula can not be expected to
give accurate results because it is based on two
incorrect assumptions: fu-st, that all suspended
particles are removed from the water as it is being
transported through the gills; second, that the dis-
persion of particles in the suspension does not
change dming the duration of the test. J0rgen-
sen's observations showed considerable differences
in the properties of new and old suspensions of
graphite, and Chipman and Hopkins (1954) demon-
strated that the efficiency of the removal of cells
changes with time and is not related to cell concen-
trations. In their experiments the rapid rate of
removal of Nitzschia or Chlamydomonas cells was
followed by a decrease in the filtering efficiency of
the gills and in the increased retm-n to the suspen-
sion of the phytoplankton cells which had passed
through the gills.

These difficulties introduce great uncertainty in
the studies of the rate of water transport by the
gills based on turbidity determinations. Some of
the problems may be solved by the use of radio-
active plankton.

Use of radioactive plankton. — The advance of
radioisotope techniques has made it possible to
employ labeled plankton algae for determining
their rate of removal by water-filtering mollusks.
Chipman and Hopkins (1954) and Chipman (1959)
applied this method in a study of the rate of water
transport in bay scallops, and Smith (1958) ex-
tended their observations to the clam, Mercenaria
(Venus) mercenaria. Single species cultures of the
diatom Nitzschia closterium (56 m in length) and a
species of Chlamydomonas (7^ in size) were made
radioactive by the incorporation of phosphorus,
P^-. The cells grown in a culture medium (modi-
fied Aliquel solution) that contained virtually no
phosphorus except the P^^, were highly radio-
active. This isotope, emitting only beta particles
of rather high energy, was found to be useful for
this purpose. The details of the method de-
veloped in the Biological Laboratory of the Bureau
of Commercial Fisheries at Beaufort, N.C., are
described in a paper by Rice (1953). The method
is extraordinarily sensitive and allows detection
of very slight changes in cell numbers which other-
wise would have remained unnoticed. The use of
radioactive plankton presents several advantages
in studies of the functions of the gills; it allows
observations without undue increases in the con-
centration of suspended material and it makes
possible recordings of changes in the rate of water
transport which could not be detected with other
methods.

CONTROL OF RATE OF WATER TRANSPORT
THROUGH THE GILLS

Estimates of the rate of water transport by an
adult oyster, made by investigators who have
studied the problem carefully, vary from several
liters to a maximum of 34 l./hr. (Loosanoff and
Xomejko, 1946). Naturally the rate of water
transport depends on the size of the oyster, its
physiological state, and environment. The abso-
lute figures are, therefore, of little significance
unless they are accompanied by data on tempera-
ture, conditions under which the tests were made,
and size of the oysters used.

It is self-evident that the quantity of water
propelled by the gills must depend on the size of
the mollusk. No comparative data of this

194

FISH AND WILDLIFE SERVICE

nature are available for the oyster, but determina-
tions by Chipman (1955) and Chipman and Hop-
kins (1954) of the rate of propulsion of water by
the bay scallop (Pecten (Aequipecten) irradians
Lamark) clearly indicate this relationship. The
data plotted in figure 178 were taken from the
table of observations bj' these authors, who used
a suspension of radioactive Nitzschia closterium
and Chlamydomonas. A similar relationship was
reported to exist in the California mussel in which
the absolute rate of water transport through the
gill was found to be a function of the weight of the
soft parts of the mollusk (Rao, 1953). The
relationship was well defined at temperatures of
20° and 16°, but was indefinite at 9° C. Rao also
referred to the activity of mussels from various
geographical regions. He stated that, regardless
of temperature, mussels from higher latitudes
transport water at a greater rate than mussels
taken from the lower latitudes. The observations
were of brief duration, lasting from 1 to 3 hours and
therefore can not be considered as representative
of typical behavior of bivalves over longer periods
of time.

In the Bureau's shellfish laboratory at Woods
Hole records taken continuously for several weeks
show considerable variability among oysters of

16
15-
J4
13
12-
II
a:'°

09
K e

UJ

U)
UJ ^

4
3
2

41 43 45 47 49 51 53 55 57 59 61 63 65 67
AVERAGE SHELL HEIGHT IN MM

Figure 178. — Mean rate of water transport of scallops
(Aequipecten irradians) of different shell length kept in
sea water at room temperature ranging from 21.9° to
25.8° C. The plotted values represent the averages of
6 to 11 speOimens. (From the data of Chipman and
Hopkins, 1954).

equal size and origin that are kept under identical
conditions. These changes in the rate of water
transport can not be correlated with changes in the
environment. In the tests only large oysters (10
to 12 cm. in height and 6 to 8 cm. in length) were
used. The}' were healthy, free of boring sponge,
Polydora, and other commensals. Daily fluctua-
tions of temperature did not exceed 1° to 2° C.
and salinity changes were less than 0.1 %o. The
range of daily fluctuations in the rate of ventila-
tion by a single specimen varied from 9.9 to 24.3
l./hr. Ln 1 day, and from 1.1 to 24.3 l./hr. 2 days
later. The total quantity of water transported
daily by this oyster in the 2 consecutive days of
recording was 77.5 and 457 1. In the other
oyster tested within the same month of July the
range of daily fluctuations in the rate of water
transport varied from 0.2S to 3.31 l./hr. in one
day to 5.0 to 13.0 l./hr. the following day. The
total quantity of water transported in these 2 days
was 8.6 and 239 l./day respectively.

The more than 2,000 records of daily activities
of oysters accumulated in the course of many
years of my studies confirm this great variation.
Some of the records were made continuously for
33 days, others were interrupted after 2 to 3 days
of observations. All the records were obtained
using the technique shown in figures 169 and 170.

STEADY STATE

Ventilation of the gills may continue for hours
without interruption or significant changes in the
rate of water transport. This condition, which
may be called a steady state, occurs when temp-
erature, salinity, and food content of the water
remain constant and the oysters are not disturbed
by sudden changes in illumination, vibrations, or
other mechanical stimuli. The heart rhythm
during the steady state remains constant. Judg-
ing by the rate of formation of fecal ribbons, the
ingestion of food during these periods continues
without interruption, provided the water does not
contain excessive amounts of detritus, clay, or
plankton which may stimulate the formation of
pseudofeces and cause frequent snapping of the
valves. The temperature at which the steady
state was observed ranged from 15° to 25° C.
It is conceivable, however, that it takes place at
other temperatures.

An example of the steady state in oysters is
shown in figure 179. In this experiment two
oysters of approximately the same size were ob-
served simultaneously. Their activities were slightly

TRANSPORT OF WATER BY THE GILLS AND RESPIRATION

195

water transport

iiiiiiiiiiiiiiiiiiiuiiiiiiinuuiuii iiihiuiiiiiniihiiiuMmiiimiiiiii iiliiuiiiiiuiiiniiiiiiuiiiuiiiiiiiiiiiiii[iiiiiiiiii]HiMiiiiiilj|iiliiiiliiiiiiiiiimiHIMiuMiiimii»iii»i»uiiMiii

shell movement

JLi_LJuLJLJLJL_J L < .i , 1 U

water transport

1

iiiiiiiii(iiii«(i(i(iiwmmi(ii(((finmiiiii((mini(i!iii«i(i'

stiell movement

«

lUJJoU^fjOiLkKJ-JJ-J-

X

_L

_L

Figure 179. — Kymograph tracings of water transport and shell movements of two adult oysters. Each vertical stroke
of the first and third line represents the discharge of 57 ml. of water (the capacity of the dumping vessel). Temp-
erature 19.5° C. Salinity 31.2 °/oo. Oyster dimensions: 10.6 x 6.5 cm. (upper); 10.5 x 6.6 cm. (lower). Time interval,

1 hour.

different, although both were kept under identical
conditions; water was delivered at a uniform rate
from a common supply tank, and the temperature was
kept constant at 19.5° ±0.1° C. The upper
record shows that during the S hours of obser-
vations the rate of water transport of one oyster
was fairly uniform, varying between 19 and 21 l./hr.
Occasional contractions of the adductor muscle
were followed by an immediate return to the
former tonus level. In the second oyster (lower
part of figure 179) the rate of water transport
decreased slightly from 14 1. per hour at the start
to 11 l./hr. at the end of the record; its shell
movements were more frequent and less regular
than in the first oyster. It is apparent from these
and other observations of the same type that
imder identical conditions of environment the
rate of water transport may be different, depend-
ing on the intrinsic state of the organism.

Water transport by the gills is not carried on
with a machine-like performance controlled en-
tirely by such environmental factors as tem-
perature, salinity, chemical composition of water,
etc.; it is adjusted to or governed by the needs of
the mollusk. It may be assumed that the var-
iable daily requirements for food and o.xygen
and the necessity of eliminating the metabolites
determine both the duration of the activity and
the rate of water transport. In addition, the

rate of water transport is affected by irregularity
of gill activity shortly after spawning during the
summer. Oysters often maintain a nearly con-
stant rate of water propulsion for 2 or 3 days.
Invariably these periods are followed by periods
of partial or total inactivity (closed valves)
manifested in greatly reduced rates of water
transport or its complete cessation. It appears
reasonable to deduce that the needs for food,
elimination of products of metabolism, and require-
ments for oxygen determine both the number of
hours per day the oysters stay open and the rate
at which water is being transported through
the gills.

REDUCTION OF WATER TRANSPORT

Increased and irregular shell movements are
usually associated with a decreased rate of water
transport. This relationship is apparent in trac-
ings which were made shortly before the closing
of the shells or immediately after their opening.
The records of three oysters kept at a temperature
of about 20° to 22° C. (fig. 180) show that in all
of them the rate of water transport slowed down
before the valves began to close. Complete clo-
sure of the valves took place in 72, 15, and 18
minutes after cessation of the current. In the
majority of cases examined the time intervals
between the resumption of tlie cloacal current to

196

FISH AND WILDLIFE SERVICE

woter transport

shell movement

JJLljJjJLJLijJj#^

water transport

imiiiJmiiiiiiumu-

shell movement

--wJ~^

water transport

uiiMimiiiiaouuiiiiMMmi-

shell movement

I

_L

J_

Figure 180. — Tracings of the rate of water transport and
shell movements of three adult oysters, C. virginica at
Woods Hole. Temperature 20° to 22° C. July. Ver-
tical strokes of water transport hne correspond to the
discharge of 247 ml. of water by the dumping vessel.
Time interval, 1 hour.

full velocity and the opening of the valves were
about equal (fig. 181).

A reduction in the rate of water transport may
also be caused by the slowing down of the lateral
cilia of the gill filaments. To studythe activity
of these cilia it is necessary to eliminate the
effects caused by the movements of the shell and
mantle. A record of the activity ol the lateral
cilia can be obtained with an electric drop counter
placed under the small tank (fig. 168) to receive
water discharged through the cloaca. Parts of
such a record made in the summer at Woods Hole
are reproduced in figure 173. Interruption of a
steady state shown on the second pair of lines in
figure 173 was caused by slight tapping against the
side of the experimental tank. The lateral cilia

are very sensitive to minor mechanical stimuli
and slow down at the slightest disturbance. These
interruptions were of brief duration, and the
preceding rate was restored within a few seconds
(line three).

By using the drop counting technique it is
possible to observe minor fluctuations in the ac-
tivity of the lateral cilia and to demonstrate their
responses to changes in temperature, salinity,
different concentrations of drugs, food particles,
etc. In a graphic summary of one such record
(fig. 182) the average number of drops per 5 sec.
was plotted against time shown at 5-sec. intervals.
The rate of current remained fairly constant with
the exception of the period between 110th and
145th sec. when a suspension of eggs was added
to the gills. The reduction was temporary, and
the normal rate was soon resumed.

The dilation and constriction of the ostia and
the expansion and folding of the gill lamellae also
affect the rate of water transport. The effect of
these minor adjustments can not be measured
separately from the activity of the lateral cilia.
I have noticed, however, that changes in the
position of the gill lamellae and the expansion or
constriction of the ostia are usually associated
with the muscular activities of the adductor
muscle and the mantle.

DISSOLVED ORGANIC SUBSTANCES AND
RATE OF WATER TRANSPORT

Collier and his associates (1050, 1953) reported
that sea water of the Gulf of Mexico contains cer-
tain organic substances which have the general
chemical cliracteristics of carbohydrates. These
substances respond to analytical tests with
N-ethyl-carbazole (or with anthrone, Lewis and
Rakestraw, 1955) and were reported to occur in
concentrations up to 50 mg./l. The figm-e greatly
exceeds the concentrations found in sea water
by others. According to the reliable studies of
Krough (1934) the content of dissolved organic
matter in sea water remains fairly constant at the
level of about 5 mg./l. It was further claimed
by Collier that the carbohydrates occur in variable
concentrations in coastal water near Pensacola,
Fla., and that their presence greatly influences the
shell movements and the rate of water transport
by C. virginica. Since in their experiments the
response of the oysters was not consistent with the
concentrations of carbohydrates determined by
carbazole reagent, the authors tried to overcome

TRANSPORT OF WATER BY THE GILLS AND RESPIRATION

197

water transport

jjd^JMjimM^

shell movement

-L

_L

X

Figure 181. — The shell movements and rate of water transport of an adult C. virginica preceding the closing (leftside)
and following the opening (right side) of the valves. Temperature of water 22.5° C. Time interval 1 hour.

the difficulty by assuming that "each oyster ap-
pears to have a threshold limit to the carbohydrate
below which it will not pump."

The existence of a general chemical factor which
controls the principal activity of the oyster would
be of great importance to the study of feeding and
nutrition of marine invertebrates. It seems that
if such a factor actually exists then Putter's
theory of the significance of dissolved organic
substances in the feeding of marine invertebrates
should be reinvestigated, especially in view of the
assertion made by Collier and his colleagues that
"the oysters remove variable quantities (up to
50 mg./l.hr.) of the carbohydrates from sea water."

a 4
o 3

50 75 100 25

75 200 25 50 75 300 25 50 75 400 25
SECONDS

Figure 182. — Effects of egg .suspension on the water
transport by the lateral cilia of the gill of a ripe male
oyster, C. virginica. Data from the drop counting record
are plotted as averages of 5 sec. intervals. Temperature
of water 22.5° C.

An attempt to identify the substances concerned
was made by Wangersky (1952), who reported
the isolation of a compound having the absorption
spectrum of dehydroascorbic acid and the presence
of a substance which "gives some indication of
rhamnoside" and is found in the inshore waters
of the Gulf of Mexico in concentrations up to

0.1 g./l.

A description of the reagents and methods for
carbohydrate determination can be found in the
paper of Lewis and Rakestraw (1955) who remark
that in general, the N-ethyl-carbazole method
(used by Collier, Ray, Magnitzky, and Bell) is
"considerably less satisfactory" than the anthrone
method.

The N-ethyl-carbazole method is as follows: 1 g.
of the reagent recrystallized from ethanol and
water is dissolved in one liter of 90 percent sulfuric
acid cooled in ice. The acid should be of highest
purity, stored in glass-stoppered bottles. The so-
lution is stored in a dark bottle and kept refriger-
ated. Exposure to air and sunlight must be
avoided as much as possible. Under these condi-
tions the reagent is stable for at least 2 days.

A 2.5-ml. sample of filtered (or centrifuged) sea
water is transferred to a 60-ml. bottle, 22.5 ml. of
N-ethyl-carbazole reagent is added, and the sample
thoroughly mixed. The sample is immediately
placed in a water bath at 70° C. ( ± 0.2° C.) and
left for exactly 30 minutes. After 15 to 20 min-
utes in a refrigerator the sample is allowed to come
to room temperature. The optical density at 562
mix is determined between 30 to 60 minutes after
the removal of the sample from the water bath.

198

FISH AND WILDLIFE SERVICE

Standard curves and reagent blanks are deter-
mined daily with double distilled water. The
reddish-violet reaction product is unstable. It is
easily destroyed by sunlight and oxidizing agents
with a resultant dark green coloration. In filter-
ing the sample it is necessary to establish that
passage through the filter does not introduce ex-
traneous carbohydrates.

Collier, Ray, Magnitzky, and Bell (1953), ex-
pressed the results of their determinations in terms
of concentrations of arabinose (in mg./l.) although
the substance or substances present in the water
were not definitely identified. They state that
they "may not be true carbohydrates."

A series of analyses using the N-ethyl-carbazole
method were made at Woods Hole of samples of
sea water pumped from the harbor to the labora-
tory. In July 195.3, the carbohydrate content was
low, varying from 0.3 to 1.8 mg./l. Many deter-
minations giving values of about 0.1 mg./l. were
almost below the threshold of sensitivity of the
method and many others had to be discarded be-
cause of contamination of the samples or deteriora-
tion of the reagent.

A study of the records of shell movements and
water transport of oysters in relation to the natural
fluctuation of the carbohydrate content of sea
water in which they were kept gave negative re-
sults. No evidence was found to support the view
that the opening of the valves and the resumption
of water transport were associated with the in-
crease of carbohydrate concentration from 0.3 to
1.8 mg./l. The oysters opened and closed spon-
taneously regardless of the slight fluctuations in
the content of the substances which react with
carbazole. Several excerpts from the laboratory
records presented in table 20 illustrate this point.
Within the range of concentrations found in the
water, the high and low degrees of activity were
not correlated with the changes in the carbohy-
drate content (compare the behavior of oysters B,
B' and C, D'). In each case the recording was
continued for a period varying from 6 to 8 hours.
After periods of inactivity the oysters B', D, and
D' opened and resumed water transport, while
there was no change in the concentration of carbo-
hydrate. I deduce from these observations, which
were repeated several times with similar results,
that the presence of carbohydrates within the
range found naturally in Woods Hole sea water
had no effect on the oysters and is not a factor
which controls the function of their giUs.

Table 20. — Carbohydrate concentration in sea water {mg./l.),
shell movements and water transport in C. virginica at
Woods Hole

[Active shell movement means frequent opening and closing of the valves]

Oyster Xo.

Temper-
ature

"Car-
bohy-
drate"

Shell movement

Water
trans-
port

A

" C.
16.5
16.5
15.6
16.0
21.5
21.5
21.5

mg.ll.
1.3
1.1
1.8
1.8
0.8
1.0
0.6

Active

l.lhT.
0

Ai

Open

5 0

B

Active

12 0

B'

C _

Active

15 0

D.__.

Closed

Di_

Closed

0

The determinations of the concentration of
carbohydrates in Woods Hole water are in agree-
ment with the findings of Lewis and Rakestraw
(1955) for the Pacific Coast, where they encoun-
tered carbohydrates in quantities varying from
0.1 to 0.4 mg./l. and as high as 8 mg./l. in coastal
lagoons.

If carbohydrates did in fact influence the rate of
water transport it would be reasonable to expect
that the addition of these substances in quantities
exceeding their concentration in natural water
would produce a measurable effect. Quantities
of various carbohydrates were added at a known
rate to the mixing chamber from which the water
was supplied to the constant level tanks with
oysters (Galtsoff and Arcisz, 1954). The fol-
lowing substances were used in concentrations
ranging from 10 to 100 mg./l.: arabinose, fructose,
dextrose, maltose, and ascorbic acid. The tests
were continued for several hours. In all cases
there was no evidence that the increase in carbo-
hydrate concentration in any way affected the
rate of water transport or shell movements of the
oysters.

In some instances (first column, table 21) there
was a gradual increase in the activity of the gills
which cannot be attributed to the presence of
arabinose since the rate of water transport con-
tinued to increase for several hours after the return
of the oyster to natural sea water.

Similar results were obtained in another set of
experiments using arabinose and fructose. The
following rates of transport of water were recorded:

Before adding arabinose

9.5 to 11.4 l./hr. during 2 hours
In arabinose solution (65 mg./l.)

7.6 to 13.3 l./hr. during 4 hours
Return to natural sea water

19.0 to 26.6 l./hr. during 3 hours

TRANSPORT OF WATER BY THE GILLS AND RESPIRATION

199

Table 21. — Rate of water transport, in liters per hour, of
three adult C. virginica in natural sea water and in xoater
containing 1-arabinose in the concentrations of 65 mg.,
80 mg., and 100 mg./l. Salinity 31.2 "/co

[In all three experiments the shift from natural sea water to arabinose solution
in sea water and again to sea water was made at the end of tlie hour shown
in the first column without disturbing the oyster. Fifteen minutes were
needed to fiush the experimental tank with natural sea water.]

Water transport in sea water and in arabinose solution in sea water

Time

in
hours

Water
transport

Temp.

Water
transport

Temp.

Water
transport

Sea
water

Arab.

65
mg./l.

Sea
water

Arab.

80
mg./l.

Sea
water

Arab.

100
mg./l.

Temp.

1

2

3

l.lhT.
5.7
4.7

l./tir.

"'i'.j'

3.8
6.7
5.7
5.7

°C.
19.8
19.8
19.9
20.1
20.2
20.3
20.4
20.8
20.9
21.2
21.2

l.lhr.
3.8
2.4

"'zi'

4.1
4.1

l./hr.
1.6

° C.

18.2
18.2
18.3
18.3
18.0
17.9
17.9

l.lhr.
6.6
6.6
0.3
3.8
3.8

""e'V

l./hr.

"'h'l'
6.7

17.5
17.5
18.0

4

18.5

5

20.0

6

19.8

20.0

g

9.0
11.4
13.3
10.4

20.1

10

In another test using an oyster with a h)\ver
rate of water transport the results were as follows:

Before adding arabinose 3.9 l./hr. during 2 hours

After addition of arabinose (85 mg./l.)

4.3 to 4.6 l./hr. during 2 hours
Return to natural sea water

5.6 to 6.0 l./hr. during 2 hours

During both tests the salinity of the water was
.31.2 °/oo and the temperature varied between
18.0° and 19.5° C. In the concentration of 102
mg./l. of fructose the following rates were
observed:

Before adding fructose

3.9 to 4.1 l./hr. during 2 hours

In fructose solution 2.8 to 4.7 l./hr. during 3 hours

Return to natural sea water

4.3 to 4.5 l./hr. during 2 hours

No effects were observed even when the con-
centrations of carbohydrates were increased to
0.5 percent. The rate of water transport in
natural sea water varied between 8.4 and 10.8
l./hr. and from 7.2 to 10.8 in the arabinose solu-
tions. Similar negative results were obtained in
1 percent maltose and 1 . percent fructose in sea
water and in various concentrations of ascorbic
acid.

Additional tests with ascorbic acid were made
with the carmine cone method for measuring the
velocity of the cloacal current. The results
(table 22) show lack of any effect on tlie efficiency
of the lateral cilia in concentrations var\-ing from
10 to 50 mg./l. Velocity of the cloacal current is

given in table 22 as an average of 10 consecutive
i-eadings made at 2-minute intervals. Fifteen
minutes elapsed between each group of 10 readings.
The concentration of 400 mg./l. (0.04 percent)
completely inhibited the current.

My observations are in full agreement with the
results obtained by Butler and Wilson (1959),
who presented conclusive evidence that the
increases and decreases in the rate of water trans-
port by oysters (C virginica) at the Bureau's
Biological Laboratory at Gulf Breeze, Fla., (the
place where CoUier's experiments were conducted),
are not correlated with the changes in the con-
centrations of carbohydrates in the water and that
there is no "minimal threshold level of carbo-
hydrate concentration below which oysters fail
to pump."

The experimental studies show that the organic
substances which give an N-ethyl-carbazole reac-
tion have no effect on the water transport of oysters
in the concentrations in which these substances
are encountered in the tidal waters of Cape Cod.

RESPIRATION

Exchange of gases takes place primarily in the
gills, but the mantle also has a role of lesser
importance in the respiration of bivalves. Obser-
vations on the comparative rate of oxygen con-
sumption by various tissues of the oyster have not
been made, but the data on oxj'gen consumption
by the gills and mantle of the hard clam Mer-
cenaria (Venus) mercenaria are available. Hop-
kins (1946) compared the oxygen consumption of
the mantle of that species with that of the gills
and found that the oxygen uptake by the gills
varied from 815 to 912 and that of the mantle
only from 11.73 to 15.52 cu. mm./hr./g. of dry

T.4BLE 22. — Effect of ascorbic acid on the velocity of cloacal
current of C. virginica

(Carmine cone method. Temperature 19.6° to 20.6° C. Each figure of
velocity is an average of a group of 10 consecutive readings. Time inter-
val between each group— 15 minutes.]

Ascorbic acid

Velocity of cloacal current

In normal
sea water

In water
with acid

pH

10

Mg.ll.

cm.jsec
2.1
2.6
3.3
4.3
2.5
2.6
2.1
3.3
4.7

cm.lsec
2.0
2.9
3.7
4.0
2.6
2.8
2.0
3.7
no current

8.1

10

8.1

10

8.1

15

8.2

15

8.0

15

8.0

50

8.0

50 -

8.0

400

8.0

200

FISH AND WILDLIFE SERVICE

tissue. Similar differences in ratio were observed
during all seasons although the absolute figures
of oxygen consumption varied. It is probable
that a similar ratio may be found in oysters and
other bivalves.

METHODS OF STUDY

In the old method of determining the rate of
oxygen consumption, oysters were put in a small
container filled with sea water wliich was analyzed
for oxygen content at the beginning and at the end
of the test. The method was crude since no atten-
tion was paid to the increase in the concentration
of metabolites in the water or to the opening and
closing of the shells during the test period. The
results obtained under such conditions were
erratic. Some of the defects of this method were
eliminated by using the open-chamber technique
(Galtsoff and Whipple, 1931). The oyster was
mounted on a support and placed in an open jar
in sea water under a layer of paraffin oil. Shell
movements were recorded on a kymograph. The
water in the jar was gently stirred, and the volume
drawn off for oxygen determination was replaced
by fresh sea water of known oxygen content. The
temperature was kept constant by placing the
respiratory chamber in a water bath equipped
with proper temperature control. The sea water
used for the test was filtered to eliminate the effects
of photosynthesis and respiration of plankton.

More reliable results were obtained by using the
modified respiration chamber of Keys (1930a,
1930b) which was designed originally for studies
of oxygen consumption by fishes. The method
was used in my laboratory to determine the
fluctuations in the rate of oxygen consumption by
oysters that were kept for several hours in slowly
running water of constant oxj^gen content (Galt-
soff, 1947). During the period of testing the shell
movements were recorded and the position of the
borders of the mantle was observed. The appa-
ratus (fig. 183) consists of a respiratory chamber A
submerged in a large water bath in which the
temperature is kept constant within ±0.2°C.
Filtered sea water of known oxygen content is
supplied by gravity from a battery of carboys, and
a uniform rate of delivery is controlled by head
pressure kept constant in a small delivery vessel
H. The water from the carboys is fed to this
vessel at a constant rate; and excess water is
voided bj' suction (on the right side of vessel H)
in order to maintain the constant level in H. The
tubing that leads from vessel H is divided at I into

two branches of equal diameter, one leading to the
collecting vessel L, the other to the respiratory
chamber A. The constant rate of flow is main-
tained by means of two capillary glass tubings of
equal diameter O inserted in the delivery tube.
Stainless steel valves, pinch and glass stopcocks
were found unsuitable for this purpose because of
the slight shifting of their moving parts. Glass
tubings of appropriate diameter were selected for
each test from a set of several calibrated capil-
laries kept on hand, and the rate of flow of water
was carefully checked at the beginning and end
of each test. Before entering the respiratory
chamber A, the water passes through a glass coil
B (shown in figure 183 in a vertical position but
actually lying flat on the bottom) which is com-
pletely submerged in water bath C. The bath is
equipped with a constant temperature controller
(not shown in figure 183). The water leaves the
respiratory chamber through an outlet on the top
and runs to one of the collecting cylinders K;
by using a three-way stopcock J the flow of water
may be shifted from one collecting cylinder to the
other. The cylinders are suspended from pulleys
and are counterbalanced by weights N. When
empty the cylinders are raised above the water
tank M; as they are filled with water they descend
until they are partially submerged. Heavily
paraffined wooden floats prevent direct contact of
water collected in the cylinders with the air. For
taking samples the cylinder is disconnected from
the respiration chamber by turning off the stop-
cock J so that water is diverted to the second
cylinder. Then the closed cylinder may be lifted
out and the water taken through the drain cock at
the bottom. Glass stoppered Erlenmeyer flasks
of 100-ml. capacity were used for sampling. The
sample for analysis is taken at the middle level of
the cylinder between the 300- and 500-ml, marks.
Water which runs directly from the supply carboy
is sampled in the same manner. The difference
in the o.xygen content of the water running in and
out of the respiratory chamber multiplied by the
rate of flow through the chamber gives the quantity
of oxygen consumed by the oyster in a unit of
time.

Each oyster was prepared carefully for the tests.
Shells were scrubbed with a wire brush, rinsed in
fresh water, dried, and covered with melted
paraffin applied with a small brush. During this
treatment the oyster was kept vertical with its
hinge at the bottom, and paraffin was applied in

TRANSPORT OF WATER BY THE GILLS AND RESPIRATION

201

Centimeters

Figure 183. — Metabolism chamber for determining the oxygen uptake of an oyster kept in continuously renewed filtered
sea -water. A — respiratory chamber (for details see figure 184); B — glass coil (placed horizontally on bottom) to
bring the sea water to a desired temperature; C — constant temperature water bath; D — thermometer for recording
the temperature of water before it enters the respiratory chamber; E — thermometer for recording the temperature
of water in the bath; F — slow-motion kymograph; G — supply of filtered sea water of known oxygen content; H—
constant level tank which regulates the flow of sea water through the chamber; I — T-tube connection leading to the
sampling cylinder; J — two-way stopcock; K — two sampling cylinders which receive water from the respiratory
chamber; I, — sampling cyhnder which receives sea water directly Irom supply G; M — water bath in which the sampling
cylinders are suspended; N — counter weights to balance the sampling cylinders; O — capillary tubing for regulating
the rate of flow of sea water. Constant temperature regulator, heater or cooler in bath 0, are not shown.

short strokes directed away from the edge of the
shell to avoid accidental sealing of the valves.
After careful examination and removal of superflu-
ous paraffin the oj'ster was placed in the oval
respiratory chamber (figure 184), which was built
of hea\^ plastic with a removable slanted top (E)
kept in place by two metal clamps (F). The
capacity of the chamber, which rests on two heavy
lead bars, is about 800 ml. When it is in opera-

tion, filtered sea water of known oxygen content
is delivered through inlet B and is discharged
through outlet C on the top.

The chamber is filled with filtered sea water,
the oyster is placed inside, and the cover clamped
down. All air bubbles are carefully evacuated.
To record the shell movements a glass test tube
(H) is lowered through the wide neck of the
cover E until it rests on the oyster valve; the

202

FISH AND WILDLIFE SERVICE

Centimeters

Figure 184. — Metabolism chamber, details ot construction. A — oyster resting on heavy concrete base ready for me-
tabolism test; B — inlet for sea water of known oxygen content; C — outlet; D — lead bars; E — slanted removable top
underlined with a rubber gasket; F — clamps; G— rubber balloon which serves as a flexible gasket for test tube H
which rests on the valve of the oyster; I — laboratory brush inserted into the test tube and attached to the writing
lever of a kymograph.

small rubber balloon G tied with a silk thread
acts as a fleyible, watertight gasket witii enough
slack to permit slight vertical movements of the
tube as the oyster opens and closes its valves.
The connection with the writing lever of a kymo-
graph is made by inserting a small laboratory
brush in the glass tube and attaching the metal
handle of the brush to the arm of the lever (fig.
183). Outlet C is connected to the tubing which
leads to collecting cylinders K (fig. 183). The

chamber without oyster must be checked first by
taking simultaneous samples of water from the
control and from one of the collecting cylinders.
When the samples give identical values of oxygen
content a second test is made with the closed
oyster in place in the respiratory chamber. If the
difiference in the two samples exceeds the probable
titration error, a search for trouble should be
made. Usually it can be traced to a defective
paraffin coating of the shell or to the growth of

TRANSPORT OF WATER BY THE GILLS AND RESPIRATION

203

fungi and bacteria inside the rubber connections.
To avoid this growth all rubber tubings and joints
should be periodically cleaned, dried, and steri-
lized. With these precautions duplicate deter-
minations in the Bureau's shellfish laboratory
gave consistent results, the error not exceeding
±0.01 nig. of oxygen in the 100-nil. sample.

Oysters were kept in the respiratory chamber
from 4 to 9 hours, and samples of water were
taken at half-hour intervals. The duration of the
test was limited by the available quantity of
filtered and aerated sea water. For o.xygen
determination the Winkler titration was used; in
some tests the Van Slyke volumetric method was
employed for determining the carbon dioxide and
the oxygen content of the water.

To obtain data corresponding to the level of
basic metabolism, the oysters were starved for
24 hours by placing them in filtered sea water.
This period was found to be long enough to
cleanse the intestinal tracts and to discard the
feces and pseudofeces. Throughout all the tests
the temperature of water was maintained at
25°C.±0.1°C.

MICRODETERMINATION OF OXYGEN

Oxygen content in very small volumes of water
can be found by using one of the microdetermi-
nation methods developed by Lund, 1921; Thomp-
son and Miller, 1928; Kawaguti, 1933; Krogh,
1935b; and Van Dam, 1935a. The volume of
water used for analysis in these methods varies
from a few ml. to a fraction of 1 ml. Samples
can be taken simultaneously or nearly simul-
taneously from the inhalant and exhalant currents
of a bivalve. It is obvious that such a procedure
requires great precision of sampling. This is
made possible by Krogh's syringe pipette (Krogh
and Keys, 1931) or its modification nuide by Van
Dam (1935a). The syringe pipette designed by
Krogh and suitable for delivery of small quantities

of fluid with a high degree of accuracy is shown in
figure 185. It is a glass cylinder with a carefully
ground plunger and a heavy-walled glass capil-
lary welded to the tip instead of the conventioiuil
metal injection needle of a hypodermic syringe.
Tuberculin syringes with blue plungers are suit-
able for this purpose. The glass capillary is
about 5-cm. long with a small bore of inner
diameter of about 0.15 to 0.20 mm. The syringe
is mounted on a frame of two steel rods set in a
bakelite or ebonite base. The notched bar, N,
determines the highest position of the plunger.
The volume delivered by the syringe is adjusted
to any desired fraction of its capacity by a metal
collar which may be pushed into one of the
notches and set in a fixed position by set screw S.
The pipette with the special tip answering Van
Dam's specification is not available from stock at
any scientific supply store in this country and
has to be made to order by an experienced glass
blower.

For taking samples two syringes are mounted on
an adjustable screw stand that allows fine and
independent movement in the vertical and hori-
zontal planes. The syringes are attached to the
arms of the stand by ball bearing holders H, so
that they can be set at any angle to the horizontal
plane. The stand must be heavy and must have
adjustments fine enough so that the tips of the
collecting sjTinges can be introduced into the
cloacal region of the oyster or the branchial and
anal siphons of clams without touching or disturb-
ing the sensitive tissues. The type of stand
suitable for this purpose is shown in figure 1 of
Van Dam's paper (1935a).

Samples of water for microanalysis also can be
taken by means of a siphon; the tip is introduced
deep into the cloacal chamber of a bivalve mol-
lusk, as shown diagrammatically in figure 186.
This device was used by Van Dam (1954) in his

K

^Illlllllllll.lllllWItWJttlhlw

V-2. 2CC

0 50

Centimeters

FiGi'RE 185. — Van Dam's modification of the syringe pipette for taking .small samples of fluid with a high degree of accu-
racy. H — clamp holder; N — notched bar; S — set screw.

204

FISH AND WILDLIFE SERVICE

Figure 186. — Van Dam's method of sampling sea water
from the exhalant current of sea scallop. Samples are
taken by syringe pipette P (shown in part, left side)
from the end of siphon with the lower part introduced
into the cloaca, CI. The horizontal arm of the siphon
is about 5 mm. inside the cloaca. S — scallop. From
Van Dam, 1954.

study of respiration of the scallop. The presence
of the tip of the siphon in the cloaca does not in-
terfere with the normal propulsion of water, pro-
vided the needle does not touch the scallop. Be-
fore the first samples were taken Van Dam allowed
the water from the cloaca to pass through the
siphon for about 1 hour at the rate of 1.5 ml. per
minute. Such a slow rate of collecting was con-
sidered a guarantee that the sample was not con-
taminated with outside water. The difference
between tne oxygen content of the water that
entered the gill and of the water of the exhalant
current showed the percentage of utilization of
oxygen.

In bivalves with long and narrow siphons, as
for instance Alya or Mercenaria, water leaving the
exhalant aperture probably has a uniform content
of oxygen. In species lacking siphons the cloaca
opens as a wide cone-shaped slot and the stream
of water escaping from the cloaca contains con-
siderable and variable amounts of outside water
depending on the distance from the epibranchial
chamber. This introduces uncertainty in inter-
preting the results of the test. Van Dam (1954)
found that in bay scallops the oxygen content of
the samples taken at different positions within the
cloacal current varied from 28.5 to 66.6 percent
of the oxygen content of the inhaled water. Be-
cause of this uncertainty the method does not
appear to be suitable for measuring tne true oxygen

utilization of bivalves unless the tip of the col-
lecting syi'inge is introduced deep inside the
epibranchial chamber of the gills. Since the rate
of propulsion of water and the volume of water
transported are not known, the total quantity of
oxygen used cannot be determined. This greatly
limits the usefulness of the method. The main
advantage of microdetermination methods is that
the mollusks may be kept in running sea water
under conditions which closely approach their
normal environment.

The selection of a method for respiratory studies
must be governed by the purpose and conditions
of the experiments. All closed system methods
are suitable for tests that should be completed
before the depletion of oxygen begins to affect the
respu-atory rate. The microanalytical methods
are suitable for determining the utilization of
oxygen by bivalves (i.e., the percent of oxygen
removed by tissues during the transport of water)
provided the samples of the exhalant current are
not contaminated with outside water. This is
nearly impossible to avoid in species like scallops
and oysters, which have no siphons. The use of
the respiratory chamber with a constant rate of
flow of water seems to be the most satisfactory
technique for long-term observations on oysters.
The method gives reproducible values of the
oxygen uptake over a period of many hours. The
following discussion of the respiration of the
oyster is based primarily on results obtained in
the Bureau's shellfish laboratory with this method.

OXYGEN UPTAKE

The rate of oxygen uptake is influenced by
several extrinsic and intrinsic factors. The first
group includes seasonal and diurnal changes in
the temperature and salinity of water, and the
occasional presence of contaminants or other
envu'onmental changes, such as an abundance of
unicellular algae which may depress the rate of
respiration. The existence of the intrinsic factors
becomes apparent in observations of differing
metabolic rates in oysters of known origin and
uniform size and age kept under constant condi-
tions (see: p. 207). Some of the intrinsic factors
are associated with differences in the contents
of water and glycogen in the tissues; with the
loss of solids due to the discharge of sex cells
during spawning; and with generally poor condi-
tion of the oysters. In a comparative study of
respiratory rates several precautions are necessary
to minimize the extent of individual variations.

TRANSPORT OF WATER BY THE GILLS AND RESPIRATION
733-851 O— 64 14

205

Oysters should be taken from a healthy population;
they should be devoid of parasites and commensals;
they must be of uniform size and age. The
metabolism tests should be made at constant
temperature and salinity.

Observations described below were made in
accordance with these requirements. Oysters
obtained from grounds near the laboratory varied
from 9.6 to 10.3 cm. in height and from 6.5 to
7.0 cm. in length. They were fully adjusted to the
salinity of the laboratory water. The temperature
of the water in the respiratory chamber was kept
between 24° and 25° C. but changes during each
test did not exceed ±0.1° C. The salinity of
water was kept constant at the concentration
corresponding to the salinity of their natural
environment. Tests were performed at Woods
Hole and in Milford, Conn.*

Shell movements were recorded continuously
during tests which lasted from .3.5 to 8.5 hours,
depending on the behavior of the oyster. If the
oysters remained closed for more than 30 minutes
the test was discontinued, since it was reasonable
to expect on the basis of previous experience that
the period of closure would continue for several
hours. Samples of water for oxygen determina-
tion were taken at half-hour intervals. For the
study of seasonal changes of respiratory rates the
oysters were marked by engraving a serial number
on their left valves. Between tests they were
kept in the harbor or in a large outdoor tank with
circulating sea water. The data of oxygen uptake
are expressed either as cu. ml. of oxygen (at 0° C.
and 710 mm. barometric pressure) or as mg. of
oxygen consumed per oyster per hour. (To
convert the number of ml. into mg. of oxygen the
first value should be multiplied by 1.4292.)

Oxygen uptake of animals is usually expressed
per unit of their body weight. In the case of the
oyster the use of the total weight may be mis-
leading because of the great variations in the
weight of metabolically inert shell material. It
is, therefore, more sensible to refer to the o.xygen
uptake per unit of either wet or dry tissues. The
use of dry weight gives more consistant results
because in this way the variability caused by
changes in the water content of the tissues is
eliminated. The rate of oxygen uptake by a
single oj^ster of knowni size is, however, of interest

to ecologists who are concerned with the oxygen
requirements of an entire oyster community.
Furthermore, in a study of seasonal metabolic
changes the experimental oysters could not be
sacrificed at the end of each test. Their weight
at the end of the entire series of observations
would be meaningless because of the changes in
solids. The data on seasonal variations in res-
piration are, therefore, given in the amounts of
oxygen consumed by a single adult oyster in 1
hour.

The range of individual variations in the rate of
oxygen uptake by adult oysters of approximately
equal size is fairly large. Wide fluctuations fre-
quently occur during a single test until the oyster
reaches a steady state and remains open with a
minimum of shell movement. Therefore, esti-
mates of oxygen demand based on one or two
readings made shortly after placing the mollusk
in the respiratory chamber are meaningless. As
the observations described below indicate, the
study of the respiratory rates should be based on
a series of readings continued for several hours
and made at regular intervals.

Table 23 presents a summary of observations
made on 11 adult Long Island Sound oysters,
which prior to the tests were kept for at least 4
weeks in Woods Hole harbor and were adjusted
to the salinity of the laboratory water. With a
few exceptions the o.xygen uptake of each oyster
remained fairly constant during the test. The
mean oxygen consumption per oyster per hour
varied from 3.0 to 5.8 mg. The mean value for
the entire group was 4.08 mg. of oxygen per hour
per oyster. The group apparently divided into
two classes of oysters, those with the low
metabolic rate of 2.5 and 3.6 mg. of oxygen per

Table 23. — Oxygen uptake in mg. per hour per oyster of 11
adult Massachusetts oysters during 3.5 hours at half hour
-intervals at the temperature of 34° to 25° C.

The tests were made at the Woods Hole laboratory in June before the be-
ginning of spawning. Nos. 4, 8, and 11 are females, the others are male.
Dimensions of oysters: height 9.6 to 10.3 cm.; length 6.6 to 7.0 cm.]

' I gratefully acknowledge the valuable cooperation of Walter .\. Chlpman
in conducting for me a series of tests at Milford; and to two medical students,
now doctors, John F. Reppun and George Mishtowt, who assisted me at
Woods Hole.

Hours
after

Oyster Number

start

1

2

3

4

5

6

7

8

9

10

11

0.5

4.5
5.4
4.3
4.2
4.0
3.9
3.7

1.8
2.8
3.0
3.2
3.7
3.8
3.8

2.9
2.9
2.9
2.9
2.9
3.4

5.1
5.0
5.2
5.1
5.2
5.1
5.0

3.9
4.2

'4^5"

1.9

2.5
2.0
3.7

6.3

6.7
5.7
5.9
6.6
6.8

4.8
4.7
6.0
4.8
5.1
6.2
4.6

3.6
3.3
3.8
3.1
2.9
3.0
3.1

3.7
3.8
3.7
3.4
3.5
3.7

4.8

1.0

4.8

1 5

6.1

2.0 ._..

5.3

2.5

4. 1 2. fl

6.7

3.0

3.9
3.9

2.9
2.8

5.4

3.5..

4.9

Mean

Std. dev....

4.29
±0.56

3.16
0.72

3.0
0.25

5,13
0.07

4.01
0.11

2.54
0.42

5.84
0.26

4.89
0.24

3.26
0.33

3.64
0.16

5.17
35.0

Mean of means 4.08.
Standard deviation ± 0.337.

206

FISH AND WILDLIFE SERVICE

hour, and those in which the mean oxygen con-
sumption was higher varying from 4.0 to 5.8 mg.
of oxygen per hour per oyster. These differences
could not be associated with sex or sexual maturity.
The three females of the group (Nos. 4, 8, and 11)
had a high metabolic rate, but an even higher
value was recorded for one of the males (No. 7).
Subsequent tests showed that all 11 oysters were
sexually mature and upon stimulation spawiied
copiously.

For comparison the metabolic rates of six
oysters from Onset, Mass., were determined.
The same technique was used but the duration
of each observation was extended to 7.5 to 8.5
hours (table 24). The tests were completed
during the last week of July and the first week
of August. The mean oxygen uptake in these
oysters varied from 2.99 to 4.24 mg. per hour per
oyster, and the mean value for the entire group
was 3.47. After the test was finished the oysters
were examined and found to be partially spawned,

Table 24. — Oxygen uptake in mg. of oxygen per hour per
oyster of six adult oysters from Onset, Mass.

[Tests made between July 12 and .\ugust 7 after the completion of spawning.
Temperature 25.1° C]

Hours after start

Oyster Number

11

2

3

42

5

6

0.6

4.2
2.2
2.5
1.3
3.2
3.5
3.5
3.8
4.6
3.4
3.4
3.6
3.3
3.8
3.3

5.4
3.4
3.8
3.6
3.6
3.7
3.9
3.3
3.0
3.5
3.7
3.7
4.0
3.4

3.5
3.9
5.7
3.8
6.2
4.5
3.8
3.8
3.9
3.6
4.2
3.8
3.8
4.3
4.2
4.0

4.6
4.3
4.3
4.2

4.2
3.8
4.1
4.0
4.0
2.6
1.5
1.8
1.4

4.2
3.6
3.6
3.4
3.5
3.2
3.9
3.4
3.4
2.1
1.4
1.7
1.3

3.6

1 0

2.0

1.5 -

4.8

20 - .

1.0

2.5

2.0

30

3.1

3.5

3-1

4 0 - . . _

3.0

4.5

3.4

5.0

4.4

5.5 -

3.4

6.0

3.4

6.5 -

3.4

7.0

3.0

7 5 ...

3.4

8.0

3.1

8.5

3.1

Mean ..

3.31

0.80

3.71

0.67

4.24
0.76

3.46
1.33

2.99
0.98

3.12

Standard deviationi...

0.75

whicli suggested that their lower metabolic rate
possibly was associated with the loss of sex cells.

To clarify this point three tests were made with
11 adult New England oysters of approximately
the same sizes as those of the Onset animals.
After the fu-st test, made early in July, the oysters
were induced to spawn several times in the labora-
tory and were tested again 2 weeks and then 1
month after spawning. The data shown in table
25 are the mean values of six consecutive readings.

A significant decrease in the uptake of oxygen 1
montli after spawning was found in all these
oysters. The difference was less pronounced 2
weeks after spa\vning, possibly because of the
incomplete discharge of sex cells. The rapid
decrease in the metabolic rate is shown by plotting
the rates of oxygen consumption as percentages
of the initial rate observed before spawning (fig.
187). This inference that basal metabolic rate
decreases after spawning is substantiated by data
on seasonal changes in the composition of oyster
meat (ch. XVII) which shows that the lowest

WEEKS AFTER SPAWNING

NOTE: Mean of all observations 3.47±0.48 mg. oxygen per oyster per hour .

' Partially closed from 1.5 to 2.0 hours; fully open at 2.6 hours.
' Oyster partially closed from 6.0 to 6.6 hours.

Figure 187. — Decrease in the rates of oxygen con.sumption
in 11 adult C. virginica from Long Island Sound after
spawning. The changes are plotted as percentages of
the initial oxygen uptake before spawning.

T.A.BLE 25. — Mean oxygen uptake, in mg. per hour, per oyster, determined early in July before spawning, at 2 weeks and then

1 month after spawning
The figures are the mean values of each test and were computed from six consecutive readings made at half hour intervals at temperature 24.0 to 25.0° C .

Oyster Number

Standard
deviation

1

2

3

4

6

6

7

8

9

10

11

Mean

Before spawning (July) .

6.1

6.8
5.7

4.8
4.3
3.4

4.3
4.1

2.8

7.3
5.6
4.8

5.7
4.6
3.2

3.6
4.4
4.3

8.3
5.9
4.2

7.0
6.7
4.5

4.4
4.4
3.6

5.2
4.0
3.2

7.4
7.3
6.2

5.8
5.2
4.2

±1.51

Two weeks after spawning (August)

One month after spawning (August)

±1.14
±1.08

TRANSPORT OF WATER BY THE GILLS AND RESPIRATION

207

solid content of oysters occurs shortly after the
spawning; season. It will be shown later (ch.
XIV) that the gonads of sexually mature oysters
may constitute as much as 40 percent of the body
weight and volume, exclusive of the shell. The
loss of a considerable portion of gonad tissues may
acct)unt for the lower oxygen uptake.

The amount of oxygen consumed by an orga-
nism during a unit of time depends on its weight.
In the oyster this relationship is obscured by
wide fluctuations in the proportion of solids to
water. In a series of tests made during the
second half of August at Woods Hole oysters of
different weights were selected from an oyster
bottom at Onset at the head of Buzzards Bay,
Mass. The total weight of individual oysters
varied from 80 to 203 g. and the wet weight of
their tissues ranged from 11.35 to 23.25 g. The
oysters had already spawned but still retained a
substantial amount of sex cells, with the exception
of one oyster in which the gonad was empty and
its sex was not recognizable. The data given in
table 26 are the mean values of oxygen uptake
computed for each oyster from 10 consecutive
readings made at half-hour intervals. The rate
of oxygen consumption per oyster per liour
varied from 3.97 to 7.29 mg. of oxygen. The
o.xygen consumption of the three heaviest oysters
(Nos. 1, 2, and 3) was higher than for the others,
but there are no significant dift'erences in oxygen
uptake per unit of dry weight according to oyster
size (fig. 188). The oxygen demand expressed in
this way varied between 2 and 3 mg. per hour.

In tlie majority of tests the initial oxygen
consumption measured within tiie first hour after
placing the animal in the respiration chamber
was noticeably higher than in the successive
samples. This phenomenon recorded for five

T.\BLE 26. — Oxygen uptake per hour of adult C. virginica
from Onset, Mass.

(End of .August.

Salinity of water 31.2 to 31.3°/oo. Temperature 24.0° to
25.0° C.)

Sex

Wet
weight

Dry

weight

Solids

Oxygen uptalce

Oyster

Per

oyster

Per 1 g.
dry tissue

1.

9

M
F
M
M
M
M
M
M
M

<?.

23.25

21.81

21.07

18.34

16.04

14.52

14.38

12.06

11.48

11.35

0.

2.65
3.19
2.63
2.57
2.36
1.86
2.01
2.00
1.73
1.68

Percent
11.4
14.6
12.5
14.0
14.7
12.8
14.0
16.5
15.1
14.8

Mg.lhr.
5.88
6.76
7.29
4.59
4.06
4.40
5.56
4.32
3.97
4.43

Mg.lhr.
2.22

2.12

3

2.77

4 .

1.79

5

1.72

6

2.37

2.77

S

2.16

9

2.29

10

2.63

3 7

a 6

,0, UPTAKE PER OYSTER

Oj UPTAKE PER GRAM OF DRY WEIGHT

13

15 17 19 21

WET WEIGHT IN GRAMS

Figure 188. — Mean uptake of oxygen expressed in mg.
per oyster (circles) and mg. per 1 g. of dry weight per
hour (triangles) in relation to the wet weight of tissues.
Each item is a mean value of 6 to 10 consecutive deter-
minations of oxygen consumption of a single oyster.
Temperature 24.5° C.

out of the total of six oysters (table 24), represents
the effect of an oxygen debt incurred during the
time the oysters were closed while being prepared
for the test. The rate of oxygen uptake usually
reaches a more or less stable level after a variable
period of adjustment to the new situation. In
figure 189 the rate of metabolism recorded con-
tinuously for 8.5 hours is plotted against time.
Partial closure of the valves was accompanied
by a decrease in oxygen consumption (fine B).
Both oysters A and B reached a steady level of
oxygen uptake after the initial periods of adjust-
ment, which in the case of oyster A required
4 hours.

The rate of oxygen uptake decreases with a
partial closing of the valves, presumably because
of the decrease in the rate of water transport.
In the test summarized in figure 190 the two
oysters A and B remained in a steady state for
nearly 4^2 hours. One of them (oyster A, solid
line) then began to reduce the opening between
the valves and completely closed them at l)i
hours. The decline in oxygen consumption cor-
responded to the shell movement and registered
zero at the moment the shell was closed. No
measurable changes in o.xygen content in the
water were noted after the valves remained
tishtlv closed for some time.

208

FISH AND WILDLIFE SERVICE

HOURS AFTER START

Figure 189. — Oxygen uptake, in mg. of oxygen per oyster,
per hour, measured at half-hour intervals. Tempera-
ture 24.5° C; sahnity of water 31.9°/oo. Oyster A
(solid line): wet weight of tissues 16.1 g. ; dry weight
2.5 g. Oyster B (broken line): wet weight of tissues
12.5 g. ; dry weight 1.4 g. Before the tests were made
these 6-year-old oysters from Long Island Sound were
kept for 35 days in Woods Hole Harbor. August.

Oxygen Uptake Under Continuous Pull of the
Adductor Muscle

Increased shell movements of oysters are asso-
ciated with an increased oxygen uptake. This has
been reported by Galtsoff and Whipple (19.31),
who found that oxygen consumption by the oysters
wliich made only five or less shell closures per hour
was about 20 percent lower than that of oysters
which closed and opened their valves 30 times or
more per hour. The effect on the metabolic rate
of the oyster of the resistance of the adductor
muscle to a continuous pull and of the mainte-
nance of a forced muscle tonus has been studied
for large New England oysters using the equipment
designed to determine the power of the adductor
muscle (see: p. 176, ch. VIII). The entire plat-
form upon which the oyster was mounted was
lowered into a tank of 3 1. capacity filled with
filtered sea water and covered by a layer of light
mineral oil about 0.5 inch thick. The water was
stirred gently, and the temperature was kept con-
stant at 24° ±0.3° C. Samples of water, of 100-
ml. capacity, withdrawn from the tank were
immediately replaced by an equivalent volume of
filtered sea water of known oxygen tension. An
accurate record was kept of tiie total volume of
water in the tank, and its oxj'gen content was re-
computed after every addition. During the tests,
which lasted from 3 to 6 hours, sampling was made
every 30 miimtes. The initial oxygen tension

varied in different experiments from 4 to 5 ml.
per 1. As the muscle was stretched by the pulling
force of 4 kg. (fig. 191), the oxygen uptake, which
ordinarily was 3.7 to 5.0 mg. per hour per oyster,
decreased to 2.2 mg. Similar results were ob-
tained in several tests in which the pulling force
varied from 2 to 4 kg. per oyster. In all cases the
adductor muscle was stretched but continued to
maintain tonus at a new level. In many instances
the up and down shell movements persisted but
on a noticeably reduced scale. In the majority of
cases the oxygen uptake decreased markedly to
almost zero as the shell movements were reduced.
During the test shown in figure 192 the shell move-
ments were limited and their character did not
change after the pulling force of 2 kg. was applied.
The new tonus level was accompanied by an im-
mediate decrease in the uptake of o.xygen to about
one-half its preceding rate. In 2 hours the con-
sumption of oxygen almost stopped.

In several instances a sudden increase in oxygen
consumption was observed when the oj'ster was in
the respiratory chamber despite a pulling force of
2 kg. (fig. 193). Examination of the water re-
vealed the presence of eggs or sperm released from
the gonad. The metabolic rate of the sex cells
contained in the gonads was suddenly increased as
soon as the cells were free in water. Within the
gonad tubules the sex cells are tightly packed and

AFTER START

Figure 190. — Oxygen uptake, in mg. of oxygen per oyster
per hour, measured at half hour intervals. Temperature
24.5° C: salinity of water 31.8°/c,o. Oyster A (solid
line): wet weight of tissues 15.2 g. ; dry weight 2.1 g.
Oyster B (broken line): wet weight of tissues 12.8 g. ;
dry weight 1.5 g. Before the test these 6-year-old
Long Island Sound oysters were kept for 30 days in
Woods Hole Harbor. August.

TRANSPORT OF WATER BY THE GILLS AND RESPIRATION

209

(r

UJ

H

cn

>-
o

(£
UJ

O

X

q:
ui
a.

d

UJ

<

H
3

Figure 191. — Shell movements (upper line) and oxygen
uptake (vertical bars) of an adult C. virginica before and
during the application of a pulling force of 4 kg. Sharp
inflection of the curve at about .3^4 hours corresponds to
the point of tearing off the muscle. July experiment.
Temperature 23.7° C.

their surface is not in direct contact with sea water.
The high oxygen uptake after they are shed into
the water is probably due to the greatly increased
free surface areas of these cells.

In these observations the forcible stretching of
the adductor muscle was always associated with
a decrease and eventual cessation of ciliary current.
Under the conditions of the tests the velocity of
the cloacal current could not be measured, but
decrease in current velocity was observed by the
movements of particles suspended in the water or
by the slanted position of the fecal ribbons, which
in actively feeding oysters are horizontal to the
axis of the cloacal current.

Two inferences can be deduced from these obser-
vations: First, the uptake of oxygen is dependent
primarily upon the rate of water transport by the
gills; and second, in maintaining a tonus level the
locking meclianism of the adductor muscle is not
dependent on the uptake of oxygen from surround-
ing water.

These observed rates of oxygen uptake are
considerably greater than those found by J0rgensen

2 KILOGRAMS

5 6

Figure 192. — Shell movements and oxygen uptake of an
adult C. virginica before and after the application of a
pulling force of 2 kg. Oxygen uptake in mg. per oyster
per hour is shown by vertical columns. July. Tem-
perature 22.4° C.

(1952) for adult C. virginica of Woods Hole. The
oysters used in his experiments were supplied from
the Bureau's shellfish laboratory and were ap-
proximately of the same size as those which I
tested in the preceding summers. The rate of
o.xygen uptake determined by J0rgensen was less
than 1 ml. per hour (1.5 mg. of o.xygen per oyster).

UI

I-
cn

UJ
Q.

CC

O

I

a:

UJ
CL

4-

C9

5 2

<
I-

3

CM

O

0

•b

O

2 KILOGRAMS

z
z

»
<
a.

Hi 1 _i I _ I

1 1

0.5

I 1.5 2

HOURS

2.5 3.0

Figure 193. — Oxygen uptake of an adult oyster under
normal conditions and after the application of a pulling
force of 2 kg. Sudden increase of o.xygen consumption
at 254 hours is due to the discharged sperm. Upper line
indicates shell movement. July. Temperature 24.8° C.

210

FISH AND WILDLIFE SERVICE

Unfortunately, J0rgensen does not mention the
dimensions, weight, or conditions of his oysters,
and does not describe the details of his technique.
He states, however, (1952, p. 362) that "the oyster,
Ostrea uirginica, and the ascidians Ciona intestinalis
and Alolyula manhattennis filter about 10 to 20 1.
of water for each ml. of oxygen consumed."

The low o.xygen uptake reported by J0rgensen
may have been due to the experimental conditions
and particularly to the presence in the water of
graphite particles used bj' him in determining the
rate of water filtration.

Environmental effects

Seasonal changes in the rate of oxygen uptake. —
After spawning the New England oysters pass
through a period of lowered activity and tend to
keep their valves closed, sometimes as long as
2 to 3 days; when they open again the rate of
water transport is lower than it had been before
the start of the reproductive period. Through
the cold season of the year, from October to
April, the oxygen uptake remains at a low level.
In order to obtain comparable results and to
eliminate the effect of temperature, tests of
metabolic rates were made at 25° C, using oysters
that were kept outdoors and brought into the
laboratory for 3 to 4 days before testing to adjust
gradually to the higher temperature. Exam-
ination of the data summarized in table 27 shows
that the period of lowered metabolic activities
occurred primarily during the winter.

EJfect of change in salinity and pH. — No sig-
nificant change in the respiratory rate was no-
ticed in water of lowered salinity to which the
oyster had become adjusted. In these tests
the metabolic rate was fu'st measured in water
of 31.6 7oo salinity. After the first test, which
lasted 6 hours, the oyster was transferred for 3
days into running sea water diluted with fresh
water to the salinity of 24.1 °/oo (approximately
76 percent of the previous concentration of salts).

T.\BLE 27. — Seasonal changes in the oxygen uptake in mg.
of oxygen per oyster per hour of adult Long Island oysters
about 10 cm. long and 7 cm. wide

Oyster No.

>5

a

o

>
o

Z

s

d

03

1

C3

a.

<

>>

s

3

60

5.0
3.8
3.5
4.4
5.2

4.2

2.6

2.6
2.9
2.4

2.3

....

....

2.0
2.1
1.7

•)

51

52

1.6

....

1.8

1.9

1.6
3.6
4.0
2.9
3.1

53

55

56

4.1'

3.0

i'i

3."i"

3.6

2.7

4.7
4.4
5.4

4.3
o q

62

4.7

2.6

2.0

4 3

The rate of respiration was measiu-ed on the
4th day under standard conditions. The results
of the test (figure 194) show that the rate of
o.xygen uptake in the water of lower salinity
was not significantly different from that observed
in 31.18 °/oo salinity. The tests were repeated
several times with different oysters with identical
results. In all experiments the oysters were
left in water of lower salinity for at least 3 days
to adjust to the new conditions. The effects of
gi-eater dOution of sea water have not been
studied because of the technical difficulty in
providing sufficient food to the oysters during the
prolonged periods of adjustment.

HOURS AFTER START

Figure 194. — Oxygen uptake of an adult Long Island
oyster recorded at normal salinity (solid line) of Woods
Hole (31.58 °/oo) and 4 days later at lowered salinity
(broken line) of 24.1 °/oo. All tests were made in
August under standard experimental conditions at the
temperature of 25.0° C.

Tlie pH has a very pronounced effect on the
rate of oxygen uptake. The water used in
metabolism tests was acidified by adding a quan-
tity of 0.1 N hydrochloric acid. After six or
eight readings with normal sea water, the acidified
water was turned on and the testing continued
for another 3 to 4 hours. The curve in figiu-e 195
summarizes the results of all 10 tests performed
in July to September using Long Island and Mas-
sachusetts oysters. At pH 6.5 the oxygen uptake
drops to about 50 percent of the normal rate and
rapidly decreases to less than 10 percent at
pH 5.5. At pH 5.8 the oxygen uptake may
continue for several hom's at a greatly reduced
rate (fig. 196).

TRANSPORT OF WATER BY THE GILLS AND RESPIRATION

211

8.0 7.5 7.0 6.5 6.0

pH OF SEA WATER

5.5

Figure 195. — Effect of lowered pH on the rate of oxygen
uptake of adult oysters. Summary of summer ex-
periments at Woods Hole at 25° C.

RESPIRATORY QUOTIENT, R.Q.

The respiratory quotient (R.Q.) is the ratio of
the volume of carbon dioxide produced to the
volume of oxygen consumed at the same time.
Definite values of R.Q. have been recognized for
the principal types of food (Vernon, 1895; Richard-
son, 1929; Krogh, 1941). The R.Q. for carbo-
hydrate is 1.00; for protein abotit 0.79; and for fat
0.71. On an average mixed diet the R.Q. of man
is about 0.80 to 0.85. The herbivorous animals
tend to have a higher R.Q., while the carnivorous
have a lower one.

5

\

z>
o

X

^

A

pH 8 1

^

-^

-

^-^

o

= 2

13

>'

0^

1

p~

1

1

.0,

pH 58

1 1

"O''

1

o

1

2 3 4 5

HOURS AFTER START

Figure 196. — O.x^'gen uptake of two oysters of approxi-
mately equal size (about 6 cm. in lieight) at pH 8.1
and 5.8. Salinity 31.6 °/oo. Measured for 6'^ con-
secutive hours. Temperature 24.5° C.

212

It is of interest to find out whether the R.Q. of
the oyster, which is an herbivorous mollusk,
changes after the breeding season when it begins
to acctimulate and store glycogen. Orton (1927a,
1927b) suggested, without presenting supporting
evidence, that during the reproductive cycle of
O. edulis there is a shift from predominantly
protein to carbohydrate metabolism and that this
shift is correlated with the completion of the male
sexual phase.

In conducting observations on carbon dioxide
production it is necessary to keep in mind that
deposits of calcium carbonate in the bodies of
some marine invertebrates may suddenly release
large quantities of this gas which would give a
false R.Q. (Bosworth, O'Brien, and Amberson,
1936). For instance, the reported R.Q. 1.39 of
lobster was found to be false since in lobsters
coated with collodion the R.Q. was only 0.92.
The shell of the bivalves is the principal storage
place of carbonates which act as buffers when the
valves are closed. In metabolism studies this
possible soiu-ce of error should be eliminated by
coating the shells with paraffin.

The values of respiratory quotients vary during
the conversion of food substances witliin the
organism. Fattening of livestock and birds by
forced carbohydrate feeding is usually accompanied
by high R.Q., while the utilization of fats and
proteins and their possible conversion to carbo-
hydrates lowers the R.Q. values. It is, therefore,
possible to expect that seasonal variations in the
R.Q. of the oyster would give a clue to changes in
the titilization of its food.

A Van Slyke constant volume apparatus for
gas analysis was used in a series of observations of
a group of marked oysters kept in live boxes in the
liarbor. Xo definite trend in the changes of R.Q.
could be detected. It varied throughout the year
from 0.51 to 1.44 as may be seen in the summary
of observations (table 28) made under standard
conditions in water containing no plankton.
Addition of water soluble food in the form of
dextrose resulted in an increase in R.Q. The
latter determinations (table 29) were made in
filtered water to which dextrose was added.
There was no increase in R.Q. in water containing
0.0025 percent dextrose, but in 0.005 and 0.01
percent the R.Q. \alues were significantly higher.

RESPIRATION IN OTHER SPECIES OF OYSTERS

Comparison between the results described for
C. liryinica and the data published by others for

FISH AND WILDLIFE SERVICE

Table 28. — R.Q. of C. virginica through the year
[Long Island Sound oysters]

Oyster
No.

Jan.

Feb.

Mar,

Apr.

May

June

July

Aug.

Sept.

Oct.

Nov.

Dec.

M 1

0.97
0.84
0.97

1.18

0.81

M 1

1.00
0,83
0,89

\I "^

M-4.—

0.92
0.79

0.97

1.06
0.79

0,84
0,69

1.19

0.67

M 9

0.51
0.71

0.57

0.82

0.50

M-9

M-10...
R-50

0.61

6' 72"
0.72

0.71

0.55
0.75

R-52....

0.61

0.80

1.13
0.97

R-55....
R 56

0.66

0.86
1.35
0.54
0.44
1.41

0,92

0.74
0.84

0.89
0.64

R 59

R-62....

1.44

0,82

0.86

other Ostreidae is difficult because of the different
conditions under which the metaboUsm tests were
made. In a discussion of the relation between
the metabolism and temperature and its zoogeo-
graphical significance Sparck (1936) makes the
statement that, "0. edulis consumes more
oxygen than Gryphaea (Crassostrea) angulata of
which it is shown that it is able to supplant 0.
edulis in several locahties." This conclusion based
on a few single determinations is not well sub-
stantiated.

Pedersen (1947) studied the respiration of 0.
edulis living in the small salt-water ponds along
the Skagerrak coast in Norway. The summer
temperature in these ponds rises to 25° C. and
higher, while in winter ice covers the ponds for
about 5 months. Prior to making the test
Pedersen kept the oysters for a few days in filtered
sea water, brushed them, washed the shells with
40 percent alcohol, and wrapped them in pieces
of gauze to prevent small bits of shell from being
broken off. For measuring the oxygen uptake
the oysters were placed in hermetically closed

Table 29. — Effect of dextrose in sea water on R.Q. of C.
virginica

R.Q. values in-

Percent-

Date

Filtered

sea

water

Filtered
sea water
+ dextrose

ages of
dextrose

Nov 14 . . ..

0.87
.57
.82
.92

0.85
1,56
1,56
1,38

0, 0025

Dec 2 -..

,005

Dec 10

,01

,01

glass containers filled with 2 1. of unfiltered sea
water. The containers had to be turned over in
order to mix the water. Closing and opening of
shells were not recorded. Undoubtedly the turn-
ing of vessels caused the oysters to close their shells
and discontinue the ventilation of the gills. For
control Pedersen used blanks that contained no
oysters. The difference between the blanks and
the samples taken from the experimental con-
tainers was considered equal to the quantities of
oxygen consumed and carbon dioxide produced by
the oysters. The consumption of oxygen was
expressed in mg. of oxygen per 100 g. of total
weight or per 10 g. of net weight (presumably
the wet weight of the meat) per 24 hours. Under
these conditions and at temperatures around 24°
to 25° C. the oxygen consumption of the oysters
varied from 15.0 to 48.9 mg. of oxygen per 10 g.
of weight per 24 hours or from 0.62 to 2.0 mg.
per 1 hour. Pedersen's technique had serious
drawbacks since the time the oysters were open
is not known; the mollusks were disturbed by
violent motions (turning over of the containers);
and the animals probably were affected by accum-
ulation of metabohtes. The reported R.Q. of the
Norwegian oysters varied from 0.8 to 1.0, but
in some instances it was as low as 0.6 or as high
as 2.6 and 3.0. The abnormally high and low
values are probably fictitious because of some
deficiency in technique.

In a study of the energy-metabolism of 0. edulis
Gaarder and Eliassen (1955) used a closed chamber
system (desiccators) of 750-ml. capacity. The
shells of the oysters were kept open by glass rods
inserted between the valves. The sahnity of
water was 32 °/oo, and the temperature was kept
constant within ±0.05° C. The results were
expressed in ml. of oxygen consumed per 1 g.
of wet weight per hour. To facilitate the com-
parison I have recomputed the data of the Nor-
wegian investigators to mg. of oxygen per 10 g.
of wet weight of oyster tissues. At 25° C the
oxygen consumption by 0. edulis computed on
this basis was about 2.0 to 2.5 mg. of oxygen per
hom- per oyster.

As one may expect from observations on the
effect of temperature on ciliary motion of the
gill epithelium of the oyster, the oxygen consump-
tion increases about 1.5 times for every 10° rise
(Qio) of temperature between 10° and 25° C.
The max-imum is reached at about 25° C. Below
5° C. the oxygen uptake decreases rapidly but

TRANSPORT OF WATER BY THE GILLS AND RESPIRATION

213

measurable values were recorded by Gaarder and
Eliassen even at temperatures approaching 0° C.
It may be assumed that under normal conditions
the valves would be closed at this low temperature
and ventilation of the gill stopped.

Another experiment by Gaarder dealt with the
effect of oxygen tension on oxygen consumption.
The "critical oxygen tension" at which a decrease
in the oyster oxygen consumption becomes
apparent was found to be 4 ml. of oxygen per 1.
(at 22° C). If the figure is correct, it would
indicate that 0. edulis has a higher "critical
point" than the one reported for C. virginica in
which the rate of oxygen consumption begins to
diminish when the oxygen tension is reduced to
2.5 cm.^ per 1. or lower (Galtsoff and Whipple,
1931). Gaarder and Ehassen disagree with Peder-
sen's (1947) conclusion that 0. edulis can hve for
quite a while in water poor in oxygen. They
think this species shows a rather high "critical
point" of oxygen tension. In both 0. edulis and
C. virginica the uptake of oxygen is independent of
oxygen tension above the respective critical points.

0. circumpicta, observed in a closed chamber
system of about 8-1. capacity containing a thick
layer of liquid paraffin, was found by Nozawa
(1929) to consume oxygen at the rate of about 3.2
ml. of oxygen per hour per 10 g. of wet tissues.
This rate is computed from Nozawa's published
data with an assumption that his figures of oxygen
uptake represent the cm.' of oxygen. The R.Q.
values of this species gradually increased during
the 22 hours of observations from 0.85 to 2.8.
The vahdity of the latter figure is questionable
and is probably due to accumulation of metab-
olites. Nozawa claims that oxygen consump-
tion of 0. circumpicta is independent of oxygen
tension until the latter is reduced to 0.1 percent
of its normal content in water. The figure
appears to be too low to be accepted without
further verification.

UTILIZATION OF OXYGEN

Bivalves use only a small portion of the oxygen
dissolved in the water which they transport
through the gills. The percentage of oxygen
consumed is the measure of the intensity of
utilization of oxygen. In most cases less than
10 percent of the oxygen available is removed
from the water (Hazelhoff, 1938). In comparison
to gastropods and cephalopods, which utilize up
to 80 percent of the available oxygen, the oxygen

demand by bivalves is very low. The actual ■
figures of utilizaton vary depending on the condi- \
tions of the moUusks. In Mya arenaria and in i
fresh-water Anodonta the normal utilization ranges
from 2 to 10 percent (Van Dam, 1938). The
low rate of utilization is due to the rapid transport
of water which both mollusks have to maintain
in order to obtain a sufficient supply of food.
Van Dam reports (1937, 1938) that in many
cases when the respiratory current was slowed
down or when before making the test the mollusks
were left in the air for 20 hours, as much as 97
percent of the oxygen was utilized.

The rate of oxygen consumption is usually
higher after a period of interruption of respiratory
current or after exposure of mollusks to air. This
compensation by oysters for an oxygen debt has
also been observed in Mya arenaria, and in
Anodonta cygnea (Koch and Hers, 1943). The
authors maintain that the rate of ventilation of
the gills of Anodonta is regulated both by the need
of the animal for oxygen and by the availability
of oxygen in water. Oxygen determination in
their experiments was made by means of a polaro-
graph. By this method is was possible to record
photographically the continuous changes in the
oxygen content of water of the exhalant current.
The inference the authors draw from their observa-
tions is that the regulation of the branchial current
in Anodonta by contraction of the exhalant siphon
has relation to the intensity of metabolic processes.
They found that the periods of closure of siphons
are longer in water rich in oxygen and become
shorter when the oxj'gen content is low. The
technique of dropping mercury electrodes
(Petering and Daniels, 1938; F0yn, 1955; Brezina
and Zuman, 1958) appears to be promising and
should be applied in further study of respiration
in mollusks.

The coefficient of oxj-gen utilization in the
oyster (percent of oxj'gen removed from the water
as it passes through the gills) has not been deter-
mined. The data of the metabolism tests cannot
be used for this purpose because the actual rate of
water transport cannot be measured in an oyster
kept in the respiratory chamber. The flow of
water through the chamber was maintained at a
rate lower than the expected rate of water transport
through the gills and, consequently, it is reasonable
to expect that the water in the chamber passed
through the gills several times before it reached
the outlet.

214

FISH AND WILDLIFE SERVICE

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gen dissolved in one cc. of water. Journal of
Experimental Biology, vol. 12, No. 1, pp. 80-85.

1935b. On the utilization of oxygen by Mya arenaria.
Journal of E.x'perimental Biology, vol. 12, No. 1,
pp. 86-94.

TRANSPORT OF WATER BY THE GILLS AND RESPIRATION

217

V^AN Dam, L.

1937. t)ber die Atembowegungen und das Atemvolu-
men von Phryganea-Larvcn, Arenicola marina und
Nereis virens, sowie uber die Sauerstoifausnutzung
bei Anodonta cygnea, Arenicola marina und Nereis
virens. Zoologischer Anzi-iger, Band 118, pp. 122-
128.

1938. On the utilization of oxygen and regulation of
breathing in .some aquatic animals. Drukkerij
"Volharding", Groningen, Holland, 143 pp.

1940. On the mechanism of ventilation in Aphrodite

aculeata. Journal of Experimental Biology, vol. 17,

No. 1, pp. 1-7.
1954. On the respiration in scallops (Lamellibranehi-

ata). Biological Bulletin, vol. 107, No. 2, pp.

192-202.
Vernon, H. M.

1895. The respiratory exchange of the lower marine

invertebrates. .Journal of Physiology, vol. 19,
pp. 18-70.

VlALLANES, H.

1892. Recherches sur la filtration de I'eau par les
MoUusques et applications a I'ostreiculture et i
rOc6anographie. Comptes Rendus Hebdomadaires
des Stances de rAcadSmie des Sciences, tome
114, pp. 1386-1388.
VoN Skramlik, Emil.

1941. tleber den Kreislauf bei den Weichtieren.
Ergebnisse der Biologic, Band 18, pp. 88-286.
Wangeesky, Peter J.

1952. Isolation of ascorbic acid and rhamnosides
from sea water. Science, vol. 1 15, No. 2999, p. 685.
Wells, N. A.

1932. The importance of the time element in the
determination of the respiratory nietaboUsm of
fishes. Proceedings of the National Academy of
Sciences, vol. 18, No. 9, pp. 580-585.

218

FISH AND WILDLIFE SERVICE

CHAPTER X
ORGANS OF DIGESTION AND FOOD OF THE OYSTER

Page

Mouth — - - — - 219

Esophagus and stomach - 219

Gastric shield - - - 222

Crystalline style. 223

Formation --- -- - --- 225

Chemical composition... 225

Midgut and rectum 226

Digestive diverticula 226

Alimentary tract and formation of feces 226

Digestion 228

The pH content of the gut andstomach 230

Absorption of food by the gills and mantle .- 231

Food and feeding. 231

Artificial feeding. 234

Bibliography 235

The system of organs concerned with the in-
gestion and digestion of food and elimination of
feces consists of the mouth, short esophagus,
stomach, crystalline style sac, digestive diver-
ticula, midgut, rectum, and anus (fig. 197). With
the exception of a short section of the rectum, the
entire alimentary canal lies within the visceral
mass and is completely immobilized by the sur-
rounding connective tissue. In the absence of
peristaltic movements the food is moved from the
mouth toward the anus exclusively by the strong
ciliary motion of the epithelial lining of the system.
It is difficult to reveal all anatomical details of the
alimentary tract by dissecting the tissues. Better
preparations can be obtained by making casts of
latex or other suitable material injected through
the esophagus or via the rectum and left until it
sets. Yonge (1926a) used for this purpose a warm,
concentrated solution of gelatin. Awati and Rai
(1931) employed a mixture of paraffin and resin
colored with carmine. Satisfactory results were
obtained in the Bureau's shellfish laboratory by
using red or yellow latex mjected into the esop-
hagus through a wide mouth pipette with a rubber
balloon. The preparation was immediately placed
in 5 percent formalin in which the latex sets and
remains tough and flexible for a long time. Casts
of the entire alimentary canal or of its parts can
be obtained in this way.

FISHERY bulletin: VOLUME 6 4, CHAPTER X

MOUTH

The mouth is a compressed U-shaped slit be-
tween the two lips (fig. 104) and is lined with
columnar ciliated epithelium set on a narrow basal
membrane. The epithelial cells of the mouth are
taller than those of the labial palps and contain
only a few mucous glands. In the surrounding
connective tissue are large vesicular cells, num-
erous muscle fibers, and blood spaces which are
occasionally filled with leucocytes. Leucocytes
are also found in narrow spaces between the tissue
cells and on the surface of the mantle lining from
which they are discarded.

ESOPHAGUS AND STOMACH

The esophagus, a short, funnel-shaped, and
dorso-ventrally compressed tube, is lined with
epithelium similar to that of the mouth. It leads
to the stomach, which occupies a central position
within the visceral mass (fig. 197). The stomach
is an irregularly shaped, large sac (figs. 198 and
199) with several outgrowths or pouches. At the
entrance of the esophagus the wall of the stomach
forms an anterior chamber, a, which leads into a
broader posterior chamber, b (figs. 198 and 199).
An oblique outgrowth or pouch called the caecum,
c, is the most conspicuous structure which arises
from the ventral side of the anterior chamber.
Both the anterior and posterior ends of the caecum
are curved and form the anterior and posterior
appendices (a.ap., p.ap.). The larger posterior
appendix is a strip curved ventrally and toward
the right of the stomach. The configuration and
relative sizes of the appendices vary but the
structures are recognizable in all the casts. A
groove along the wall of the caecum leads to the
opening of the midgut (m.g.) and serves for sorting
of food (Yonge, 1926a). On the left side below
the caecum the wall of the stomach bulges out to
form a broad pyloric caecum (p.c), which leads
to a long outgrowth alongside the midgut, the
crystalline sac (cr.s.).

219

oe.

diq. di.

cr 3.

Centimeters

Figure 197. — Digestive system of the oyster, C. virginica, drawn from the dissected preparation after the injection of
latex. The right outer labial palp was cut off to expose the esophagus. The parenchymal tissues over the stomach and
intestine were removed, an. — anus; cl. — cloaca; cr.s. — crystalline style sac; dig. div. — digestive diverticula; int. —
intestine (mid-gut); oe. — esophagus; r. — rectum; st. — stomach.

Three groups of wide ducts emerging from the
wall of the stomach lead to the digestive diver-
ticula. Two of them (fig. 199. di, d2) originate
at the anterior chamber and one (da) from the
posterior chamber.

The internal lining of the anterior chamber
forms a number of irregular ridges and furrows
covered with ciliated epithelium. A broad ridge
separates the anterior from the posterior chamber
and apparently directs the food particles. The
left ventral wall of the posterior chamber is
covered by a translucent meml)rane, the gastric
shield (fig. 200), which lies directly opposite the

opening of the long sac occupied by the crystalline
style (cr.s.).

Ciliary tracts of the stomach lining are very
complex. Detailed observations on the course
followed by food particles after they enter the
stomach were made for 0. edulis and Mya arenaria
by Yonge (1923, 1926a), who studied them by
carefully cutting off the wall and adding fine
powdered carborundum or aquedag to the exposed
surface. In general the pattern of ciliary move-
ments in the stomach of the American oyster is
similar to that of 0. edulis. The direction of
ciliary beat along different ridges and channels

220

FISH AND WILDLIFE SERVICE

a^ap. j^

0 ^ .. , 10

Centimeters

Figure 198. — Latex cast of the stomach, crystalline style
sac, and esophagus of a large C. virginica, viewed from
the left side. Upper part of the esophagus is slightly
distended by injection. The visceral mass was dissected
and the injected parts left in their natural position.
Drawn from a preparation preserved in 5 percent for-
malin, a. — anterior chamber; a.ap. — anterior appendix
of the caecum; b. — posterior chamber; c. — caecum;
cr.s. — crystalline style sac; d.p. — dorsal pouch; d. —
group of ducts of the digestive gland; m.g. — midgut;
p.ap. — posterior appendix of the caecum; p.c. — pyloric
caecum.

Centimeters

Figure 199. — Latex cast of the stomach, crystalline style
sac, and esophagus of a large C. virginica viewed from
tlie right side. The visceral mass was dissected, and the
injected parts were left in their natural position. Drawn
from a preparation preserved in 5 percent formalin,
a. — anterior chamber; b. — posterior chamber; cr.s. —
crystalline style sac; di, d2, da — ducts leading to the diges-
tive diverticula; m.g. — midgut; oe. — esophagus.

brings the food from the esophagus to the caecuiii
where the food materials are separated according
to size. Some of the larger particles entering the
midgut may be voided without being digested
while the smaller particles are pushed toward
the gastric shield. Other groups of cilia conduct
the particles toward the ducts leading to the
digestive diverticula. The ducts branch out into
a large number of smaller passages that ramify and
extend deep into the mass of diverticula.

Nearly the entire inner surface of the stomach

ORGANS OF DIGESTION AND FOOD OF THE OYSTER
733-S51 O— 64 15

is covered by ciliated epithelium; only the areas
imder the gastric shield and near the posterior
end of the stomach are nonciliated. The epithe-
lium is of columnar type with very long cilia,
which are particularly prominent on the ridges
(fig. 201). The height of the cells gradually
decreases toward the caecum. Mucous cells are
abundant, particularly near the junction with the
midgut, and phagocytes are numerous between
the epithelial cells and in the underlying connec-
tive tissue. There is no well-developed muscular
layer under the epithelial lining, but a few smooth

221

Centimeters

Figure 200. — Gastric shield viewed in its natural position
in a dissected stomach. The position of the crystalline
style is indicated by the dotted line. Drawn from an
unpreserved preparation.

muscle fibers may be found under the basement
membrane. In general, the histological picture
of the stomach of an adult C. inrginica is similar
to that described for the spat of this species by
Shaw and Battle (1957), C. angulata by Leenhardt
(1926), and 0. chilensis and 0. edulis by Dahmen
(1923) and Yonge (1926a) respectively.

GASTRIC SHIELD

The stomach wall in front of the openings to
the midgut and style sac is covered by a thin
but tough, irregularly shaped membrane (fig. 200)
made of translucent and slightly striated material.
The structure, named the gastric shield by Nelson
(1918), rests on a prominent epithelial ridge of
narrow colunmar cells with oval nuclei, rich in
chromatin (fig. 202). The cells are devoid of
cilia. The shield is made of two portions of
different size, joined together by a narrow middle
piece (fig. 200). The thicker portion of the shield
lies over the peak of the ridge and is underlined by
tlie tallest cells in the area. On both sides of
the peak the epithelium flattens and at the edges
changes into the typical ciliated lining of the
stomach. The surface of the sliield is roughened
by the remnants of food particles embedded in it.

The origin of the shield has not been fully
e.xplained. Obviously, it is tlie product of the
underlying cells, but the process of its formation
has not been studied sufficiently. One view,
advanced by Gutheil (1912) and shared by some

investigators, assumes that the shield is formed
by the droplets secreted by the epithelial cells.
No evidence in support of this view can be found
in the histological preparations of the stomachs
of 0. edulis and C. virginica. No droplets could
be seen in the sections of stomach, and no other
indication of the secretory activities of these cells
could be found. Yonge (1926a) thinks that the
shield is very likely formed by the fusion of the
cilia and in support of this view points out that
the structure is attached to the epithelium by fine
threads which transverse the substance of the
shield and resemble the cilia. Indistinct trans-
verse striation can be seen in the sections of the
stomachs of C. mrginica fixed in osmic acid and
stained with iron hematoxylin (fig. 202). The
question could be settled by electron microscopy,
which would reveal the structure of the cilia if the
latter are present within the shield substance. So
far no such studies have been made.

The shield is not destroyed by boiling in a 40
percent solution of potassium hydroxide. Treat-
ment with iodine followed by a strong solution of
zinc oxide gives the deep violet coloration that is
characteristic of the color reaction for chitin
(Zander reaction) . These facts support Berkeley's
(19.35) findings that the material of the shield of
the common Pacific coast clam, the Pacific gaper
{Schizothaerus mdalli nutalli Conrad), is made of
chitin and contains no chondrinlike constituent.

In C. angulata Leenhardt (1926) described the
torch bearing cells near the edges of the area
occupied by the gastric shield. The function of
the cells is not known. They are not found in my
preparations of C. virginica and are not mentioned

Microns

Figure 201. — Cross section of the wall of the stomach.
Kahle, Hematoxylin-eosin stain.

222

FISH AND WILDLIFE SERVICE

0

Microns

200

Figure 202. — Two cross sections of the wall of the stomach of C. virginica under the gastric shield. A — the thickest
portion of the shield. Benin, hematoxylin-eosin stain. B — cross section near the periphery of the shield. Osmic
acid, iron hematoxylin. The surface of the shield is rough due to embedded and partially ground food particles.
Note cross striation of the shield visible in B.

by Shaw and Battle (1957) in their work on the
microscopic anatomy of the digestive tract of this
species.

The function of the shield is to provide a base
for grinding of food by the rotating head of the
crystalline style.

ORGANS OF DIGESTION AND FOOD OF THE OYSTER

CRYSTALLINE STYLE

The posterior wall of the stomach leads to an
elongated outgrowth or sac which extends a con-
siderable distance along the ventral arm of the
visceral mass (fig. 197, cr.s., and fig. 203) on the
antero-ventral side of the adductor muscle. A

223

narrow slit joins tlie sac over nearly its entire
length to the midgut; near the entrance to the
stomach the two structures are separated. The
sac is slightly twisted around the midgut and
occupies a somewhat dorsal position, while the
midgut forms the ventral portion of the common
structure (figs. 198, 199). A cross section of the
sac and midgut shows (fig. 204) that the two
channels are separated in the middle by a narrow
slit compressed by the two protruding lobes or
typhlosoles. In figure 204 the style sac is at the
top; its lumen is usually larger than that of the
midgut (lower part of the figure). This relation-
ship between the style sac and midgut is similar
to the topography of this organ in 0. chilensis
(Dahmen, 1923), 0. edulis (Yonge, 1926a), Mya
(Edmondson, 1920), Ensis (Graham, 1931a),
Mytilus edulis (Sabatier, 1877), M. latus and M.
magellanicus (Purdie, 1887), and Anodonta (Nel-
son, 1918). In the old literature the structure was
called a "tubular stomach" by Sabatier (1877),
and "pyloric appendix" by Purdie (1887), names
which have not been accepted in malacological
literature.

Centimeters

Figure 203. — Crystalline styles of C. virginica (left)
and C. gigas (right) in their natural position. Drawn
from live preparations. The wall of the stomach and
of the crystalline style sac has been dissected.

J

/ VT^--

&

1T.5

Mil I i meters

Figure 204. — Transver.se section of the style sac (upper
part) and midgut (lower part). The crystalline style
is absent. Kahle, hematoxylin-eosin.

The style sac is lined with densely packed
cylindrical cells that have large oval nuclei and
long cilia measuring about 20 ix. The intracellular
fibrillar apparatus is well developed. Phagocytes
and mucous cells are scarce. The basal membrane
rests on a thin layer of collagenous fibers; circular
muscles are sparsely arranged, as in the stomach.

and there is no distinct muscular layer. The
epithelial cells of the two lobes (typhlosoles) of
the sac and midgut gradually change from robust,
long cells to shorter cells with smaller cilia, typical
for the lining of the midgut. The mucous cells
are more abundant in the midgut than in the sac.

224

FISH AND WILDLIFE SERVICE

The connective tissue around the sac and under
the typhlosoles consists of typical vesicular cells.

In actively feeding oysters the lumen of the sac
is occupied by a gelatinous rod with a bulging head
protruding inside the stomach (fig. 203) and the
pointed tail extending to the distal part of the sac.
The color of the style varies from greyish white
to deep yellow and brown, depending on the type
of food consumed by the oyster. The head is
usually darker than the rest of the style because
of the food particles ^v^apped around it.

Inside the sac the style is rotated by the ciliary
action of the epithelium. The rotary motion
was originally suggested by List (1902) in his
work on mussels, but the demonstration that the
rotation actually takes place in Anodonta and
Modiolus was made by Nelson (1918). According
to Yonge (1926a), the large cilia of the groove of
the sac of 0. edulis move in such a way as to
produce a slow clockwise rotation of the style
when seen from the stomach. There is, however,
a tract of cilia on the side of the larger typholsole
which beats in the direction of the stomach and
presumably pushes the style forward. Food
particles that enter the sac are carried by the
currents down the gut but some of them tangle
in the substance of the style, are wrapped around
it, and carried back to the stomach. This process,
observed by Nelson (1918, 1925), Allen (1921),
and Orton (1924), may be significant for the bi-
valves in which, like in Ostrea, the style sac is in
direct communication with the midgut.

As the style rotates and rubs against the gastric
shield, aiding in mixing and grinding food particles
it slowly dissolves in the gastric juice and yields
digestive enzymes.

FORMATION

The crystalline style is not a permanent struc-
ture. In oysters removed from water and left in
the ah- the style dissolves in a short time. This
observation, reported for 0. edulis by Orton (1924),
has been confirmed for C. virginica and C. gigas.
At room temperature of 21° to 22° C. the crystal-
line styles of the American species removed from
the sac and left exposed to air completely dissolve
wdthin 45 to 60 minutes. In the body of the
oysters {C. virginica) taken out of water the style
disappears in 2 to 3 hours. The absence of the
style is frequently observed in nonfeeding oysters.
The symptom is useful, but not entirely reliable
because under certain conditions the style may be
present in oysters which do not take food. Ob-

servations made in winter in the Woods Hole
laboratory showed that in late December, at tem-
peratures varying from 5.4° to 5.7° C. about 4 out
of 10 oysters had crystalline styles. No trace of
food was found in these oysters, which were ex-
amined within a few minutes after they had been
taken out of water.

Yonge (1926a) states that in 0. edulis the style
is always present in healthy oysters, even when
they are starved, and is absent only under ab-
normal conditions and in diseased specimens.

The style must be the product of secretion but
investigators do not agree on the manner and site
of its formation. List (1902), Nelson (1918),
Edmondson (1920), and Mackintosh (1925) think
that the style is secreted by the narrow cells of the
smaller typhlosoles but do not present conclusive
evidence in support of this view. For fresh-
water Anodonta, Gutheil (1912) demonstrated the
presence of vesicular granules around the nuclei
of the epithelial cells of the sac and probably
interpreted them correctly as a sign of active
secretion. No such granules were found, however,
in the histological preparations of 0. edulis (Yonge,
1926a) and in my slides of the sac of C. virginica.
Evidence of the secretory activity of the style sac
was produced by Yonge (1926a) by injecting 0.5
percent solution of iron saccharate into the ad-
ductor muscle, washing the animals, and then
dissecting and fixing the sac at 2-hour intervals.
The sections were treated by potassium ferricy-
anide in acid solution to demonstrate the presence
of iron by Prussian blue reaction. Fine blue
granules indicative of the presence of iron salt were
found in the cytoplasm above the nuclei and be-
tween the cilia of the epithelial cells. No iron
was detected in the epithelium of the midgut or
of the larger typhlosoles, although some of the
metal was present in the epithelial cells of the
minor typhlosole. The experiments may indicate
the secretory function of the epithelial cells, but
they cannot be considered as evidence of the
formation of the style from the secreted granules.

CHEMICAL COMPOSITION

Analysis of the crystalline style of Cardium
made by Barrois (1889, 1890) showed the following
composition: water 87.11 percent; solid organic
matter 12.03 percent; sohd inorganic matter 0.86
percent. The organic component of the style was
considered to be a globulin with traces of mucus
or chondrinlike substance. Berkeley (1935) dem-

ORGANS OF DIGESTION AND FOOD OF THE OYSTER

225

onstrated that the styles of four species of bivalves
(Crassostrea gigas, Mya arenaria, Schizothaerus
nuttalii, and Saxidomus giganteus) in addition to
protein, yield, on acid hydrolysis, glucionic and
sulphuric acids and a hexamine, the essential
constituents of both mucin and chondrin. The
ease of the hydrolysis and the solubility of the
style materials indicate that mucin rather than
chondrin is involved. The variations in the solu-
bility and the quantitative differences in the
chemical composition of the styles suggest, ac-
cording to Berkeley, that the less readily soluble
styles contain larger quantities of mucin.

All examined styles are carriers of certain
enzymes which they yield upon dissolution. The
role the styles play in digestion is discussed later
(see p. 230 of this chapter).

MIDGUT AND RECTUM

The portion of the intestine between the stomach
and the rectum is called the midgut. It begins
at the ventral wall of the stomach next to the
opening of the crystalline sac and runs parallel to
the sac as far as its distal end, then tui'ns sharply
backward parallel to its previous com'se (fig. 197,
int.). The ascending branch of the intestine
makes a loop that completely encu-cles the stomach
and continues as a descending branch which ends
with the rectum and anus (r., a.).

Throughout its entire length the midgut is
characterized by a well-developed typhlosole
which extends along its inner wall (fig. 205).
The gut is lined with columnar ciliated epithelium;
there is an abundance of mucous cells and of
wandering leucocytes. The muscular layer is
absent.

The rectum (fig. 197, r.) runs along the dorsal
side of the heart. In this respect the oyster
differs from many other bivalves (sea mussels,
clams, fresh-water mussels) in which the rec-
tum runs through the heart. The structure of
the rectum is similar to that of the midgut; the
main difference is the disappearance of the well-
developed typhlosole near the anal region where
the lining is thi-own into numerous small folds
(fig. 206). A distinct feature of the rectum is a
circular layer of smooth muscles which, however,
do not form a sphincter at the anus (fig. 207).
According to Leenhardt (1926), the anal sphincter
is present in the Portuguese oyster. The surface
epithelium of the anal region is well developed
and abounds in mucous cells.

DIGESTIVE DIVERTICULA

The stomach is surrounded by an irregularly
shaped mass of dark tissue which has been called
the "liver" or "hepatopancreas." Its color varies
from light yellow to dirty green and dark brown.
In most cases the color is noi visible through a
white or creamy layer of connective tissues rich
in glycogen.

Yonge (1926b) has shown that assimilation and
intracellular digestion takes place in this mass of
darkly colored tissues; it has none of the known
functions of the liver or pancreas. He named it
"digestive diverticula", a term that correctly
describes its role.

The digestive diverticula are made of a large
number of blind tubules emptying into several
large ducts which lead to the interior of the
stomach. The structure of the tubules is simple.
In cross section (fig. 208) they are usually round
with a lumen in the form of a cross. The tubules
are surrounded by connective tissue in which
muscle fibers are absent. The digestive cells
which form the interior of a tubule are large and
well vacuolated, with large nuclei at their base.
Food vacuoles can be seen in them during feeding.
At the corners of the "cross" of the lumen one
usually finds crypts of young cells with dark
staining protoplasm, large and compact nuclei,
and indistinct cell boundaries. Cells from these
crypts replace the old cells that are cast off. The
digestive cells of the American oyster are non-
ciliated, but the cells in the diverticula of other
bivalves {Teredo) have been reported to have
ciha (Potts, 1925). Yonge (1926a, 1926b) believes
the cilia are present in the tubules of edulis but
probably retract so rapidly that they cannot be
seen in the fragments of tissues pressed by a
cover slip. I was not able to detect them in C.
virginica. Phagocytes are very abundant between
the cells and in the surrounding connective
tissue.

The ducts that connect the tubules with the
stomach are circular in cross section and are
fined with cifiated epithelium (fig. 209). Their
lumen is, however, irregular due to the variations
in the height of the epithelial cells. The epithe-
lium is similar to that of the stomach and con-
tains many mucous glands and phagocytes.

ALIMENTARY TRACT AND FORMATION
OF FECES

Food ingested by the oyster is moved through
the alimentary canal by the ciliary action of the

226

FISH AND WILDLIFE SERVICE

VJ^Ari

I I I I I 1

0 Microns ^50

Figure 205. — Cross section of the midgut. Bouin, hematoxylin-eosin.

epithelium. There is no peristaltic motion since
the muscular layer of the intestines is either
absent or poorly developed, and the feces are
discharged in a continuous ribbon which is carried
away by the cloacal cm'rent and eventually settles.
The time required for food to pass through the
entire intestinal tract can be measured by record-
ing the time between addition of a suspension of
carmine or yeast to the gills and the appearance
of the red or white particles in the feces. The
rate of passage naturally depends on the length
of the intestinal tract and the rate of feeding.

In large oysters (about 10 by 6 cm.) kept in run-
ning sea water of about 15° to 16° C. the time
required for food to pass through the entire intes-
tinal tract varied from 90 to 150 minutes. The
length of the intestinal tracts of the oysters used
in these tests was measm-ed on latex casts which
were left in situ and exposed by dissecting the
tissues above them. The lengths of the alimentary
tracts were as follows:

In an oyster measuring 1 1 by 6 cm 14.5 cm.

In an oyster measuring 10.0 by 7.5 cm 11.1 cm.

In an oyster measuring 1 1 by 6 cm 12.9 cm.

In an oyster measuring 11.5 by 5.5 cm 12.6 cm.

ORGANS OF DIGESTION AND FOOD OF THE OYSTER

227

Microns

150

Figure 206. — Cross section of the rectum near the anal
region. Boiiin, hematoxylin-eosin.

The rates of discharge of fecal ribbons observed
on actively feeding oysters in laboratory sea water
of 15.0° to 15.7° C. are given in table 30. The
average of the observed rate was 8.1 cm. per hour.
Assuming that the average length of the intestines
was 12.8 cm., the estimated time of passage of
food through the entire alimentary tract was 95
minutes.

The feces of the oyster are voided from the
rectum as a compact and slightly flattened ribbon
of sufficient consistency to withstand the velocity
of the cloacal current. In an actively feeding
oyster the ribbon is maintained in a horizontal
position along the axis of the current, but being
heavier than the sea water it sinks down to the
bottom as soon as the cloacal current slows down
or ceases. Large masses of fecal ribbons accumu-
late on the bottom a short distance from the
opening of the cloaca. The ribbon remains intact
for 2 to 3 days untU it is disintegrated through
decomposition and mechanical disturbance. The
appearance of the fecal masses of the oyster is
typical and can be recognized by their shape. It
was shown by Moore (1931) that specific identi-
fication of fecal pellets can be made for a number
of marine invertebrates.

Fecal ribbons of oysters contain many live
cells — diatoms, dinoflagellates, yeast, and others
which are not killed by the gastric and intestinal
juices and can be recultured.

Table 30. — Rate of formation of fecal ribbons {in cm.) in
C. virginica during feeding in laboratory sea water, Woods
Hole

Date

May 18
18
18

May 19
19

May 23

Time

Tempera-

Length of

ture

ribbon

Minutes

"C

Cm.

108

15.6

23

17

15.0

3.2

102

15.0

9.8

70

16.2

14.2

123

15.0

9.3

60

15.7

7.2

Rate of
formation

Cm.lhr.

.7
11.3
6.8
12.2
4.6
7.2

DIGESTION

The digestion and absorption of food in the
oyster are primarOy an intracellular process which
takes place in the digestive diverticula. This was
demonstrated by Yonge (1926a, 1926b) in a series
of carefully executed feeding experiinents in which
the solutions of iron saccharate, suspension of
carmine powder, oil emulsion, and dogfish blood
corpuscles were fed to European oysters. He
produced convincing evidence that very small
food particles are absorbed by the cells of the
digestive diverticula, while the diatoms and other

Mil limeters

Figure 207. — Longitudinal section of the anus and ad-
jacent portion of the rectum. Note the fecal mass
inside and the absence of a sphincter. Bouin, hema-
toxylin-eosin.

228

FISH AND WILDLIFE SERVICE

Microns

40

Figure 208. — Cross section of a single digestive divertic-
ulum. Note the crypts of young cells at the corner of
the cross of the lumen. Kahle, hematoxylin-eosin.

algae of larger size are ingested by phagocytes.
Yonge's work fully confirmed the idea, first ex-
pressed by Saint-Hilaire (1893), that the digestive
diverticula are the organs of absorption. He
found no evidence of any secretion from the di-
verticula and demonstrated the importance of
phagocytes in the digestive processes. Since the
work of the earlier investigators is fully discussed
by List (1902) and more recent investigations are
summarized in several papers of Yonge (1926a,
1926b), the reader interested in the history of the
problem is referred to these publications.

The digestion of food also takes place in the
stomach where several digestive enzymes are
present. On the basis of our knowledge, which
admittedly is not complete, the process of di-
gestion seems to take the following course. After
being sorted several times by various mechanisms
of the gills and labial palps, the food particles
enter the stomach where the sorting continues
and the larger particles are broken by the com-
bined action of the crystalline style rotating
against the gastric shield and the chemical action
of enzymes which dissolve from the style. Very
small particles are pushed by the cilia, through
the ducts into the digestive tubules where they
are taken into the vacuoles of the digestive cells

and are acted upon by the enzymes of these cells.
Usable material is ingested by the phagocytes or
is stored in the surrounding connective tissue.
Indigestible substances like colloidal carbon of
india ink are expelled. Some of the food particles,
especially of larger size, are engulfed by the
phagocytes which abound in the digestive tract.
Circulation of food in the ducts is maintained
by the ciliated cells.

The stomach contains free enzymes which are
dissolved from the crystalline style. The most
active among them are the amylase and gly-
cogenase which digest starch and glycogen.
Yonge's experiments (1926a, 1926b) showed that
the optimum activity of oyster amylase is at
approximately pH 5.9. Purification by dialysis
or with absolute ethyl alcohol inactivates the
enzyme, but its action can be restored by the
addition of chlorides or broirddes. Besides these
two enzymes, the style contains a complete
oxidase system. The presence of oxidases in
the extract of styles was first demonstrated by
Berkeley (1923) in the Pacific coast clam, Sax-
idomus giganteus, in rock cockle, Paphia staminea,
and in soft-shell clam, Mya arenaria. This
finding lead Berkeley to advance a theory that
the crystalline style represents a reserve of oxygen
and is a factor in the anaerobic respiration of
mollusks. The theory is not supported by
sufficient evidence and has not been accepted by
the students of moUuscan physiology.

The presence of oxidases in the styles of Ostrea

Microns

Figure 209. — Cross section of the duct leading to the
digestive diverticula. Kahle, hematoxylin-eosin.

ORGANS OF DIGESTION AND FOOD OF THE OYSTER

229

was confirmed by Yonge (1926a) and Graham
(1931a, 1931b). The enzymes were obtained by
grinding the styles with sand and extracting for
2 to 3 days in distilled water with a small amount
of toluol as an antiseptic. For testing, the
5-ml. samples of 1 percent extract of style were
treated with 2 ml. of hydrogen peroxide and 12
drops of 1 percent pyrogallol. After 5 minutes
the sample turned dark red-brown. The ex-
tract produced color even in the absence of hydro-
gen peroxide, indicating the presence of a complete
oxidase system. Reactions with guaiacum and
2 percent hydroquinine were less pronounced
than with pyrogallol.

Sawano (1929) reported the presence of buty-
rase, an enzyme that clots milk, in the styles of
0. circumpicta but his observation remains un-
confu-med.

Extracts of digestive diverticula contain a
large array of sucroclastic enzymes which act
on starch, glycogen, sucrose, maltose, lactose,
rafRnose, and on some glucosides. The amylase,
which converts starch into dextrin and dextrin
into maltose, is present both in the style and in
the digestive diverticula of the oyster. It has,
however, different optima; the style amylase
acts best at pH 6.0, whereas the enzyme from
the diverticula has an optimum at pH 6.4 (Sawano.
1929).

The proteolytic enzyme of 0. edulis is absent
in the gut but can be found in the extract of the
diverticula. It acts very slowly and has two pH
optima at 3.7 and 8.5 when casein is used as a
substrate. With gelatin the optima are 4.1 and
8.5.

Cellulase, the enzyme which hydrolizes cellulose,
has not been found in the digestive extracts of the
oyster. It must be assumed, therefore, that the
oysters are unable to digest cellulose. The
possibility is not excluded, however, that this
enzyme may be present in the bacteria and fungi
which happen to be in the gut. The presence
of cellulase in mollusks has been established for
the gastropods Helix and Linnaea and for the wood
boring bivalve Teredo.

Fats are hydrolized to fatty acids and alcohols
by the action of lipase. Yonge (1926a) dem-
onstrated the presence of this enzyme by
feeding the oysters an emulsion of olive oil stained
red with Nile blue sulphate and watching the
change of red color into blue as the digestion
proceeded. Oil is ingested by phagocytes and is

carried by them through the tissues, the gradual
change of color serving as an index of the action
of lipase. From the observation that the droplets
of oil found free in the stomach retain the red
color, Yonge deduced that free lipase is absent
in the gastric juice. These findings are contra-
dicted by the observations of George (1952) who
showed that in C. virginica and in Mytilus the
hydrolysis of neutral fats takes place extra-
cellularly in the stomachs and that lipase can be
extracted from the crystalline style. According to
his observations, droplets of olive or peanut oil
stained scarlet red with Sudan I or Sudan III
are not deposited in the tissues. It is known
that in mammals and birds the stained fat may
be stored in the bodies (Gage and Fish, 1924).
Several possibilities may be considered: (a) that
the stained fat is rapidly metabolized; (b) that
it may be deposited in connective tissue in minute
quantities undetectable under the light microscope;
and (c) that the mollusks are unable to utilize
the peanut and olive oil because of the differences
between the fatty acids of these oils and the
unsaturated fatty acid of their natural food. So
far no experimental evidence has been presented
in support of any of these possibilities (George,
1952) and further studies of the problem of fat
digestion in bivalves are needed.

pH CONTENT OF GUT AND STOMACH

The digestive fluids found in the alimentary
tract are acid. The most acid conditions exist in
the stomach (average pH 5.5) due to the dis-
solution of the crystalline style, which has a pH
of 5.2 (5.4 in starved animals), and, according
to Yonge {1926a), is the most acid substance in tho
gut. In the absence of the style the pH of thfl
stomach fluids increases. This has been demon-
strated on oysters with clamped shells, kept for 6
days out of water. Under these conditions the
pH of the stomach rose from 5.67 to 6.14 while the
pH of the liquid in the mantle cavity decreased
due to the accumulation of carbon dioxide from
6.7 to 6.14. It is significant that the acidity in
the stomach caused by the dissolution of the style
approximates the optimum (pH 5.9) for the action
of the style's amylase. The pattern of pH dif-
ferences in various parts of the alimentary tracts
as shown by Yonge is as follows: esophagus 5.6-
6.0; stomach 5.4-5.6; style 5.2; midgut 5.5-6.0;
rectum 5.8-6.3. The pH of the extracts of the
styles of C. virginica, determined by placing the

230

FISH AND WILDLIFE SERVICE

styles across the one-drop electrode, was found by
Dean (1958) to vary between 5.8 and 6.0.

Extracts of the digestive diverticula of 0.
edulis have pH values from 5.6 to 5.9; the varia-
tions are probably associated with the resting and
active stages of digestion. The styles of C.
rirginica contain a heat-labile substance, probably
an enzyme, which has the ability to attack certain
algal cells only during the dissolution of the style
(ir within a very short period after the dissolution.
This has been reported by Dean (1958) who
observed rapid disintegration of Cryptornonas cells
in buffered sea water (pH 6.0) containing style
extract. Monoehrysis sp. were immobilized by
the extract while Isochrysis sp. were not affected
and were able to swim near or even touch the
style. Dean thinks the "enzyme" may be a
protease, lipase, or amylase. The observed results
may be interpreted as the differences in the resist-
ance to digestion by different species of algae used
by the oyster as food.

It has been pointed out in support of the im-
portance of extracellular digestion that fragments
of partially disintegrated large diatoms (Coscin-
odiscm, Melosira, Skeletonema) are frequently
found in the stomachs of C. virginica (Nelson,
19.34), but the question of the significance of
extracellular digestion in bivalves has not been
settled. Weak proteolytic action was found in
the stomach of the giant clam, Tridacna, the
pearl oyster, Pinctada (Mansour-Bek, 1946,
1948), and in the crystalline style extract of C.
virginica (Chestnut, 1949) and strong amylo-
lytic activity in the stomach of the oyster was
demonstrated by a number of investigators.
Oysters apparentl}^ have a great capacity to
utilize materials rich in carbohydrates.

ABSORPTION OF FOOD BY GILLS AND
MANTLE

The idea that the exposed surfaces of bivalves,
particularly the gills, palps, and mantle, absorb
the organic matter dissolved in sea water (Ranson,
1926, 1927) is not substantiated by experimental
evidence. In experiments with 0. edulis Yonge
(1928) has shown that the oyster absorbs glucose
from the water but that this absorption takes
place tlu'ough the alimentary canal and digestive
diverticula. No absorption was recorded in the
animals in which the access of water to the esopha-
gus was prevented by stuffing the mouth with wax
plugs. Glucose may be absorbed, however, by

the phagocytes which accumulate on the surface of
the mantle. The results of Yonge's observations
were confirmed by Roller (1930) in his experiments
with MytUus edulis and Mya arenaria.

Since phagocytes normally aggregate on the
surface of the mantle and gills, it is possible that
the oyster may absorb the substances present in
the surrounding media by means of these wander-
ing cells. Yonge admits this possibility in the case
of oysters fed u-on saccharate, and I observed that
the particles of iron oxide added to the water in
which I kept C. virginica were ingested by the
phagocytes of the gills and transported to the
deeper parts of the body.

FOOD AND FEEDING

The study of food of the oyster has attracted
the attention of many investigators who examined
the stomach contents and recorded the variety of
organisms found in it. One of the earliest obser-
vations was made more than a century and a half
ago by Reade (1844, 1846), who was "induced" to
examine the contents of the stomach of British
oysters and the "well known ciliary currents In
the fringes of the oyster." His curiosity was well
satisfied, for he found "myriads of living nomads,
the Vibrio also in great abundance and activity,
and swarms of a conglomerate and ciliated living
organism, which may be named Volvox ostrearius,
somewhat resembling the Volvox globator, but so
extremely delicate a structure, that it must
be slightly charred to be rendered permanently
visible." He listed also a number of common
diatoms, silicoflagellates, and desmids which he
called "Infusoria." It is impossible to guess the
true identities of the "Vibrio" and "Volvox."

Since the oyster is a filter feeder it is natural
to expect that the contents of its alimentary canal
would reflect the material suspended in water.
Many of the investigators were unduly impressed
by the occurrence of one or several species in the
stomach and because of their abundance considered
them to be of primary importance in the oyster
diet. Opinions based on such examinations re-
ferred to the following forms found in the European
oyster as important food materials: Navicula
fusiformis v. ostreari, Griin. (Puysegur, 1884);
desmids, minute animals, and dead organic matter
(Hoek, 1883); bottom diatoms Nitzschia punctata,
N. acuminata, N. sigma, Grammatophora oceanica,
and Diploneis bombus var. densestriata, the latter
species being considered of special importance for

ORGANS OF DIGESTION AND FOOD OF THE OYSTER

231

fattening of oysters (Hinard, 1923). American
biologists made similar observations in C. virginica.
McCrady (1874) concluded that "diatoms and
spores of algae" constitute the food of Carolina
oysters; Lotsy (1893) found that in the James
River, Va., "oyster lives almost exclusively on
diatoms"; according to Smeltz (1898), the natural
food of Florida oysters "can be supplemented
by . . . the pollen of our pine trees and the bloom
of our palmetto", (p. 307) but no evidence vs^as
presented that pollens were found in the stomachs
or that they can be digested by the oysters. The
flourishing and fattening of oysters in Delav^are
Bay was attributed by Nelson (1947) to the abun-
dance of the diatom Skeletonema, which he called
"the most valuable of all diatoms in the food of
oysters in New Jersey watei-s." In an earlier
paper (1923b) and in the Report of the New
Jersey Agricultural Experiment Station for the
year 1924 (Nelson, 1925), he emphasized the
significance of nannoplankton which "comprise by
far the largest part of the food of the oyster" and
at times is composed of small flagellates and other
minute forms which may comprise 80 to 90 percent
of the stomach's contents. Since no plankton
analysis was made by Nelson of the Delaware Bay
water at the time of the Skeletonema bloom, the
conclusion that the species is "the most valuable"
requires corroboration.

Moore (1910) found that eight species of diatoms
constituted 98 percent of the total amount of food
in the alimentary tract of Texas oysters and that
organic detritus also might play an important part
in nutrition. Experimental studies of the feeding
of oysters made by Martin (1927b) showed no
significant differences between the average in-
creases in size of young oysters which were fed
pure cultures of the diatoms — Nitzschia jmlea,
Amphora cojfeaeformis, Nitzschia -poleaceae, Amphora
cofeaejormis var. lineata, and one species of green
alga, Gloeocystis vesiculo.m. No check was made on
the amount of food added to the water and the
experiment lasted only 4 weeks. Water was
changed only once during this period. Because of
the obvious deficiencies in the experimental
technique no definite conclusions could be made
from these observations. Martin also suggested
that zoospores of Enteromorpha and other algae
{Ulva, Monostroma, Ectocarpus, and Pylaiella)
form an important element in the food of plankton
eaters (Martin, 1927a). A comprehensive in-
vestigation of the food of the Em'opean oyster

drfS> G

Figure 210. — Moore's method for washing out the stomach
and intestinal content of the oyster. A — reservoir with
sea water; B — canule inserted in the rectum; C —
aspirator; D — collecting vessel; E — canule inserted into
mouth of the oyster; F — siphon of the reservoir; G^
siphon of the aspirator; O — oyster. Upper insert O —
shows the details of the method of inserting canules B
and E into the oyster.

was made by Savage (1925), whose work remains
the most valuable contribution to the study of
the problem. He used Moore's (1910) method of
washing the entire alimentary canal; this technique
is diagrammatically shown in figure 210. Two
canules are introduced, one into the anus, B, and
the wider one, E, into the mouth. Rubber tubing
connects the anal canule with the siphon F in-
serted in glass container A filled with sea water.
The oral canule leads to a small collecting vessel
D, which is connected to the aspirator bottle C.
By regulating the flow of water from the aspirator
C the alimentary canal may be washed out without
damaging the digestive tract. The volume of the
collected material is measured and the collected
microorganisms identified and counted. By this
method Savage (1925) sampled at regular intervals
the stomach contents of British oysters and analyzed
throughout the year the seasonal fluctuations in
the abundance of different species of algae. He
considered that the following diatoms were the
most important food items of the British (Oxford)
oysters: Nitzschiella parva, PleurosigmasTp., Coscin-
odiscus sp., Rhizosolenia sp., and Melosira sp.
The most significant conclusion made by Savage
is that the greater part of the food found in the
oysters examined by him consisted of organic

232

FISH AND WILDLIFE SERVICE

detritus and that "the animate food (i.e., living
microorganisms) never exceeded 10 percent of the
total" (by volume). He also advanced a hypoth-
esis which, however, lacks experimental con-
firmation, that growth of Oxford oysters was due
mainly to the inanimate food (detritus) and that
fattening was caused by diatoms (Nitzxchiella
longwH'ima f. parva). He found no evidence of
selection of food by the oysters and commented
that the actively feeding oyster appears to ingest
anj^thing that it can capture.

The extreme view that phytoplankton is of no
direct significance as food of 0. edulis in Danish
water was expressed by Blegvad (1914), who
classified tliis moUusk as a "pure detritus eater."
Phytoplankton, according to liis view, contributes
tf) the food only as part of the detritus after tiie
death of the algae.

Petersen and Jensen (1911) attributed great
importance to eel grass, Zostera, as a possible
source of food for bottom organisms. On the
basis of their observations Sparck (1926) ex-
perimented with 0. edulis, which he kept in a
tank with sea water to which he added a liberal
supply of old brown Znstera. Examination of tlie
stomach contents of these oysters showed many
species of flagellates and some Zostera detritus,
but the cjuaiitity of the latter was by no means
greater than in the oysters from the natural
bottoms in the fjord. Decaying Zostera probably
fertilized tlie water and stimulated the growth of
the plankton. Danish investigators emphasized
the fact that pentosan released from the decaying
Zostera is a principal source of organic food for
bottom invertebrates. The substance is ap-
parently useless to oysters because they are unable
to digest it, as has been shown by Yonge's experi-
ments (1926a). The cjuestion of tlie extent of
utilization by the oyster of the organic detritus
which is always present in its natural environment
has not yet been settled.

Naked flagellates and infusoria are frecjuently
found in the contents of the alimentaiy tract.
Under tlie influence of gastric fluids these forms
are rapidly destroyed and, therefore, cannot be
enumerated with any degree of certainty. The
same problem applies to the bacteria which reacli
the alimentaiy canal. That tliey may play a
considerable role in the feeding of lamellibranclis
is indicated by the experiments of ZoBell and
Landon (19:57), and ZoBell and Feltham (193s).
with the California mussel, which was fed known

amounts of red coccus and a spore-forming
bacillus. Within 3 hours the mussel removed
about 200 million bacteria per 1 ml. of water.
The microorganisms were actually ingested and
after 6 hours disappeared from the digestive
tract. In 9 months the mussels which were fed
red coccus gained an average of 12.4 percent, the
bacillus fed animals gained 9.7 percent, and the
fasting mussels, kept as controls, lost about 6.8
percent. These experiments suggest an explana-
tion of tlie observations by Kincaid (1938) that
oysters kept for several months in water with
nothing to feed on except bacteria appeared to be
normal and even increased their glycogen content.
Kincaid's experiment should, of course, be re-
peated and the cpiestion of the role of bacteria
should be adequately studied before a conclusion
can be made of their significance in the feeding of
oysters and other bivalves.

By feeding the oyster known concentrations of
coliform bacteria, Galtsoff and Arcisz (1954)
found that 15 minutes after the start of addition
of the culture the two oysters retained from 21 to
49 percent of Esrherischia coli available in sea
water. The accumulation of bacteria soon I'eached
the point at which no more microorganisms were
retained and the effluent leaving the oysters
contained more E. coli than the surrounding
water. Retention and elimination of microorgan-
isms are probabty associated with the secretion
and discharge of mucus by the gill epithelium.
These results confirm the previous observations
by Galtsoff (1928) that over 50 percent of the
bacteria pass through the gills and that only a
fraction of then- total number is retained.

The organisms found in the stomach of the
oyster reflect the composition of plankton and
nannoplankton present in the surrounding water.
Selection is made primarily by the size and shape
of food particles, although the ability of the
oyster to discriminate between two suspensions
of microorganisms of different colors but of the
same size was suggested by I^oosanoff's experi-
ments (1949). A more detailed study should be
made, however, before the discriminating ability
of the oyster is confirmed.

There are several weaknesses common to all the
studies on the feeding of oysters. The conclusions
are based on examinations of the contents of the
stomach and composition of feces without giving
proper consideration to the nutritive value of
different forms and their digestibility. The simple

ORGANS OF DIGESTION AND FOOD OF THE OYSTER

233

test of feeding the oysters inert materials such as
carmine powder, carborundum, clay, pulverized
williamite, and colloidal carbon would show that
these undigestible nuiterials, if fed gradually and
not in excessive quantities, are swallowed and pass
through the digestive tract. The fluorescent
mineral williamite, which I used extensively in my
studies, is particularly suitable for this purpose
because it permits easy detection of the most
minute granules of the mineral inside the intestinal
tract or in the feces when illununated by ultra-
violet light. The fact that some of the micro-
organisms found in the stomach are not destroyed
and can be recaptured alive in the feces has been
known for a long time. The dinoflagellate
Prorocentrum micans was seen by Blegvad (1914,
p. 47) to pass unharmed. Living Chlorella and
Nitzschia closterium given to C. virginica in large
quantities can be recovered alive from the feces
and recultured (Loosanofl" and Engle, 1947). In
studies of the effect of feeding oysters in the labora-
tory I frequently used a light suspension of
Fleishmann's yeast, and observed that such a
large number of yeast cells passed undigested that
the feces acquired a milky color. Thus, the
presence of an organism or its remnants in the
alimentary tract in itself is not a proof that it is
being used by the oyster as food and that it has
nutritive value. Neither the enumeration of the
organisms found in the stomach nor the deter-
mination of their volume gives satisfactory quanti-
tative data. It is at present impossible to judge
whether, for instance, one cell of Coscinodiscus
equals or differs in nutritive value from a single
cell of Pleurosigma, Skelefonema, Nitzschia, or
other forms. Information is lacking about the
caloric value and chemical composition of various
forms and, therefore, it is impossible to determine
the number that should satisfy the energy re-
quirements of the oyster.

Through trial and error oyster growers know
that certain grounds in their possession are partic-
ularly suitable either for the growth or for
fattening and conditioning of oysters for market.
Sometimes a great difference in the productive
capacity of grounds may be found in the two
areas located a short distance apart. In an
ecological survey of the bottom it is relatively
easy to detect conditions which are unsuitable for
growth. It is, however, impossible at present to
evaluate the potential productivity on the bottom

234

because of the inadequacy of our knowledge of the
nutrition of the oyster.

ARTIFICIAL FEEDING

So far only a few experiments on artificial
feeding reported in the literature were successful
in producing an increase in the weight of the
oysters. As a rule oysters kept in the laboratory
show lack of nutrition and die sooner or later.
Better results may be obtained by keeping them
in large outdoor tanks adequately supplied with
sea water which has not been stored for any
length of time. Experiments by Martin (1927a,
1927b) in feeding oysters with pure cultures of
plankton forms resulted in very poor growth.
Sparck (1926), experimenting with Zosiera as a
potential food for the European oyster, emphasized
the fact that oysters "may thrive, increase in
size and even spawn in very small limited water
volumes without any renewal of water worth
mentioning." Such conditions occur in the Nor-
wegian oyster basins and in the French "parks"
which, however, must contain "some source
producing nourishment in sufficient quality and
quantity." This material presumably may derive
from the organic detritus. He also reports
that in his experiments the "development of
bacteria did not seem in any way to hurt the
oyster, rather the opposite."

A unique experiment, unfortunately not well
known to biologists, was made by Gavard (1927,
quoted from Korringa, 1949) in Algiers. He fed
the oysters an artificial detritus prepared from
animal and plant material and obtained an increase
of 15 kg. per 1,000 oysters per season. Korringa
states that these results demonstrate the ability
of the oysters to gi-ow without using living organ-
isms as food. Without access to Gavard 's
original paper it is impossible to judge if the
detritus was directly consumed by the oysters
as food or whether it stimulated the growth of
bacteria and nannoplankton.

Artificial enrichment of sea water by adding
commercial fertilizers at one time seemed to be
a simple answer to the problem of providing in-
creased food supply to the oyster. To test the
idea a series of experiments was conducted in the
Bureau of Commercial Fisheries Biological Labora-
tory at Milford, Conn., which resulted in the
interesting discovery that an excessive concentra-
tion of microorganisms {Chlorella sp., Nitzschia
closterium, Prorocentrum triangvlatnm, Euglena
viridis) adversely affects the feeding of oysters

FISH AND WILDLIFE SERVICE

(Loosanoff and Engle, 1947). A large-scale
"natural" experiment along the same line took
place in Great South Bay where unbalanced fer-
tilization of sea water by manure from the duck
farms located along the banks of the bay boosted
such reproduction of Chlorella-like organisms that
the heretofore prosperous shellfish industry of the
bay suffered a serious setback (Redfield, 1952).

Nelson (1934) made a series of tests of several
substances as artificial foods for oysters. He
used corn starch, ground alfalfa, soybean meal,
and ground meat of the king crab. It is not clear
in his report if the criterion of results was the
weight of the oyster meat. Nelson states that
only with corn starch "was any success obtained."
The details of these experiments have not been
disclosed.

In spite of doubtful results the artificial feeding
of oysters appears to be a definite possibility which
should be carefully investigated. Since oysters
are able to absorb glucose dissolved in sea water
(Yonge, 1928), it seems desirable to explore more
thoroughly this method of feeding. Furthermore,
the diet of the oyster and the nutritive value of
different diatoms and flagellates should be investi-
gated together with the methods of their cultiva-
tion. It is reasonable to expect that certain forms
richer in protein, may be more useful for obtaining
better growth of oysters; others, richer in car-
bohydrates, may prove more valuable for their
fattening. Research along these lines offers
many interesting possibilities that may prove
useful in the artificial culture of oysters.

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733-851 0—64 16

237

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238

FISH AND WILDLIFE SERVICE

CHAPTER XI
THE CIRCULATORY SYSTEM AND BLOOD

Page

General characteristics 239

The pericardium 239

The heart 240

Physiology of the heart 242

Automatism of heart beat 242

The pacemalier system.. 245

Methods of study of heart beat 247

Frequency of heat ___ 248

Extracardiac regulation 250

Effects of mineral salts and drugs 251

Blood vessels _. 253

The arterial system 253

The venous system 254

The accessory heart 258

The blood 259

Color of blood 261

The hyaline cells 261

The granular cells 262

Specific gravity of blood 265

Serology. 265

Bibliography 266

A heart, arteries, veins, and open sinuses form
the circuhitory system of oysters and other bi-
valves. The sinuses, or lacunae, are irregular
spaces of varying size in the tissues and have no
walls of their own other than the surrounding
connective tissue. They are interposed between
small arteries and veins and function in place of
the capillaries of vertebrates. Blood cells are not
confined to the vessels; they wander throughout
the tissues, aggregating in the sinuses. A large
number of them accumulate on the surface of the
mantle and gills and are discarded. Diapedesis,
i.e., slow bleeding tlirough the surface of the body,
is a continuous and normal process which is ac-
celerated by adverse conditions, by injuries to the
tissues, and by removal of an oyster from its sliell.

The open sinuses within the circulatory system
present a mechanical puzzle. It is difficult to
visualize how the pressure of the systolic contrac-
tion forces the blood to leave the open spaces and
enter the venal system, which has no valves, go
through a complex net of branchial vessels and
finally enter the heart. To a great e.xtent the
mechanical deficiency of the circulatory system is
compensated by the pulsating vessels of the mantle
and by the contractions of two accessory hearts on
the walls of the cloacal chamber. The pulsations

FISHERY bulletin: VOLUME 64, CHAPTER XI

of these organs are independent of the beating of
the principal heart, and their primary function is
to oscillate the blood within the pallial sinuses.

THE PERICARDIUM

The heart is located in the pericardium, a thin-
walled chamber between the visceral mass and the
adductor muscle (fig. 71). In a live oyster the
location of the heart is indicated by the throbbing
of the wall of the pericardium on the left side.
Here the pericardium wall lies directly under the
shell. On the right side the promyal chamber
extends down over the heart region and tlie mantle
separates the pericardium wall from the shell.

The cavity in which the heart is lodged is slightly
asymmetrical ; on the right side it extends farther
along the anterior part of the adductor muscle
than on the left. The pericardium is large enough
to accommodate the heart and to retain a supply
of the fluid in which the heart is bathed. The
volume of the pericardium can be measui-ed by
the following metliod. A solution of plastic or a
thin mixture of plaster of paris is poured into the
pericardium from which the heart has been re-
moved; after the material has set, the plaster
molds are waterproofed by immersing them in a
hot mixture of beeswax, rosin, and turpentine.
The volumes are measured by displacement. In
an adult Crassostrea virginica about 12 to 14 cm.
in height, the capacity of the pericardium varied
from 2.4 to 2.7 ml. ; approximately the same
\'olunie of blood and pericardial fluid could be
withdrawn from the cavity by hypodermic syringe.

Two reno-pericardial canals open on the right
and left side of the ventro-posterior wall of the
pericardium and provide direct communication
with the excretory system (see: ch. XII). The
wall of the pericardium is formed of connective
tissue similar to that in the mantle; the tissue is
well supplied with blood vessels, blood sinuses
(figs. 211 and 212), and branches of the cardiac
nerve (fig. 213). The epithelium lining of the side

239

0

Microns

200

Figure '211. — Transverse section of the pericardium wall of C. virginica. Surface epithelium is rich in mucous (light) and
eosinophilic cells (dark granules). Large vein (right) and blood sinus (left). The epithelium of tlie inner sides
(lower side of the drawing) faces the heart. Bouin, Mallory triple stain.

facing the heart consists of small flattened cells
and a few scattered eosinophilic and mucous cells;
on the opposite side, facing the shell, the peri-
cardium wall is covered with large columnar
epithelial cells with oval nuclei and many eosino-
philic and mucous cells. Basal membrane on the
upper side of the wall is well developed.

THE HEART

The three-chambered heart is suspended
obliquely in the pericardium and is held by the
root of the aorta on one side and by tlie common
efferent veins on tlie other. The ventricle is
larger and bulkier than the two auricles; a con-
striction between the ventricle and auricles marks
the partitition between them (fig. 214). The
auricles are darkened by pigment cells in tlieii'
walls. The degree of pigmentation varies from

light brown to almost black. The ventricle is a
pear-shaped structure slightly constricted along
tlie middle. Its walls are formed by tliick bundles
of nonstriated muscle fibers which traverse the
ventricular cavity and incompletely divide it into
two chambers.

In the majority of bivalves the rectum passes
tlu-ough the lieart, but in the oyster the rectum
lies behind the heart (fig. 71).

The fibers of the heart muscle cross one another
in many directions, fref}uently branch and anas-
tomose, and are surrounded by delicate connec-
tive tissue. In general the muscle tissue has a
spongy appearance (fig. 215). In the ventricle
the nmscle fibers are thicker and stronger than in
the auricles.

The wall of tlie ventricle and the septum be-
tween the two parts of the heart are formed by a

240

FISH AND WILDLIFE SERVICE

0 .„■ 200

Microns

Figure 212. — Transverse section of a portion of the peri-
cardium wall of C. virginica with an artery surrounded
by large vesicular cells, nematopsis cysts on upper right
and lower left sides. Bouin, hemato.xylin-eosin.

framework of muscle fibers and connective tissue
cells forming an irregular trabecular structure
(fig. 216), with amoebocytes in the spaces between
the fibers and in the connective tissue. The outer
surface of the ventricle is covered with epithelium
of a single layer of flat and thin cells with con-
spicuous nuclei.

The walls of the auricles, thinner and lighter than
those of the ventricle, also form a trabecular
framework supported by connective tissue (fig.
217). 'Amoebocytes are numerous between the
connective tissue cells and along the muscle fibers.
On the outside the auricles are covered with tall
colunmar epithelium which contains many glan-
dular and dark pigment cells; this epithelium
constitutes a part of the excretory system in
bivalves (Franc, 1960, p. 2016). Neither the ven-
tricle nor the auricles has an inner epithelial
lining.

The movement of blood from the auricles to
the ventricle is controlled bj' the two auriculo-
ventricular valves which appear as circular bands
of tissue surrounding small openings (fig. 218).
In longitudinal section the auriculo-ventricular
valve (fig. 219) resembles a convoluted cylindrical
tube. The walls of the valves consist of several
layers of muscle fibers arranged obliquely and
supported by connective tissue. When the auricle
(left part of fig. 219) contracts, blood is propelled
into the ventricle (right portion of the figure),
which in turn contracts, compressing the walls
of the valves and forcing the blood forward into
the aorta (not shown in fig. 219).

The heart is well supplied with ganglion cells
and nerve fibers which end in the muscles. Prep-
arations of heart tissue of C. virginica stained
with methylene blue and examined in glycerin
under oil immersion showed a great abimdance of
these elements (fig. 220). These observations
support the findings of Suzuki (1934a, 1934b),
who described the ganglion cells in the hearts of
Ostrea circumpicta Pils., 0. ijifias Thunb., and
Pinctada martensi. According to his data, the
ganglion cells in these oysters are particularly
abundant at the septum separating the auricles
from the ventricle where they form a ring at
the narrowest portion of the heart. Direct con-

Microns

Figure 213. — Transverse section of the pericardium wall

, of C. virginica with the branch of the cardial nerve (cut

at a slightly slanted angle). Bouin, hematoxylin-eosin.

CIRCULATORY SYSTEM AND BLOOD

241

^ Millime-ters ^

Figure 214. — Heart of the oyster C. virginica viewed
from the ventro-anterior side. Part of the heart's wall
was removed to show the auriculo-ventricular septum
and the musculature of the heart. Upper part — ventri-
cle and root of the aorta; lower part — two auricles and
common efferent veins. Drawn from an unpreserved
preparation.

nections between the nerve cells scattered in the
heart muscle and nerve fibers entering the heart
have not been demonstrated.

A summary of the results of many investigations
of the innervation of the bivalve heart was
given by Esser (1934), who denied the e.xistence
of the cardial ganglia in tlie heart of Anodonta
cyijnea and stated that tlie so-called nerve cells of
the nioUusk's myocardium have none of the typical
features of the nerve cells. He thought that
these cells were identical with certain amoebocytes
of the blood of Anodonta. It is true that the

amoebocytes found in the heart muscle of C
virginica have a certain similarity to the cells
depicted by Esser. In structure and in general
outline they differ, liowever, from the nerve cells
and can be recognized in the preparations stained
with methylene blue. Under high magnification
the ganglia cells in the myocardium of C. virginica
appear to be oval-shaped and bipolar (fig. 221)
rather than unipolar as described by Suzuki
(1934a) for 0. circumpicta. Their cytoplasm con-
tains granules deeply stained with methylene
blue. Round granules of larger size distributed
along tlie axis of the nerve are visible in vitally
stained preparations (fig. 220). Similar structures
are shown by Suzuki in his figure 4 (1934b) of the
preparation of the heart muscle of the Japanese
oyster (C. gigaii and 0. circumpicta). The nature
of the granules is not known.

PHYSIOLOGY OF THE HEART

Contributions to the study of the physiology of
the heart of bivalves have been made by Carlson
in a series of papers published during the years
1903-09 (Carlson, 1903, 1905a, 1905b, 1905c,
1905d, 1906a, 1906b, 1906c, 1906d, 1907, 1909);
by Ten Cate (1923a, 1923b, 1923c, 1929); Jullien
(1935a, 1935b, 1935c, 1935d, 1936a, 1936b, 1936c);
Jullien and Morin (1930, 1931a, 1931b); JuUien
and Vincent (1938); Jullien, Vincent, Bouchet,
and Vuillet (1938) ; JulHen, Vincent, Vuillet, and
Bouchet (1939); Takatsuki (1927, 1929, 1933,
1934a, 1934b) ; Oka (1932) ; Suzuki (1934a, 1934b) ;
Prosser (1940, 1942); and many others. The
literature up to 1933 is adequately reviewed by
Dubuisson (1933), and more recent investigations
are sunmiarized by Krijgsman and Divaris (1955).
The studies cited above were made primarily on
the fresh-water mussel Anodonta, on Mytihts,
Pecten, and Mya. A relatively small number of
observations were made on oyster heart.

AUTOMATISM OF HEART BEAT

Most of tlie experimental work on bivalve
hearts has been done with excised preparations
of the organ kept in a perfusion chamber supplied
with the van't Hoff or Ringer solutions or with
natural sea water. Few observations were made
on the heart in situ.

An automatic rhythmical beating of the excised
oyster heart continues for a long time if the heart
is kept in an isotonic solution, preferably in sea
water, at normal pH of about 8.0 or in the peri-
cardial fluid, and the heart muscle is slightly

242

FISH AND WILDLIFE SERVICE

0

Microns

200

Figure 215. — Small piece of heart wall of C. virginica showing spongy appearance of muscles. Slightly compressed whole

mount. Formalin, 5 percent, hematoxylin-eosin.

stretched by the pull of a light lever to which the
aorta end of the ventricle is attached; the opposite
end of the ventricle is tied to an immobilized
glass rod. Gentle stretching is sufficient to pro-
vide the necessary stimulus. Takatsuki (1927)
claimed that under these conditions the isolated
heart of the Japanese oyster, 0. circumpicta, may
remain active for 16 days. Observations in the
Woods Hole laboratory show that tiie excised
hearts of C. virginica kept in sea water at room
temperature continued to beat for 2 to 3 days,
but the frequency and the amplitude of beat
decreased noticeably after the first 24 hoiu-s.

The molluscan heart functions as a pressure
pump which must develop considerable power

to propel the blood through the circulatory system.
The mechanical force during the systole is pro-
duced by the contraction of a trabecular wall
made of many anastomosing fibers. This arrange-
ment, also present in 0. edulis (Jullien, 1935b), is
shown in figures 214 and 215.

In a number of bivalves {Anodonta, Mytilus,
Osfrea) the peristaltic wave in the ventricle
starts at the posterior end and progresses forward
(DeBoer, 1929; Ten Gate, 1923a, 1923b, 1923c).
The contraction of the ventricle compresses the
auriculo-ventricular valves (fig. 218) and prevents
the reflux of blood into the amides. There is an
interval between the contractions of the ventricle
and auricles which may be noticed by visual

CIRCULATORY SYSTEM AND BLOOD

243

Millimeters

Figure 216. — Cross section through auriculo-ventricular
septum of C. virginica. FormaHn 5 percent, hema-
toxj-lin-eosin.

inspection. The electrocardiogram of the oyster
heart {0. edulis) published by Eiger (1913) shows
that the interval is about 0.5 second. A similar
condition in the heart of C. virginica was demon-
strated on an electrocardiogram (fig. 222) made
in the Bm'eau's shellfisii laboratory by removing
part of one valve and placing the electrodes on
the pericardium wall and on the adjacent tissues.
Action currents observed by Taylor and Walzl
(1941) in the ventricle of the excised heart of
C. virginica consist, according to their interpre-
tation, of two components, a major diphasic
wave preceding the contraction, and a slow wave
at the time of contraction.

The refilling of the heart during the diastolic
phase is dependent on pressure mechanism in the
pericardiimi. Krijgsman and Divaris (1955) pro-
pose the following probable explanation which
requu-es further corroboration. Tlie change in the
hydrostatic pressure in the pericardial chamber,
caused by systolic contraction, is compensated by
the expansion of the auricles. At the moment the
ventricle starts to contract it exerts a suction
which brings in blood through the reno-pericardial
canal and venous sj-stem. Thus, the contraction

Microns

200

Figure 217. — Cro.ss section of the wall of the auricle of
C. virginica. Outside wall is covered with glandular
epithelium. Rouin, hematoxylin-eosin.

of the ventricle automatically results in the expan-
sion of the auricles. This interesting hypothesis
may be corroborated by observations on hydro-
static pressure inside the heart and in the peri-
cardial cavity and by motion pictures of the
sequences of ventricular and auricular ])eat. To
my knowledge these have not yet been made.

Observations on bivalve hearts in situ show
that the ventricle and auricles alternately increase
in size while they are being filled with blood.
Both auricles of the oyster heart contract simul-
taneously (Skramlik, 1929).

Experimental e\'idence indicates that the autom-

0m^^

Millimeters

100

Figure 218. — Cross section of the heart at the auriculo-
ventricular valves of C. virginica. Bouin, hematoxylin-
eosin.

244

FISH AND WILDLIFE SERVICE

Millimeters

Figure 219. — Auriculo-ventricular valve of C. virginica seen in longitudinal section. Auricle on the left. Bouin,

hematoxylin-eosin.

atism of the bivah'e heart is of diffuse nature.
Berths and Petitfrere (19;34a, 1934b) showed that
contractions of the lieart of Anodonta originate at
any point of tlie ventricle whether it is observed
in situ, or on isolated and even sectioned pieces.
In these studies the authors used optical methods
to record the beats of the hearts, which were fully
submerged in Ringer solution or in Anodonta blood
and were not stretched by writing levers. They
found that such distension of the ventricle removed
the asynchronism in automatic acti^^ty, increased
the amplitude of the contraction, and diminished
the rhythm. Jullien and Morin (1931a) reported
that the pulsations in dissected strips of heart
muscles of 0. edulis continue for some time. One
may conclude that the hearts of the oyster and
other bivalve mollusks are myogenic, i.e., their

intrinsic automatism originates in the muscular
tissue. In the myogenic hearts of bivalves the
beat can start at any point and the contraction
can be local or involve the entire organ (Berthe
and Petitfrere, 1934b). This type of activity
differs from that of the neurogenic hearts, such as
those of arthropods, in which the excitation wave
of the beat originates from the nerve cells of the
ganglia.

THE PACEMAKER SYSTEM

We know that the rhythmic activity of the
hearts of bivalves originates in the heart itself and
is not provoked by impulses from the central
nervous system. Whether this automatism is
produced by localized pacemakers or is a general
property of all muscle fibers has not been adequate-

CIRCULATORY SYSTEM AND BLOOD

245

0 ^„ 60

Microns

Figure 220. — Nerves in the heart muscle of C. trirginica vitally stained in methylene blue. Glycerin-jelly.

ly studied. The presence of nerve ceUs in the heart
has been confirmed for many bivalves, gastropods,
nudibranchs, and cephalopods (Dogiel, 1877;
Suzuki, 1934a, 1934b; Dubuisson, 1933). On the
other hand several investigators deny the presence
of nerve cells in the heart of moUusks and consider
that connective tissue cells were mistakenly
described as nerve cells (Krijgsman and Divaris,
1955). Motley (1933), Esser (1934), and Prosser
(1940, 1942) were unable to find them in Anodonta
and VenuK. Inconsistencies in the results are
probably due to the uncertainties encountered in
staining nervous elements of the heart with the
usual histological technique and frequent failures
in using some brands of methylene blue.

It is known that in Anodonta and Mytilus the
wave of ventricular contraction starts at the

posterior end. Furthermore, by applying heating
to various places of the hearts of Anodonta, Unio,
and Mytilus DeBocr (1929) was able to^how that
warming the posterior part of the ventricle
increases the beat frequency, whereas the heating
of the anterior part has no effect (Krijgsman and
Divaris, 1955). In the heart of a dying oyster
(0. edulvi), the aortic region continues to beat for
a longer time than do the other parts of the organ;
the isolated hearts seldom beat if the aorta is
completely cut off from the preparation (JuUien
and Morin, 1931a). This is also true for the
longitudinal fragments of the heart, which con-
tinue to beat if they contain a piece of aorta.
These observations seem to support the opinion
that in most cases the bivalve heart possesses a
diffuse myogenic pacemaker.

246

FISH AND WILDLIFE SERVICE

0

10

Microns

Figure 221. — Nerve cells in the heart muscle fiber of C. virginica. Methylene blue vital stain.

Pharmacological evidence of tlie effect of drugs
on heart, described later (p. 252), and particularly
the action of acetylcholine and the antagonism of
curare to acethycholine, support the view that
the pacemaker system in the oyster heart is of a
diffuse mj^ogenic nature.

METHODS OF STUDY OF HEART BEAT

In order to count the number of beats per unit
of time a portion of the left valve must be removed
without injury to the adductor muscle and tlie
underlying tissue. The oyster is then kept in
sea water at constant temperature, and tiie num-
ber of beats is recorded. The method was used
by Federighi (1929) and by Koehring (1937),
who drilled a small round window in the valve
and with sharp scissors dissected the pericardium
to expose the heart. These oysters lived for several
weeks in running sea water in the laborntory of
the Bureau of Commercial Fisheries at Woods
Hole without noticeable ill effects.

Stauber (1940) modified the technique by
cutting windows in both valves without injury to
the pericardium wall and cementing them over
with pieces of glass or cellophane. For observa-
tion the operated oysters were illuminated from
underneath. In a few days both were covered by
new shell and had to be replaced. Shell material
that covered the window of the left side, where the
pericardium wall touched the valve, probably
spread from the adjacent areas of the mantle.

Figure 222. — Electrocardiogram of C. virginica tal^en in
situ. A gentle wave corresponding to auricular con-
traction A precedes by approximately one-half sec-
ond the contraction of the ventricle. Temperature 22.(3
°C Time intervals, 1 second.

Pulse records can be obtained without touching
the heart itself by removing a portion of the valve,
using the pericardium wall as a sphygmograph
tambour, and providing a small stand made of
light plastic to support one arm of the writing
lever. The disadvantage of this method used in
the shellfish laboratory at Woods Hole was that
the heart became fatigued after several hours of
recording.

There is another technique to study heart
contraction in situ. The pericardium wall is
exposed by cutting off the valve above the adductor
muscle. A small S-shaped glass hook connecting
the heart with the kymograph lever is placed
under the auriculo-ventricular junction or under
the ventricle. A silk thread tied to the upper
part of the hook is connected to a writing lever,
which is carefully balanced so that the tension on
tiie heart does not exceed 100 mg. Care must be
taken to adjust the tension so that the pull of the
hook will not displace the heart from its normal
position (fig. 2215).

There will be a minimum of damage to the
nervous system and adjacent organs if only part
of the vah'e between the adductor muscle and the
hinge is removed. This leaves the muscle itself
intact, and only the pericardium wall is dissected
to expose the heart. The oyster is kept in a
known volume of water in a finger bowl, which is
placed in a large crystallizing dish to permit the
rapid change of water or of experimental solution
without disturbing the setup. Temperature in
the larger dish (not shown in figure 223) is
thermostatically controlled at any desired degree.
Under such conditions the beating of the heart
continues for about 2 days.

The perfusion chamber method is frequently
employed (fig. 224) in the pharmacological
studies of the effects of drugs on bivalve hearts.
In this method tiie heart is cut off at tlie levels of
the auricles and the aorta, ligatures are applied at

CIRCULATORY SYSTEM AND BLOOD

247

<Z)-

Centimeters

Figure 223. — Method of obtaining tracings of oyster heart
in situ. ad.m. — adductor muscle; h. — glass hook under
the ventricle, V; w.l.— water level. The upper valve
has been removed, the pericardium dissected, and the
oyster placed on a suitable base in a finger bowl.

both ends and the organ is placed in the perfusion
chamber filled with sea water or with Ringer
solution. The aorta end of the heart is connected
to the writing lever, and the auricular end is
attached to the base. The chamber is a glass
tube about 2 cm. in diameter with an overflow
arm near the top (fig. 224). The length of the
tube may be adjusted to obtain the desired
volume, usually 10 or 20 ml., between the bottom
and the overflow. The liciuid (perfusate) is
delivered through an inlet A at the bottom; it
fills the chamber to the level of the overflow and
runs out through outlet B. The preparation
may be aerated through a second glass tubing
inserted in the bottom. Under this condition
the heart remains alive and active for several
days.

A very delicate technique to study the nerves
which stimulate the oyster heart {0. circrimpicta)
was developed by Oka (1932). The preparation
was made in the following manner: the shell was
carefully cut off without any injury to the peri-
cardial region and visceral ganglion, the greater
part of the gills with the mantle were removed;
the adductor muscle was dissected; and the
oyster was fastened to a small board in the manner

248

shown in figure 225. In this way the visceral
o-andion with its nervous connection and the
heart were exposed and made accessible for stimu-
lation. The heart was kept in water, but the
ganglion was e.xposed to air. The rhythm was
recorded for the heart in situ and separately for
the ventricle and two auricles. For the latter pur-
pose the heart was cut at the auriculo-ventricular
junction and the cut end tied with a silk thread.
The free end was connected to a writing lever of
a kymograph (upper right part of figure 225).

FREQUENCY OF BEAT

The heart beat of all bivalves is so gi-eatly
affected by the envu'onment that reports of the
rates of beat are of little value unless the conditions
under which the observations were tnade are
completely and accurately described. Frequency
of heart beat increases with the rise of temperature
and decreases with its fall. According to Federighi
(1929), the response follows Arrhenius equation
from which the so-called temperature coefficient
(designated as ju) can be calculated, using the
technique developed by Crozier. Discussion of
temperature characteristics of biological processes
in general and the application of the Arrhenius
equation of the effect of temperature on chemical
reactions to heart physiology is beyond the scope
of tliis book. The reader interested in the problem
is referred to Barnes' (1937) Textbook of general
physiology, chapter XIII, or to chapter I in
Crozier and Hoagland's (1934) Handbook of
general experimental psychology. There is, how-
ever, serious reason to question the validity of

PERFUSATE

OUTLET

Centimeters

Figure 224. — Wait's perfusion chamber for recording the
activity of an excised heart of mollusks. From Wait,
1943.

FISH AND WILDLIFE SERVICE

Centimeters

Figure 225. — Oka's method of exposing the visceral ganglion for study of heart stimulation in the oyster. Reproduced

from Oka, 1932.

tlie underlying theory of temperature coefficients
of biological reactions (Belehradek, 1935).

In experiments with C. in.rginica at the Woods
Hole laboratory Federighi (1929) found the values
cif M equal to 16,000 and 1.3,600. It is rather
difficult to convert his data into conventional
terms of number of beats per minute since his
experimental results are presented only as plots
of logarithms of the frecjuencies (time required for
10 beats) multiplied by 100 against the reciprocals
of absolute temperature. x\t my request Federighi
in a personal communication supplied excerpts
from his laboratory notes which show the following
rates:

Temperature

Beats per
minute

Temperature

Beats per
minute

25°

47
35

16°

01

210-22''

10°

n

The rates appear to be much higher than those
observed by others. In Federighi's experiments

the upper critical temperature above which there
was rapid decline in pulse rate was approximately
30° C.

In Koehring's (1937) observations on C. irirgin-
ica the heart rate averaged 20 beats per minute at
20°. She found also that in the oysters with one
valve completely removed the heart action was
inhibited for several hours and there was no ciliary
motion of the gill epithelium. Inhibition of the
heart's activity when the shells are closed was
reported by Stauber (1940) in oysters uninjured
except for perforation of both valves. He found
that the heart rhythm of C. virginica slowed down
and became irregular when the oyster closed the
valves. In some of the closed oysters the heart
remained inactive for 2 to 3 minutes, then resumed
beating at low frequencies of about two to three
times per minute, only rarely exceeding six beats
per minute at the temperature of 17.5° C. As
the valves began to open, the heart beat increased
to 14 to 16 times per minute. These results are
in accord with observations on Anodonta and

CIRCULATORY SYSTEM AND BLOOD

249

Sphaerium (Cyclas) by Gartkiewicz (1926), who
described the suppression of heart beat and of
ciliary motion during the periods of shell closui'es.
Because of the high transparency of the shell of
Sphaerium- the behavior of the heart of this mol-
lusk could be observed under normal conditions.
Gartkiewicz calls the inhibition of cardiac and
ciliary activity the "sleep" of the bivalves. The
cause of the heart's inhibition is not known; it is
probable that in the case of Sphaerium the lowered
pH of body fluids and the accumulation of carbon
dio.xide may have contributed to the suppression
of cardiac activities. This, however, does not
account for the observed temporary cessations of
heart beats in the oysters and clams kept in sea
water but with their valves partly removed.
Apparently the stoppage associated with the con-
traction of the adductor muscle was due to inhibi-
tion originated from the nervous system.

The heart beat in 0. circumpicia of Japan
reaches a ma.ximum of 30 beats per minute at 35°
C. and slows down to three beats per minute at
5° C. and to 14 at 40° C. No heart action was
recorded by Takatsuki (1927) at 0° and at 45° C.
Climatic conditions apparently influence the heart
rhythm since it was shown by the same author
(Takatsuki, 1929) that the heart pulsation of 0.
circumpicta P. from the waters of the northern part
of Japan (Anomori Prefecture) is about 14 times
per minute at 20° C. In contrast, the pulse of (K
dendata Kuster from the bay of Palau, .South Sea
Islands, where the temperature ranges from 28° to
29° C. throughout the j^ear, was only eight times
per minute, and the ma.ximum rate of 22 times per
minute was observed in the laboratory at 45° C.
The pulsation in the northern species at tempera-
ture of 28° to 29° C. was 24 times per minute, and
the critical temperature was 35° C. These obser-
vations may indicate differences in thermic adjust-
ments of oysters inhabiting cold and warm waters.
No general conclusions can be drawn at present
from Takatsuki's observations because other fac-
tors such as degree of se.xual maturity and general
conditions of the oyster, which were not reported,
may affect the heart beat.

Visual observations can be carried on for short
periods of time only, and their usefulness is, there-
fore, rather limited although their distinct ad-
vantage is that the heart is not affected by experi-
mental manipulations. The pulse curve of the
heart beating inside the intact pericardium may
be obtained by the sphygmogi-aph tambour tech-

nique. Continuous recording may be made for
several hours before the heart is fatigued by the
weight of the wTiting lever pressing on the pericar-
dium wall and the rhythm and amplitude decrease.
The wave-line curve sllo\\^l in figure 226 repre-
sents the changes in the hj-drostatic pressure inside
the pericardium, the increase in pressure corre-
sponding to systolic contraction of the ventricle
which is followed by the falling of pressure dm'ing
the diastole when the auricles expand and are
gradually filled with blood. The method is not
sensitive enough to record separately the con-
tractions of the auricles, which beat shortly before
the contraction of the ventricle. In the experiment
shown in figure 226 the oj'ster was kept in about
3 1. of sea water at 22.5° C. ; its pulse rate was 18 to
20 times per minute.

Figure 22G. — Pulsu of an adult C. virginica at 22.5° C.
recorded by transmitting the motion of the pericardium
membrane to the writing lever. Time interval: 3
seconds.

Tlie contractions of auricles interposed between
the two ventricular contractions are clearly seen
on the tracings of the beats of an exposed heart
witli the hook connecting the writing lever placed
under the auriculo-ventricular junction (fig. 227,
two lower lines). In the upper line, the hook was
under the ventricle near the emergence of the aorta
and the auricular contractions were not registered.
The increase in frerjuency of beat shown in the
third (lowest) curve was due to an increase in the
water temperature from 20.5° to 24.5° C.

Tracings obtained with the excised heart are
similar to those made by the heart in situ with
the hook under the ventricle since no contraction
of the auricles can be registered in such prepara-
tions (fig. 228).

EXTRACARDIAC REGULATION

Carlson (1905a, 1905b, 1905c, 1906a, 1906b,
1906c, 1906d, 1907) has shown that stimuhuion
of the visceral ganglion of Cardium, Ptden,
Mytilua, and other bivalves produces an inhibitory
effect on the heart. Using faradic stinuiiation,
Diederichs (1935) demonstrated that a single
shock applied to the visceral ganglion of Afytilus
prockices diastolic arrest. By separating the
ganglia he obtained evidence that both the ac-

250

FISH AND WILDLIFE SERVICE

A/WWWWWWAAAAAAAAA/

Figure 227. — Three records of heart beat of C. virginica
in situ. The upper curve was obtained by placing the
connecting hook of a kymograph lever under the ven-
tricle. The two lower curves were made when the hook
was placed under the auriculo-ventricular junction. In-
creased frequency of the lowest curve is associated with
an increase of temperature of sea water from 20.5° to
24.5° C. Time interval, 2 seconds.

celerating and inhibiting nerves lead from the
visceral ganglion to the heart and that the two
other ganglia affect the heart by way of the visceral
ganglion. Oka (1932) found that stimulation of
the visceral ganglion inhibits both auricular and
ventricular rhythms, and Irisawa, Kobayashi, and
Matsubayashi (1961) determined the action po-
tentials in 0. laperousi and found that oyster
heart rela.xes through anodal current.

The cardiac nerve is a small branch of the
visceral nerve which emerges from the cerebro-
visceral connective near the x-isceral ganglion. Its
branches enter the auricles at their base and
regulate only the auricular rhythm. The ventri-
cular rhythm, according to Oka's view, is regu-
lated by the cardiac nerves which enter the
ventricle at tlie aorta end. This finding is not in

Figure 228. — Tracings of the beating of the excised heart
of C. virginica at 20° C. Salinity 31.7 °/oo. Time
interval, 5 seconds.

agreement with Carlson's observations that the
cardiac nerves enter the heart of a bivalve at
the base of the auricles and not at the aortic
end. Experimentation with the oyster heart is
difficult because exposure of the ganglion causes
profuse bleeding and collapse of the heart. Ftu-ther-
more, the cardiac nerves in C. virginica are ex-
tremely small and difficult to observe in the living
tissue.

Investigations by Carlson did not demonstrate
the presence of acceleratory nerves in the hearts of
bivalves. Oka (1932) thinks that possibly both
kinds of nerves, the acceleratory and the inhibitory,
are present in the heart of 0. circumpicfa but that
the action of the inhibitory nerve predominates.
The suggestion is based on the observation of old
heart preparations of lowered vitality in which the
beat of the auricles was slightly accelerated by
stimulation of the ganglion. The evidence is not
con\'incing and requires verification.

Krigsman and Divaris (1955) arrive at the
following conclusions which appear to be applicable
to the oyster heart: 1) The systolic mechanism
is situated in the heart's muscle fibers; and 2)
extrinsic regulatory nerves infiuence the pace-
maker system. The inhibiting fibers are probably
cholinergic, and the accelerating fibers may have
adrenergic properties. The latter statement needs
further verification.

EFFECTS OF MINERAL SALTS AND DRUGS

Bivalve he^irts respond readily to changes in
the chemical composition of water and to the
presence of low concentrations of various drugs
and poisons. Because of this sensitivity the hearts
of several common species such as Anodonta, Mya,
Mercenaria, Ostrea, and others often have been
used in pharmacological bioassays. The test is
usually made with a preparation of an excised
entire heart (or ventricle) in the perfused chamber.
Increased acidity slows the beat of the excised
heart of C. virginica; a pH of 4.0 and lower
causes diastolic arrest and from pH 4 to 9 the
rate increases with the increase of pH values.
Above pH 9 the contractions become irregular
(Otis, 1942).

A change in the balance of metallic ions in the
surrounding water affects cardiac activity. Small
excesses of potassium stimulate the heart by in-
creasing the frec|uency of beat (positive chrono-
tropic effect) and by changing the tonus (tono-
tropic effect) of the myocardium (.Tullieii and
Morin, 1930, 1931b).

CIRCULATORY SYSTEM AND BLOOD

251

The action of sodium is similar to that of the
potassium, but response is less pronounced. Small
excesses of calcium cause negative chronotropic
and positive tonotropic effects, and magnesium
acts in a way similar to that of calcium, i.e.,
produces negative chronotropic effect and causes
diastolic arrest of the heart. Lack of magnesium
results in a systolic arrest (JuUien and Morin,
1931b; Jullien, 1936a).

Among the effects of various drugs the most
interesting is that of acetylcholine, a chemical
agent in neuromuscular transmission which de-
presses heart action of oysters and other mollusks
(Jullien, 1935c; Jullien and Vincent, 1938; Jullien,
Vincent, Vuillet, and Bouchet, 1939; Prosser and
Prosser, 1938; Prosser, 1940, 1942; and Wait,
1943) and is particularly effective on the heart
of the clam {Mercenaria mercenaria) . Prosser
(1940) has shovm that inhibition of the heart of
this species can be obtained with a concentration
as low as 10"'^ Recent investigations by Pilgrim
(1954) and Greenberg and Windsor (1962) showed
that in the hearts of many bivalves acetylcholine
produces a "combination response", depressing
the cardiac activity in low concentrations and
exciting it at high concentrations. The authors
used ventricle strip preparations of the hearts of
40 American (in the Greenberg and Windsor ex-
periments) and 8 New Zealand species (in Pil-
grim's tests). Preparations which remained qui-
escent when first set up attained regular rhythm
in 2 to 3 hours, a condition which was also observed
in tests made in the Woods Hole laboratory on C.
mrginica. In Greenberg's and Windsor's experi-
ments the quiescent preparations were induced
to beat with 10"' to 10'^ molar concentrations of
5-hydroxytryptamine.

There exists great variability in the responses of
different bivalve species to acetylcholine. In some
of them only the depressing effect of the drug was
recorded. This group includes oysters (C. virgin ica
and C. gigas), several clams of the family Veneridae
{Mercenaria mercenaria, Tapes philipinarum, Saxi-
domus giganteus, and others), Mya arenaria,
Entoderma saxicola, and Prododesmus macro-
schisma. The excitor effect was demonstrated for
Mytilus calijornianus and M. canaliculus (in Pil-
grim's tests), thus confirming previous observa-
tions on Mytilidae by JulHen and Vincent (1938).
In Pectinidae, Matridae, Carditidae, and other
families, both types of responses were recorded.
The following explanation of the "combina-

tion response", i.e., depression in low concentra-
tion and excitation in high concentration, was sug-
gested by Pilgrim (1954): the low concentration
tends to inhibit pacemaker activity; at high con-
centration, while the pacemaker is inhibited, the
drug acts directly on the nmscle causing a steady
contraction. Further research is needed to cor-
roborate tliis hypothesis.

Greenberg and Windsor (1962) remark that "a
reasonable mode of acetylcholine action on bi-
valve hearts should involve either two separate
sites of action or two modes of attachment to the
same site at high and low concentrations".

Sensitivity of bivalve hearts to acetylcholine
varies in different species. The most sensitive
ones, reported by Pilgrim, are Dosinia, Amphi-
derma, and Mercenaria mercenaria. Oysters are
less responsive to the drug. Jullien (1935c) re-
ported that in C. angulata the frequency and the
amplitude "f heart beat are decreased in a con-
centration of 10"^ with diastolic arrest following
at two times 10"^ to two times 10"'* concentration.
In New Zealand species, Ostrea hejferdi, the
cardiac activity is depressed with a diastolic arrest
at concentrations varying from 10"' to 10"^
(Pilo;rini, 1954). In C. virginica the decrease in
the frequency and amplitude of isolated heart was
apparent at concentration 10"^ (fig. 229) and the
effect persisted for several minutes after the
preparation was flushed with fresh sea water
(second line). The effect of tlie drug can be
noticed even in extremely low concentrations of
10"** and 10"'. Under normal conditions the
iiearts of bivalves contain little acetylcholine
(Jullien and Vincent, 1938), but the heart of the
gastropod Murex is very rich in this compound.

Eserine causes periodical alterations in tlie
amplitude of heart beat and slight increase in the
rate of beating (fig. 230). The significance of the

I

i

Fr S W.

FiiiURE 229. — Effect of acetylcholine in the concentration
10-5 on the beat of isolated heart of C. virginica. ACh—
acetylcholine added; Fr.S.W.— perfusion chamber
fluslied with fresh sea water. Temperature 23.7° C.
Time interval, 5 seconds.

252

FISH AND WILDLIFE SERVICE

f\j\f[l\jV[M\[\!\imiWyw^

Figure 230. — Tracings of the heart beats (in situ) of C.
virginica in sea water (upper line) and after the addition
of eserine, (second line) in concentration of 10^*, to the
pericardial chamber. Temperature 21.5° C. Time
interval, 5 seconds.

drug in heart physiology is the fact that it prevents
the destruction of acetylcholine by the enzymes of
the organism.

Veratrine has a temporaiy stimulating effect on
the heart of 0. edulis (Jullien, 1936a). In my
experiments with isolated heart of C. drginica, a
slight stimulating efl'ect on the frequency of
ventricular contraction was recorded in the con-
centration of veratrine of 1:10,000. Within a
few seconds tlie number of beats increased from 1 2
to 18 and 20 times per minute at 20.5° C. (fig. 231).
Navez (1936) described the depressive action of
pilocarpine on the heart of Anomia.

High concentrations of curare inhibit the heart
activity of the oyster; in lower concentrations the
drug has a strong positive tonotropic effect
(Jullien, 1936a) and also counteracts the inhibitory
effect of acetylcholine. Jullien found that heart
action stopped by acetylcholine was restored by
subsequent applications of curare.

Adrenaline accelerates the heart beat of 0.
circumpicta, (Takatsuki, 1933) in a concentration
of about 1.8 times 10"'. Similar activating action
has been reported for C. virjiinica (Otis, 1942) and
for 0. edulis (Jullien, 1935d, 1936a, 1936c).
Stronger concentrations produce irregular beating
and some times systolic arrest.

y\/V\/lA/l/i/VWW\AAA/lA/ViAAAM/V

Figure 231. — Effect of veratrine (cone. 1: 10,000) on
ventricular contractions of the isolated heart of C.
virginica. Temperature 20.5° C. Time interval, 5 sec.
Upper line — in sea water; lower line — immediately after
the perfusion with veratrine in sea water.

CIRCULATORY SYSTEM AND BLOOD
"33-851 O— 64 17

BLOOD VESSELS

Lack of continuity between the arteries and
veins due to the presence of sinuses is the charac-
teristic featm'e of the open circulatory system of
bivalves. The spaces which function as capillaries
have no distinct walls, are of irregular shape, and
appear as slits in the tissue (fig. 79). Their pres-
ence imposes difficulty in the maintainance of
effective circulation of blood through the organs
and tissues. The deficiency is partially overcome
by the presence of pulsating vessels and accessory
hearts, which assist in the moving of blood through
the mantle.

All blood vessels of the oyster have very thin
and delicate walls that are easily ruptured by a
slight increase in pressure. In anatomical prepa-
rations of the circulatory system, it is, therefore,
difficult to obtain complete penetration of arterial
and venous sj^stems by injection. Partial success
may be obtained by using a warm gelatine solu-
tion stained with appropriate dyes; by injecting
borax or lithium carmine and immediately placing
the preparation into 95 percent alcohol in which
the stain is precipitated; or by injecting vinyl
resin solution diluted with acetone (Eble, 1958).
For more detailed study the preparation may be
dehydrated and clarified in oil of cloves or in
cedarwood oil. Very small vessels may be in-
jected through a capillary tubing using aquaeous
solution of methylene blue, toluidin blue, or some
other suitable d.ye. Although no permanent prep-
aration can be obtained in this way, the method
is useful for tracing the connection between the
small vessels.

Because the injection of the venous system is
even more difficult tlian that of the arteries,
knowledge of venous circulation in bivalves is
less complete than that of the arterial system.
Attempts to observe the movement of blood in-
side the veins usually are not successful because
the tissues are either too contractible or contain
so much glycogen that the vessels are obscured.
The description of the principal blood vessels of
the oyster given below is based on the examina-
tion of many specimens injected by various
methods and studied under a low power of
magnification.

THE ARTERIAL SYSTEM

The arteries can be recognized in microscopic
preparations by their well-developed walls lined
with a single layer of flattened endothelial cells

253

(fig. 81). They have a distinct layer of circular
and longitudinal muscles surrounded by connec-
tive tissue.

The arterial system described liere is shown
diagrammaticaUy in fig. 232 from the right side,
after the partial removal of the mantle and some
of the visceral mass. The right wall of tlie peri-
cardium is cut off to expose the heart. This
diagrammatic drawing is based on examination
of several specimens injected through the ventricle.

Two large arteries emerge from the posterio-
dorsal side of the ventricle. Tlie largest one is the
anterior aorta (ant.ao.), which upon leaving the
lieart forms a short enlargement or a bulb leading
to the large visceral arteiy (vise. a.) with its nu-
merous branches and small pericardial arteiy
(small unmarked vessel under the visceral artery),
which supplies blood to the wall of the pericardium.
The much smaller posterior aorta (post.ao.) sup-
plies blood to the adductor muscle and rectum (r.).
Near the point of emergence of the posterior aorta
it gives off a small rectal artery (r.a.), which fol-
lows the wall of the rectum.

The visceral artery (vise. a.) emerges from the
anterior aorta as a wide vessel that supplies blood
to the organs of the visceral mass. Its upper
branch reaches the level of the lalnal palps and of
the cephalic hood. The lower l>ranch extends
along the wall of the crystalline sac and forms the
reno-gonadial artery (r.g.a.) ; numerous small
branches of this vessel supply blood to gonads and
kidneys.

In its course toward the anterior part of the
body, the anterior aorta (ant.ao.) passes under the
intestinal loop (not shown in fig. 232) and gives off
several small vessels which bring blood to the
digestive diverticula (gastric arteries, g.a.), man-
tle, and the labial palps. At the anterior end of
the body the aorta forms a common trunk of the
pallial artery (co.p.a.), which divides into two
short branches corresponding to the left and right
side of the body, each branch giving rise to the
ventral and dorsal circumpallial arteries (cr.p.a.).
Each of these continues along the peripheiy of the
mantle lobes, supplying blood to the mantle
through a large number of sliort vessels which entl
in the mantle lacunae. A very small subliga-
mental artery emerges from the end of the common
pallial arter\- and leads to the subligamental glanfl
(fig. 78). Tlie cephalic artery- (cph.a.) and labia!
arteiy (l.a.) supply blood to the anterior end of the
body and to the right and left labial palps.

254

THE VENOUS SYSTEM

Since the presence of irregular sinuses prevents
the filling up of the entire venous system with one
injection it is necessary^ to make separate injec-
tions of the principal vessels and to supplement
the study with an examination of sectioned mate-
rial. The course of small veins may be traced by
injecting a water soluble dye and watching its
penetration in tlie tissues of the visceral mass and
gills.

The venous system comprises the sinuses, af-
ferent and eft'erent veins and small vessels of the
gills. It is diagrammaticaUy sliown in figure 233.
Ramifications of the vessels are omitted for the
sake of clarity.

The sinuses occur throughout the entire visceral
mass, in the pallium, along the adductor muscle,
and around the kidneys. Their outlines are
highly irregular, and tlie area thej' occupy varies,
depending on the degree of distension by blood.
Tlie renal sinus (r.s.) consists of several smaller
sinuses which surround the main part of the
kidneys and open into the efferent branchial
vessel and into the sinuses between the adductor
muscle and the heart at the posterior side of the
body. The renal sinus spreads into the connec-
tive tissue of tlie adjacent area and is in communi-
cation with the inter-nephridial passages leading
to the pericardium. The renal vein (r.v.) carries
blood from the sinus into the common afferent
vein. The visceral sinus, v.s., not definitely
outlined in the diagram, spreads over the surround-
ing tissues and drains its blood through the
gastric (g.v.), hepatic (h.v.), and other veins into
the common afferent vein (c.af.v.). The muscle
sinus (m.s.) is a small area below the renal sinus
on the surface of the adductor muscle under the
pyloric region. The system of afferent veins
consists of a single common afferent vein (c.af.v.)
and two lateral afferent veins, 1. af. v. (fig. 233 and
fig. 73). The common afferent vein runs on the
ridge formed by the fusion of the two inner
ascending lamellae of the gills. The blood
received by this vein conies from tlic deeper
parts of the Ixidy and is l)rought by a number of
veins which can be identified as the ceplialic
veins (c.v.) from the cephalic region; tlie labial
veins (l.v.) ; the gastric and hepatic veins (g.v.,
h.v.); the network of small reno-gonadial veins
(r.g.v.); short renal vein (r.v.) and the adductor
muscle vein (not shown in the diagram). In
thin, wateiy specimens most of these veins can 1

FISH AND WILDLIFE SERVICE |

0

_L

Centimeters

Figure 232. — Diagram of the arterial system of C. rirginica. A — right auricle; ad. a. — adductor muscle artery; an. —
anus; ant.ao. — anterior aorta; cl.ch. — cloacal chamber; co.p.a. — common pallial artery; cph.a. — cephalic artery;
cr.p.a. — circumpallial artery; g. — gills; g.a. — gastric arteries; l.a. — labial palp artery; l.p. — labial palps; m. — mantle;
post.ao. — posterior aorta; r. — rectum; r.a. — rectal artery; r.g.a. — reno-gonadial artery; vise. a. — visceral artery. For
the sake of clarity profuse ramifications of the vessels are not shown.

CIRCULATORY SYSTEM AND BLOOD

255

Centimeters

Figure 233. — Diagram of the venous system of C. virginica viewed from the right side. The right demibranch is open
and pulled out to show the water tubes and the vessels of the descending and ascending lamellae. The left demi-
branch is not visible. Vessels carrying oxygenated blood are shown in solid black; others are open. The diagram

256

FISH AND WILDLIFE SERVICE

be seen from the surface. In sexually mature and
"fat" oysters they are obscured by the deposits
of glycogen and by the accumulation of sex cells.
The paired lateral afferent veins (l.af.v.) arc of
smaller diameter than their ct>mmon partner.
They are located along tlie axis of tlie outer
ascending lamella where the lamella fuses with
the mantle lobe. The lateral afferent veins
receive the blood fr(im the mantle through the
pallial veins (p. v.).

At regular intervals the common afferent vein
is connected with the lateral veins by short trans-
verse (horizontal) vessels (t.v.). These vessels
can be seen in injected preparations of tlie gills
and in sectioned material. The communication
between the horizontal vessels in the gill tissues
is maintained bj* means of vertical vessels which
emerge from the walls of the three afferent veins
as a series in a double row, one following tlie inner
and the other the outer lamella of the demibranch.
At each interfilamentar shelf the vertical vessels
empty into a lacuna and eventually into the tubes
of the gill filaments. There is no special path for
the return of the blood from the interfilamentar
lamellae and the tubes because the filaments end
blindly. The walls of the common afferent vein
contain a layer of elastic fibers arranged circularly;
they are scarce in the walls of other veins. Endo-
thelium is absent in all these vessels. The walls
of the vertical vessels of the lamellae have a layer
of muscular fibers which are able at intervals to
constrict the lumen of the vessels along their
length. In this way the flow of blood inside tlie
gills is regulated (Elsey, 1935).

The blood channels in the interlamellar junctions
are in communication with the vertical vessels and
provide for the passage of blood from one lamella
to the other. This rather inefficient circulation
of the blood in the gill vessels is influenced by the
contraction of the entire gill musculature and by
contractions of the major afferent and efferent
veins. The pulsations of these vessels have not

been observed in vivo, but their histological
structure suggests that they are capable of con-
stricting their lumen. A tangential section of the
common afferent vein preserved in a relaxed state
(fig. 2.34) shows a well-developed layer of circular
muscles flanked on both sides by thin bands of
longitudinal muscles.

The system of efferent vessels comprises two
short common efferent veins (c.ef.v.) which open
into the auricles, a pair of branchial efferent
veins (br.ef.v.) which run along the axis of the gill
lamellae (fig. 73), pallial efferent veins (not shown
in fig. 233), and the interlamellar and interfila-
niental vessels (il.v.) of the gills. The branchial
efferent veins (br.ef.v.) run along the giU axis
parallel to the branchial nerves (fig. 73) at the
junctions of the ascending and descending lamellae.
In their course they receive blood from the renal
sinuses and empty into the common efferent vein.
Blood which circulates in the mantle is carried
to the heart through pallial sinuses and veins,
but part of the blood from the posterior portion
is drained back to the gills and to the branchial
efferent vein (br.ef.v.).

^'■'^riry

Figure 234. — Photomicrograph of a tangential section of
the wall of the common afferent vein of C. virginica
preserved in fully relaxed state. Narcotized in mag-
nesium sulfate. Kahle, hematoxylin-eosin.

was drawn from a number of preparations of partially injected venous sj'stem. Only the approximate position of
various vessels is indicated. The diagram does not intend to show the actual appearance and distribution of veins.
A — auricle; V — ventricle; a. — anus; ad. m. — adductor muscle; br.ef.v. — branchial efferent vein; c. af. v. — common
afferent vein; c. v. — cephalic vein; c. ef. v. — common efferent vein; g. — gills; g. v. — gastric veins; h. v. — hepatic
veins; il. v. — interlamellar veins of the gills; 1. af. v. — lateral afferent vein; 1. p. — labial palps; 1. v. — labial vein;
m — mantle; m. s. — mantle sinus; p. v. — pallial vein ; py. c. — pyloric caecum; p. d. v. — posterior dorsal vein; p. v. v. —
posterio-ventral vein; t. v. — transverse veins of the gills; r. — rectum; r. s. — renal sinus; r. g. v. — reno-gonadial veins;
r. v. — renal vein; v. s. — visceral mass; w. t. — water tubes of the gills.

CIRCULATORY SYSTEM AND BLOOD

257

c, p. a.

vessels

Figure 235. — Diagram of the circulation of blood in C. virginica. The position of various sinuses marked with capital
letters is indicated by broken lines; only one demibranch and one accessory heart are shown. A — auricles; A.cH. —
accessory heart of one side; P.S. — pallial sinuses; R.S. — renal sinuses; V — ventricle; V.S. — visceral sinuses; br.ef.v. —
branchial efferent vein; c.af.v. — common afferent vein; c.ef.v. — common efferent vein; ce.a. — cephalic artery; cp.a. —
circuinpallial artery; c.v. — cephalic veins; ga.a. — gastric artery; g.v. — gastric vein; h.a. — hepatic artery; h.v. —
hepatic vein; l.a. — labial artery; l.af.v. — lateral afferent vein; l.v. — labial vein; m.a. — adductor muscle artery; m.v. —
adductor muscle vein; p. a. — pallial arteries; p.af.v. — pallial afferent vein; p.ef.v. — pallial efferent vein; py.a. — pyloric
artery; r.a. — renal artery; r.g.a. — reno-geonadial artery; r.g.v. — reno-gonadial vein; r.v. — renal vein; tr.v. — tran.s-
verse veins of the gills.

In visualizing the circulation of blood within
the gills one must keep in mind the location of the
five horizontal vessels at the top of the duplicated
W-shaped junctions of the gill lamellae (fig. 73).

The course of circulation presented schemati-
cally in fig. 235 shows that the arterial blood goes
to the sinuses (P.S., V.S., R.S.) and then is con-
veyed through the afferent veins to the gills and
reaches the auricles via two common efTerent
veins. Some of the blood from the pallial sinuses
(P.S.) and from tlic renal sinus (R.S.) bypasses
the gills and is directly delivered to the auricles
through the common efferent veins.

The deficiency in circulation caused by the
presence of large smuses is counteracted by the
pulsations of radial vessels of the mantle and by
a pau- of accessory hearts (Ac.H.), which function
independently of the principal heart of the oyster.

The red and blue colors of the diagram show that
only oxygenated blood fills the heart.

THE ACCESSORY HEART

The accessory heart is a paired tubular struc-
ture along the inner surfaces of the right and left
mantle folds where they join together to form the
cloacal chamber. Its position on the wall of the
cloaca and its relation to the adjacent organs are
shown in figure 236 drawn from life.

The accessory heart of the oyster is not the
simple tubular structure described by Hopkins
(1934, 1936) and Elsey (1935). It consists of
three branches of almost equal size, joined to-
gether at a common center (fig. 237). The entire
structure has the shape of the letter Y. The
lower or ventral branch (v.br.) extends along the

258

FISH AND WILDLIFE SERVICE

p<t/,

ac h

Figure 236. — The position of the accessory heart on the
left of the cloacal wall of C. virginica. The epibranchial
chamber was dissected, and the demibranchs of the
right and left side pulled apart to expose the ventral
side of the adductor muscle. The oyster was fully
narcotized. The accessory hearts on both sides
were fully expanded (only the part of the right accessory
heart is shown), a. — anus; ac.h. — accessory heart
on the left side; ad.m. — adductor muscle; m. — mantle;
pal. or. — pallial organ; r. — rectum.

wall of the cloaca to the pallio-branchial junction
(p.br.j.).

Under slight mechanical stimulation the delicate
wall of the accessory heart collapses and the
structure becomes invisible. This explains why
the earlier investigators did not recognize it as an
active organ and mistook it for ridges on the inner
wall of the mantle (Rawitz, 1888; Kellogg, 1892).

The structure of the accessory heart of C.
idrginica (fig. 238) resembles that of the arteries
of the mantle. The walls have a well-developed
layer of longitudinal and circular muscles, but the
endothelium lining is indistinct and is probably
absent.

The pulsation of accessory hearts of C. virginica
observed in winter at the Woods Hole laboratory
was very irregular, not exceeding two to three
times per minute at room temperature of 20° to
22° C, and was independent of tlie heart beat.
During the summer the rate of contraction was six
to seven times per minute. Hopkins (1934)
states that in C. gigas the accessory heart beat
at a slower rate than the average heart rythm of
this species and the frequencies for right and left
organs averaged 6.0 and 7.5 times per minute
respectively.

The connection of the accessory heart to other
vessels was studied by the following method of
injection. Live oysters were kept for 24 to 48
hours in a refrigerator, tlien placed overnight in
cold sea water with 5 percent magnesium sxilphate.
About 2 ml. of litliium carmine was injected,
using the finest iiypodermic needle. The prepara-
tion was rapidly rinsed in fresh water and im-
mediately immersed in 95 percent ethanol, which
precipitated the dye. The injected material
remained inside the vessels and was not diffused
or washed away by dehydration and clarifying
agents (cedar oil or xylene). In this way several
permanent preparations were obtained.

Dye injected into the ventral branch (fig. 237,
v.br.) penetrated some distance into the circum-
pallial arteries of the right and left mantle lobes
and into the small branches and capillaries of the
efferent vein of tiie gills (ef.v.). The dorsal
branch (d.br.) was found to extend along the wall
of the cloaca: it does not "disappear into the
excretory organs," as stated by Hopkins, but
extends under the renal sinus to the dorsal part
of the cloacal wall. The ramifications of the
branch end in a number of capillaries which
connect them with the dorsal portion of the
efferent vein. The third or posterior branch
(p.br.) follows the ventro-lateral border of the
adductor muscle and gives many ramifications
inside the cloacal wall.

Blood carried by the ventral branch of the ac-
cessory heart enters the pallial artery against the
pressure produced by the principal heart. Under
these conditions its penetration inside the artery
must be limited, and at the end of the contraction
wave some of the blood probably re-enters the
branch. Movement of the blood inside the cir-
cumpallial artery can not be seen, but through the
thin wall of the accessory heart one can observe the
flushing of blood cells back and forth. Ramifica-
tions of the ventral and dorsal branches form
capillaries which are in direct connection with the
side vessels of the efferent vein. It can be assumed
from the direction of the contraction waves that
blood from the accessory heart moves toward the
efferent vein of the gill and that part of the blood
is flushed back as the impulse wave progresses
along the wall of the branch.

OsciUation of the blood in the mantle is the
primary function of the accessory hearts. Their
oscillatory movements facilitate the gaseous ex-
change and provide a means for efficient respira-
tion. The location of the accessory hearts con-

CIRCULATORY SYSTEM AND BLOOD

259

Centimeters

10

Figure 237. — Accessory heart of C. virginica. Drawing made from an injected preparation, ad.m. — adductor muscle;
cap. — capillaries; cr.p.a. — circumpallial artery; d.br. — dorsal branch; ef.v. — efferent vein; p.br. — posterior branch;
p.br.j. — pallio-branchial junction; r.s. — renal sinus; v.br. — ventral branch.

firms the opinion that the mantle and the wall of
the cloaca play significant roles in tlie respiration
of oysters.

The pulsation of the accessory hearts makes it
possible for the blood of the pallial sinuses to enter
the branchial efferent veins or to be forced into
the gills through the lateral afferent veins. The
pacemaker system and the nervous control of the
accessory hearts have not yet been studied.

THE BLOOD

There are two distinct groups of blood corpuscles
in bivalve mollusks, the hyaline cells and the
gi-anular amoeboid cells. The latter are fre-
quently called granulocytes because of the large
number of granules in their cytoplasms, or

amoebocytes and phagocytes because of their
ability for amoeboid movements and phagocytosis.
Tlie hyaline cells are not entirely devoid of
granules but tliey are very sparse. These cells
also display amoeboid movement but are much
less active than the granular cells. Both types of
cells are present in the oyster.

Samples of blood for e.xaniination may be
obtained by puncturing the pericardial wall with
a fine glass pipette and drawing tlie desired
volume of blood. In the same manner blood may
be obtained directly from the ventricle or auricles.
vSome blood cells are always present in the shell
liquor and on the surfaces of the gills and mantle.
A fair sample of cells can be obtained by scraping
these tissues with cover slips or by drawing the
pipette along them. For examination of live

260

FISH AND WILDLIFE SERVICE

0

Mi lllmeters

0.5

Figure 238. — Transverse section of the accessory heart of C. virginica preserved in widely expanded state. Kahle,

hemato.xyhn-eosin.

cells the sample may be placed in a moist chamber
or a small quantity of blood may be dropped in
a glass dish with sea water of the same salinity
from which the oysters were taken. Under these
conditions the cells of C. virginica may remain
alive for about 6 daj^s and can be used for studies
or classroom demonstration (Breder and Nigi-elli,
1933).

For smear preparations drops of blood should be
left on slides until the cells begin to expand.
When a desired state of expansion has been at-
tained, the preparation is fixed in Bouin III for a
few minutes or in chromic or osmic acid (liquid or
fumes). Satisfactory preparations may be ob-
tained by using Romanowsky's, Leishmann's,
Giemsa's, and McNeal's tetrachrome stains made
in a solution of absolute methyl alcohol. These
reagents fix and stain the cells in one operation.

COLOR OF BLOOD

The blood of the oyster is colorless alid contains
no respiratory pigments such as the hemoglobin
in vertebrates or hemocyanin found in snails and
cephalopods. In semipopular books on oysters a
statement is sometimes found about the presence
of hemocyanin in oyster blood. To clarifj' this
question, a composite sample of blood and peri-
cardial fluid was collected from six adult C.
virginica and submitted for spectrophotometrical
analysis, which was kindly performed in George
Wald's laboratory at Woods Hole. The following
is the report received from Wald:

"The pH (of the sample) was 7.33. The ab-
sorption spectrum showed specific absorption in
the visible region corresponding to the hemocyanin
band at about 570 ni/z. Hemocyanin possesses
also a very high, sharp absorption peak at about
340 m/j., some 20 to 30 times as intense as the
absorption in the visible spectrum. This therefore
constitutes a very delicate test for the molecule.
This also did not appear in the spectrum though
a small band was found at lower wavelengths, at
about 327 m^.

"The 570 and 340 m^. absorptions are found in
oxyhemocyanin; both are abolished in the reduced
condition. As an added test therefore this sample
of oyster blood was reduced with sodium hydro-
sulfite. The ultraviolet absorption at about 327
ii\fi. instead of being depressed, rose greatly. I
do not know what this substance is, but quite
certainly it is not hemocyanin."

THE HYALINE CELLS

These cells with clear cytoplasm containing but
few granules are of uniform shape, varying only in
size from 5 to 15 ti. When examined alive they
are usually spherical (fig. 239), have a distinct
cell membrane, and are of pale yellow-green color.
Because of their high refractive quality they stand
out sharply in the field of view of the microscope.
The slow movenaent of the cells can be noticed if
the preparation is watched intently for 30 minutes
or longer. One of these cells, under continuous
observation in the Woods Hole laboratory for 45

CIRCULATORY SYSTEM AND BLOOD

261

0

Microns

30

Figure 239. — The hyaline blood cells of C. virginica. Very small cells on the left; normal cells on the right. Camera

lucida drawing of live cells on glass.

minutes changed its shape foui' times from round
to oval and back again. The movement is ex-
tremely gradual and consists mainly in bulging of
one side of the body. The nucleus is not visible
in the live cells and rarely can be seen in stained
preparations. The cells are basophilic, staining
reddish-purple with Romanowsky's stain. The
nucleus stains the same color as the cell.

The hyaline cells comprise about 40 percent of
the total number of blood cells in a sample. This
is an average of a number of samples taken from
the oysters of Long Island Sound and of Chesa-
peake Bay in which blood was drawn from the
pericardium, heart, and shell liquor. In the
oysters in good, healtliy condition, the proportion
of hyaline cells varied from 25 to 64 percent, but
the differences were not consistent and did not
seem to be affected by the origin of the oysters
or by the part of the body from whicli the sample
was taken.

THE GRANULAR CELLS

The granular cells or the amoebocytes vary
greatly in shape, size, and behavior. This un-
doubtedly is due to their pronounced ability for
amoeboid movement. In live contracted state
tliey measure about 6 /x in diameter, but they
expand and spread to a much larger size. When
fresh blood drawn from the oyster by a pipette
is spread on a glass slide, many blood cells form
aggregates or clumps. This aggregation or ag-
glutination results from the adhesiveness of the
cell membranes, which stick on contact with one
another (Drew, 1910). In a quiescent stage the
cells are usually round and motionless. In about
half an hour they begin to expand and separate
from the clump. By the end of the first hour

262

the amoeboid movement becomes active and the
cells disperse themselves and form concentric
rings around the clump.

The cytoplasm and the granules of a moving
amoebocyte (fig. 240) flow slowly from the center
of the cell out to the edge and push the cell
membrane out, forming a pseudopodium. During
the formation of very narrow pseudopodia the
cytoplasm appears to flow out with the granules
arranged in single fUe. Contraction seems to be
aflfected all at once over an entire cell area, and the
action can be quite sudden. In withdrawing, the
cytoplasm sometimes leaves behind it a colorless
and empty membrane. Fine hyaline projections
called "bristle pseudopodia" (fig. 240, right) may
remain extended from the membrane and some
can be traced back to it. This seems to confirm
the argument of Goodrich (1920) that the bristle
type pseudopodium is a fold or thickening in the
membrane and not a physiologically active part of
tlie cell body.

Clots of blood cells are often observed in injured
blood vessels and the connective tissue surrounding
small arteries of the mantle, and can be produced
by intercardiac injection of tissue extracts.
Infiltration of connective tissue by amoebocytes
and intravascular blood clots is usually found in
watery green oysters from polluted water (fig. 24 1 ) .

There is no true coagulation of the oyster blood.
The coalescence and clot formation of blood cells
outside of the body is the result of the entangle-
ment of amoebocytes by the bristlelike pseudopodia
or by tlie strands of hyaline ectoplasm (fig. 242).

The granules of live amoebocytes are usually
yellowish-green, with the color much more pro"
nounced in green oysters. The staining aflinities
of blood cells have been studied by several in-

FISH AND WILDLIFE SERVICE

0

30

Microns

Figure 240. — Amoebocytes (granular cells) of C. virginica observed in vivo. Camera lueida drawings of live cells on glass.

Figure 241. — Intervascular blood clot and infiltration of amoebocytes in the mantle of green C. virginica. Bouin,

hematoxylin-cosin.

vestigators with somewhat different results. Koll-
mann (1908) found that marine hmiellibranchs
have acidophilic granules, while those of fresh
water moUusks are amphophilic. The granules of
0. eihdw (Takatsuki, 1934a) are neutrophilic with
a tendency to become stained vitally by basic

CIRCULATORY SYSTEM AND BLOOD

dj-es. The amoebocytes of C. circumjncfa (Ohuye,
1938) liave eosinophilic or amphopliilic cytoplasm
and basopiiilic granules. In a blood smear
preparation of C. virginica examined in the
Bureau's shellfish laboratory the granules stained
reddish-purple to dark blue with polyclirome

263

Microns

Figure 242. — Beginning of coalescence of blood cells of
C. virginica. Camera lucida drawings of live
preparations.

methylene blue mixture (Ramanovsky stain).
Methylene blue alone stained the granules very
poorly. In Ehrlich triacid stain a few granules
were blue, indicating a neutrophilic reaction. In
my preparations the blood cell granules never took
up eosin, which is very acid stain.

The oval-shaped nuclei of the amoebocytes can
be seen easily in a stained preparation. The
nucleus is usually located slightly off the center of
the cells in a pocket devoid of granules.

Some of the amoebocytes accumulate iron,
copper, zinc, and manganese. The presence of
hea^'y metals can be detected by treating the
sectioned tissues with ammonium sulfide, which
blackens the metals inside the cells (see: cliapter
XVII).

The following enzymes have been found in
extracts of amoebocytes: amylase, glycogenase,
lipase, protease, and a complete oxidase system
(Yonge, 1926; Takatsuki, 1934a).

Phagocytic activity of amoebocytes is very
pronounced. It can be demonstrated by injecting
into the mantle or gill cavity various suspensions
such as olive oil (stained with Sudan), carborun-
dum, colloidal carbon, carmine, saccharated iron
oxide, and cultures of diatoms or f'hlorella. Some
of the suspended particles may be picked up by
the amoebocytes which are always present on
the surface of the gills and the mantle. Ingestion
of iron particles was observed in the Woods Hole
laboratory by adding a suspension of iron sac-
charate to the shell liquor and treating the samples
of tissues or smears with ferricyanide solution to
produce Prussian blue reaction. Phagocytosis
can also be observed in live amoebocytes placed
in sea water on glass slides. Frequently under

this condition the amoebocyte approaching a
bacterium reverses its movement and turns aside.
The cause of this failure of phagocytosis has not
been determined. According to Bang (1961),
who described the phenomenon in C. rirginica,
it was impossible to assign the failure to a parti-
cular combination of bacteria and amoebocytes
because repeated observations gave inconsistent
results. He concluded that there was probably
an undiscovered factor in phagocytosis in oyster
blood which was responsible for this variation in
behavior.

Tripp (1960) found that various species of living
bacteria and yeast cell injected in the tissues of
C. virginica were rapidly destroyed extracellularly
and by phagocytes. Bacterial spores were re-
moved from tissues at a nmcii slower rate.

At the beginning of phagocytosis of an uni-
flagellate bacterium, observed by Bang with the
electron microscope (fig. 243), many filamentous
pseudopods extend from the cell's surface and
entangle the flagellum which is coiled around
them while tlie bacterium remains outside the
amoebocyte's body.

Figure 243. —Electron micrograph of a periphery of one
amoebocyte which spread out on a collodion film and
was fixed with osmium vapor. Courtesy of F. B.
Bang.

264

FISH AND WILDLIFE SERVICE

SPECIFIC GRAVITY OF BLOOD

The osmotic pressure of body fluids of bivalves
is about equal to that of the surrounding water
so it may be expected that the specific gravity
of blood approximates tliat of the water. For
determining the specific gravity of blood or of
pericardial fluid, the falling drop method of
Barbour and Hamilton (1926) has been used.
The procedure consists of timing a drop of fluid
of uniform size as it falls a distance of ,30 cm.
thi-ough a mixture of xylene and bromobenzene
in a vertical glass tube of exactly 7.5 mm. in
diameter. The time is recorded with a stopwatch
accurate to one-tenth of a second. The speed of
falling of a drop of the sample is compared with
that of a drop of the same size of standard potas-
sium sulfate (K2SO4) solution of known density.
By using an alignment chart (supplied with the
instrument), correction is made for room tem-
perature; the specific gravity of the sample can
be calculated with an accuracy of 1 times 10~*.
The source of error caused by variations in the
size of drops is minimized by using an automatic
Guthrie pipette controller. The method is simple,
rapid, and gives consistent results. In tliis way
the specific gravity of blood was determined foV
oysters taken from various environments.

A series 01 tests was also made to record changes
that occurred in oysters placed in diluted sea water
and in those exposed to air. The blood collected
from the ventricle with a glass pipette was centri-
fuged for 20 minutes at 1,200 r.p.m. to separate
blood cells from plasma. For brief storage the
sample of plasma was kept in a paraffin coated
container from which portions were taken for
determination. Observations were made at the
time of full sexual maturity of the oysters in the
middle of July and were repeated 2 weeks later
at the completion of spawning. All tests were
made at 22° C. and salmity 31.0-31.5 7oo. The
oysters were collected from Wellfleet Harbor,
Mass., but remained in the laboratory tanks for
about 3 weeks before the tests. The specific
gravity of blood during the July 15 to 18 period
varied from 1.0252 to 1.0262; in the tests made
after spawning between July 28 and 31 the specific
gravity of blood varied from 1.0258 to 1.0259.
The results are close to those reported by Yazaki
(1929) for 0. circumpicUi in which the specific
gravity of blood in the summer specimens varied
between 1 .025 and 1 .029.

CIRCULATORY SYSTEM AND BLOOD

No significant changes were found in the blood
of oysters kept for 72 hours in the refrigerator at
temperatures varying from 4.5° to 7.5° C. At
the end of the test the specific gravity of the blood
of the refrigerated mollusks was 1.0258; and in the
controls which were kept in running sea water at
21° to 22° C. the blood was 1.0259.

A gradual decrease in specific gravity occurred
in the oysters kept in running sea water of
diminishing salmity. The results of this experi-
ment are shown in table 31.

In highly diluted water shell movements of
some of the oysters were abnormal and most of
the time they remained closed. In these oysters
the specific gravity of the blood after 72 hours of
exposure to salinity of 9 to 12 °U^ was relatively
high (1.0138 and 1.0178) compared to the specific
gravity of 1.0092 in the oysters which stayed open
for more than 50 percent of the total tune. It
may be deduced from these e.\-periments that the
oysters kept in water m which the salinity was
reduced from 31-32 %„ to 16.7-17.7 °U^ at-
tained the osmotic equilibrium of blood in about
120 hours.

Table 31.— Z)ecrease in the specific gravity of cell-free blood
of the oyster, C. virginica, in water of lowered salinity

Salinity (°/„^)

Time

24 hours

48 hours

72 hours

120 hours

31-32, control.... .

1.0259
1.0145
1. 0199

1.0259
1.0143
1.0103

1. 0259
I. 0143
1.0092

1.0259
1.0127

16.7-17.7

9-12

'Observations discontinued after 72 hours.

SEROLOGY

Serological reactions between several moUusks
were studied by Makmo (1934), who experimented
with the following species; bivalves — Meretrix
meretrix, Paphia philipinarum, Ostrea (Cras-
sostrea) gigas, Area injiata; g&stro pods— Turbo
cernutum, Haliofis gigantea, Rapana thomasiana;
cephalopods — Sepiella japonica and Pohjpus
variabilis. In these tests the extracts of tissues
m physiological saline solution were injected
intraperitoneally or subcutaneously into rabbits
to obtain the antisera. Injections were repeated
for 7 days using doses which increased from 0.5 to
5 grams. One ml. of extract and 0.1 ml. of anti-
serum were used in performing precipitation tests,
and the tube was set aside for 1 hour at 37° C.
Positive reaction was obtained with all the species.
Ostrea antiserum reacted very strongly with
Meretrix and Paphia and less strongly with

265

Turbo, Haliotis, and Bapana. It is interesting to
note that Area, which belongs to the phj^logenet-
ically low order of Protobranchia, reacted very
strongly not only with Meretrix and Ostrea, but
also with the gastropods Turbo, Haliotis, and
Rapana.

Wilhelmi (1944) applied the precipitation reac-
tion to the problem of determining the relationship
of the mollusca to other invertebrates. Using a
technique similar to that employed by Makino,
he made tests between two species of Busycon,
Pecten irradians, Nereis, Limjil.us, and Asterias
forbesi and concluded that, serologically, mollusca
are more closely related to annelids than to any
other group. At present this work has historical
interest only, since it is obvious that no broad
speculations about the relationship of various
phyla should be made on the basis of a few tests
made with only six species belonging to four
different phyla.

The existence of serological differences in five
bivalves {Anadara inilata, A. larelu, Pecten yes-
soensi^, Ostrea (Crassostrea) yessoe/tsis, and 0.
circumpicta) was demonstrated by Tomita and
Koizumi (1951). In this work the serum was ob-
tained by centrifuging the blood withdrawn from
the auricles of the moUusk. Antisera were ob-
tained by injecting rabbits with increased doses,
starting with 1 ml. and adding 1 ml. each time
until 5 ml. were given on the 5th day. Blood was
taken on the 9th day after the last injection. In
homologous precipitation tests with C. gigas, i.e.
using the antiserum against the antigen of the
same species, positive reaction occurred in 1:16
dilution of antiserum with 1:12S0 dilution of
antigen.

Finer differences between closely related species
were detected by absorption tests. When a cross
reaction is obtained in a test of an antiserum of
one species against the serum of a related organism,
it is assumed that the second organism possesses a
chemical substance connnon with the homologous
substances of the first one. If after the absorption
the serum still reacts with homologous sub-
stance, it is considered that the antiserum con-
tained antibodies to two or more chemical com-
ponents including the one which is common to
both. Using this method Tomita and Koizumi
found that absorption with C. gigas antigen re-
moved from C. circumpicta serum all antibodies
for gigas but not for circvmpicta. In another test
circumpicta removed from gigas antiserum all anti-

bodies for circumpicta but not for gigas. The
authors' interpretation is that tliere are some com-
mon antigens between C. gigas and C. circumpicta
but that each also has its own specific antigen.
The investigators also found that Anadara {Area)
has all the antigens possessed by C. gigas plus its
owTi specific antigen. This is in accord with the
generally accepted view that Anadara (Area) is a
phylogenetically primitive form. Fresh-water
Anodonta showed no affinities with any other
species tested in this work.

The application of absorption technique enabled
Numachi (1962) to show that the four local races
of C. gigas — Hokkaido, Miyagi, Hiroshima, and
Kumamoto — have some antigenic differences that
are in accord with their geographic isolation.

Application of serological tests is a very promis-
ing method for studies of racial differences among
oysters. At present it is not known whether the
observed antigenic differences are hereditary
characteristics or are caused by differences in local
environment and particularly in the diet of oysters
from different localities.

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1908. Recherches sur les leucocytes et les tissu
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1938. On the coelomic corpuscles in the body fluid
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1953. Osmotic relations in moUuscan contractile
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270

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CHAPTER XII
THE EXCRETORY SYSTEM

Page

Anatomy of the excretory system _ 271

Histology __ _ _ _ 273

Physiology 274

The waste products 276

Osmoregulation _ 278

Bibliography _. ___ __ 279

End products of bivalve catabolism are excreted
by the nephridia, pericardial glands, wandering
phagocytes, and the mantle epithelium. The
urinary function, which is the principal activity
of the excretorjr system, is performed by the paired
nephridia situated on either side of the visceral
mass near the heart. The pericardial glands, as
the name indicates, are located on the wall of the
pericardium but in the oysters and some other
species are represented by special cells on the outer
wall of the auricles. The wandering phagocytes
may be found throughout the tissues of the visceral
mass and the gills. They accumulate on the sur-
face of the body by diapedesis and are discarded.
Mucus or goblet cells of the surface epithelium
are, in addition to their primary- function of
secreting mucus, involved in excretion and cany
within their bodies various granules which contain
pigments and hea\y metals (see: ch. XVII).

The principal part of the excretory- system of
bivalves is similar to that of annelids. It consists
of a pair of tubular nephridia (fig. 244, 1. neph.,
r. neph.) which retain direct communication with
the pericardium and sex glands on one side and
open to the outside through a short passage. The
coelom of bivalves is reduced to two separate
spaces, the pericardial cavity and the inside of tlie
gonad tubules. The ducts leading from the
nephridia and gonads open to the outside either
independently or through a common reno-gonadial
vestibule.

Comprehensive reviews of the anatomy and
histology of the excretory system in mollusks may
be found in the papers of Odhner (1912), Strohl
(1924), Haas (1935), Spitzer (1937), and Franc
(1960).

FISHERY bulletin: VOLUME 64, CHAPTER XII

ANATOMY OF THE EXCRETORY
SYSTEM

The excretory organ of the oyster occupies an
indistinctly outlined triangular area on either side
of the visceral mass. On the surface its location
is marked by light brownish pigmentation. The
organ consists of a central part which lies between
the pericardium and the adductor muscle and two
branches or limbs which extend along both sides of
the body. The right limb is slightly longer
than its opposite member. Excretory- tissues are
found directly under the surface epithelium; they
are surrounded by branchial vessels and numerous
blood sinuses.

The relation between the different parts of the
excretory system, the heart, and the adductor
muscle is shown diagrammatically in figure 244.

Each nephridium of a bivalve is bent into a
U-shaped tube which forms many convoluted
branches penetrating the surrounding tissues.
The narrow central part of the excretory system
extends across the body from one side to the other
and contains a narrow and twisted inter-nephridial
passage (i.r.p.) which connects the two limbs.
Fingerman and Fairbanks (1958) think that this
passage "does not appear to be present in
Crassostrea virginica." Reconstruction of the
structure of this area from serially sectioned
material shows that there is a communication
between the right and left nephridia although the
passage is narrow and greatly twisted (fig. 245)
and, therefore, appears in the preparations as a
series of circles and irregular cylinders. Exami-
nation of a number of consecutive sections shows
an uninterrupted connection between the right
and left nephridia.

The nephridium of each side begins inside the
pericardium by the reno-pericardial opening (fig.
244, r.p.o.), which leads through a ciliated funnel,
the nephrostome and a short reno-pericardial
canal (fig. 244, r.p.c, and fig. 246), to the body of
the kidney. Both limbs of the kidney are formed

271

0

Centimeters

Figure 244. — Diagram of the excretory system of C. rirginica based on examination of a series of transverse sections.
Viewed from the anterior side. Gonads and their ducts, blood vessels, and blood sinuses are not shown. The peri-
cardium wall is dissected and pulled down. A — auricle; a. a. — anterior aorta; Ad.M. — adductor muscle; a. 1.1. —
anterior left limb; a.r.l. — anterior right limb; ef.v. — efferent vein; i.r.p. — internephridial passage; 1. neph. — left
nephridium; p. 1.1. — posterior left limb; p. r.l.— -posterior right limb; r.d. — renal duct; r. neph. — right nephridium;
res. — reservoir; r.g.v. — reno-gonadial vestibule; r.p.c. — reno-pericardial canal; r.p.o. — rono-pericardial opening;
V — ventricle; v.g. — visceral ganglion.

by numerous branching and twisted tubules lined
with excretory cells.

Much of the posterior limb of the kidney is
occupied by a wide vesicle or reservoir (fig. 245,
R.) for the storage of urine. A short renal duct
(fig. 244, r.d., and fig. 247) leads from the reservoir
to the outside and opens into the reno-gonadial
vestibule, a shallow indentation on the surface of
the pyloric caecum, behind the opening of the
gonad (fig. 247). Numerous anastomosing tubules
of the nephridia form a spongy tissue which ex-
tends into the visceral mass (fig. 248). Odhner
(1912) pointed out that regardless of the apparent

complexity of the nephridia, it is always possible
to distinguish the two limbs or branches of the
excretory system.

Topography and relationship of the excretory
and reproductive systems vary greatly within the
class of bivalves. Certain topographical features,
however, remain constant. One of them is the
position of the renal opening in relation to the cere-
bro-visceral connective. Lacaze-Duthiers (1855),
one of the early students of molluscan anatomy,
pointed out that the renal opening (also called
"ncphroprokt") is always located beyond the
cerebro-visceral connective. Pelseneer (1891) at-

272

FISH AND WILDLIFE SERVICE

irp

Centimeters

0.5

Figure 245. — Caniora lucida drawing of a left portion of tlie central part of the excretory organ of C. virginica seen on
cross section. The beginning of the inter-nephridial passage (i.r.p.) at the junction with the reservoir, R, is on the
left side; Ad.M. — adductor muscle. Bouin, hematoxylin-eosin.

tached considerable importance to this morpho-
logical relationship for which he coined the term
"ektaxial."

In the absence of a generally accepted ana-
tomical termuiology for the excretory system of
bivalves, considerable confusion exists in the lit-
erature because of the great variety of names and
descriptive terms aoplied to identical parts. The
nephridium was discovered m 1817 by Bojanus
(1819) who mistook it for the organ of respiration.
.Since then the excretory system of mollusks has
been known in zoological literature as the organ of
Bojanus. There seems to be no advantage in the
conthiued use of this name, because the organ is
frequently called kidney, nephi-idium, renoperi-
cardial or nephropericardial passage (Renoperi-
cardialgang, in German), and nephridial sac in
cephalopods. The dorsal limb of the nephridium
may be called the proximal part, inner limb, peri-
cardial part, nmer sac, excretional part, antero-
posterior hmb, and kidney sac. The ventral (or
distal) limb is also knowii as outer sac, efferent
part, and kidney passage. The reservoir is some-
times referred to as vesicle and bladder. The
reno-pericardial opening is called nephrostome,
nephridial funnel, and "Nierenspitze" or kidney
syringe (Haas, 1935).

HISTOLOGY

The glandular nephridial tubes of the oyster are
lined with a special epithelium; the cells are

THE EXCRETORY SYSTEM

characterized by clear cytoplasm and absence of
granules. Three kinds of epithelial cells are found
in the different parts of the excretory system. In
the tubules of the anterior limb the cells are of
medium size, cylindrical, and vacuolated (fig. 249).
The lining of the internephridial passage and of
the tubules originating from its wall is made of
short, almost cubical cells with inainspicuous
vacuoles (fig. 250). In places the epithelium
lining is two or three cells deep. Tall columnar
cells with large terminal vacuoles are found pri-
marily in the tubules of the posterior limb and in
the reservoir (fig. 251 and fig. 252). The cells
are about seven to nine times longer than their
width. The vacuoles have no granules visible
under the light microscope; they appear to contain
liquid as if they are ready to burst. Some of the
vacuoles seem to be empty, and many separated
vacuoles are found in the lumen. .

The lining of the reservfiir consists of columnar
cells of different heights; some of them are twice
or three times taller than the others and protrude
into the lumen (fig. 252). The epithelium rests
on a basal membrane with a well-developed layer
of cu-cular muscles which extends to the renal duct.
There is no organized sphincter for the control of
the flow of urine. The contraction of the bladder
in response to the fluid flowing into it from the
pericardial cavity was observed by Fingerman and
Fairbanks (1958).

273

Microns

200

Figure 246. — Longitudinal section through the reno-
pericardial opening and reno-pericardial canal, which
connects the inner part of the kidney with the auricle
in C. virginica. Part of the auricle at upper left and the
adjacent portion of the kidney at lower right. Bouin,
heniatoxylin-eosin .

The epithelium of the reno-pericardial passage
and of the renal duct is ciliated (fig. 246 and fig.

247).

PHYSIOLOGY

No comprehensive account of the biochemical
and physiological processes of excretion in bivalves
can be written at present because of the inadequate
state of our knowledge, however, a review is
possible.

During the past three-cjuarters of the century
many studies have been made of the function of
the organ of Bojanus and of the role of the peri-
cardial glands. Most of these investigations were
carried on for gastropods, cephalopods, and fresh-
water mussels. The reader is referred to the review

of this subject found in the papers of Marchal,
1889; Letellier, 1889; Cuenot, 1900; Fosse, 1913;
Turchini, 1923; Delaunay, 1924, 1931, 1934;
Spitzer, 1937; and Franc, 1960. Very little work
has been done, however, witli marine bivalves and
particularly with oysters for wliich physiological
studies of the excretory system present consider-
able technical difficulties. The nephridial organs
of oysters do not form a sharply outlined unit.
They are surrounded by delicate and loose connec-
tive tissue which contains numerous blood vessels
with very thin walls and spacious sinuses. Con-
sequently, it is very difficult to obtain a sample
of the liquid from the various parts of the kidney
system without contaminating it with blood or
with the sea water which surrounds the organ.
Such difficulties are not encountered in the studies
of land snails and cephalopods.

An experimental approach to the study of the
function of nephridia was made by Grobben
(1888), Kowalevsky (1890), and Emeljanenko
(1910) wlio injected various dyes in the foot,
mantle, or visceral mass of the mollusk. It was
shown by this method that the excretory organs
of bivalves excrete indigo-carmine and that
ammonia carmine injected in physiological solu-
tion is concentrated in the pericardial glands.
In Ostrea the carminates are concentrated in the
walls of the auricles, while in Peden maximus
they are spread throughout the connective tissue
(Cuenot, 1900).

Attempts to determine the rate of filtration of
fluid into tlie pericardium of fresh-water Unionidae
were made by Turchini (1923) and Picken (1937).
Picken studied the hydrostatic and colloid osmotic
pressm-e of the body fluid. He supposed, probably
correctly, that the pericardial fluid is a filtrate of
the blood througli the wall of the heart. His
observation that the pericardial fluid of Anodonta
is isotonic with blood seems to support this view.

On the basis of the experiments by these authors
and by Robertson (1949, 1953), the following
course of mine formation may be visualized: The
blood is filtered through the wall of the heart into
the pericurthum, and this fluid, together with the
secretion of the pericardial glands, passes through
the reno-pericardial opening into the nephridial
tubes. The secretion from the tubules is added to
the liquid. Its final composition wifl depend on
how far and how rapidly the secretion and possibly
the reabsorption of certain constituents proceed
in the secretory epithelium.

274

FISH AND WILDLIFE SERVICE

Millimeters

0.5

Figure 247. — Section through the reservoir and renal duct of the right nephridium C. virginica. Gonad at the lower left
corner and cerebro-visceral connective at the upper right corner. Bouin, hematoxylin-eosin.

The experimental procedures for separate collec-
tion of blood, pericardial fluid, and urine from the
reservoir are uncertain, both because the tissues
are fragile and because a rich supply of blood is
received by the nephridia directly from the visceral
sinuses. Prosser (1950, p. 36) states correctly that
urine volume cannot, therefore, be calculated from
the rate of accumulation of the pericardial fluid ;
neither can the composition of urine be inferred
from an analysis of the fluid collected from the
interior of the kidney.

The rate of filtration of blood from the ventricu-
lar wall into the pericardium was studied in Ami-
(lonta by Picken (1937) using the following method :
A hypodermic needle of 1.5 mm. bore was inserted
in the pericardial wall previously exposed by
cutting off the adjacent portion of the shell. A
\ length of about 2 cm. of glass tubing was sealed

THE EXCRETORY SYSTEM

to the hypodermic needle, which was tilted so
that there was a difference of about 0.5 cm. in
the levels at each end of the needle. The fluid
from the pericardium dropping through the needle
was collected for periods of 5 minutes at 10-minute
intervals. The average rate of filtration deter-
mined in this manner was about 25 ml. in 4 hours.
The method probably gives a higher rate of blood
filtration than occurs under normal conditions be-
cause of a loss of pressure inside the pericardium
resulting from the punctm'e of the wall. Fm'ther-
more, it does not necessarily follow that the rate
of the discharge of fluid by the kidney is con-
trolled by the rate at which the pericardium fills
up with blood. The method does not seem ap-
plicable to the oyster because of the position of
the excretory organs on the oyster body and the
inherent technical difficulties.

275

Ml lllmeters

0.5

Figure 248.— Tightly packed and twi.sted blind tubules
of the excretory organ of C. virginica form "spongy"
tissue which extends on both sides into the visceral
mass. Adductor muscle at upper right. Bouin, hema-
toxylin-eosin.

0

Microns

40

Figure 250. — Cubical cells of the epithelium lining of the
inner wall of the internephridial canal of C. virginica.
Oil immersion. Kahle, hematoxylin-eosin.

Microns

40

Figure 251. — Tall columnar cells of the lining of the tubu-
les of the posterior limb of C. virginica. Oil immersion.
Kahle, hematoxylin-eosin.

0

Microns

40

Figure 249. — Medium-sized cylindrical epithelial cells
lining the lumen of the nephridial tubules of the anterior
limb of C. virginica. Oil immersion. Kahle, hema-
toxvlin-cosin.

276

THE WASTE PRODUCTS

Relatively little is known about nitrogen excre-
tion in the oyster or in other marine bivalves.
As in other organisms the waste products are
various nitrogenous compounds. The presence of
a low concentration of ammonia, the principal
nitrogenous product of amino acid breakdown in
moUusks (0.051 mg. per 100 ml.) was demonstrated
by Florkin and Houet (1938). In the excreta of
the mai-ine clam {Mya arenaria) ammonia com-
prises 21.5 percent of the total nonprotein nitrogen
(Delaunay, 1924), and in Mytil%f< the figure varies
from traces to 10.8 percent (Spitzer, 1937). Small
quantities of urea amounting to 4.5 percent of the
nonprotein nitrogen excreted in different forms
were found in Mya and only traces of it in Mytilus.
No uric acid was found in the latter species

FISH AND WILDLIFE SERVICE

(Spitzer, 1937) and only traces were reported in
Alya. In both organisms amino acids in the ex-
creted material amounted to 18 percent in Mya
and from 17 to 35 percent in Mytilux.

Two different methods were used in the studies
of nitrogen secretion. The nephridia with adja-
cent tissues were ground with sand and extracted
with distilled water, and the extracts analyzed;
alternately, the entire animals were placed in
small quantities of distilled water, kept at 16° to
18° C. for a period of time varying from 24 to
72 hours, and the metabolites accumulated in
the water were analyzed. It appears from the
experiments in which both fresh-water and marine
bivalves were used that uric acid is not present in
the nephridia. of Anodonta, Unio, Mytilus, and Mya
(Spitzer, 1937; Przylecki, 1922a). Przylecki
found that in Anodonta up to 60 percent of the
total excreted nitrogen is represented by ammonia,
and that the amount of ammonia excretion may be
greatly increased by placing the animals in acidi-
fied water for a short time. He also found that
ui'ease, the enzyme which converts m'ca into

ammonia, is present in abundance in Mytilus edvlis
and Helix (Przylecki, 1922b). In a study of the
urinary functions in bivalves Letellier (1887,
1SS9) arrived at the conclusion that in the
nephridia of Mytilus, Anodonta, and Cardium
urea takes the place of the uric acid; and Marchal
(1889) was not able to find uric acid in the excre-
tion of 50 mussels he tested. Spitzer (1937)
demonstrated, however, the presence of uric acid
in the middle intestine of Unio and Mytilus.
Delaunay (1931) found traces of uric acid in Mya
arenaria and small quantities of it in the products
of nitrogen excretion in the Portuguese oyster
Crassostrea (Grypfuiea) angulata.

Among the identified nitrogenous metabolites
excreted by Mya arenaria and Mytilus edulis
the largest proportion belongs to the amino
acids. Next in importance is ammonia, followed
by purine, urea, and uric acid. The numerical
values expressed in grams of nitrogen of a given
compound per total nitrogen in excreta are given
in table 32.

Microns

50

Figure 252. — Columnar cells of the lining of the kidney reservoir of C. virginica. Basal membrane with a well-developed
layer of circular muscles. Oil immersion. Kahle, hematoxvlin-eosin.

THE EXCRETORY SYSTEM

277

Baldwin (1935) suggested that the enzyme
arginase may be involved in the elaboration of
both urea and uric acid in gastropods. He found
no evidence of its presence in Mytilus edulis or
Pecten (rpercularis although the enzyme was found
in fresh-water Anodonfa. Brunei (193S) points
out that many investigations have shown that
uric acid and allantoine (the product of oxidation
of uric acid) are not found in bivalves, and
Needham (1935) considers that its absence in
that class and its presence in land snails are
adaptations to terrestrial life by uricotelic orga-
nisms, which often can not find sufficient water for
their needs and avoid toxemia by converting
poisonous ammonia into the insoluble and rela-
tively innocuous uric acid. The body of a marine
bivalve is, as a rule, permeable to water and to
small molecules. Consequently, the ammonia
formed during catabolism easily escapes by
diffusion into the external environment.

Table 32. — Excretion of nitrogen in three species of bivalves
in grams of nitrogen per total nitrogen excreted

[From Handbook of biological data, 1956, Spector, ed., p. 43]

Species

Ajiilno
acid N

Ammonia

N

Purine

N

Urea

N

Uric
acidN

Un-
identi-
fied

N

Craisosirea an-
gulata . .

13.2

18
17-36

7.2

21.6

Trace-11.4

3,2

4.3

Trace

1.6
Trace

75

Ml/a (irenaria...
Mutiltt.^ edulis, .-

5
Trace-16

51
39-79

OSMOREGULATION

It is generally considered that marine bivalves
have little power of osmoregulation when placed
in diluted sea water (Morton, 1958), and can pre-
vent loss of salts only by closing their valves.
There is, however, an indication tiiat the excretory
system may serve as an osmoregulating organ.
Kumano (1929) reported almost complete agree-
ment between the ionic concentrations of the
blood and pericardial fluid of O. circumpicta and
the sea water in which the oyster was kept. The
exception was reported only for magnesium and
sulfate ions, which were higher in the blood liv 1 1
and 7 percent respectively than in the sea water.
The corresponding values for the pericardial fluid
were 5 percent higher for magnesium and 2
percent lower for sulfate.

Robertson (1949, 1953) found that marine bi-
valves and gastropods {Pecten, Mya, Ensis, Pleu-
rohranchus, Neptiinca) accumulate potassium and
calcium and eliminate sulphate to a small degree.

The range of values expressed in percentage of
ionic concentration in the surrounding sea water
was as follows: sodium, 97 to 101; calcium, 103 to
112; magnesium, 97 to 103; chlorine, 99 to 101;
sulphate, 87 to 102.

Robertson defined ionic regulation as the main-
tenance of ionic concentrations in the body fluid
differing from those found in a passive equilibrium
witii the external medium. Accordingly, he made
an analysis of a body fluid obtained from a mol-
lusk and determined its ionic concentration. A
second determination was made using the same
fluid after it had been dialyzed in a collodion sac
against the original sea water. The results repro-
duced in table 33 show that Mytilus and Ostrea
exert little ionic regulation in their coelomic fluid
or plasma, apart from the accumulation of potas-
sium. Magnesium remains within 3 to 4 percent
of the equilibrium while calcium sometimes ex-
ceeds this by a few percent. The accumulation of
sulphates by Mytilus galloprodmialis was re-
garded by Robertson as a rare feature in marine
invertebrates. The protein content of blood
plasma was found to be low (0.2 to 0.3 percent) in
0. edulis and significantly higher in M. gallopro-
vincialis.

Table 33. — Ionic concentrations in plasma as percentage
of concentrations in dialyzed plasma {according to
Robertson, 1953)

[Data based on pooled samples]

Na

K

Ca

Mg

01

SOi

Protein
mg./ml.

Ostrea edulis _..

Mijtilus edulis,

.\/. galloprovineialis..

. 99.6
. 100.0
. 100.9

129, 1
134,7
120,8

100, (i
99,5
106,6

102.3
99.5
96,5

99,8
100,6
98,6

99.7
98.2
120. 0

0.2
0.3
0.8

The suggestion that some sort of osmoregu-
lation is present in C. virginica was made by
Fingerman and Fairbanks (1957, 1958), who
placed Louisiana oysters in sea water of three
different salinities. After periods up to 14 days,
samples were taken from the ventricles, the peri-
cardial cavity, the reservoir of the excretory
system, the secretory portion of the nephridia,
and the mantle. The autliors claim that at the
highest concentration of 515.6 milliequivalents of
chloride per 1. all fluids were hypotonic and at low
salinities (161.7 milliequivalents of chloride per 1.)
all body fluids were hypertonic to the environ-
ment. The original data are not given, and the
results are presented as average differences. The
"t" test of sia;nificance ransrins; in the values of P

278

FISH AND WILDLIFE SERVICE

from 0.20 to 0.50 seems to indicate that the
difi'erences are not significant. The authors'
conclusion can not, therefore, be accepted without
further confirmation.

The relative significance of the nephi-idia, peri-
cardial glands, and phagocytes in excretion can
not be evaluated at present. The three different
systems probably supplement one another by
eliminating different waste products. Histo-
chemical studies show, for instance, that phago-
cytes play the major role in the accumulation,
storage, and discharge of u'on and copper. Un-
doubtedly the phagocytes heavily loaded with dark
granules contain other substances besides heavy
metals which are discarded by the organism. On
the other hand, other tissues also are involved in
the process of excretion. For instance, the epi-
thelia of the gills, of the edge of the mantle, and of
the labial palps under certain conditions store
iron. My observations on ('. rirginica indicate
that iron is excreted by the epithelium of the
mantle, and vStauber (1950) found that India ink
injected into the ventricle is distributed by the
phagocytes to all parts of the organism and is
eliminated through the epithelium of the alimen-
tary tract, digestive diverticula, palps, mantle, and
pericardium. Nephi-idia and shell-forming parts
of the mantle are not, however, the routes of
migTation of these cells. Observations made by
Kowalevsky (1890) and Cuenot (1900) showed that
if a mollusk with pericardial glands is injected
with a mixtm-e of indigo carmine and ammonia
carmine, the latter is concentrated in the cells of
the pericardial glands, while the indigo goes into
the nephridia. These separations lead some in-
vestigators to designate the different cells as
"carmineathrocytes" and "indigoathi-ocytes"
(Strohl, 1924), terms which have not been accepted
by biologists. The affinity of these cells to special
dyes does not necessarily indicate that they par-
ticipate in the normal process of excretion.

Pigmentation may be considered as a certain
phase of excretion. Green oysters of Long
Island Somid develop dark gi-een pigment as the
result of absorption and storage of copper by the
phagocytes (Galtsoff and Whipple, 19.31 ). Schiedt
(1904) considered that the development of black
pigment in the mantle of oysters exposed to strong
illumination is also a product of excretion. His
finding was not in agreement with the observa-
tions of Faussek (1899) on pigmentation on the
mantle of Mytilus with partially removed shells.

Excretion through diapedesis is undoubtedly of
great importance to the mollusk, but its exact
role is not fully understood.

BIBLIOGRAPHY

Baldwin, Ernest.

19.35. Problem.^ of nitrogen catabolism in inverte-
brates. III. Arginase in the invertebrates, with
a new method for its determination. Biochemical
Journal, vol. 29, No. 2, pp. 252-262.

BOJANUS, L.

1819. Sendschreiben an den Herrn Chevalier G. de
Cuvier iiber die Athem- und Kreislaufwerlvzeuge
der zweyschaaligen Muscheln, insbesondere des
Aiiodon cygneum. Isis oder cncyclopadische Zei-
tung von Oken, Jahrgang 1S19, Band 1, pp. 81-100.
Brunel, Arthur.

1938. Sur la degradation des substances d'origine
purique chez les MoUusques Lamellibranches.
Comptes Rendus Hebdomadaires des Seances de
I'Academie des Sciences, tome 206, pp. 858-860.
Cu£not, L.

1900. L'e.xcretion chez les MoUusques. Archives de
Biologie, tome 16, pp. 49-96.
Delaunay, H.

1924. Reeherches biochimiques dur l'e.xcretion azotee
des invertebres. Bulletin de la Station Biologique
d'Arcachon, Vingt et unieme Annee (21st year)
(1924), fascicule 2, pp. 41-84.

1931. L'excretion azotee des invertebres. Biological
Reviews of the Cambridge Philosophical Society,
vol. 6, No. 3, pp. 265-301,

1934. Le metabolisme de I'ammoniaque d'apres les
reeherches relatives aux invertebres. Annales de
Physiologic et de Physicochimie Biologique, tome

10, pp. 695-724.
Emel,tanenko, Paul.

1910. tJber die Ausscheidung von Farbstoffen durch

das Bojanussche Organ bei Molusken. Zeitschrift

fiir Biologie, Band 53, pp. 232-254.
Faussek, V.

1S99. tJber die Ab'agerung des Pigmente- bei

Mytilus. Zeitschrift fiir wissenschaftliche Zoologie,

Band 65, pp. 112-142.

FeRNAU, WlLHELM.

1914. Die Niere von Anodonta celknsis Schrot. Teil

11, Die Histologie der Niere. Zeitschrift fur wis-
.senschaftliche Zoologie, Band 110, pp. 303-358.

FiNGERMAN, MiLTON, and Laurence D. Fairbanks.

1957. Investigations of the body fluid and "brown-
spotting" of the oyster. Proceedings of the Nat-
ional Shellfisheries Association, vol. 47, August
1956, pp. 146-147.

1958. Ilistophysiology of the oyster kidney. Pro-
ceedings of the National Shellfisheries Association,
vol. 48, August 1957, pp. 125-133.

Florkin, Marcel, and Ren£ Houet.

1938. Concentration de I'ammoniaque, "in vivo" et
"in vitro", dans le milieu interieur des invertebres.
I. h' Anodonte. Archives Internationales de Physio-
logic, vol. 47, pp. 125-132.

THE EXCRETORY SYSTEM

279

Fosse, R.

1913. Presence de I'uree chez Ics Invert^bres et dans
leurs produits d'excretion. Coniptes Rendus Heb-
domadaires des Seances de 1' Academic dcs Sciences,
tome 157, pp. 151-154.

Franc, Andr£.

1960. Classe des bivalves. In Pierre-P. Grasse
(editor), Traite de Zoologic, Anatomic, Systema-
tique. Biologic, tome 5, fascicule 2, pp. 1845-2133.
Masson et Cie, Paris.

G.^LTSOFF, Paul S., and Dorothy V. Whipple.

1931. Oxygen consumption of normal and green
oysters. Bulletin of the U.S. Bureau of Fisheries,
vol. 46, for 1930, pp. 489-508. (Document 1094.)

Grobben, Carl.

1888. Die Pericardialdriise der Lamellibranchiaten.
Arbeiten aus dem Zoologiscben Institute der Uni-
versitat Wien und der Zoologiscben Station in
Triest, tome 7, pp. 355-444.

Haas, F.

1935. Bivalvia. Tei) I. Dr. H. G. Bronn's
Klassen und Ordnungen des Tierreichs. Band 3:
Mollusca; Abteilung 3: Bivalvia. Al^ademische
V^erlagsgcsellschaft, Leipzig, 984 pp.
Kowalevsky, a.

1890. Ein Beitrag zur Kenntnis der Exkretionsor-
gane. Biologisches Centralblatt, Band 9, pp. 65-76.
Krogh, a.

1939. Osmotic regulation in acjuatic animals. Cam-
bridge University Press, Cambridge, England,
242 pp.
Kumano, Masao.

1929. Chemical analysis on the pericardial fluid and
the blood of Ostrea circumpicta Pils. Science Re-
ports of the Tohoku Imperial Univer.sity, series 4,
Biology, vol. 4, Xo. 1, fasc. 2, pp. 281-284.
Lacaze-Duthiers, H.

1855. M^moire sur I'organe de Bojanus des Acephales
lamellibranches. Annales des Sciences Naturelles,
Zoologic, serie 4, tome 4, pp. 267-319.
Letellier, a.

1887. De la fonction urinaire chez les molhisques
acephales. Communication faite a la Seance
Publique a I'Hotel-de-Ville de Saint-Sauveur-le
Vicomte, le 25 Septembre 1887. Bulletin de la
Societe Linneennee de Normandie, serie 4, vol. 1,
pp. 316-321.

1889. Etude de la fonction urinaire chez les mol-
lusques acephales. Revue Scientifique (Revue
Rose), tome 16 (tome 42 de la Collection), serie 3,
No. 14, 6 October 1888, pp. 435-437.

Marchal, Paul.

1889. L'acide urique et la fonction renale chez les
Invertebres. Memoires de la Societe Zoologique de
France pour I'Annee 1890, tome 3, pp. 31-87.
Morton, J. E.

1958. Molluscs. Hutchinson University Library,
London, 232 pp.
Needham, Joseph.

1935. Problems of nitrogen catabolism in inverte-
brates. II. Correlation between uricotelic metabo-
lism and habitat in the phylum Mollusca. Bio-
chemical Journal, vol. 29, part I, pp. 238-251.

Odhner, Nils.

1912. Morphologische und phylogenotische Unter-
suehungen viber die Nephridien der Lamelli-
branchien. Zeitschrift fiir wissenschaftliche Zoo-
logic, Band 100, pp. 287-391.
Pelsbneer, Paul.

1891. Contribution h 1 etude des Lamellibranches. j
Archives de Biologic, tome 11, pp. 147-312.
Picken, L. E. R.

1937. The mechanism of urine formation in inverte-
brates. II. The excretory mechanism in certain
mollusca. Journal of Experimental Biology, vol.
14, No. 1, pp. 20-34.
Pilgrim, R. L. C.

1953. Osmotic relations in molluscan contractile
tissues. I. Isolated ventricle-strij) preparations
from lamellibranchs (Mytilus ediilis L., Ostrea
edulis L., Anodonla cijgnea L.). Journal of E.xperi-
mental Biology, vol. 30, No. 3, pp. 297-317.
Prosser, C. Ladd (editor).

1950. Comparative animal physiology. W. B. Saun-
ders Company. Philadelphia, Pa., 888 pp.
Przylecki, St. J.

1922a. L'excretion ammoiiiacale chez les Invertebres
dans les conditions normales et experimentales.
Archives Internationales de Physiologic, vol. 20,
pp. 45-51.
1922b. Presence et repartition de I'urease chez les
Invertebres. Archives Internationales de Physio-
logic, vol. 20, pp. 103-110.
Robertson, James D.

1949. Ionic regulation in some marine invertebrates.
Journal of Experimental Biology, vol. 26, No. 2,
pp. 182-200.

1953. Further studies on ionic regulation in marine
invertebrates. Journal of E.xperimental Biology,
vol. 30, No. 2, pp. 277-296.
Schiedt, R. C.

1904. Some ])henomena of animal pigmentation.
American Journal of Physiology, vol. 10, No. 7,
pp. 365-372.
Spector, William S. (editor).

1956. Handbook of Biological Data. W. B. Saun-
ders Company, Philadelphia, Pa., 584 pp.
Spitzer, J. M.

1937. Physiologisch-okologische Untersuchungen
iiber den Exkretstoffwechsel der Mollusken. Zoo-
logische Jahrbiicher, Abteil ungfur allgemeine Zoo-
logic und Physiologic der Tiere, Band 57, pp. 457-496.
Stauber, Leslie A.

1950. The fate of India ink injected intracardially
into the oyster, Ostrea virginica Gmelin. Biological
Bulletin, vol. 98, No. 3, pp. 227-241.

Strohl, J.

1924. Die Exkretion. E. Mollusken. Hans Win-
terstein Handbuch der vergleichenden Physiologic,
herausgegeben von Hans Winterstein, Band 2,
halfte 2, pp. 443-607.

TiRCHiNi, Jean.

1923. Contribution a I'etude de I'histologie com-
paree de la cellule renale. L'excretion urinaire
chez les molhisques. Archives de Morphologic
Generale et lOxijerimentale, fascicule 18, 253 pp.

280

FISH AND WILDLIFE SERVICE

CHAPTER XIII
THE NERVOUS SYSTEM

Page

Methods __ 281

Anatomy 283

Microscopic structure 285

The pallia! organ 289

Sensory stimulation 293

Bibliography __. 295

The nervous system of the oyster is rehitively
simple. The visceral and cerebral gangha are
joined by the cerebro-visceral connectives; the
U-shaped cerebral commissure goes around the
esophagus; tlie circumpallial nerve travels along
the mantle's edge; and a number of nerves
originate from the ganglia and extend to different
parts of the body. The pedal ganglia, well
developed in many other bivalves, and tlie cerebro-
pedal connective are absent. These retrogressive
features are associated with the sedentary mode
of Ufe of the oyster and the loss of the organ of
locomotion (foot). In the evolution of bivalves
this simplification of the anatomy represents an
adaptive change and cannot be regarded as a
primitive trait (Jhering, 1877).

The only organs of sense in the oyster are the
tentacles, along the edge of the mantle, and the
pallial organ inside the cloaca. The tentacles
are highly sensitive to changes in illumination;
they contract if a shadow passes in front of a
feeding oyster, or a beam of light is focused on
them. They also detect the presence of minute
quantities of various drugs, chemicals, excessive
amounts of suspended particles, and changes in
temperature and composition of sea water. The
function of the palUal organ is not well understood;
the organ is probably concerned with the detec-
tion of mechanical disturbances in the surrounding
water.

The eyes found in freely moving bivalves, such
as scallops, are absent in adult oysters but are
present in fully grown larvae.

There is no major control center (brain), and
in this sense the nervous system is decentralized.
The integi-ation of its various parts is accomplished
by the interconnections of the ganglia through

FISHERY bulletin: VOLUME 64, CHAPTER XIII

the cerebro-visceral connectives, cerebral com-
missm-e, and the larger nerve trunks. All these
parts contain groups or nuclei of nerve cells and
have a structure similar to that of the ganglia.

In oysters the two visceral ganglia are fused
into a single organ. Its double origin is clearly
visible on tangential sections.

The distribution of nerves in bivalves was
studied by many zoologists of the 19th century
(Garner, 1837; Duvernoy, 1854; Jhering, 1877;
Rawitz, 1887, Babor, 1896; Pelseneer, 1891;
Freidenfelt, 1896; Gilchrist, 1898) who described
the principal topographic features of the nervous
system of various species. The nerves of the
European oysters were quite accurately depicted
in the old paper of Duvernoy; a general de-
scription of the nerve system of 0. chilensis was
given by Dahmen (192.3); of C. angulata by
Leenhardt (1926) , and of 0. cucullata by Awati
and Rai (1931).

METHODS

Anatomical dissection of the nervous system of
the oyster is rather difficult. The nerves are
small, unpigmented, and are embedded in con-
nective tissue. In fully ripe or in so-called "fat"
oysters even the principal nerves of the visceral
mass are hidden under a thick layer of gonad or
are covered by large quantities of glycogen. The
lean and watery specimens usually found shortly
after spawning are most suitable for dissection. Im-
mersion in 10 percent nitric acid, followed by
washing and clarification in glycerol, may be useful
because the nerve tissue is stained by the acid a
light brown color. The entire nervous system
may be stained in toto by using the following
procedure: oyster tissue preserved in 95 percent
ethyl alcohol or in 5 percent formalin is transferred
into 1 percent aciueous solution of potassium hy-
droxide (solution No. 1) for 1 to 3 days. The
specimen is then placed in solution No. 2 made
of 1 part glacial acetic acid, 1 part glycerol and 6

281

parts of 1 percent chloral hydrate solution, and is
left in it for about 24 hours. The tissue is then
transferred into solution No. 3 consisting of 1 part
of Ehrlich acid heniato.xylin, 1 part of glycerol and
6 parts of 1 percent chloral hydrate solution.
After several days in this niixtm-e the preparation
is destained in the solution No. 2 for about 12 to
24 hours and cleared in glycerol. Time of staining
and destaining may be modified depending on
thickness and condition of tissues. In successful
preparations the dark purple nervous tissue is

visible against the semitransparent visceral mass.
In my experience tlie method proved to be capri-
cious and not entirely reliable.

In a live oyster the nerves appear as thin, white
threads which can be traced for some distance in
very thin oysters containing no glycogen. Oysters
starved for 4 to 6 weeks in filtered sea water were
found to be very suitable for nerve study. Dissec-
tion of the nervous system must be supplemented
by reconstruction of ganglia and nerves through
examination of serially sectioned material.

/ D. n.

^ Centimeters ^

Figure 253.- — Diagram of the nervous .sy.stem of C. virginica seen from the right side. Dorsal and ventral ends of the
gills are shown under the mantle; the middle portion of the gill has been cut off; the middle portion of the branchial
nerve is indicated by the dotted line. Ad.m. — adductor muscle; ad.n. — adductor muscle nerve; a.p.n. — anterior
pallial nerve; br.n. — branchial nerve; e.g. — cerebral ganglion; c.p.n. — circumpallial nerve; com.p.n. — common
pallial nerve; c.v.c. — cerebro-visceral connective; g. — gills; Ib.n. — labial nerve; lb. p. — labial palps; l.p.n. — lateral
pallial nerves; p.o. — pallial organ; p.p.n. — posterior pallial nerve; r. — rectum; v.g. — visceral ganglion. The
cerebral commissure is not visible.

282

FISH AND WILDLIFE SERVICE

Centimeter

Figure 254. — Visceral ganglion of a large C. vhginica seen through the epibranchial chamber, ad.m. — ventral side of the
adductor muscle; br.n. — branchial nerve; c.v.c. — cerebro-visceral connective; ki. — kidney; l.p.n. — labial palp
nerve; p.o. — pallial organ; py.p. — pyloric process; v.g. — visceral ganglion. The oyster was fully narcotized and
the adductor muscle completely relaxed. Drawn from life.

ANATOMY

The visceral ganglion, the largest unit of the
nervous system, is a wedge-shaped structure
embedded on the anteroventral side of the adduc-
tor muscle in a depression formed by the junction
of the translucent and opaque parts of the muscle.
To see the ganglion in its natural position one
must cut off the wall of the epibranchial chamber
and lift the tip of the pyloric process. The
location of the ganghon in such a preparation,
examined from the right side of the oyster, is
showTi in figure 253. The entire ganglion can be
examined in situ in an intact oyster. For this
purpose the oyster is narcotized until the valves

gape, and a beam of light is directed into its cloaca
with the oyster's posteroventral end held toward
the observer; when the pyloric process is raised
slightly with a probe, the ganglion becomes
visible as a white or sometimes slightly yellowish
flat organ (fig. 254). In the preparation from
which the drawing was made the muscle was
completely relaxed and the gaping distance
between the valves measured about one-half inch.
Tlie visceral ganglion is higlily developed in all
Ostreaceae (Rawitz, 1887). The right and left
components, fused into a single organ, are dis-
tinguishable, and the ganglion appears to consist
of three parts, one central section and the two

THE NERVOUS SYSTEM

283

lateral sections from which emerge six pairs of
nerves. In some bivalves the ganglion and the
nerves are pigmented, but not in the oyster.

The pair of cerebral ganglia of the oyster
(fig. 253, e.g.) is located at the bases of the labial
palps. The position of these small organs is
slightly asymmetrical in relation to the palps,
with the left ganglion slightly lower than the
right one. Because of such asymmetry, only
one ganglion is usually seen in the transverse
sections of the visceral mass made at the level
of the bases of the palps. In large and thin
("poor") oysters the cerebral ganglia may easily
be located, but in "fat" specimens they are not
usually clearly visible.

The narrow cerebral ganglion of each side is
bent at a sharp angle: this gives it the appearance
of a saddle sitting over the basal membrane of the
epithelium of the palps (fig. 255). The cerebral
commissure emerges from the end of the ganglion
(c.com.) and makes an inverted U-shaped loop
which passes dorsally over the esophagus and
connects with the ganglion of the opposite side.

The following nerves emerge from the anterior
side of the visceral ganglion: The cerebro-visceral
connectives rise from the anterior side of the
ganglion and soon are buried in the connective
tissue of the visceral mass (figs. 253, 254, 255,
c.v.c.) ; next to the connectives and slightly dorsal
to their roots are the anterior pallial nerves
(a.p.n.), which run forward through the kidney,
pass across the pericardium wall toward the dorsal
part of the body and along their course give off
branches extending to the central part of the
mantle. Two branchial nerves (figs. 253, 254,
br.n.), arise near the roots of the cerebro-visceral
connective and run parallel to it for a short
distance. These nerves are more easily recogniz-
able than the others because they form a convex
curve and enter the gill axis on each side of the
body accompanied by the efferent blood vessels.
The two distinct branches of the lateral pallial
nerves originate from the extreme points on the
sides of the visceral ganglion. The outer branch
(upper in fig. 254) almost immediately enters the
mantle, while the inner branch (lower in fig. 254)
divides into numerous smaller nerves which estab-
lish contact with the circumpalhal nerve (fig. 253,
c.p.n.). The posterior pallial nerve (fig. 253, 255,
p.p.n.) runs from the ventral end of the ganghon
along the ventral side of the adductor muscle as
far as the rectum and divides into smaller branches

com. p. ft.
Ikn

ad. n.

[.p. n.

Figure 255. — Diagram of the nervous system of C. vir-
ginica seen from the anterior side. ad.n. — adductor
muscle nerve; a.p.n. — anterior pallial nerve; br. n. —
branchial nerve; e.g. — cerebral ganglion; c. com. — cere-
bral commissure; com.p.n. — common pallial nerve;
c.v.c. — cerebro-visceral connective; Ib.n. — labial nerve;
l.p.n. — lateral pallial nerves; oe. — esophagus; p.o. —
pallial organ; p.p.n. — posterior pallial nerve; v.g. —
visceral ganglion.

which penetrate the mantle. The pallial organ
(fig. 255, p.o.) is located along the course of this
nerve. A pair of adductor nerves arises from the
dorsal side of the ganglion and immediately enters
the muscle tissue (fig. 255, ad.n.). They are not
visible from the ventral side of the muscle.

284

FISH AND WILDLIFE SERVICE

Except for the pair of cerebro-visceral connec-
tives, only a few nerve trunks originate from the
cerebral ganglia. The common pallial nerve (figs.
253 and 255, com.p.n.) enters the mantle of the
corresponding side and establishes connection
with the dorsal portion of the circumpallial nerve.
The labial nerves (Ib.n.) branch to the four
labial palps.

The circumpallial nerve (fig. 253, c.p.n.) is a
faii-ly large nerve trunk which runs parallel to the
edge of the mantle. Throughout its course it is
accompanied by the circumpallial artery and
supplies numerous branches to the tentacles and
makes connections to the radial nerves which
extend from the base of the mantle to its edge
(see: fig. 86 in chapter V). The circumpallial
nerve is connected anteriorly with the cerebral
ganglia and posteriorly ends in the visceral
ganglion. Stimuli received anywhere on the
mantle may be transmitted to the entire nervous
system of the oyster by this circular nerve.
Because of this arrangement the oyster may
respond to stimuli as a whole in spite of separation
of its nerve centers.

The cerebral and visceral ganglia also are joined
by a pair of relatively broad and long cerebro-
visceral connectives (fig. 253, 255, c.v.c), which
constitute the main nerve trunks through which
communication is maintained with all the parts
of the body.

MICROSCOPIC STRUCTURE

The ganglia are formed of a central core or the
neuropile, tightly packed bundles of nerve fibers,
and the cortex made of several layers of nerve
cells. This arrangement gives the ganglia a
resemblance to the white and gi'ay matter of the
central nervous system of vertebrates. A layer
of loose connective tissue forms the outer sheath
of the molluscan ganglia.

The visceral ganglion is a relatively large,
wedge-shaped structure embedded on the ventral
side of the adductor muscle in the depression
between its two parts (fig. 256). In the oyster
the ganglion is completely fused but its paired
origin is clearly seen on a tangential section
(fig. 257).

The cortex is made of a continuous layer of
nerve cells. Scattered nerve cells also occur in
the neuropile. The ventral side of the ganglion
facing the epibranchial chamber is covered with
a unicellular layer of epithelium.

THE NERVOUS SYSTEM
733-851 O— 64 19

The nerve cells which in the oyster and other
bivalves form the cortex were described by
Rawitz (1887), whose paper remains the major
contribution to the histology of the molluscan
nervous system. To obtain the entire nerve
cells with their axons Rawitz macerated small
pieces of ganglia in 25 percent ethyl alcohol for
4 to 5 hours or in an aqueous solution of potassium
bichromate (from 0.025 to 0.1 percent) for 8 to 24
hours.

Individual nerve cells of the oyster are either
pear- or club-shaped with one, two, or several
processes extending from their bodies. These
processes give rise to fine fibrillae which enter the
neuropile. Depending on the number of the
processes, the cells are called unipolar (fig. 258,
a, b), bipolar (e), and multipolar (c, d). The
unipolar cells are more abundant than the other
two types. The apolar cells, i.e., those without
the processes, have not been found in bivalves,
according to Rawitz.

The size of nerve cells varies. The multipolar
cells are usually the largest; their dimensions, with-
out the processes, range from 14.5 ^ by 5 m to 20 /i
by 8 M- The unipolar and bipolar cells are smaller,
varying in size from 12.5 m by 4 m to 14 m by 6 m-
The tapered ends of the cells give rise to the nerve
fibers, which enter the neuropile where they
combine with other nerve fibers to form several
compact bundles. Single bipolar and unipolar cells
are scattered throughout the neuropile, but do not
aggregate into distinct nuclei or groups. The
protoplasm of the nerve cell is dense and is deeply
stained with Ehrlich's and Delafield hematoxylin.

The nerve cells and their axons are supported by
a framework of connective tissue cells which
descend from an outer sheath of the ganglion.
These cells were observed first by Freidenfelt
(1897). Bochenek (1906) and Jakubski (1912,
1913) described tlie supporting elements in the
ganglia of Anodonta, Pinna, and several gastropods,
tunicates, and echinoderms, and regarded them
as typical glia cells. Jakubski distinguished three
groups of glia cells: (1) Star-shaped flat cells with
oval nuclei and thin processes which continue as
neuroglia fibers were found in the outer sheath of
the ganglion, (2) spindle-shaped cells with a
pointed nucleus usually have two outgrowths and
are most common in the inner portion of the
ganglion for which they form a supporting frame-
work; and (3) "neuropile glia cells", which occur
singly, scattered through the neuropile. Jakubski

285

0

lillimeters

0.5

Figure 256. — Visceral ganglion of C. virginica. Transverse section at the right angle to the fibers of the adductor

muscle. Bouin, hemato.xylin-eosin.

(1912) pointed out that the interstitial elements of
the nenropile do not isolate the nervous elements,
while in the nerves and in the commissures the
glia cells surround a number of nerve fibrillae
which form distinct tracts. Of the three different
types of glia cells described by Jakubski, the
narrow, spindle-shaped cells could be seen in the
preparation of the ganglia of f\ inrginica stained
with Delafield and iron hematoxj'lin. Nissl
granules were described in tlie multi-polar nerve
cells at the ends of siphons in Mya (Pieron, 1941).
The nerve fibers which form the neuropile are
clearly noticeable in sections of the visceral gang-

lion (fig. 256) ; some of them cross the ganglion,
while others enter the viscero-cerebral connective
on the same side where they emerged.

Rawitz (1887) described in detail the pathways
of these fibers in the visceral ganglion of Mytilus.
He found that in each half of the ganglion approx-
imately one-quarter of the fibers originates from
the nerve cells of the same section; one-quarter
arises from the opposite half; one-quarter is
derived from the cerebro-visceral connective of the
corresponding side; and one-quarter originates in
the cerebro-visceral connective of the opposite side.
It is difficult to determine these pathways in the

286

FISH AND WILDLIFE SERVICE

Millimeters

Figure 257. — Visceral ganglion of C. virginica. Tangential section. Bouin, hematoxylin-eo.sin.

a

0

Microns

50

Figure 258. — Nerve cells isolated from the visceral ganglion of 0. eduUs, according to Rawitz (1887). a, b, — unipolar

cells; c, d, — multipolar cells; c, — bipolar cells.

THE NERVOUS SYSTEM

287

sectioned material of the oyster ganglia. The
crossing of some fibers from the right to the left
side and then- extension into the connective can be
seen, but there is no way to estimate the relative
abundance of these fibers. There is not enough
evidence to establish with certainty which fibers
run from the nerve cells of the connective into the
ganglion and which follow the opposite direction.
The tissues of the cerebral ganglion are less
compact than those of the visceral ganglion, and
the entire organ is rather indistinctly separated
from the underlying connective tissue (fig. 259).
The cerebral ganglia are located on each side of
the oyster directly under the surface epithelium
but separated from it by a thin layer of connective
tissue fibers. The inner part of the cerebral gan-
glion (fig. 260) consists of bipolar cells of medium
size. The outer layer, corresponding to cortex, is
made up of large unipolar and multipolar nerve
cells with dense protoplasm, stained dark with
hematoxylin. Loose connective tissue covers the

cv.c

Millimeters

Figure 259. — Longitudinal section of a portion of thu
cerebral ganglion, (e.g.) at the base of the labial palps
of C. virginica; cv.c. the beginning of the cerebro-visceral
connective. Kahle, hemato.wlin-eosin.

TiST

Microns

50

Figure 260. — Small portion of a longitudinal section of
the cerebral ganglion of C. virginica under the epithelium
of the base of the labial palps. Large unipolar and
multipolar nerve cells form a cortex layer. Medium
sized bipolar nerve cells and nerve bundles occupy the
central part of the ganglion. Loose connective tissue
covers the structure (right side of the drawing). Kahle,
hematoxylin-eosin.

ganglion on the outer side. The entire organ is
less compact than the visceral ganglion.

The stiiicture of the cerebro-visceral connective
is similar to that of the ganglia (fig. 261). A
thick layer of large nerve cells surrounds the nem-o-
pile, which is divided into several bundles. There
is no well formed sheathing, but small connective
tissue cells are found along the periphery of the
ganglion and are scattered throughout its entire
structure.

A similar pattern of ganglionic structure repeats
itself in many nerves emerging from the ganglia.
This plan of organization is found in the circum-
pallial, branchial, and many other nerves.

The circumpallial nerve is surrounded by a
sheath of connective tissue fibers (fig. 2G2). The

288

FISH AND WILDLIFE SERVICE

Microns

Figure 261. — Transverse section of the cerebro-visecral
connective of C. virginica. Large aggregation of nerve
cells on the periphery. Kidney tubules on extreme left.
Kahle, hematoxylin-eosin.

nerve cells are predominantly at the periphery.
Along its entire course the nerve gives rise to
numerous branches which at regular intervals enter
the tentacles and terminate at their surface in a
number of small fibers which can be seen in gold-
impregnated preparations (fig. 86, ch. V).

Small peripheral nerves such as the branchial
nerves (fig. 263) and the radial nerves of the mantle
(fig. 264) are made of bundles of fibers with occa-
sional small nerve cells between them . Tlie sheath
of these nerves consists of a narrow layer of
spindle-shaped connective tissue cells. A well-
developed nerve net, visible in gold-impregnated
sections (fig. 87, ch. V), is found over the entke
mantle.

The anatomical and Iiistological picture de-
scribes an intercomnmnication system which
connects all the organs and parts of the oyster.
Stimulus received, for instance, at the dorsoan-
terior part of the mantle and transmitted to the
cerebral ganglia may reach the visceral ganglion
either througli the cerebro-visceral connective or
directly via the numerous nerve branches which
extend from the edge of the mantle and are
connectctl by the circumpallial nerve. Stinmli
transmitted to cephalic ganglia may reach the
visceral ganglion through one of the connectives

and vice versa. Thus, in spite of the absence of
a central nervous system, the nervous reactions
of the oyster are well integrated.

The major movements of the oyster are hmited
to the contraction and relaxation of the adductor
muscle. Muscular contraction may be provoked
either by the stimulation of the receptors of the
tentacles and mantle or by impulses which origin-
ate from the internal organs. An example of
the latter type of stimulation is found in the
spawning reaction of the female. It consists of a
series of rhythmic contractions of the adductor
muscle associated with the release and dispersal
of ova by the sexually mature oyster but in the
immature specimen it cannot be induced by drugs,
mechanical, electric, or thermal stimuli. The
reaction is fully discussed in chapter VIII, page 172.

THE PALLIAL ORGAN

The only sense organs of the oyster are the
tentacles of the mantle edge and the palhal (also
called abdominal) organ. The structure and in-
nervation of the tentacles have been already
discussed in chapter V, page 85. The pallial
organ is a very small structure attached to the an-
terior side of the adductor muscle inside the ex-
halant chamber of the gills. In order to see the
organ the wall of the cloaca and of the epibranchial
chamber must be dissected and the two sides
drawn apart in the manner shown in figure 236
in chapter XI, page 259. The pallial organ is
a small, colorless protuberance marked on the
drawing by the letters pal. or.

The structure was discovered by Thiele (1889)
in a number of bivalves including 0. edulis. In
tlie European oyster the organ was described as a
comma-shaped protuberance ("kommaformige
Erhebung") with the pointed end turned to the
right and the concave side oriented toward the
posterior end. The organ on the right side is well
developed but on the left side is small and degen-
erate. The description and illustration published
by Thiele apply to C. virginica. In this species
the pallial organ on the right side is also better
developed while on the left side it is much smaller
and frequently absent. A similar condition is
found in 0. cucullata (Awati and Rai, 1931).

The structure of the pallial organ of C. pirginica
is revealed in a series of sagittal sections (fig. 265).
The rounded surface of the organ is covered by
elongated epithelial cells with hairlike cilia which
are longer than the cell bodies. This type of cell

THE NERVOUS SYSTEM

289

Microns

FiGUBE 262. — Cross section of the circunipallia) norve of C. virginica. Kahlo, hematoxylin-eosin.

closely resembles the so-called brush cells (Pinsel-
zelle) described by Flcmniing (1SS4) in the sense
organs of various mollusks. Thiele (1SS9) found
such cells in the pallial organs of Pinna, Avicula
Area, Lima, and other bivalves. The epithelium
of the pallial organ of C. virginica consists of two
layers of cells: the deeper one is made of cells
with globular nuclei, while in the surface layer the
cells and their nuclei are oval-shaped (fig. 266).
This observation is in agreement with the descrip-
tions given by Thiele. The epithelial layer of the
pallial organs of Lima inflata and L. hians is made,
however, of a single layer (.Studnitz, 1931).
Thiele remarks that the cilia in Area noac and

Lima are motionless. It seems reasonable to as-
sume that this may be true for the pallial organs
of oysters. Unfortunately, the location of the
pallial organ inside the e.xhalant chamber of the
gills makes it impossible to study the function of
the organ without inflicting serious injury to the
sin-rounding tissues and nerves. From the his-
tological picture it appears that stiff, hairlike cilia
on the surface transfer the stimuli to the nerve
endings and to the nerve trunk. Figure 266 rep-
resents a small section of the epithelial covering of
the pallial organ of C. rirgitnca e.xamined at high
magnification. Here the surface layer contains
many sensory cells recognizable by their narrow

290

FISH AND WILDLIFE SERVICE

0

Microns

50

Figure 263. — Transverse section of branchial nerve of C. virginica. Gill muscles are above the nerve. Kahle, hematoxy-

lin-eosin.

Microns

Figure 264. — Radial nerve in the mantle of C. virginica.
The sheathing of connective tissue forms the periphery.
Here the nerve consists of three distinct nerve trunks.
Blood cells are scattered in the connective tissue of the
mantle. Kahle, hematoxylin-eosin.

bodies and long processes which extend I'roni tlie
surface of the organ deep into the underlying con-
nective tissues. The nuclei are elongated, and the
cells are deeply stained with iron and alum hema-

toxylin. A large nerve trunk, shown in figure
265, sends its branches to the surface of the pallial
organ. The nerve entering the organ is a branch
of one of the posterior pallial nerves which emerge
from the visceral ganglion. Typical vesicular
connective tissue forms the core of the organ.

There has been no experimental study of the
pallial organ, and its physiology is not well under-
stood. Elsej^ (1935) expressed the opinion that
its primary function is the regulation of respiratory
current. Haas (1935) thought that in Lima the
pallial organ is primarily concerned with chemical
testing of water, and this \aew is repeated by
Franc (1960) in his review of Bivalvia.

The long, stiff cilia seem to be more suitable for
detecting mechanical disturbances than for
responding to chemical stimuli. The organs of
chemical taste in the oyster are the tentacles of
the mantle. Because of their location at the edge
of the mantle they are the first ones to come in
contact with the irritating or poisonous substances
in water and give the oyster a signal to prevent
its access into the pallial cavity. It appears
unreasonable that an organ of chemical taste
should be located inside the water transporting
system near its end. Dahmen (1923) and Awati
and Rai (1931) think that the pallial organ in
bivalves is primarily the organ for detection of

THE NERVOUS SYSTEM

291

0

Microns

200

Figure 265. — Sagittal section of the pallial organ of C. virgiiuca. Long ciliated cells rest on semiglobular protuberance
of connective tissue richly supplied with blood vessels and ramifications of the posterior pallial nerve. Kahle,
hemato.xylin-eosin.

mechanical disturbance in the surrounding water.
A final answer to the enigma of the pallial organ
must await results of physiological studies which
so far have not been undertaken.

A different type of pallial sense organ of the
spat of 0. edulis was described by Cole (1938),
who found a thin-walled spherical and pigmented
sac projecting from the inside mantle sin-face

near its border, about one-third of the length of
the free edge from the mouth. The cavity of
the sac contains a comparatively large non-
calcareous and nonsiliceous concretion which
stains visibly with eosin. There are no cilia inside
the sac, which is covered by very attenuated
cpithelimn. Cole remarks that the organ is
suitably constructed and in a favorable position

292

FISH AND WILDLIFE SERVICE

wmmm^m.

'■'^:

.9

&

..■■«?

■!^-.

Microns

50

Figure 266. — Section of a small part of the ciliated
covering of the pallial organ of C. virginica showing
sensory cells between the ciliated epithelial cells.
Kahle, iron hematoxylin.

for receiving and reacting to vibration. He
found that this organ is fully developed in 4- to
S-day-old spat. It is not known whether the
organ is present in adult 0. edulis or in C. virginica.

SENSORY STIMULATION

Very little experimental work has been con-
ducted on the physiology of the nervous system of
the oyster. Most of the research on neuro-
physiology of otlier l)i valves (Pecten, Ali/tilus, Mya,
and Anodonta) dealt with the action potentials,
tonus of the adductor muscle (see: ch. VIII), and
stimulation of the siphons of Mya (Hecht, 1919a,
1919b, 1920a, 1920b; Pieron, 1941) and Pholas

(Hecht, 1928). From a study of the action
potentials along the nerve trunks of the siphon of
the soft shell clam, Pieron calculated that the
velocity of the transmission of stimuli along the
nerve of this mollusk is of a magnitude of several
meters per second. The value is probably common
to other bivalves.

Neuro-secretory cells are present in a number of
marine bivalves (Nucula, Anomia, Mytilus, Modio-
lus, Chlamys, Lima, Donax, Arcopagia, Macfra,
Cardium, Venerupis, Venus, and Paulora) and
probably may be found in other genera including
the oyster (Gabe, 1955). These cells are typical
small neurones of the cerebral and visceral ganglia
but are absent in the pedal ganglia (Lubet, 1955).
The amount of secretion they contain varies with
the season and is apparently related to or parallels
the sexual cycle for it increases with the matura-
tion of sexual products (see: ch. XIV, page 312).

Sensory stimulation of the tentacles of the
oyster by chemicals was studied by Hopkins
(1932a, 1932b). He measured their sensiti^^ty by
determining with a stopwatch the latent period,
i.e., the time elapsed between the application of a
chemical and retraction of a tentacle or a group of
them. The method is very simple. The mantle
is exposed by cutting off a portion of shell, and the
oyster is placed in sea water running at a constant
speed through a rectangular tank with two com-
municating parts, one of them shallow and the
other several inches deeper. Water in the taller
part is kept at a constant level. The oyster is
placed in the shallow portion of the tank, and a
vessel containing the solution to be tested floats in
the taller part. A three-way stopcock is mounted
on the wall separating the two parts of the tank,
one branch of the stopcock is bent horizontally and
ends in a capillary nozzle placed a short distance in
front of the tentacles. The two other branches are
fitted with flexible rubber tubing at the end; one is
lowered into sea water, the other into the floating
vessel with the test solution. At the beginning of
the test the stopcock is turned to deliver a con-
stant, gentle stream of sea water to the tentacles,
which remain in a relaxed state as long as the
ciuTent and temperature of the water are constant.
The stopcock is turned abruptly, and the sea water
is suddenly replaced by the solution to be tested.
The total time from the turning of the stopcock to
the observed retraction of the tentacles is measured
with the accuracy of one-tenth of a second. Before
making a test the time required for a test solution

THE NERVOUS SYSTEM

293

to pass from the container to the tip of the nozzle
is recorded by using a colored solution and the value
obtained is subtracted from the total time meas-
ured, giving the duration of the actual latent
period. Since the vessel in the deeper part re-
mains afloat, the level from which the solution is
withdrawTi remains constant and consequently
there is no change in the velocity of current striking
the tentacles.

All parts of this simple apparatus may be built
of plastic. The following precautions should be
taken: 1) the nozzle in front of the oyster must be
firmly mounted and placed on a solid stand to
avoid mechanical disturbance when turning the
stopcock; 2) water and test solutions should be
kept at equal and constant temperature; and 3)
levels from which the sea water or the test solu-
tion are delivered to the tentacles should be con-
stant in order to avoid a change in the velocity of
current.

This method was satisfactorily used in the Woods
Hole laboratory in testing the reaction of oysters
to various organic compounds and contaminants.

Sensory stimulation of tentacles by inorganic
salt solutions depends on the chemical composition
of the substance used and its concentration. The
relationship between the concentration of a given
substance and the latent period, presented in
Hopkins' papers, indicates that the effect, con-
sidered as the reciprocal of the latent period, is
directly proportional to concentration. Some-
times a secretion of mucus covers the tentacles and
impedes the reaction. In such cases Hopkins
found it necessary to subtract a constant from the
latent period values.

The range of latent period values in Hopkins'
experiments varied from a fraction of a second to
about 15 seconds. By using 0.5 m solutions of
several inorganic salts Hopkins arrived at the
conclusion that sensory stimulation is primarily
the function of cations, which he listed in the
following order of effectiveness: K>Na>NH4>
Li.

Tentacles respond also to chemical stimulation
by various organic compounds such as quinine
sulphate, cumarin, etc. An odorous compound
such as cumarin is detected by the oyster in a
concentration of 0.0004 percent. The oyster
responds also in a measurable reaction to a 0.004
percent solution of quinine sulfate. This con-
centration is one-eighth of the strength of the

solution of quinine that can be detected by man's
tongue (Hecht, 1918).

Cane sugar has little stimulating effect on the
tentacles (Hopkins, 1932b). Tests with fructose
(in sea water) that I made in the Woods Hole
laboratory gave the following results:

0.05 percent No reaction in 1 minute.
1.0 percent Latent period 8 to 12 seconds

in four out of 10 trials.
5.0 percent Latent period 4 to 8.4 sec-
onds.
Arabinose is more effective:

50 mg./l Latent period 26 to 49 seconds

in five out of 10 trials.
100 mg./l Latent period 12 to 34 seconds

in all 10 trials.
5.0 percent Latent period 5 to 8 seconds in
all 10 trials.

An interesting reaction was observed when
diluted sea water (one part fresh water -\- three
parts sea water) was used. The inner lobe of
the mantle curled up, but the tentacles were
not affected. The gradual curling ended in a
sharp contraction of the adductor muscle. Nor-
mal sea water added to the edge of the nantle in
a control experiment produced no such result.

A similar protective reaction was observed
when the extracts of the meat of the oyster drill
{Urosalpinx cinerea) and of the stomach of the
starfish (Asteriasf orbed) were used. The tent icles
reacted strongly in contact with the undiluted
juice of Urosalpinx with the latent periods of 1.5
to 3.6 seconds. These experiments suggest that
sensory mechanism of the oyster may be sufficient
to detect the close proximity of carnivorous
gastropods.

The mechanism of sensory stimulation has not
been adequately studied and is not fully under-
stood. Its biological significance is, however,
apparent. The warning received by the tentacles
is transmitted through the circumpallial nerve
of the mantle to the ganglia. If stimulation is
sufficiently intense either a part or the entire
mantle is withdrawn, the entrance to the gills
is closed, and the ensuing contraction of the
adductor firmly closes the shell. The three
steps outlined can be observed under experi-
mental conditions; they constitute the three
distinct phases of the defense reaction of the
organism.

294

FISH AND WILDLIFE SERVICE

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1935. XII. Sinneswerkzeuge, pp. 906-969. In Dr
H. G. Bronns Klassen und Ordnungen des
Tierreichs, Band 3: Mollusca, Abteilung 3;
Bivalvia, Teil 1. Akademische Verlagsgesellschaft,
Leipzig.
Hecht, Selig.

1918. The physiology of Ascidia atra LeSueur. II.
Sensory physiology. Journal of Experimental
Zoology, vol. 25, No. 1, pp. 261-299.
1919a. Sensory equilibrium and dark adaptation in
Mya arenaria. Journal of General Physiology, vol.
1, No. 5, pp. 545-558.
1919b. The nature of the latent period in the photic
response of Mya arenaria. Journal of General
Physiology, vol. 1, No. 6, pp. 657-666.
1919c. The effect of temperature on the latent
period in the photic response of Mya arenaria.
Journal of General Physiology, vol. 1, No. 6, pp.
667-685.
1920a. The photochemical nature of the photo-
sensory process. Journal of General Physiology,
vol. 2, No. 3, pp. 229-246.
1920b. Intensity and the process of photoreception.
Journal of General Physiology, vol. 2, No. 4, pp.
337-347.
1928. The relation of time, intensity and wave-
length in the photosensory system of Pholas.
Journal of General Physiology, vol. 11, No. 5, pp.
657-672.

THE NERVOUS SYSTEM

295

Hopkins, A. E.

1932a. Chemical stimulation by salts in the oyster,
Ostrea virginica. Journal of Experimental Zoology,
vol. 61, No. 1, pp. 13-28.
1932b. Sensory stimulation of the oyster, Ostrea
virginica, by chemicals. Bulletin of the U.S.
Bureau of Fisheries Bulletin No. S, vol. 47, pp.
249-261.
Jakubski, Antoni W.

1912. Zur Kenntnis dcs Gliagewebes im Nerven-
system der MoUusken. Verhandlungen des VIII
Internationalen Zoologen-Kongresses zu Graz, 15-
20 August, 1910. Gustav Fischer, Jena, pp. 936-
939.

1913. Studien Uber das Gliagewebe der Mollusken.
Teil 1. Lamellibranchiata und Gastropoda. Zeit-
schrift fur wissenschaftliche Zoologie, Band 104, pp.
81-118.

Jhering, Hermann von.

1877. Vergleichendc Anatomic des Nervensystems
und Phylogenie der Mollusken. Wilhelm Engel-
mann, Leipzig, 290 pp. [In other publications this
author spells his name, Ihering.]

Lbenhardt, Henrv.

1926. Quelcjues etudes sur "Gryphaea angvlaia"-
(Huitre du Portugal). Annales de I'lnstitut
Oceanographique, nouvelle serie, tome 3, fascicule
1, pp. 1-90.

LuBET, Pierre.

1955. Cycle neuros^cretoire chez Chlamys varia L.
et Mytilus edulis L. (MoUusques lamellibranches).
Comptes Rendus Hebdomadaires des Seances de
I'AcadSmie des Sciences, tome 241, pp. 119-121.
Pelseneer, Paul.

1S91. Contribution a I'etude des Lamellibranches.
Archives de Biologie, tome 11, pp. 147-312.
PiERON, Henri.

1941. Recherches neurophysiologiques sur la Mye.
Bulletin Biologique de la France et de la Belgique,
tome 75, fascicule 4, pp. 421-484.
Rawitz, Bernhard.

18S7. Das zentrale Nervensystem der Acephalen.
Jenaische Zcitschrift fvir Naturwissenschaft, Neue
Folge, Band 13, pp. 3S4-460.
Spengel, J. W.

1881. Die Geruchsorgane und das Nervensystem der
Mollusken. Ein Beitrag zur Erkenntnis der
Einheit des MoUuskentypus. Zeitschrift fijr wis-
senschaftliche Zoologie, Band 35, pp. 333-383.
Studnitz, Gotthilft v.

1931. Die Morphologic und Anatomic von Lima
inflata, der Feilenmuschel, nebst biologischen
Untersuchungen an Litna hians Gmel. Zoologische
Jahrbticher, Abteilung fiir Anatomic und Ontogenie
der Tiere, Band 53, pp. 199-316.
Thiele, Johannes.

1889. Die abdominalen Sinnesorgane der Lamelli-
branchier. Zeitschrift fiir wissenschaftliche
Zoologie, Band 48, pp. 47-59.

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CHAPTER XIV
ORGANS OF REPRODUCTION

Page

Anatomy 297

Determinations of volume and weight of gonad _._ 299

Histology 299

Spawning... 303

Spawning reaction of the female 303

Spawning reaction of the male 310

Frequency of spawning 312

Fecmidity of the oyster 313

Sex ratio, hermapliroditism, and sex change 314

Lunar periodicity 318

Biological significance of spawning reaction 319

Bibliography _ 320

ANATOMY

The ripe gonad is a massive organ located near
the sm-face of the body within a layer of connec-
tive tissue between the digestive diverticula on
the inner side and the surface epithelium on the
other. In sexually mature oysters it appears as
many branching tubules (follicles) which merge
along the dorsal side of the body to form one
continuous structm-e encompassing the visceral
mass and extending ventrally to the tip of the
pyloric process (fig. 267).

The reproductive gland is not encapsulated,
and its outlines are indistinct. At sexual maturity
the surface covering of the body becomes so thin
that a network of fine genital canals is clearly
visible through it. The diameters of the canals
gradually increase as they converge into a wider
gonoduct through which the germ cells are dis-
charged. Two separate systems of genital canals,
one on each side of the body, are the only sign of
the paired origin of the sex gland which in an
adult oyster is completely fused into a single
organ.

In many bivalves sex products are discharged
thi-ough tlie kidneys, but in the oyster the gono-
duct opens into a vestibule, or atrium, which
also receives the urinal duct. This relationship
can be seen on a series of slightly slanted sections
of the lower part of a ripe gonad. One of these
sections is shown in fig. 268. The female oyster
from which the tissue was taken was preserved
dm-ing the act of spawning. The follicles of the

FISHERY bulletin: VOLUME 6 4, CHAPTER XIV

lower part of the ovary (left side of the figure)
are almost empty of ripe eggs; a short ciliated
passage between the ovarian follicle and the
pear-shaped area of the kidney vestibule are at
the right; two spawned eggs are near the outside
opening of the vestibule, which is lined with
ciliated epithelium. A transverse section of the
kidney reservoir lined with typical secretory
cells is in the upper right portion of the figure.
The connection between the vestibule and the
reservoir is located above this section. The
folUcles in the inner portion of a gonad lie at an
angle to the genital ducts into which tliey discharge
theii' content. The structure of spermary differs
from that of the ovary only in that the follicles
are filled with spermatozoa.

The degree of sexual development can be esti-
mated roughly by measuring with calipers the
thickness of a transverse section of the gonad
layer. Since the gonad is not uniformly thick in
all its parts, the section should be made at some
selected place. In conformity with the practice
used by the Biological Laboratories of the Bureau
of Commercial Fisheries at Milford, Conn., Oxford,
Md., and Gulf Breeze, Fla., the oyster is cut with a
razor blade along a line extending from the lower
corner of the labial palps across the stomach to the
posterior end of the body. There is considerable
variability in the gonad layer of oysters of known
age and similar environment. In Long Island
Sound the average maximum thickness of ripe
gonads of 4- to 5-year-old oysters taken from three
depth levels of 10, 20, and 30 feet was, according to
Loosanoff and Engle (1942), about 2.4 mm. for the
shallow water oysters and only about L5 mm. for
those found in deeper water. Much gi-eater
gonadal development was recorded in other loca-
tions. Some fuUy mature Cape Cod oysters used
in my experimental work. had a layer of gonad
from 6 to 8 mm. thick, and a similar degi-ee of
development was noted in oysters from a small
tidal pool near the laboratory at Beaufort, N.C.

297

0

Centimeters

Figure 267. — Fully ripe C. virginica. The mantle of the right side was dissected above the epibranchial chamber and
pushed to the right to expose the pyloric process. A network of canals leads to the single gonoduct through which
the sex cells are discharged. Drawn from life. an. — anus; cl. — cloaca; d.br. — right demibranch; g.o. — genital
opening; gon. — gonad; m. — mantle; w.t. — water tubes inside the gills.

Gonadal layers of about 10 mm. thickness were
found in large C. giyas (8 inches in height) from
Willapa Bay, Wash. (Galtsoff, 1930a, 1932).

In all species of Crasf;ostrea the bulk of gonads
varies from season to season, reaching its maximum
shortly before the onset of spawning. The num-
ber of sex cells produced during one reproductive
period varies, depending on the conditions of the
environment. The greatest gonadal development
is more likely to be found in the populations of
oysters from the nortliern latitudes north of the

Chesapeake Bay rather than in the south Atlantic
and Gulf waters. This is apparently associated
with the fact that the reproductive season in the
northern latitudes is of short duration, 4 to 6 weeks,
whUe in the warmer waters of the south gonadal
formation and spawning, continue, with interrup-
tions, for several months. In both groups the
annual reproductive capacity (i.e., the number of
eggs produced annually) maybe of the same order
of magnitude or even greater in the southern
oysters because of the longer reproductive period.

298

FISH AND WILDLIFE SERVICE

but the greatest bulk of a ripe gonad is more likely
to occur in oysters which have only one spawning
period per year.

Microns

Figure 268. — Slightly slanted tangential section through
the opening and adjacent portion of the ovary of
C. virginica. Oviduct with two egg.s at the lower right
side. Kidney reservoir at upper right. Ovary follicles
at left. Drawn semidiagramniatically from a photo-
micrograph of a preparation. Kahle, hematoxyhn-eosin.

Several factors besides geography influence
gonadal development. The most significant are
temperature, depth (Loosanoff and Engle, f942),
salinity, available food, and pollution of oyster
bottoms. Many examples of the suppression of
gonadal growth by adverse conditions may be
cited. For example, oysters living in waters highly
polluted by various trade wastes have, as a rule,
poorly developed gonads. Sometimes the devel-
opment of the sex gland is suppressed to such a
degree that only traces of follicles are found in the
visceral mass, and the layer of the digestive di-
verticula is visible from the surface. These oysters
have the greenish or brownish coloration typical
for the digestive diverticula which in the sexually
ripe oyster is not noticeable under a thick layer of
gonad. Oysters with suppressed gonad develop-
ment are found in the waters which receive con-
tinuous discharge of the pollutants from pulp and

paper mills. Similar poor oysters are frequently
encountered in waters of extremely low salinity or
from areas where salinity increases to the highest
Umit of tolerance (34 to 40 7oo)- The determi-
nation of gonadal thickness described above lacks
precision because variation in the compactness of
the gonadal tissue cannot be measured, but the
method is, nevertheless, useful for the practical
purpose of estimating expected intensity of
spawning.

DETERMINATION OF VOLUME AND
WEIGHT OF GONAD

The fully developed gonad is the largest organ
of the oyster. The ovaries or spermaries can be
separated from the underlying digestive diver-
ticula by using small curved scissors. The excised
pieces are weighed, and their volume is measured
by displacement in a simple device made from a
glass cylinder of appropriate dimensions (depend-
ing on the size of the sample) with a drain pipe
at the bottom and a side glass tubing of about 5
mm. in diameter fused to the side at an angle of
about 45° to record the water level. A convenient
water level is selected and recorded, the tissue is
introduced, and the water is then drained into a
measming vessel to the previous level. The body
weights of adult New England oysters with fully
developed gonads varied in my observations from
14.2 to 23.2 g. The gonads comprised from 31.2
to 40.7 percent of the total body weight exclusive
of shell. The volume of the oysters' tissues ranged
from 21 to 24 ml., with the gonads accounting for
32.8 to 33.4 percent of the total bulk. Oysters
selected for these measurements were of the highest
commercial quality and with maximum develop-
ment of gonads. In poor oysters with light gon-
adal development the proportion of the weight of
the gonad to body weight is only a small fraction
of the figures given above.

HISTOLOGY

The gonads of the oyster originate from a group
of primordial germ cells located in the mesodermal
band on the ventral side of the pericardium in the
vicinity of the visceral ganglion (Coe, 1943a). In
embryos of bivalves primordial germ cells are
identified by their relatively large size, round
siiape, and clear vesicular nucleus with one or two
nucleoH (Okada, 1936, 1939; Woods, 1931, 1932).
Tlie primordium soon becomes separated into two
groups whicli by continuous multiplication of the

ORGANS OF REPRODUCTION

299

constituent cells extend symmetrically along both
sides of the body. Each group gi-ows anteriorly,
surrounded by vesicular connective tissue of the
visceral mass, and forms a system of profusely
branching tubular follicles. The fusion of the
branches along the dorsal side obliterates any rem-
nants of the paired origin of the gonad.

The microscopical structure of the gonad varies,
depending on the age of the oyster, degree of ma-
tiu'ity, season, and environmental condition. In
Ostreidae and in some other pelecypods (Pecten,
MytUus, Volsella) the follicles of a fully developed
gonad consist almost entirely of primitive sex cells
(gonia) at various stages of development with only
a few minute follicular cells between them. Be-
cause of the absence of a capsule or membrane
around the gonad, the sex cells are in direct con-
tact with the surrounding tissues (fig. 269). It
may be assumed that the role of follicular cells in
the growth of the gonad of oysters is insignificant
and that the gametogenic cells obtain their nour-
ishment directly from the connective tissue which
surrounds them. In Mya, Teredo, Bankia, and
other Adesmacea the follicular cells are large and
function as accessory nutritive cells.

In immature oysters and in the adults, that had
completed the spawning period, the germinal
epithelium consists of undifferentiated sex cells,
some at the early stages of gametogenesis. Their
sex can be recognized only by careful cytological
examination. As the folhcles grow and ramify
they spread throughout the surrounding layer of
connective tissue. The maturing sex cells inside

Microns

60

Figure 269. — Terminal portion of the follicle of a gonad
of C. virginica at early stage of development (male
phase). The follicle is surrounded by a large mass of
connective tissue. Redrawn from C'oe, 1936a.

Figure 270. — Cross section of one surface follicle in a fully
developed ovary of C. virginica. Bouin, hematoxylin-
eosin.

them midtiply, grow, and fill up the lumen (fig.
270). The foUicles near the surface of the gonad
are distinctly different. Their outer walls facing
the body surface are fined with ciliated epithelium,
and only the inner sides of the follicles retain the
germinal cells (fig. 271). This differentiation of
the germinal epithelium into two distinct types is
probably common to all species of oysters. The
follicles lined with ciliated cells, function as the
genital canals through which mature sex cells are
moved by ciliary action. They were first de-
scribed by Hoek (1883) for 0. edulis and subse-
quently were found in 0. lurida and in several
species of Crassostrea.

Since the transformation of germinal cells into
ova and sperm is a gradual process which does
not involve all the cells of the germinal epithelium
at the same time, numerous undifferentiated or
so-called residual cells are usually found along
the inner periphery of a follicle. Some of them
can be found even in a fully developed gonad
(fig. 270).

The bulk of a functional ovary is made up of
fully developed ova which fill up the lumina of

300

FISH AND WILDLIFE SERVICE

0

Microns

150

Figure 271. — Cross section of a surface follicle from a fully developed ovary of C. virginica. Germinal epithelium is
formed by cells at different stages of ovogenesis and by small undifferentiated cells. Bouin, hematoxylin-eosin.

K

0 Ml

100

Figure 272. — Photomicrograph of an obhque section of a ripe ovary of C. virginica shortly before spawning. Bouin,

hematoxylin-eosin.

ORGANS OF REPRODUCTION
733-851 0 — 64 20

301

Figure 273. — Photomicrograph of a cross section of a ripe spermary of C virginica. Bouin, hematoxylin-eosin.

the foUicles and of a number of cells at diflferent
stages of the development nearer to the walls.
The eggs are attached to the walls of the follicles
by elongated peduncles, giving them a pear-shaped
appearance. At the height of sexual develop-
ment the layer of undilTerentiated germinal
epithelium is reduced to a very narrow band of
small cells hardly visible at low magnification
(fig. 272). The connective tissue between the
folhcles also very nearly disappears.

The arrangement of cells in a ripe spermary (fig.
273) is similar to that in the ovary. Fully formed
spermatozoa are massed together inside the
follicle with their tails inward. They become
separated as the sperm moves through tlie genital
canals, which are frequently distended by the
accumulation of spermatozoa ready to be ejected.
In 0. edulis and 0. lurida the sperm in the lumen
of a follicle form distinct balls which retain their

shape until they are discharged by the oyster.
A number of sex cells remain in the follicles at the
completion of spawning. Consequently, at the
end of the reproductive season the gonads contain
mature ova and sperm as well as undifferentiated
cells; many of them are pycnotic and detached
from the germinal lining of the wall. Phago-
cytosis becomes pronounced as large numbers of
leucocytes invade the follicles to digest and
cytolize the remaining sex cells (fig. 274). The
connective tissue between the follicles becomes
disorganized. After the reabsorption of the gonad
is completed only a narrow band of germinal epi-
thelium remains in a few follicles and the entire
layer has shrunk to such a thin band that it is
not visible to the naked eye. The oyster is now
at an indifferent stage. Its sex can be recognized
in some individuals in which young ovocytes or
spermatocytes are present, but in many others

302

FISH AND WILDLIFE SERVICE

Microns

60

Figure 274. — Ovarj' of C. virginica shortly after comple-
tion of spawning. Unspawned ova are cytolized.
Note the invading phagocytes and the disorganization
of connective tissue. Bouin, hematoxylin-oosin.

the young sex cells are not sufficiently differ-
entiated and their sexes can not be identified.
Ovocytes, when sufficiently developed, can be
distinguished from spermatocytes by their large
nuclei and granular cytoplasm.

During spawning the sex cells are moved inside
the genital canals by ciliary motion of the epithe-
lium. How they are released from the follicles
and reach the genital canals located near the sur-
face of the sex gland is not knowm. Histological
examination discloses no contractile elements in
the tissues surrounding the follicles, and no con-
traction of the gonad or part of it could be detected
during spawning. Upon reaching the gonoduct
the released cells continue to be moved by the
powerful cilia of the duct and vestibule (fig. 275) .
Longitudinal aiiu circular muscle fibers are found
under the basal membrane of the vestibule and
appear to be better developed in the male gonad
than in the female. Roughley (1933) refers to
the presence of sphincter muscles in the gonoducts
of Ostrea (Crassostrea) commercialis , but no struc-
ture resembling a sphincter is found in C. virginica
(Galtsoff, 193Sa). It is conceivable that a contrac-
tion of circular muscle fibers of the spermiduct to
a certain degree controls the passage of sperm

through the genital opening, but no such action
has been detected under experimental conditions.
The epithelium of the distal end of the gonoduct
and of the urinogenital vestibule contains, besides
the ciliated cells, a large number of mucous cells
(MC) not present in the lining of the canals. In
the ovary the opening of the oviduct into the
vestibule is marked by a small ridge of ciliated cells
(Galtsoff, 1938a, 193Sb).

SPAWNING

The sexual apparatus of oysters is of the simplest
type for it lacks the accessory sex organs which in
some moUusks are used for mutual excitation,
storage of sex cells, copulation, and secretion of
egg capsules. In spite of the structural simplicity
of the reproductive organs, the spawning of the
female oyster is a rather complex action which in-
volves coordination of the gills, nervous system,
mantle, and the adductor muscle, while the sexual
act of the male is much simpler. It is, therefore,
more convenient to describe separately the phe-
nomena involved in the spawning of the two sexes.
Under natm-al conditions simultaneous release of
sperm and eggs, essential for successful reproduc-
tion of the species, is attained through mutual
stimulation.

SPAWNING REACTIONS OF THE FEMALE

Spawning of the female proceeds in several
consecutive steps which, in the order of their
participation, involve the ovary, the gOls, the
mantle, and the adductor muscle. The behavior
of these organs follows a distinct pattern, one
action succeeding the other in a precise sequence
which fuially termiTiates in the dispersal of eggs
in the surrounding water.

The first step is ovulation, i.e., the discharge
of eggs from the ovary into the epibranchial
chamber. The moment the eggs appear in the
epibranchial chamber the two edges of the mantle
come together and effectively seal the pallial
cavity and cloaca. This peculiar behavior of the
mantle may be observed in a spawning female
oyster placed close to the wall of a rectangular
tank. In an actively feeding animal the space
between the two mantle edges is wide open; the
pallium and the tentacles are extended outward
parallel to the surface of the valves and the gills,
exposing the side of the adductor muscle, the
rectum, and the inner part of the cloaca (fig. 276).
A different picture is seen m a spawning female.
A few minutes before ovulation the edges of the

ORGANS OF REPRODUCTION

303

° Millimeters *-*•'

Figure 275. — Section through the lowest part of spcrmiduct and the adjacent portion of the vestibule. Both organs are
grossly distended by the discharged sperm. Tissue of C. virginica was preserved during the act of spawning. G —
approximate position of the junction of spermiduct and the vestibule; CE — ciliated epithelium; CT — connective
tissue; I\I — longitudinal muscle fibers; MC — mucous cell; SB — opening of the vestibule into the epibranchial chamber;
U — lower part of the urinogenital vestibule. Bouin, hematoxylin-eosin. From GaltsofF, 1932.

Eggs trapped inside the epibranchial chamber
liave to pass througli the water tubes in order to
accuimilate in the space between the gUls and the
mantle since tliere is no other way by which they
can reach this area (Galtsoff, 1938a). This
conclusion was confirmed by microscopic exami-
nation of a section of the gills of a female preserved
during tlie act of spawning (fig. 279). Any other
minute particles suspended in water pass through
tlie ostia and water tubes into tlie exhalant cham-
bers and are swept by the outgoing current. The
eggs released tlu-ough the genital pore, however,
take the opposite course when they enter the
water tubes of the gills.

While the eggs pass through the gills the ostia
are wide open and the ciliary currents along the
filaments are neither inhibited nor reversed. The
eggs, therefore, flow against the current produced

mantle display unusual activity; they come
together and temporarily close the access to the
gills; then for a few seconds they open again.
Both the rate and range of shell movements at
this time gradually increase. Finally, the en-
trance to the gill cavity closes completely except
for one small opening or "window" as shown in
fig. 277. Soon a white cloud of unfertilized eggs
appears at the window, the adductor muscle
contracts sharply, and the eggs are discharged and
dispersed several inches away from the oyster
(fig. 278). The opening between the edges of the
mantles may be formed at any place along their
periphery but once formed its position remains
unchanged throughout the duration of spawning.
Spawning may last from a few minutes to nearlj'
1 hoirr, depending on the amount of mature eggs
in the ovary.

304

FISH AND WILDLIFE SERVICE

0

Centimeters

Figure 276. — Actively feeding C. virginica. The valves are wide open, the pallium and the tentacles are extended out-
ward and the cloaca, the adductor muscle, and rectum are clearly visible. Drawn from life. The opening between
the valves is slightly e.xaggerated.

0

Centimeters

Figure 277. — Position of the mantle and tentacles of C. virginica shortly before the discharge of eggs through a small
window left open (right side of figure). Drawn from life.

bj' the lateral cilia. Laboratory observations
show that when the valves open the gill lamellae
also expand and the water tubes dilate. xVt the
same time the closing of the cloaca and of the
mantle cavity cuts off the access of water from the
outside. As a result, a suction is produced inside
the water tubes by the expansion of gill lamellae,
forcing the eggs into the water tubes and through
the ostia to the surface of the gills.

Eggs of larviparous oysters {0. lurida and 0.
ediilis) also pass through the gills but are retained
in the pallial cavity until fully developed larvae

are formed and escape from the mother's body.
Stafford (1913) thought that the eggs of 0.
lurida are too heavy to be carried by the respira-
tory current and so fall into the water tubes and
are forced tlu-ough the ostia by the pressure of
their own mass. The correctness of such an ex-
planation seems doubtful. The act of spanning
in the larviparous species has not been studied
adequately, probably because the ovulation and
passage of eggs into the pallial chamber proceed
without any outward indication (Yonge, 1960).
Eggs f)f these species are fertilized inside the body

ORGANS OF REPRODUCTION

305

Figure 278. — Spawning of large femalo C. virginica, photographed in the laboratory.

by sperm drawn in from the outside with the
respiratory current and are extruded as well-de-
veloped larvae. The process is called "swarming"
(Korringa, 1941). Careful studies of shell move-
ments of 0. luri'la or 0. edulis during the repro-
ductive period nuiy uncover some peculiarities of
the behavior of their adductor muscles associated
with swarming.

If the shell movements of a spawning female are
prevented by cutting off a piece of valve between
the adductor muscle and the hinge, the eggs can-
not pass through the gills and are discharged
through the cloaca. This has been demonstrated
in the experiments illustrated in fig. 280, A and B.
In both cases fully mature Cape Cod oysters
were placed in finger bowls under a low-power
binocular microscope. In oyster A the gills were
exposed by cutting off a piece of the right valve
without injuring the adductor nniscle. Its shell
movement remained normal. In oyster B the en-
tire dorsal half of the right valve above the muscle
attachment was removed, and in this way shell
movements were prevented. During the spawning
of oyster A the released eggs (e) passed through

the gills, while in oyster B they were discharged
through the cloaca.

Female spawning of C. gigas and 0. cucullaia
follows the same pattern as the American oyster
(Galtsoff, 1932). It is apparent that the mantle,
gills, and adductor muscle of Crassostrea species
temporarily assume the role of accessory sex organs
and through coordination and adjustment of their
activities perform a specific role which is distinct
from their primary functions.

The release of sex cells from sexually mature
oysters often requires a stimulus which causes a
triggerlike effect and initiates spawning. Very
often this effect is associated with a sudden rise
in the temperature of the water. Numerous
ecological observations show that under natural
conditions oysters spawn at rising temperature.
This led to the concept of "critical temperature,"
and for many years the temperature of 20° C. was
considered the lowest at which spawning takes
place. It was postulated that "once this critical
temperature' of 20° C. is reached a trigger mecha-
nism is released which requires some hours for its
consummation" (Nelson, 192Sa). Further obser-

306

FISH AND WILDLIFE SERVICE

vations disproved this concept. Nelson reported
that C. virginica transplanted from the United
States to England could be induced to spawn at
19.1° C. (Nelson, 1931). Some of the oysters
of Long Island Sound spawn at 16.4° C. (Loosan-
off, 1939).

Ecological evidence shows that spawning of an
oyster population often coincides with a rapid rise
of temperature but it is not determined by a
specific "critical" temperature. Phj'siological
studies at the Woods Hole laboratory indicate that

temperature and chemical stimulation, acting
singly or jointly, may induce spawning in sexually
ripe oysters. On the other hand it is apparent
that certain internal and external conditions in-
hibit spawning.

The effect of temperature on spawning can be
observed by placing a sexually mature oyster in a
tank of water, connecting its right valve to the
writing lever of a kymograph, rapidly warming
the water and then maintaining the temperature
at a desired level. Shell movements of the

FiouRE 279. — Transverse section of the gills of female C. virginica preserved during spawning.

eggs inside the water tube (center) and in the ostium.

Hematin-eosiu. Note the

ORGANS OF REPRODUCTION

307

Centimeters

Figure 280. — Experiment showing the role of shell movements in the discharge of spawned eggs of C. virgivica through
the gills. A. — Portion of the right valve was removed to expose the gills; the adductor muscle was not injured and
shell movements during spawning were normal. Eggs (e) pass through the gills. B. — Portion of the right valve
between the adductor muscle and the hinge was cut off to prevent shell movements. Eggs (e) pass through the
cloaca. Drawn from life.

spawning oyster are recorded on a kymograph,
and the presence of discharged eggs or sperm in
the water is ascertained by microscopic examina-
tion of samples taken at frequent intervals. In
the case of heavy spaw^ning so many se.x cells may
be shed that the water becomes milky and opaque;
when there is light spawning the presence of eggs
should be checked by collecting material which
settles on the bottom of the tanks.

Spaw'ning of sexually ripe females of C. mrgrnica
may be induced by warming the water from 18°
to 20° C. to 22° to 23° C. and maintaining this
temperature for several hours. Relatively few
oysters respond to this mild stimulation. A more
effective method, which in my experience gave
positive results in about 40 percent of the oysters
tested, consisted in rapidly raising the temperature
of the water from about 20° C. to 33° to 34° ('.
The remaining 60 percent of the oysters which
did not respond to thermic stimulus required

additional stimulation by live sperm. Using this
technique I found that the population of oysters
from a single small bed in Onset, tested within a
few days, consisted of individuals which greatly
varied in the degree of their response to spawning
stimuli. The tests were made at intervals of
2° C. The females that failed to spawn at 22° to
23° C. spawmed at this temperature when sperm
was added to the water. Some of the oysters
spawned at 2.5° to 27° C, but still others required
the addition of sperm to induce ovulation at this
temperature level. Similar results were obtained
at 29° to 31° C. and 32° to 33° C. In each of the
groups tested there were specimens which did not
respond to the rise of temperature and required
additional stimulation by live sperm. All the
oysters used in these experiments were mature;
they had fully developed gonads, the eggs were
lertili/.able, and spawning, when induced, was
Copious.

308

FISH AND WILDLIFE SERVICE

The threshold temperature of spawning is not
a "critical" temperature in the sense that it
automatically induces the discharge of eggs in all
physiologically ripe oj-sters. The success or failure
of thermic stimulation depends on the responsive-
ness of the organism. It would be more appro-
priate to speak of the "critical condition" of the
organism which makes it responsive to stimulation
rather than of critical temperature of spawning.
Within broad limits between 15° and 32° to
34° C, spawning of C. lirginica may occur at any
temperature; mass spawning of an oyster popu-
lation is more likely to take place in warm water
above the 22° to 23° C. level.

Stimulation by live sperm is of great importance
in the reproduction of Crassostrea oysters. In
the Woods Hole experiments the time elapsed
between the addition of sperm suspension and the
beginning of shedding of eggs varied between 6
and 38 minutes. At about 20° C. the sperm added
to the pallial cavity passed through the giUs and
was expelled from the cloaca within 7 to 8 seconds.
The latent period of spawning reaction lasts
several minutes. This suggests that possibly the
sperm acts upon the female organism after it has
been absorbed by the cells of the water transport
system or by the digestive tract. Direct evidence,
however, is absem since attempts to prevent the
penetration of sperm into the digestive tract by
plugging the mouth and esophagus were not
successful.

Rhythmic contractions of the adductor muscle
are associated with the release of eggs from the
ovary and are not directly stimulated by tempera-

ture or by any known chemical agent. This
becomes clear from the observations which show
that spawning contractions proceed in the same
manner whether the spawning was induced by
temperature or by sperm. Two kymograph
tracings of shell movements of the two females
shown in fig. 281 are similar in spite of the fact that
in one of them (upper line) spawning was induced
by the addition of sperm, while in the other by
rapidly warming the water from 21.6° to 30.2° C.

Experiments were made to determine whether
some substances causing the contraction of the
adductor muscle are released into the blood stream
during spawning. A female was induced to
spawn by thermic stimulation, and a sample of its
blood withdrawn from the pericardium was
immediately injected into the visceral mass and
into the circulatory system of a sexually mature
but nonspawning female. Six e.xperiments of this
type were made with negative results.

Shell movements during female spawning are so
typical that they cannot be nristiken from any other
type of muscular activity. Spawning reaction is
recognized by the duration of the latent period of
not less than several minutes; the uniformity of
the tonus level at the points of relaxation; regular
rhythm of the contractions, particularly at the
beginning of the reaction; and the presence of a
small "plateau" about half-way on the relaxation
curve (see fig. 281). The plateau is indicative of
the slowing down of the relaxation phase; its
significance is due to the fact that it coincides
with the oozing out of eggs through the ostia of
gill filaments (Galtsoff, 1938b). This type of

22.1

Figure 281. — Kymograph records of shell movements during spawning of two female C. virginica. Upper line — spawning
induced by the addition of sperm at 22.1° C; lower line — spawning induced bv rapid rise of water temperature from
26.1° to 30.2° C. Time interval, 1 minute.

ORGANS OF REPRODUCTION

309

spawning curve appears in hundreds of records
obtained in the laboratory in the course of several
years of studies. It does not occur after the
spawning season is over and cannot be provoked
by temperature or chemical stimulation of the
oysters devoid of mature eggs. Injections of low
concentrations of adrenalin cause rhythmical con-
tractions of the adductor muscle but of an entirely
different type. The spawning reaction is always
followed by a refractory period of two to several
days during which the female is not responsive
to stimulation.

SPAWNING REACTION OF THE MALE

Spawning of the male does not involve the par-
ticipation of the mantle and adductor muscle.
Sperm is discharged from the spermary into the
epibranchial chamber by ciliary motion inside the
genital ducts and is swept away by the respiratory
current (figs. 282 and 283). The pallium remains
wide open and quiescent. Muscular contractions
of the adductor play no role in the release and dis-
charge of sperm, and there is no visible change in
the velocity of the cloacal current during ejacu-
lation (Galtsoff, 1938a) . Shedding of sperm occurs
sometimes in sudden outbursts of brief duration
which may be repeated at frequent intervals.
Toward the end of the reproductive season the dis-
charge of sperm may continue for several hours

without interruption until the male is completely
spent. Ejaculation proceeds either from one or
from both spermiducts simultaneously. In the
latter case the flow of milky water containing
suspended spermatozoa can be seen emerging from
the cloaca and from the promyal chamber simul-
taneously. The males of C gigas and C. coin-
mercialis behave in a manner similar to the males
of C. virginica.

Males of C. virginica are more responsive to
spawning stimuli than the females of the species.
They are more readily stunulated by rising
temperature, and shedding of sperm is easily
induced by various substances; a suspension of
eggs or filtered egg water (sea water in which eggs
were kept for some time) ; eggs of various bivalves
{Pecten irradians, Mercenaria mercenaria, Mytilus
edulis) ; and eggs of starfish, Asteriasforbesi.

The latent period of stimulation varies depend-
ing on the substance used and its concentration,
but in general it is much shorter than in female
spawning. Suspension of eggs or egg water of
C. virginica induces spawning of the male within
5 to 6 seconds at 24° to 25° C; eggs of Pecten
irradians are more effective, provoking a response
in a male oyster in 4.6 to 4.8 seconds; the latent
period in the case of clam eggs (Alya arenaria,
Mercenaria mercenaria) is from 8 to 9 seconds at

^^^^^ Jk.

_A_-_k^

_jw^

vWt4aUJvl4v^^

Figure 282. — Shell movements of three males of C. lirgimca recorded during the shedding of sperm. There was no
change in muscular contraction before, during, or after spawning. Temperature 23.5° C. Time interval, 1 minute.

310

FISH AND WILDLIFE SERVICE

V1&'.---

,,.»^

Figure 283. — Photograph of the spawning male, C. virginica. The V cut in the left valve was made several weeks before
the experiment. Temperature 2.3.0° O. Sperm ejected through the opening of the promyal chamber is seen as a
white jet opposite the V cut.

24° to 25° C. Many other substances including
various hormones (thyroxin, adrenalin, estrogen),
desiccated anterior and posterior pituitary, thy-
mus, thyroidin, cysteine, glutathione, peptone,
egg albumen, urea, different sugars (dextrose,
maltose, d-arabinose) , starch, and yeast stimulate
ejaculation in various degrees of effectiveness.
Suspension of desiccated thyroid gland (thyroidin)
in sea water was found to be the most effective

stimulant, and it has been used in the Woods
Hole laboratory in preference to egg suspension or
egg water.

Other substances may also provoke sexual
response. Miyazaki (19.38) found that the ex-
tracts from several algae, Ulva pertusa, Entero-
morpha sp. and Monostroma sp. induce spawning
in the males of C. gigas.

Mature males (C virginica) respond also to live

ORGANS OF REPRODUCTION

311

sperm of the species. In this case the latent
period of spawning reaction is much longer, vary-
ing from 6 to 27 minutes at 20° to 21° C. The
interesting fact is that in the case of sperm
stimulating male spawming the latent period is of
the same order of magnitude as that of the female
spawning reaction. Possibly the sperm acts as a
stimulant only after it has been absorbed by the
oyster, while eggs and egg water act upon the
receptors located on the body surface.

The active principle of sperm of C. virginica can
be extracted with ethyl alcohol and benzene. The
residual powder of the e.xtract can be mixed with
sea water and added to the gills of the female to
induce a typical spawning reaction (Galtsoff,
1940). Spermatozoa of C. virginica carry another
hormonelike substance which may be recovered
after alcohol extraction. The substance was
named "diantlin" by Nelson and Allison (1940),
who found that it dilates the ostia and stimulates
the increase of water flow through the gills.

FREQUENCY OF SPAWNING

Under laboratory conditions male oysters may
be induced to spawn many times at very brief
intervals. It is, therefore, reasonable to assume
that they behave in a similar way in their natural
environment. The females spawn only a limited
number of times within one breeding period. Out
of several hundreds of oysters tested in the
laboratory, the majority of the females w-ere
induced to spawn two or three times within a
6-week period (July-August), and only one
spawmed seven times. Similar conditions exist
with C. gigas. Frequently a substantial percent-
age of females of these two species fail to shed
eggs in spite of a ripeness of their ovaries and
favorable environmental conditions. The spawn
may be retained in the gonads until late fall w^hen
it is reabsorbed.

The number of times the adult female oysters
spawn under natural conditions can only be
surmised from examination of gonads and the
occurrence of larvae in plankton. It is very
difficult to decide from plankton observations
whether the entire oyster population spawned
several times or if different groups of oysters pro-
duced the larvae appearing at intervals in plank-
ton. In Long Island Sound, for instance, four or
more "waves" in tlie occurrence of straight hinge
larvae were recorded (Loosanoff and Nomejko,
1951a) in 1943, but the described periodicity may
have been due to spawning of different populations

living in shallow and deep water. Inasmuch as
the laboratory experiments show that repeated
spawning may be induced in the same female, it is
reasonable to infer that in their natural habitat
oysters spawn more than once during every
breeding season.

Laboratory observations show that spawning of
sexually mature C. virginica is sometimes inhibited
and that the oysters fail to respond to all known
methods of stimulation. Similar conditions exist
with C. gigas, which sometimes fail to spawn in
spite of full gonad development. Artificial stimu-
lation by suspension of sex cells may facilitate
spawning but is not always successful. The reason
for this inhibition of spawning in sexually ripe
oysters has not been established, but the work of
Lubet (1955) on Chlamyft and Mytilus throws some
light on the problem. Lubet discovered that the
excision of cerebral ganglia in these mollusks
provokes precocious spawning and that the muti-
lated animals spawn much earlier than the con-
trols. Excision of the visceral ganglia seems to
retard spawning. These experiments suggest that
spawning is under the control of the nervous
system. It also appears significant that neuro-
secretion in the ganglia cells precedes gameto-
genesis and that maximum accumulation of the
neurosecretory products occurs at the time of the
maturation of sex cells. In the species studied by
Lubet the neurosecretory granules were absent in
the ganglia of the recently spawned out animals.
Whether Lubet 's findings on neurosecretion apply
to sexually mature oysters is not known, but his
work seems to indicate that in the bivalves he
studied, the release of sex cells was facilitated by
the removal of internal inhibition (excision of
cerebral ganglia) and that the disappearance of
the neurosecretory products from the cerebral
ganglia was necessary for the moUusk to become
receptive to spawning stimuli. The latter infer-
ence is based on the observation that partial
disappearance of neurosecretory granules always
occurs a few days before spawning. After the
completion of spawning all neurosecretory cells
are empty. These findings are not in accord with
the results of studies conducted by Antheunisse
(1963) on zebra mussels {Dreifsena poh/morpha
Pallas) from the Amstel River near Amsterdam.
The mussels were collected once a month, from
November 1957 to November 1958, for histological
examination. For extirpation experiments only
adult females were used during the spring and

312

FISH AND WILDLIFE SERVICE

summer of 1959. Antheunisse states that in spite
of the parallelism between the neurosecretory and
reproductive C3'cles in zebra mussels spawning and
neurosecretion are not interrelated. Extirpation
experiments were difficult to perform, and most of
the mussels died some days after the operation.
It is therefore apparent that further studies are
needed tf> determine the role of the neurosecretion
in reproduction of mussels, oysters, and other
biv^alves.

FECUNDITY OF THE OYSTER

The intensity of spawning as judged by the
number of eggs or spermatozoa discharged in each
instance is variable. In both sexes the number
of sex cells produced by a ripe female or male
depends on the size of the oyster and the degree
of development of the gonad. The range of
variation is enormous. If the female gonad is
poorly developed, only a few thousands of eggs
may be released. On the other hand, the number
of eggs produced and discharged by a well-
developed gonad may reach many millions.
Potential capacity of the ovary, i.e., the total
number of eggs produced by a female during the
breeding season, is not indicative of its actual
reproductive ability which is expressed by the
number of eggs actually spawned. The following
procedure is used for estimating the number of
eggs released by the female. The oyster is placed
in a 20 1. tank and spawnuig is stimulated by
warming the water and adding sperm suspension.
After the completion of spawning five samples of
100 ml. each are taken while the water is agitated
by an electric stirrer. Eggs in the sample are
killed by adding two to three drops of 1 percent
osmic acid, allowed to settle, and are counted
in a Sedgwick-Rafter chamber. The oysters used
in four separate tests varied from 9.2 to 13.3 cm.
in height. The number of eggs (in millions)
discharged in one spawning were 15, 30.3, 70.3,
and 114.8 (Galtsoff, 1930b). After discharging
over 100 million eggs the last oyster had a gonad
about 5.5 mm. thick containing vast numbers of
eggs.

The results of these counts were questioned on
the basis that the computed volume of the
discharged eggs exceeds the total volume of the
body (Burkenroad, 1947). Rechecking the data
confixmed my estimate. The counts are correct
within ± 10 percent, the principal source of error

being the difficulty in obtaining uniform distri-
bution of eggs in the tank.

In the ovaiy the eggs are tightly packed and
compressed; upon their release the diameter of
their rounded part is increased. The spawned
eggs in the above tests averaged 40 /i in diameter.
The vf)lume of a given number of eggs can be
computed by using the conversion table from
diameters to volumes of spheres given in Perry
(1941). Since the volume of one egg of 40 m
diameter is 33,510.3 M^ the volume of 115 millions
of eggs, solidly packed would correspond to 3.8
cm.' With an allowance of 25 percent for inter-
spaces the volume of spawned eggs in the ovarj^
would be about 4.8 cm.' The latter figure is
within the range of magnitude of the volume of
the gonad obtained by the displacement method.

Not all the ovocytes become mature at the
same time. During the intervals between spawn-
ing some of them grow and replace those
discharged by the preceding ovulation.

The fecundity of C. gigas is even greater. The
five large oysters of this species forced to spawn
in the laboratory averaged 55.8 million eggs per
oyster; post mortem examination showed that
after ovulation they retained the major part of
the gonadial material. In comparison to C.
virginita and C. gigas the fecunditj- of the larvi-
parous European oyster is rather low. Estimates
of the mean number of larvae per oyster were
made by Dantan (1913), Cole (1941), Cerruti
(1941), and Millar (1961). In British waters the
mean number of larvae vary from 90,000 for a
1-year-old oyster to over a million for a 4-year-old
oj'ster. French oysters relaid in West Loch
Tarbert, Scotland, after 1 year produced as
many larvae as the native oysters on English beds.
The number of larvae is dependent, of course, on
the size of the oyster, as can be seen from the table
given by Millar.

Mean

Mean

number of

number of

Uianiftcr in cm.

larvae

Diameter in cm.

larvae

estimated

estimated

from five

from Ave

samples

samples

6.2-7.3

733, 400

7.4-7.6

1.155,000

7.3-7.4

1. 543. 000

8.3-8.5

616.000

The fully grown 0. lurida bear broods of 250,000
to 300,000 larvae, the number depending generally
upon the size of the maternal oyster (Hopkins,
1936, 1937).

ORGANS OF REPRODUCTION

313

SEX RATIO, HERMAPHRODITISM,
AND SEX CHANGE

The oviparous species of oysters of the genus
Crassostrea usually are not hermaphroditic; speci-
mens in which functional eggs and sperm are
found together are relatively rare. This condition
exists in C. virginica, C. gigas, C. angidata, C.
rhizophorae, and is probably common to all
members of the genus. The frequency of her-
maphroditism in Crassostrea oysters varies with age
and envu-onment. The earliest record was made
by Kellogg (1892), who found one hermaphrodite
among the many adult C. virginica he kept in
breeding tanks. Burkenroad (1931) reported
that about 1 percent of the oyster population on
the coast of Louisiana were hermaphrodites.
Needier (1932a, 1932b) found only four her-
maphrodites (less than 0.4 percent) among the
1,044 oysters of various ages growing on beds in
the waters off Prince Edward Island. The
hermaphrodites were found only among the
2-, 3-, and 4-year-old oysters; none were en-
countered in oysters from 5 to 8 years old. In
the course of my studies I found only two her-
maphrodites among several thousand se.xually ripe
oysters from 5 to 7 years old.

Amemiya (1929) reported only one l)ermaphr(>-
ditic specimen among 120 sexually mature C.
r;i>a.s (0.8 percent). The percentage is appa.-ently
higher in the Bombay oyster, 0. cucuUata, for
Awati and Rai (1931) reported 23 hermaphroditic
specimens (2.9 percent) among the 794 oysters they
examined

The larviparous oysters of the genus (Mrea {0.
edulis, 0. luriila, 0. eqiie.^tri.% and others) are as a
rule ambisexual, i.e., they undergo rhythmical
changes in sexuality. The initial phase in these
species is usually nuxle, followed by alternating
female and mde phases.

Orton (1927) distinguishes several arbitrary
categories of sexual changes in 0. edulis from pure
male or female to hermaphrodites which contain
an equal abundance of ripe spermatozoa and ova.
Different transitional pliases of sex changes which
take place during the life of the European oyster
are discussed later in this chapter.

Oysters have no secondary sexual cliaracters,
and their sex can be recognized only during tlie
reproductive periods by microscopic examination
of gonads. Sperm suspension, which can be forced
"Ut by gentle pressure on the surface of the gonad,
is viscous and white; the suspension of eggs is

314

creamy and has a granular appearance. Sex
determination made with the naked eye should be
verified by microscopic examination of smears.

In many species of bivalves sex is unstable, and
hermaphroditism and alternation of sex are
common. With respect to sex change oysters fall
into two groups: oysters in which sexual phases
cliange regidarly in a definite rhythm, as in 0.
edulis, 0. lurida; and tliose belonging to the
Cras.sostrea type, in which the sexes of the adults
are separate, as in C. iirginica, C. gigas, C.
angidata, and 0. cucullata. The gonads of the
oysters of the first group contain functional ova
and spermatozoa simultaneously. These oysters
are hermaphrodites. In the second group her-
maphroditic individuals are relatively rare.

The difference between the two groups is not as
explicit as it appears since the primary gonad of
Crassostrea is bisexual, i.e., it contains the germinal
cells of both sexes.

As early as 1882 the outstanding Dutch natural-
ist, Hoek (1883), in his studies of 0. edidis made
the important observation that "at the time when
an oyster is sexually mature, it always functionates
as a male as well as a female; it is, therefore,
physiologically dioecious." The significance of
this important discovery was appreciated nearly
half a century later after Orton (1927, 1933)
showed experimentally that maleness developed
in 97.3 percent of young or adult females which
carried eggs, embryos, or larvae. He further
established the fact that the earlier state of male-
ness was always found in the more recently
spawned females. Great advances in the under-
standing of sex changes in 0. edulis and other
species were made by the works of Stafford (1913)
on 0. lurida, by experimental research conducted
by Sparck (1925), and p<articularly by observa-
tions on the American species made by Coe
(1932-41). It was clearly established by these
investigations that the young oysters of the larvip-
arous species {0. edulis, O. lurida) become se.xually
mature first as males then gradually change into
functional females; later they become males again,
and such alternation with some modification con-
tinues tliroughout life. Comparable phases of
sex changes occur in the Crassostrea species al-
though the rhythm of sex alternation is different.
At the age of 12 to 16 weeks the primary gonad
of C. virginica is bisexual (ambisexual, according
to Coe's terminology) since both ovogonia and
spermatogonia are found in the same follicles

FISH AND WILDLIFE SERVICE

Figure 2S4. — Portion of bisexual gonad in young male
C. virginica. gc — genital canal; oc — ovocytes; spt —
spermatid; spc — primary spermatocyte; spz — sperma-
tozoa. Photographically reproduced from Coe, 1934,
fig. 5.

(fig. 284). At this stage the C. virginica gonad
resembles that of 0. lurida at the completion of
the male phase and transition to female (fig. 285).
In C. virginica the spermatogonia proliferate more
rapidly than do the ovogonia and soon the young
gonad attains a predominantly male appearance.
Variation in the rhythm of gonad development
in the oysters from different localities and even
among those occupying the same bed residts in
different "categories" or "phases" of maleness
or femaleness.

Development of the primary bisexual gonad in
young C. virginica in New England waters is
checked by the approach of winter when the
growth of ovocytes is inhibited while the number
of spermatogenic cells increases. A small number
of spermatids may be formed early in November
when the oysters are about 4 months old. The
spermary of these secondary males contain scat-
tered ovocytes, many of which degenerate, but
some of them continue to develop into ova capable
of fertilization. Even at the stage designated by
Coe (1934) as "true male" the sperms^ry at sexual
matm'ity still retains a small number of ovocytes
on the walls of the follicles.

At the close of the first breeding season many
undifferentiated cells remain in the gonad to form
the germinal cells of the following year.

Transformation of a bisexual gonad of C. vir-
ginica into an ovary begins before the formation
of spermatozoa. At this stage the spermatogen-
esis is inhibited by the growth of ovocytes and the
female phase is attained in a certain percentage of
young oysters. The protandry, i.e., the develop-

ment of maleness before the female phase, is well
pronounced in C. virginica.

At their first breeding season yoimg oysters form
several sex classes: immature individuals in which
the sex cells have not differentiated; males; her-
maphrodites in which functional spermatozoa and
ova are foimd in the gonad; and females. Her-
maphroditic oysters are capable of self-fertilization
and produce apparently normal larvae. The rela-
tive abundance of different sex phases of young
oysters varies greatly, as can be seen from table 34,

Table 34. — Frequency of different sex phases among C.

virginica at first breeding season

[According to Coe, 1934]

Locality

Im-
mature

Her-
maph-
rodite

Males

Fe-
males

Total
exam-
ined

W. Sayville, N.Y

77
21
17
3
373

4
0
4
0
3

154
197
389
129
9

48
7

13
7
4

283

Great South Bay, N.Y

New Haven, Conn. (I932)-__
New Haven, Conn. (1933)...
Woods Hole, Mass

225
423
139
389

Figure 285. — Transition from male to female phase in
0. lurida. Lower left — genital canal filled with sperm
balls ready to be discharged; spermatogonia on the sur-
face of large ovocytes. Right — advanced male phase in
an older oyster; ovarian follicle is packed with cells in
the later stages of spermatogenesis. Upper left — female
phase preceding ovulation; many spermatogonia in the
lumen of the follicle. Redrawn from Coo, 19.32c.

ORGANS OF REPRODUCTION

315

which summarizes Coe's observations (1934) made
on oysters from four different places along the
coast of Massachusetts, Connecticut, and New
York.

During the second breeding season the number
of males may still exceed that of the females, but
generally the sex ratio approaches ec}uality. Great
differences in the degree of protandry among the
1-year-old oysters is associated with the differ-
ences in the growth rate. Coe's observations
suggest that there is a correlation between the
development of ovocytes in the bisexual gonad and
the rate of body growth. At the first breeding
season the average size of the females is much
larger than that of males (Needier, 1932a, 1932b).
Coe (1934) found that the mean height of 389
yearling males from the New Haven area was
31.28 mm. (Std. dev. ±6.33) and that of the 13
females of the same age 38.54 mm. (Std. dev.
i8.12). The difference does not seem to be
statistically significant, and several interpreta-
tions were advanced by Coe. He suggested that
the females require more favorable conditions in
order to mature, that tliey are metabolically more
active, and that at the critical period of sex
differentiation the metabolic factor determines
the predominance of the male or female cells in
the primary bisexual gonad. These proposals
require corroboration. Since it is known that the
growth rate of young oysters is accelerated by
keeping them suspended above the bottom, there
apparently would be no difficulties in conducting
a comparative test using slow and fast growing
oysters selected from a single population.

After spawning the gonad of C. virginica retains
its bisexual potencies and its sex may alternate in
either direction. Needier (1932a, 1932b) was the
first to demonstrate that such a change actually
occurs among adult C. virginica. She found that
out of 24 siu-viving oysters which were known to
be males during one simamer, 5 became functional
females the following year, and out of 12 females
5 changed to males. Among the 57 C. virginica
studied by Needier (1942) for a period of 4 years
there was a high proportion of males which re-
mained unchanged while other oysters changed
sex at least once. Some of the individuals
changed sex every year. Needier suggested,
without providing corroborative evidence, that
the sex of the males which remained unchanged
was geneticallv determined and that the other

oysters in which sex alternation occurred air
random were hermaphrodites.

Adidt Japanese oysters, C. gigas, may change
their sex during the interval between the two
breeding seasons. Amemiya (1929), who estab-
lished this fact, found that the rate of change was
higher in the males (60 percent) than in the
females (25 percent). Sex change occurs also in
C. commercialis; 95 percent of the very young
oysters of this species were found by Roughley
(1933) to be males, but among the adult specimens
of large size the females predominated at the rate
of 270 to 100 males.

The sex of the oysters used in the investigations
by Needier and Amemiya was determined by
drilling a hole in the shell and pinching off a piece
of gonad for microscopy. The injury caused by
the operation constitutes a factor which may affect
the mistable gonad and influence its sex change.
By removing about one-third of all gill lamellae
in adult C. gigas at the time when the gonad was at
the indifferent phase after spawning, Amemiya
(1936) demonstrated that the percentage of males
in the mutilated group in all cases was larger than
those in the control. Removal of the gill tissue may
have indirectly influenced the development of male
sex by reducing the rate of feeding and growth.
This assumption also needs further corroboration.

Injuiy to the oyster used for observation on sex
change can be avoided by inducing spawning in
each individual oyster, obtaining kymograph
tracings of muscular contractions, and examining
the discharged sex cells. This technique was em-
ployed in the Woods Hole laboratory, fn a test
which continued for 5 consecutive years, 4-year-
old oysters were obtained from one of the private
oyster beds near Onset, Mass. During the first
summer 202 oysters were induced to spawn, their
sex was recorded, and an identifying number was
engraved on the right (upper) valve. Upon com-
pletion of the tests the oysters were returned to
outdoor tanks or were placed in the harbor and
remained there until the next reproductive season.
The testing was repeated every summer (Galtsoff ,
1961).

Because the males respond to spawning stimuli
more readily than the females, their number at the
beginning of the experiment was greater than that
of the females. The disparity does not represent
an actual sex ratio of the popidation of 4-year-oId
oysters which was found to be about 1 to 1. The
mortality, especially among the 7-year-old oysters

316

FISH AND WILDLIFE SERVICE

used in the test, was high, and the number of non-
spawning oysters gradually increased toward the
end of the experiment (table 35).

When oysters failed to respond to spawning
stimulation, the testing was repeated at 4- to
5-day intervals for 5 consecutive weeks. Nega-
tive results were assumed to indicate the oysters
were nonfunctional sexually, and they were re-
turned to the holding tanks for another year. In
several instances the oysters that failed to spawn
became sexually active the following breeding
season. It is not known at present whether the
increased number of failures to spawn and in-
creased mortality (table 35) should be attributed
to aging or to unfavorable conditions in the win-
ters. On several occasions the holding tanks and
live cars in which the oysters were kept were
swept by stormy waters and everything inside was
covered with a deposit of mud.

Table 35. — Changes in the percenlage of sexes in a selected
group of C. virginica tested consecutively for 4 years

[Initial number of males and females 4 years old does not represent the normal
sex ratio which was about 1 to 1]

Age in years

Males

Females

Non-
spawning

Total
survivors

4

Number

Percent
64.4
65.2
61.2
50.1
44.1

Percent
35.6
32.6
31.6
40.3
41.2

Percent

0

2.2

7.2

9.6

14.7

Number
202

5

181

6

139

7

104

8

68

During the 5-year period of observations the
number of spawning males decreased from 119 to
15 in 9-year-old inollusks. The number of females
decreased from 63 to 18. Consequently the sex
ratios of males to females of the experimental
oysters changed from 1.9:1 at the beginning to
0.8:1 at the end of the observations. The pre-
dominance of oysters of female sex in the surviving
oysters can not be attributed to more frequent sex
changes from male to female. In table 36 no
significant differences were recorded in the rates of
sex alternation in the two groups. The predomi-
nance of females at the end of the test could be
explained, therefore, by greater sm-vival rate of
oysters at the female phase. This interesting
point requires further corroboration.

Out of the 68 survivors at the end of the breed-
ing season of the fourth year, 31 had alternated
their sex at least once during the period of testing

ORGANS OF REPRODUCTION
733-851 O— 64 21

(Galtsoff, 1961). The frequency of changes were
as follows:

One change 18 instances.

Two changes 10 instances.

Three changes 2 instances.

Four changes 1 instance.

The oyster which changed sex every year was a
male at the beginning and returned to the male
phase at the last test. No distinct pattern is
apparent in the rhythm of changes except the
greater persistence of the female phase. The sex
ratio within the sex-reversed oysters changed from
23 males and 8 females at the beginning to 1 1 males
and 20 females at the end of the observations.

Table 36. — Frequency of sex alternation in adult C.
virginica from 5 to 9 years old

( The figures in the table indicate the number of males (column 2} and females
(column 5) of each age and the number and percentage of changes to females
(columns 3 and 4) and to males (columns 6 and 7) that occurred during each
year)

Age in years

Males

Changes to
females

Females

Changes to
males

Number

Number
119
88
65
25
15

Number

10

10

15

3

1

Percent
9.2
11.3
23.1
12.0
6.7

Number
63
38
33
24
18

Number
11
9
3
6
1

Percent
17.4

6

23.7

7. --

9.1

8 - -

25.0

9

6.6

The cause of sex instability and the factors
which may influence the shift of a gonad from one
sex to another are not known. Coe believed that
the physiological state of the organism in each
breeding season is the key to the determination of
the sexual phase of the oyster. No concrete proof
to substantiate this idea can be found in his ex-
periments. Egami (1952) attempted to trans-
plant pieces of gonad of C. gigas to another oyster
of the same or of the opposite sex. He found no
evidence that the sex of the host affected the sex
differentiation of the graft and concluded that the
sex of the grafted pieces has been determined at
the operating season (December) at their mor-
phologically undifferentiated state. In another
work (Egami, 1953), he corroborated the results
of Amemiya's observation on the decrease of
growth rate of C. gigas by the removal of gill tis-
sue and the increase in the percentage of males
among the mutilated oysters. He concluded that
maleness could not be attributed directly to mu-
tilation but was associated with the decreased
growth rate of oysters without gills. Egami found
tiiat among normal individuals of C. gigas those
growing more rapidly during the autumn tended

317

to develop into females the following reproductive
season.

The conclusions may be considered only tenta-
tive since they are based on a small number of
experiments which need to be repeated on a larger
and more comprehensive scale. Sex alternation
in oysters offers fascinating possibilities for further
research on this fundamental biological problem.

The spawning reactions of sex-reversed oysters,
as reflected in the type of shell movements and
in the manner of dispersal of sex cells, are in every
respect identical to those of the reactions of true
males and females (fig. 286). In some sex-re-
versed oysters the change from male to female
behavior is however delayed. Examination of
records shows that in several instances females
which were males during the preceding year
spawned at the beginning of the reproductive
season in a male fashion, discharging the eggs
through the cloaca. Full female reaction in-
volving rhythmical contractions of the adductor
muscle was fully developed toward the end of the
spawning season (fig. 287). It can be inferred
from these observations that the mechanism of
female sexual behavior develops at a slower rate
than the morphological changes in the gonads.
There was no retention of female behavior in
oysters which returned to the male phase. The
male reaction was apparent in them at the be-
ginning of the season.

First summer Typical male spawning

Following summer —female

j.jjjjjii!il^^

First summer — femole

Following summer — mole

Following summer, June2l Eggs discharged through clooca
I month loter.July 20 ■ Typical female reoction

Figure 287. — Spawning of the sex-reversed male C.
virginica. First line — spawning at the male phase.
Second line — sex of tlie gonad changed to female;
spawning proceeds in the male fashion. Third line —
typical female spawning. Oyster was 5 years old during
the "first" summer. Time interval, 1 minute.

In one instance the spawning of an hermaphro-
ditic oyster was recorded (Galtsoff, 1961). Both
eggs and sperm were discharged simultaneously
through the cloaca, and the rhythmical contrac-
tions of the adductor muscle were not fully
developed (fig. 288). A small portion of the gonad
of this oyster is shown in fig. 289. Microscopic
examination of the tissue revealed the presence of
relatively few mature eggs in the follicles occupied
by spermatozoa. Spawning of this oyster was
induced by raising the water temperature. Eggs
removed from the spawning tank were found to
be fertilized and their development traced to
trochophore stage was normal.

LUNAR PERIODICITY

The modern zoologist may disregard the early
popular beliefs and superstitions which endowed
the moon with mysterious effects on human
affairs, on animals, and plants; nevertheless, he is
confronted with several undeniable instances of
lunar periodicity in the reproduction of marine
invertebrates. Probably the most famous and
generally known examples are the swarming and
breeding of the Palolo worm {Eunice viridis Gray)
of the South Pacific at the moon's last quarter of

Figures 286. — Kymograph records of sex-reversed male
(two upper lines) and sex-reversed female (two lower
lines) C. virginica recorded at two consecutive breeding
seasons. Both oysters were 5 years old at the "first"
summer. In both instances eggs were dispersed through
the gills, the sperm through the cloaca. Time interval,
1 minute.

Figure 288. — Shell movements of a spawning hermaphro-
ditic C I'zVguMca. Temperature 24.5° C. Time interval,
1 minute.

318

FISH AND WILDLIFE SERVICE

vS^

■J-iV^'.- , l',V«'. ;'■■.■■.:!■■-},•■.'',

MB::

''•', ■'■. ..'W.''^t'.- •■■'■■■'-■

ilps ^ .

f^?-'-

I I I I L.

Microns

200

Figure 289. — Section of a small portion of an hermaphro-
ditic gonad of C. virginica. Bouin, hematoxylin-eosin.

October and November; the swarming of the At-
lantic Palolo [Odontosyllii enopla Verrill) at
Bermuda and Eunice fucata Ehlers, at Tortugas,
Florida (Mayer, 1908); and the breeding habits of
Heteronereis form of Nereis limbata at Woods
Hole (LilHe and Just, 1913). Legendre (1925)
gives an interesting historical account of the
effect of the moon on marine organisms. A
comprehensive review of the instances of lunar
periodicity of breeding among many marine
invertebrates, including several species of pele-
cypods, is given by Korringa (1947).

Evidence of a relationship of breeding of 0.
edulis to moon phases was first presented bj^
Orton (,1926), who examined weekly samples of
adult oysters from Fal estuary and found two
important maximums in spawning ,at the full
moon spring tides in the year 1925. He further
concluded that the population as a whole gave
maximal percentage of spawn (based on presence
of embryos in the oysters) in the weeks after the
July and September full moons. Later observa-
tions by Korringa (1941, 1947) in the commercial
oyster district of Oosterscbelde, Holland, con-
firmed the existence of a relationship between
breeding of 0. edulis and moon phases. He
found that the full moon exercises the same in-
fluence on the breeding of oysters as does the

new moon. Korringa based his studies on deter-
minations of the time and abundance of oyster
larvae in plankton and found a marked periodicity
in the maximums of oyster larvae occurring about
10 days after full and new moon. Fluctuations
in water temperatm-es, according to Korringa's
view, are apparently of little or no importance in
causing the periodicity in swarming which appears
to be correlated with the spring tides. Unfor-
tunately no experimental evidence is available to
substantiate this inference which is based entirely
on the concurrence of the two phenomena.

Spawning of C. virginica has no relationship to
lunar phases. The existence of such a relation-
ship was postulated by Prytherch (1929), who
stated that Long Island Sound oysters spawn
"at the end of full moon tidal period, or eight
days after the time of full moon," but the correla-
tion could not be corroborated by careful studies
of Loosanoff and Nomejko (1951a), who continued
the observations in Long Island Sound over a
period of 13 years after the termination of
Prytherch's work. Negative results were also
reported by Hopkins (1931) in Galveston Bay,
Tex., and by R. 0. Smith in South Carolina waters
(unpublished reports on file in the Bureau of
Commercial Fisheries).

BIOLOGICAL SIGNIFICANCE OF
SPAWNING REACTION

The most outstanding single factor in oyster
reproduction is the difference in spawning behav-
ior of the two sexes. The males are more respon-
sive to sexual stimulation than the females and
are easily stimulated to spawn by rising tempera-
tures and by a great variety of organic sybstances,
some of them not found in natural sea water.
The spawning response of the male is nonspecific.

The less responsive, sexually mature females
require stronger stimuli and are highly specific
to chemical stimulation; they respond only to
suspensions of sperm of the same or related
species and are indifferent to the sperm of other
bivalves and various chemical substances tested.
The specificity of females is an insurance that
eggs cannot be discharged when there is no sperm
in the water.

Males are usually the first to initiate spawning;
the discharge of sperm by even one individual
induces spawning by those next to it, and the
process spreads over the entire oyster community.
This sequence has been observed among oysters

ORGANS OF REPRODUCTION

319

;:::m^^

Figure 290. — Spawn of C. gigas carried down by the tidal river near Vancouver, British Columbia. Courtesy of D.

Quayle. Black and white enlargement of Kodachrome slide.

kept on floats and among specimens living under
natural conditions on oysters beds near low tide
level. The spawning of an entii'e oyster popula-
tion can be artificially initiated by mincing tlie
meats of several sexually mature oysters and
spreading them into the waters of the oyster bed.
This method, based on my laboratory experiments,
has been applied on a commercial scale by oyster
growers in British Columbia (Elsey, 19.36).

Simultaneous spawning of oyster populations is
essential for the production of a large brood of
oyster larvae and for obtaining setting of com-
mercial value. In the estuaries where the ma-
jority of oyster beds are located, the tides carry
the released spawn for some distance before the
eggs sink to the bottom. A transport of oyster
spawn by a strong ciurent can be seen in the
photogi'aph (fig. 290) taken by Quayle near Van-
couver, British Columbia, and kindly given to me
for reproduction. The white streak in the fore-
ground of the clear river water was formed bj-
billions of eggs and sperm discharged by a popula-
tion of C. gigas several miles up river.

The method of discharging sperm and eggs is
also of considerable significance. vSpermatozoa
carried away by the respiratory cmTent remain in
suspension for several hours. When eggs are dis-
charged in the same manner by some sex-reversed
oysters, they rapidly sink to the bottom only a
few inches away from the fenude. Lalioratory
observations indicate that under such conditions

only a very small percent of them are fertilized
or have even a slim chance of developing. On the
other hand, eggs discharged in the usual manner
through the gills and forcibly ejected from the
mantle cavity, have a much better chance to be
fertilized and survive. Furthermore, because of
the specificity of female response to sperm, eggs
are ejected only when the water contains free
spermatozoa of the same species. The female
spawning reaction is an adaptation of an oviparous
organism to the conditions of its existence and
assures the survival of the species.

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1951. Sur remission des gametes chez Chlamys raria
L. (Moll, lamellibr.). Comptes Rendus Heb-
domadaires des Seances de I'Academie des Sciences,
tome 233, pp. 1680-1081.

1955. Cycle neurosecretoire chez Chlamys varia L.
et Mytilus edulis L. (Mollusques lamellibranches).
Comptes Rendus Hebdomadaires des Stances de
I'Academie des Sciences, tome 241, pp. 119-121.

1956. Effets de I'ablation des centres nerveux sur
remission des gametes chez Mytilus edulis L. et
Chlamys varia L. (Mollusques lamellibranches).
Annales des Sciences Naturelles, Zoologie et
Biologic Animale, serie 11, tome 18, pp. 175-183.

Mayer, Alfred Goldsborotjgh.

1900. An Atlantic "Palolo", Stauroccphalus gregari-
ous. Bulletin of the Museum of Comparative
Zoology, Harvard, vol. 36, pp. 1-14.
1908. The annual breeding-swarm of the Atlantic
Palolo. Papers from the Tortugas Laboratory of
the Carnegie Institution of Washington, vol. 1, pp.
105-112. (Carnegie Institution of Washington,
D.C., Publication No. 102.)

Merrill, Arthur S., and John B. Burch.

1960. Hermaphroditism in the sea scallop, Placto-
pecten magellanicus (Gmelin). Biological Bulletin,
vol. 119, No. 2, pp. 197-201.

Millar, R. H.

1961. Scottish oyster investigations, 1946-1958.
Department of Agriculture and Fisheries for Scot-
land, Marine Research, 1961, No. 3, 76 pp.

Mitazaki, Itiro.

1938. On a substance which is contained in green
algae and induces spawning action of the male
oyster (Preliminary note) . Bulletin of the Japanese
Society of Scientific Fisheries, vol. 7, No. 3, pp.
137-138.
Montalenti, G.

1958. Perspective of research on sex problems in
marine animals. In A. A. Buzzati-Traverso
(editor). Perspectives in marine biology, pp. 589-
602. University of California Press, Berkeley and
Los Angeles, Calif.
Needler, Alfreda Berkeley.

1932a. Sex reversal in Ostrea virginica. Contribu-
tions to Canadian Biology and Fisheries being
studies from the Biological Stations of Canada,
new series, vol. 7, No. 22 (Series A, General, No,
19), pp. 28.3-294.

1932b. American Atlantic oysters change their sex.
Progress Reports of the .Atlantic Biological Station,
St. Andrews, N.B., and Fisheries Experimental
Station (Atlantic), Halifax, N.S., No. 5, pp. 3-4.

1942. Sex reversal in individual oysters. Journal of
the Fisheries Research Board of Canada, vol. 5,
No. 4, pp. 361-364.
Nelson, Thurlow C.

1928a. Relation of spawning of the oyster to tem-
perature. Ecology, vol. 9, No. 2, pp. 145-154.

1928b. On the distribution of critical temperatures
for spawning and for ciliary activity in bivalve
molluscs. Science, vol. 67, No. 1730, pp. 220-221.

1931. Stimulation of spawning in the American
oyster by sperm of the Portuguese oyster. Ana-
tomical Record, vol. 51, No. 1 (suppl.), p. 48.

322

FISH AND WTLDLIFE SERVICE

Nelson, Thurlow C, and James B. Allison.

1940. On the nature and action of diantlin; a new
hormone-like substance carried by the spermatozoa
of the oyster. Journal of Experimental Zoology,
vol. 85, No. 2, pp. 299-338.

Okada, Katsuhiro.

1936. Some notes on Sphacrium japonicurii biwaense
Mori, a freshwater bivalve. IV. Gastrula and
fetal larva. Science Reports of the Tohoku
Imperial University, series 4, Biology, vol. 11,
No. 1, pp. 49-68.

1939. The development of the primary mesoderm
in Sphacrium japonicum bitvaense Mori. Science
Reports of the Tohoku Imperial University, series
4, Biology, vol. 14, No. 1, pp. 25-48.
Orton, J. H.

1920. Sea-temperature, breeding and distribution
in marine animals. Journal of the Marine Biologi-
cal Association of the United Kingdom, vol. 12,
No. 2, pp. 339-366.

1926. On lunar periodicity in spawning of normally
grown Falmouth oysters (0. cdulis) in 1925, with a
comparison of the spawning capacity of normally
grown and dumpy oysters. Journal of the Marine
Biological Association of the United Kingdom, vol.
14, No. 1, pp. 199-225.

1927. Observations and experiments on sex-change
in the European oyster (0. edulis). Part I.
The change from female to male. Journal of the
Marine Biological Association of the United King-
dom, vol. 14, No. 4, pp. 967-1045.

1933. Observations and experiments on sex-change
in the European oyster (0. edulis). Part III.
On the fate of unspawned ova. Part IV. On the
change from male to female. Journal of the Marine
Biological Association of the United Kingdom,
vol. 19, No. 1, pp. 1-53.
Perry, John H. (editor).

1941. Chemical engineers' handbook. 2d ed.
McGraw-Hill Book Co., Inc., New York, 3029 pp.

Prytherch, Herbert F.

1929. Investigation of the physical conditions
controlling spawning of oysters and the occurrence,
distribution, and setting of oyster larvae in Milford
Harbor, Connecticut. Bulletin of the U.S. Bureau
of Fisheries, vol. 44, for 1928, pp. 429-503. (Docu-
ment 1054.)

ROUGHLEY, T. C.

1933. The life history of the Australian oyster
{Ostrea commercialis) . Proceedings of the Linnean
Society of New South Wales, vol. 58, pp. 279-333.

Sparck, R.

1925. Studies on the biology of the oyster (Ostrea
edulis) in the Limfjord, with special reference to
the influence of temperature on the sex change.
Report of the Danish Biological Station to the
Board of Agriculture, 30, 1924. 84 pp.

Stafford, Joseph.

1913. The Canadian oyster, its development,
environment and culture. Committee on Fisheries,
Game and Fur-bearing Animals, Commission of
Conservation, Canada. The Mortimer Company,
Ltd., Ottawa, Canada, 159 pp.

Stauber, Leslie A.

1950. The problem of physiological species with
special reference to oysters and oyster drills.
Ecology, vol. 31, No. 1, pp. 109-118.

Woods, Farris H.

1931. History of the germ cells in Sphaerium striati-
num (Lam.). Journal of Morphology, vol. 51,
No. 2, pp. 545-595.

1932. Keimbahn determinants and continuity of the
germ cells in Sphaerium slrialinum (Lam.). Journal
of Morphology, vol. 53, No. 2, pp. 345-365.

Yonge, C. M.

1960. Oysters. Collins Clear-type Press, Ijondon,
209 pp.

ORGANS OF REPRODUCTION

323

CHAPTER XV

EGG, SPERM, FERTILIZATION, AND CLEAVAGE

Pafje

Ovogenesis - --- - '^^

Spermatogenesis -- - --- ^^^

Structure of the mature egg -- -- -- 327

Cytoplasmic inclusions 328

Structure of the mature spermatozoon -— - - 336

Fertilization 338

Acrosomal reaction 341

Fertilization of egg --- --- 342

Aging of eggs and sperm 343

Polarity of egg 344

Cleavage -- - 344

Bibliography - - 361

As early as 6 to 10 weeks after setting, young
C. virginica of New England waters, then 6 to 8
mm. in height, develop primordial gonads of pro-
fusely branching tubules (Coe, 1932a). At this
stage the germinal epithelium is a layer of morpho-
logically undifferentiated cells; some of them will
transform into larger cells to become ovocytes,
i.e., the cells destined to develop into mature eggs.
The smaller cells of the epithelium proliferate very
rapidly and are recognizable as the male germ line,
and eventually develop into spermatozoa. For
several weeks the immature, or primary, gonad of
an oyster remains nonfunctional and bisexual
(ambisexual), for it contains both male and fe-
male germ cells which will transform into mature
spermatozoa or ova during the following summer.
In some individuals the primary bisexual gonad is
retained until the second year, a delay which Coe
(1932a, 1938) attributes to poor nutrition.

The more rapid multiplication of male germ
cells suppresses the development of ovocytes and
results in a predominance of males among the
1 -year-old oysters and in the appearance of dif-
ferent degrees of intersexuality (predominance of the
cells of one sex over the other) . In the same brood
which contains also distinctly ambisexual oysters
there are, however, other young individuals in
which the primary gonad develops directly into
ovary or spermary. Local conditions on oyster
beds apparently influence the tempo of changes.
In the warmer waters at Beaufort, N.C., young
oysters are more apt to develop directly into fe-
males than in the northern cold waters of New

England. Coe (1938) found that the proportion
of females to 100 males varied at the first breeding
season between 37.1 and 48.8 at Beaufort; 5.6 and
24 at Milford, Conn.; and 3.3 and 12.5 at New
Haven Harbor. The differences are not consistent
with geographical latitude since the female to male
ratio at West Sayville, Long Island, N.Y., was
31.2; at Delaware Bay 41.9; and at Apalachicola,
Fla., 7.1. It is obvious that these variations can-
not be attributed to temperatm-e alone and are
probably caused by a combined effect of envii'on-
mental conditions.

Toward the end of the second breeding season
the primary gonad is transformed into a definite
ovary or spermary (fig. 291). The gametogenesis,
i.e., complete transformation of the primordial
germ cells into mature ova (ovogenesis) or sperma-
tozoa (spermatogenesis), is a very complex process.
The differentiation is accompanied by rapid multi-
plication of the new generations of cells which

Microns

Figure 291. — Section of a gonad of C. virginica at an early
stage of differentiation. End of Marcli, Woods Hole,
Mass. The larger, clear cells are ovogonia, the smaller
ones are undifferentiated cells of germinal epithelium.
Bouin, hematoxylin-eosin.

324

FISHERY bulletin: VOLXJME 64, CHAPTER XV

extend inward and fill up the lumen of the follicles.
Early at this stage the sex cells become dense and
opaque, a condition which interferes with cyto-
logical study.

Gametogenesis of C. virginica and 0. lurida has
been studied by Coe (1932a, 1932b, 1934, 1936,
1938) and that of the Australian rock oyster,
C. commercialw, by Cleland (1947). Only the
main points of this process were disclosed by these
investigations.

OVOGENESIS

In all animals the primordial germ cells become
distinguishable as primary ovogonia in the females
or spermatogonia in the males after a certain num-
ber of divisions. After a period of quiescence they
begin to divide again and give rise to secondary
ovogonia or spermatogonia. After several genera-
tions the cells stop dividing and enter a growth
period, which is more prolonged in the females than
in the males. The growth period is characterized
by a series of cytological changes, each differing
from the preceding stage. The cells which \vill pro-
duce gametes are at this stage called auxocytes,
from the Greek "auxesis" meaning growth, and are
referred to as ovocytes in the female and sperma-
tocytes in the male.

Ovogenesis in oysters begins with the appearance
of enlarged cells in the germinal epithelium. These
are the ovogonia, which in C. virginica and 0.
lurida are distinguished from other cells of the
germinal epithelium by their relatively large nuclei
with conspicuous nucleoli and loose chromatin
network (fig. 292, og). The ovogonia usually lie
next to tlie follicle wall, and their distal sides do
not protrude into the lumen. Differences between
the early ovogonia and indifferent residual cells (I)
are not conspicuous. Examination of a series of
sections and study of the sequence of changes in the
appearance and structure of the cells are necessary
to assure a positive identification.

After one or two divisions the ovogonia change
in appearance as well as in size. This generation of
female sex cells called ovocytes can be recognized
by the presence of fibrillar mitochondrial bodies
(sometimes called yolk nuclei) , and by tlie spiremes
of densely packed chromosomes (figs. 293 and 294).
Their nucleoli become very conspicuous.

Dm-ing the last stage of ovogenesis the ovocyte
begins to grow rapidly, and the distal part, gi'ossly
enlarged and rounded, protrudes into the lumen
of a follicle. At the same time the connection

0C5

Figure 292, — Follicle wall of the ovary of C. virginica.
I — indifferent residual cell; oc — residual ovocyte; ocs —
two young ovocytes in synaptic phase; og — group of
ovogonia. Redrawn from Coe, 1932a, fig. 9. Highly
magnified.

oc'

Figure 293. — Portions of two follicles of a bisexual gonad
of 4-month-old C. virginica. A — predominantly female,
and B — predominantly male follicle; gc — genital canal
lined with ciliated cells; oc — large ovocyte; oc' — young
ovocyte in spireme phase; spcl — primary spermatocytes
in spireme phase; spell — secondary spermatocytes;
spt — spermatides. Photographically reproduced from
Coe, 1932a, fig. 6. Highly magnified.

Figure 294. — Two young ovocytes at spireme stage in a
mature ovary of C virginica. Redrawn from Coe, 1932a,
fig. 9. Highly magnified.

EGG, SPERM, FERTILIZATION, AND CLEAVAGE

325

with the basal membrane of the follicle wall is
narrowed to an elongated stem. The nucleus
increases gi-eatly in bulk, and the developing egg
assumes a pear-shaped form. An accumulation
of dark granules (mitochondria) at the proximal
end of the cells may indicate that food for the
growing ovocyte is obtained through the wall of
the follicle. The granules are not pronounced in
the ovocyte of C. lirginica but are conspicuous in
some other bivalves, particxilarly in Sphaerium
(Woods, 1932). From the beginning of sexual
differentiation to the final maturity of an ovum,
the early ovocyte increases in volume more than
3,000 times.

Ovogenesis in the Sydney rock oyster C. com-
mercialis, described by Cleland (1947), is some-
what different from the ovogenesis of the American
species. At the earliest stage before the start of
the growth phase an ovocyte of the rock oyster
is a small cell, 4 m to 5 m in diameter. Two-thirds
of the cell is occupied by a clump of chromosomes
siu-rounded by a rim of clear cytoplasm. Cleland
identifies this stage as a definite auxocyte, i.e.,
an ovocyte just before entry into the growth
phase. At ths stage the cell has no nucleolus.
The definite auxocyte begins to grow and passes
through three stages (called by Cleland Auxocyte
I, II, and III) which differ in size and nuclear
structure. Auxocyte I has a diameter of about
9 M. with a relatively large germinal vesicle (6 /x)
and a nucleolus of about 1.7 m- The nucleus is
centrally placed in the homogeneous cytoplasm
with an excentric nucleolus which is not in con-
tact with the nuclear membrane. Auxocyte II
has diameter of 12.6 m, with the germinal vesicle
(nucleus) about 7 n and nucleolus 2 m to 3 m-
The cell is usually spherical with a centrally
located germinal vesicle and chromosomes spaced
more widely than in Auxocyte I. At this stage
a group of granules appears at one pole of the
nucleus. Auxocyte III is a spherical cell 20 ti in
diameter, with the germinal vesicle measuring 11
^ and eccentrically located nucleolus of about
4.2 II in diameter. The cell is separated from the
wall and is free in the lumen of a follicle. Pro-
tein granules are abundant along the periphery
of the cell where they are found in a matui-e egg.
The matm-e ovocyte of C. commercialift has a
diameter of about 38 m- The nucleus is large,
about 21 M across; the nucleolus is 4.6 /i. The
chromosomes are paired and are usually placed
peripherally in the germinal vesicle but not in

326

contact with the nuclear membrane. Crossing-
over is frequently seen at this stage but the cliromo-
somes are not coiled.

SPERMATOGENESIS

Spermatogenesis in the oyster is known pri-
marily from the studies by Coe (1931) on the
development of the gonad of young 0. lurida.
Comparison with the gonads of C. virginica shows
that there is close agreement in the general features
of the process in both species. Progressive stages
of the formation of sperm begin with the undif-
ferentiated gonia which line the inner wall of the
gonad follicles. After a large number of descend-
ants have been produced the spermatogonia can
be distinguished from the ovogonia by their smaller
size and position within the follicles. Ovogonia
lie in a single row along the wall; the primary
spermatogonia of C. virginica are found either
singly or in groups between the ovogonia lining
the wall and in the lumen (fig. 295).

In the hermaphroditic gonad of 0. lurida a
single primary spermatogonium divides several
times to form a cluster of cells which become
separated from the folhcle wall and occupy a
position toward the center of the lumen (fig. 296).

The number of divisions of spermatogonia
presumably depends on the amount of nourishment
available to the gonad. Coe estimates that in 0.
lurida each primary spermatogonium divides six
to nine times to produce a cluster of 64 to 500
cells. In spite of the close contact the adjacent
cells of the clusters are separate but are held
together by a dehcate noncellular secretion. Its

Figure 295. — Portion of bisexual gonad of young C.
virginica. gc— genital canal; oc— ovocytes with sper-
matogonia filling the lumen ; spc— spermatocytes ; spt—
spermatids; spz— spermatozoa. Photographically re-
produced from Coe, 1934, fig. 5A. Highly magnified.

FISH AND WILDLIFE SERVICE

Figure 296. — Portion of an hermaphroditic gonad of O.
lurida. gc — genital canal; I — indifferent cells; oc —
ovocytes; spc — primary spermatocytes; spc> — secondary
spermatocytes; spz — spermatozoa united into a sperm
ball. Photographically reproduced from Coe, 1934, fig.
5B. Highly magnified.

chemical nature has not yet been determined. In
poorly preserved preparations the clusters some-
times have the appearance of syncytia with nuclei
embedded in a common matrix. In both species
all spermatogonia have a conspicuous nucleolus
and loose chronuitin reticulum. As the divisions
proceed the diameter of the spermatogonia di-
minishes from about 6m to 3m or less at the last
stage leading to the formation of primary sperma-
tocytes. In C. virginica these cells are globular,
each with a large nucleus resolved into slender
threads (spiremes). This leptonema stage is
frequently observed in the developing spermary
but the conjugation of chromosomes (synapsis)
has not been described with any detail. However,
in reference to the spermatogenesis in 0. lurida,
Coe (1931) states that the leptotene stage "is
followed by the usual process of synapsis."

The appearance of secondary spermatocytes is
similar to that of the primary. In C. virginica
they can be distinguished by their riwlial orienta-
tion in the lumen and small size. Different pliases
of spermatogenesis in C. virginica are shown in a
semidiagrammatic drawing published by Coe
(1932a) and reproduced in fig. 297. Meiotic
divisions and the transformation of spermatids
into mature spermatozoa have not been fully
described for C. virginica. In a mature spermary
the spermatozoa are always oriented with their
tails toward the center of the lumen. The plioto-
micrograph shown in fig. 298 shows the gradual
increase in the number of male sex cells from the

wall of the follicle toward the center. Successive
stages of the spermatogenesis of 0. lurida drawn
by Coe are shown in fig. 299.

Secondary spermatocytes of 0. lurida are held
together in spherical masses. Close contact by
the spermatids continues during their transforma-
tion into spermatozoa; in the sperm baU of a ma-
ture oyster the tails radiate from the center. Each
sperm ball is composed of from 200 to 2,000
spermatozoa originating from a single spermato-
gonium (Coe, 1932b). During mitotic divisions
the "prophase, metaphase, and telophase are aU of
typical appearance, with a delicate spindle of the
usual form" (Coe, 1931). Because of the crowded
condition of the metaphase and anaphase plates,
Coe was unable to determine the chromosome
number which he states "is not very large." In
two diagrammatic drawings of spermatocyte di\4-
sion Coe (1931, fig. 3, E and F) figures 10 chromo-
some pairs. This is the most common number of
chromosomes found by Cleland at the two- and
four-cell stage of cleavage in the fertilized egg of
C. commercialis (Cleland, 1947). The number of
chromosomes seen during the cleavage of C.
virginica eggs is discussed later (p. 345).

STRUCTURE OF THE MATURE EGG

Eggs in the mature ovary of C. virginica are
pear-shaped and compressed. Many of them are
attached to the follicle wall by long, slender
peduncles; others are free in the lumen ready to
be moved to the genital canals and discharged
(fig. 300). The long axis of the eggs varies from
55m to 75m depending on their shape; the width
at the broadest part measures from 35m to 55/i,
and the diameter of the nucleus is from 25m to 40/i.
The oblong shape is retained for some time after
the discharge of eggs into water but gradually the
egg becomes globular and denser. Under the
transmitted light of a microscope the nucleus
appears as a large, transparent area surrounded
by densely packed granules (fig. 301). In a
globular egg the nucleus cannot be seen unless it
is cleared in glycerol or other clarifying reagents
(fig. 302).

Eggs of oysters li^dng under marginal conditions
in water of salinity less than 10 %o frequently
become cytolyzed upon their removal from the
o-^'ary; the nuclei appear larger than those of
normal eggs. Only 1 or 2 percent of these eggs
is fertilizable. The delicate primary' or "vitelline"
membrane smTounding the unfertilized egg is

EGG, SPERM, FERTILIZATION, AND CLEAVAGE

327

tnci.

/in

Figure 297-

- Mature spermary of C. virginica. ind — indiflferent cells; spg' and spg- — primary and secondary spermato-
cytes; spt — spermatids; spz — mature spermatozoa. Redrawn from Coe, 1932a, fig. 8.

secreted by the egg itself while it is still in the
ovary. Raven (1958) states that in some cases
the vitelline membranes of Ostrea, Alytilus,
Dreissensia, and Dentalivm are thrown off soon
after shedding. I have not seen this happen in
the eggs of C. virginica.

CYTOPLASMIC INCLUSIONS

Cytoplasmic components of an oj'ster egg are
not well known primarily because the ultrastruc-
ture has not been studied by electron microscopy.
Certain types of minute granules can be seen,
however, in examination of live eggs under liigh

magnification of phase contrast lenses; by applying
vital and metachromatic stains; by centrifuging
whole eggs or their homogenates in order to sepa-
rate various components and study their staining
reactions. For descriptions of the techniques
used in modern cytology the reader is referred to
the textbooks on cytology and microscopic histo-
chemistry (Gomori, 1952; DeRobertis, Nowinski,
and Saez, 1960; and others). The yolk constitutes
the major part of the eggs of marine bivalves.
Quantitative data on the amount of yolk in oyster
eggs are lacking, but for fumingia teUinoidex and
MytiluK californianus Costello (1939) found that

328

FISH AND WILDLIFE SERVICE

M i c rons

Figure 298. — Photomicrograph of a cross section of one follicle of a fully mature spermary of C. virginica. HematoxyJm-

eosin.

yolk forms 35 and 31 percent respectively of the
total egg volume. The estimates were obtained
after a centrifugal force of 20,000 {Cumiiujia) and
4,800 (Mytilus) times gravity had been applied
to the unfertilized eggs. In the cytoplasm of the
eggs of the two species the relative volumes of
hyaline zone were 42 and 55 percent and of the
oil 10 and 14 percent.

The yolk of molluscan eggs is made of two

types of granules, one of proteid and the other of
fatty materials. In cytological literature the
distinction between the proteid yolk and fatty
yolk is not always made clear. In descriptions of
the cytoplasmic inclusions of an egg based on
light microscopy some authors apply the term
exclusively to protein granules, while others, in-
cluding Gatenby (1919), Gatenby and Woodger
(1920), and Brambell (1924) in their studies of the

EGG, SPERM, FERTILIZATION, AND CLEAVAGE

329

Figure 299. — Diagram of successive stages of spermatogenesis in O. liirida. A — two indifferent germ cells on the wall
of the gonad; B — small group of spermatogonia, with reticular chromatin and conspicuous nucleoli; C — small group
of secondary spermatogonia; D — division of secondary spermatogonia to form spermatocytes; E — primary spermato-
cytes with slender chromosomes; F to K — division of primary spermatocytes; L to R — division of secondary spermato-
cyte; S to T — transformation of spermatid into the mature spermatozoon. Redrawn from Coe, 1931, fig. 2.

gametogenesis in the gastropods Helix, Limnaea,
and Patella, identify the protein granules as
"mitochondria" and restrict the term yolk to fatty
inclusions.

The role of the mitochondria in the formation of
yolk has not been fully resolved. According to
Rai (1930), the fatty yolk in the eggs of C. cucullata
is formed directly from the Golgi vesicles as it is
in ascidians, Helix, and other invertebrates;
mitochondria do not participate in the vitellogen-
esis, and albuminous yolk is absent in the egg of
the Indian oyster {('. cucullata). This view is in
agreement with the conclusion of Worley (1944),
who found no protein yolk in the eggs of Alijtilus
and Ostrea. The question is not settled because
apparently the cytologists have no clear agreement
on the difference between the protein yolk and
mitochondria.

A study of cytoplasmic inclusions was made by
Cleland (1947, 1951), who separated the granules
found in egg cytoplasm of the oyster by diiferen-
tial centrifugation following homogenization.
Mature eggs were suspended in a solution of
0.2 M potassium chloride and 0.02 n sodium citrate

buffered to pH 7.5. Homogenates were obtained
by blending the suspension in an electric blender
surrounded by an ice jacket. By centrifuging
the samples of homogenates at different speeds the
following types of granules were obtained: P gran-
ules or protein yolk; L granides or lipid yolk;
M granules or mitochondria; and S or submicro-
scopic granules. The P granules obtained by
Cleland's technique are spherical and can be
stained by Janus green B in the test tube. In the
living egg these granules are located along the
periphery and absorb Nile blue stain. After
being centrifuged at 5,000 times gravity for 5
minutes they form a thick centrifugal layer with
a sharp upper boundary. Alpha or lipid yolk
granules are also spherical. They occupy the
central part of the living egg. In the centrifuged
egg they form a centripetal layer with a sharp
lower boundary. Alpha granides of phospholipid
and neutral fat can be recovered from the super-
natants of homogenate suspensions. M graimles
or mitochondria in tlie live centrifuged egg form
a thin, rather loose layei' above the P granules
and stain both with Janus green B and Xile blue.

330

FISH AND WILDLIFE SERVICE

Microns

Figure 300. — Photomicrograph of eggs in the follicles of the ovarv of C. virginica at the beginning of the spawning season.
Ovocytes and small indifferent cells line the wall; mature eggs are either free or connected to the wall with long
peduncles. Kahle, hematoxylin-eosin.

Cleland states that in the uncentrifuged mature
egg they are unrecognizable. From hontogenates
the M granules can be separated by centrifuging
for 10 minutes at 10,000 times gravity. Cyto-
phism also contains submicroscopic or S granules
(according to Cleland's terminology), which are
separable by appljang a centrifugal force of
20,000 times gravity for 30 minute?. These S
granules are probabh' homologous to mammalian
microsomes, i.e., the submicroscopic ribonucleo-
protein particles whicli are considered to be the
major sites of protein synthesis (a discussion of
this problem is found in Shaver, 1957, and
Novikoff, 1961b).

With the exception of pure lipid granules, the
cytoplasmic components of the egg of C. com-
mcrcialis show an increasing content of nucleic
acid with decreasing size of granules, the ground
cytoplasm containing the highest concentration

of nucleic acid. Cleland's observations need to
be corroborated, using the eggs of different species
of oysters.

The formation and composition of yolk in the
eggs of animals other than bivalves liave been
studied by many investigators, frequently wdth
different and sometimes contradictory results.
As Brachet (1944) stated nearly 20 years ago, the
problem cannot be resolved at present. This
uncertainty about yolk and other granules still
persists and probably will continue until the
ultrastructure of the marine egg is thoroughly
explored by electron microscopy.

Examination with the light microscope of ripe,
unfertilized, and unstained eggs of C. virginica
discloses a multitude of tightly packed minute
granules in the cytoplasm which obscure the inner
portion of the egg. The granules appear to be
uniforndy distributed around the nucleus (fig.

EGG, SPERM, FERTILIZATION, AND CLEAVAGE

331

Microns

Figure 301. — Camera lucida drawing of live unfertilized
egg of C. virginica in sea water. Germinal vesicle not
visible under yolk granules.

301). The eggs are devoid of pigment. OU
globules of different sizes can be made visible
under high magnification by gently pressing the
egg under a coverslip; by using fat-staining dyes
(Sudan II, III, or Black Sudan B) they become
conspicuous (fig. 303). Under the effect of dye
(dissolved in weak alcohol) the small granules of
lipid yolk, stained dark red or black, gradually
fuse into large globules and penetrate the vitelline
membrane and a slight pressure will force them
through it (fig. 304). The size of the globules
increases during the time that the preparation
remains under the microscope. These artifacts
are due to the fusion of globules under the effect
of dye.

The mitochondria of ( '. virginica can be stained
by a 0.5 percent solution of Jaims green in sea

water. They appear as small rodlike structures
uniformly distributed in the subcortical layer of
the egg (fig. 305). The origin of fatty or lipid
yolk in C. virginica has not been studied. In
Myiilus eggs the lipid of the yolk apparently
arises in an intimate association with the Golgi
apparatus (Worley, 1944). In Lymnaea (Bret-
schneider and Raven, 1954) they are formed in
certain parts of the protoplasm independently of
cell structures visible under the light microscope.

In the eggs of the Bombay oyster, C. cucuUata,
which are similar to those of C. virginica, the fatty
yolk, according to Rai (1930), is formed directly
from the Golgi vesicles. Mitochondria exist in the
eggs of this species in the form of very minute
granules forming a circumnuclear ring. Later they
grow in size and are more or less uniformly dis-
tributed. This conclusion is in agreement with the
observations of Gatenby and Woodger (1920), who
found that in Helix and Limnaea the Golgi ele-
ments gradually spread throughout the ovocyte
and probably take part in the formation of yolk
bodies. They found no evidence that part of the
mitochondrial constituents of cytoplasm meta-
morphose into yolk.

Oyster eggs placed for 5 minutes in a dilute solu-
tion (1 to 25,000 or 1 to 30,000) of toluidin blue 0
and washed in sea water are colored metachro-
matically. Pasteels and Mulnard (1957) found

Microns

20

FiGDRE 302. — Camera lucida drawing of live egg of
C. virginica a few minutes after fertilization.

332

FISH AND WILDLIFE SERVICE

• •.

Microns

40

Figure 303. — Oil globules in the unfertilized egg of C.
virginica after staining in Sudan III. Whole mount.
Drawing made from a photomicrograph.

Microns

40

Figure 304. — Unfertilized egg of C. virginica stained with
Black Sudan B; slight pressure on a coverslip forces
the oil globules through the vitalline membrane. Drawn
from life.

that the development of eggs of the Portuguese
oyster, C. angulata, is not affected by tohiidin blue
used in such dilute solution for only a short time.
The dye is fixed at the level of the small granules,
which the cytologists designate as alpha granules,
uniformly distributed in the cytoplasm between
the yolk vesicles. Later in the development of a
fertilized egg, new and larger granules, called beta
granules, appear at the time of prophase. Their
higher metachromasy is acquired at the expense
of the alpha granules. Subsequent studies (Mul-
nard, Auclair, and Marsland, 19.59) have suggested
that the beta granules are related to the Golgi
complexes of the eggs.

The alpha granules of the unfertilized eggs of C.
angulata can be separated from the yolk vesicles by
centrifuging; they are displaced in the direction of
the centrifugal force (Pasteels and Mulnard, 1957),
while the beta granules at the pronucleus stage of
the fertilized egg are moved in the centripetal

EGG, SPERM, FERTILIZATION, AND CLEAVAGE
I733-S51 O— &4 22

direction. The alpha and beta particles of Pasteels
and Mulnard probably correspond to the P and L
granules of Cleland. Personal observations show
that in ripe but unfertihzed eggs of New England
C. virginica stained with toluidin blue, elements
corresponding to the alpha particles of Pasteels
assume a lavender color while mitochondria and
other smaller granules are bluish. The nucleolus
is also of bluish color. After 10 minutes of centri-
fuging at 4,000 times gravity the yolk granules of
the stained eggs concentrate at the lower (centri-
fugal) pole, while the alpha particles of lavender
hue and slight!}^ bluish mitrochondria are at the
opposite pole (fig. 306).

Metachromatic granules have been described in
the eggs of various bivalves. They were found
in Barnea Candida (Pasteels and Mulnard, 1957) ;
Mactra (Kostanecki, 1904, 1908; Mercenaria
{Venus) mercenaria, Alytilus edulis, and Spisula
solidissima (Worley, 1944; Kelly, 1954, 1956;

333

Microns

Figure 305. — Photomicrograph of an unfertilized egg of C. virginica. Janus green. Whole mount. Small rodlike
inclusions seen along the periphery are mitochondria stained green; larger yolk granules are dark.

Allen, 1953; and Rebhun, 1960). The role of
these almost submicroscopical bodies is not clear,
but there is no doubt of their importance in the
physiology and development of eggs. Recent
publications of Dalcq (1960), Brachet (1960), and
Rebhun (1960), and the reviews given by NovikofT
(1961a, 1961b) should be consulted for ideas con-
cerning the possible role of these elements in the
morphogenesis of mosaic eggs in which they are
concentrated in the posterior blastomeres.

Eggs of the surf clam S. solidissima contain a
heparinlike blood anticoagulant which was also
extracted from the tissues of this clam (Thomas,
1954). Whether substances with sunilar activity
are present in oyster eggs is not known.

The nucleus of a mature egg is surrounded by a
nuclear membrane which can be clearly seen on
sectioned and stained preparations of the ovary
(fig. 300). A spherical, dense nucleolus is excen-
trically located; its diameter varies from 4 /i to 6 m-

334

FISH AND WILDLIFE SERVICE

0

Microns

40

Figure 306. — Unfertilized egg of C. virginica centrifuged
for 10 minutes at 4,000 times gravity after staining with
toluidin b!ue. Dark yolk granules are at the lower
(centrifugal) pole while the mass of small inclusions con-
sisting of lavender particles and bluish mitochondria are
at the opposite end of the egg.

The chromosomes appear as fine threadlike struc-
tures near the nuclear membrane. Using methyl
green and pyronin B stains, Kobayashi (1959)
found that the nucleolus in the eggs of C. gigas and
0. laperousi consists of two parts, the karyosome.

Microns

30

Figure 307. — Ovocyte of 0. laperousi stained with methyl
green and pyronin B after Xavashin fixation. Dark
globule is the karyosome, light shaded area is the
plasmosome. From Kobayashi, 1959.

shown in solid black in fig. 307, and the plasmo-
some, lightly shaded, in close contact with each
other. The definite KP axis (Karyosome-Plasmo-
some) of the unfertilized egg changes after fertil-
ization by the turning of the karyosome around
the plasmosome. The existence of this axis in the
eggs of other species of oysters has not been
described.

STRUCTURE OF THE MATURE
SPERMATOZOON

The spermatozoon of bivalve mullusks appears
under the microscope (Franzen, 1956; Lenhossek,
1898; Retzius, 1904) to consist of an oval or
round head with a pointed front, a middle piece
at the lower end of the head, and a long tail
(flagellum) with a narrow "end piece" which is
longer than the width of the head. The middle
piece consists of fom*, sometimes five, oval-shaped
bodies clustered around the tail; and a minute
"central granule" or centriole located in the cen-
ter at the point of attachment of the tail. The
sharply outlined oval bodies are mitochondria;
they are strongly osmiophilic and can be deeply
stained with rosanilin. The head of the sperma-
tozoon is formed by a compact nucleus capped
with the apical body or acrosome with a pointed
tip (perforatorium), which apparently assists the
spermatozoa in penetrating the egg membrane at
fertilization (Wilson, 1928). The features listed
above may be seen in properly fixed and stained
preparations of the sperm of C. inrginica and in
live spermatozoa examined with phase contrast
oil immersion lenses. In live preparations the
nucleus appears to be dark while the acrosome
and middle piece are light (fig. 308). The center
of the spermatozoon head is occupied by an axial
body, a relatively large, light-refracting structure
which is separated from the acrosome.

The dimensions of normal, uncytolyzed sperma-
tozoa have been measured by means of an eyepiece
micrometer of a light microscope. The head varies
from 1.9 M to 3.6 m in length (median value 2.7 ^)
and between 1.0 ai and 2 m in width. The tail is
from 27 m to 39 m long (median value 36 y). The
tails are usually slightly curved; specimens with
straight tails are rarely found.

Electron microscopy reveals much greater com-
plexity in the structure of the spermatozoon
(Galtsoff and Philpott, 1960). Study was made
of small sections of ripe spermary preserved in
cold 1 percent osmimn tetroxide buffered to pH

EGG, SPERM, FERTILIZATION, AND CLEAVAGE

335

^t.

eP-

0

Microns

Figure 308. — Live spermatozoon of C Virginica examined
under light microscope with phase contrast oil immersion
lens. A — acrosome; ax. b. — axial body; C — cenlriole;
e.p. — end piece of tail; h. — head; m.p. — middle piece;
mt. — mitochondrial bodies; N — nucleus; t. — tail.

7.2-7.4 iind embedded in a mixture of three parts
butyl and one part methyl methacrylate. To
increase the contrast, some of the material was
placed in 1 percent alcoholic phosphotungstic acid
for 5 to 6 hours before embedding. The embedded
material was sectioned on PhUpott's microtome

(Philpott, 1955), using a diamond knife. Since
the preserved pieces consisted of a multitude of
spermatozoa arranged in a central mass with their
tails pointing outward, the individual spermatozoa
were always cut at random in different planes,
regardless of how the embedded tissue may have
been oriented on a microtome block. The re-
sulting electron micrographs showed a number of
sperm heads cut at different planes and many
transverse sections of tails (fig. 309). The entire
structure of the head was diagrammatically re-
constructed by bringing various elements together
and placing them in their relative positions (fig.
310). The oval-shaped head consists of slightly
granular, homogeneous material covered with an
osmiophilic membrane made of two layers. The
apical portion of the nucleus is occupied by a
caplike acrosome of higlily osmiophilic substance.
The acrosome is clearly separated from the nucleus
by a sharply defined membrane. An egg-shaped
body in the central part of the nucleus extends
from the apex of the acrosome almost to the base
of the nucleus. This structure, named axial body
(Galtsoff and Philpott, 1960), has a central stem
of fibrous material which emerges from the
flattened bottom and extends about two-thirds of
the total length of the axial body. The indented
base of the nucleus is near the base of the axial
body. The caved-in space formed by this in-
dentation consists of material of lesser electron
density and extends under the nucleus to the
upper surface of the centriole, which is siu-rounded
by four mitochondrial bodies. Only two of them
are shown in fig. 310.

The centriole of the sperm of C. virginica is a
hollow, cylindrical structure wdth walls made of
nine bands; these can be seen in cross section (fig.
311). The side \-iew (fig. 310) shows that the
centriole is formed in several alternating and
slightly constricted layers which connect with the
four mitochondrial bodies. These bodies have
the typical appearance of twisted lamallae enclosed
in a membrane which encompasses the centriole
and continues over the tail.

The tail consists of a pair of axial filaments
surrounded by a ring of nine double filaments
spaced at equal intervals along the periphery
(fig. 312). The filaments are interconnected by
delicate strands. The axial filaments begin near
tlie basal plate (fig. 310) where the tail emerges.
Radial Iralieculae connect the ring filaments to
the outside wall of the tail and form nine separate

336

FISH AND WILDLIFE SERVICE

^ty^A^iA ^ dH & i& irS?<iCc

i

Microns

Figure 309. — Electron micrograph of a section of ripe spermary of C. virgirnca. Longitudinal and transverse sections of
sperm heads and tails can be seen in various parts of the micrograph. A — acrosome; ax. b. — axial body; c.t. — cross
section of tails; l.s. — longitudinal section of sperm head; l.t. — longitudinal section of part of a tail; m.b. — mitochon-
drial bodies; N — nucleus; t.s. — transverse section of head.

EGG, SPERM, FERTILIZATION, AND CLEAVAGE

337

•1.0 }JL

Figure 310. — Reconstruction of the sperm head of C.
virginica made from a large number of longitudinal
sections, a. — aorosome; ax. b. — axial body; ax. f. —
axial filament of the tail; b.p. — basal plate of the tail;
c. — centriole; c. ax. b. — core of the axial body; d.f. —
double filament on the periphery of the tail; m.b. —
mitochondrial body; n. — nucleus; t. — proximal part of
the tail.

compartments filled with material of lesser electron
density. The central radial strands are similar
to the "spokes" described by Afzelius (1955) for
the sperm of the sea urchin Psammechinu.s miliaris.
They are not present in the proximal portion of
the tail where there are no axial filaments, but
otherwise the ultrastructure of the sperm tail is
similar to that of cilia and flagella of various
animals and plants.

FERTILIZATION

The spawned eggs of C. trirginica and C. gigas
are heavier than water and quickly sink to the
bottom. The time tliey remain in suspension may
be prolonged by horizontal currents and upward
movements of the water, and consequently the

338

chances of fertilization are increased. Because
spawning is usually initiated by the males, the
water into which the eggs are discharged already
contains active spermatozoa and fertilization takes
place within a few minutes following ovulation.
It is obvious that the success of reproduction of
an oyster population in which spawning is mutually
stimulated by the discharge of sex cells is depend-
ent on close proximity of the sexes and their
simultaneous response to spawning stimuli.

Eggs and sperm secrete substances called
gamones which play an important role in fertili-
zation. Secretion from an unfertilized egg has a
significant effect on spermatozoa. This effect can
be observed if a suspension of eggs is permitted to
stand for 15 to 20 minutes and the supernatant
fluid is decanted or filtered and added to the sus-
pension of sperm. The resulting so-called "egg
water" (Lillie, 1919) causes the agglutination of
sperm. To observe the agglutination reaction
with the naked eye, a drop of egg water must be
added to a sperm suspension, which shortly forms
ii-regular lumps (fig. 313). Under a high-power
light microscope one sees that the heads of agglu-
tinated spermatozoa stick together to form large
aggregates (fig. 314).

0

Micron

I

Figure 311. — Drawing based on electron micrographs
of the cross section of the lower part of the middle
piece of the spermatozoon of C. virginica. Centriole,
at the center, is surrounded by four mitochondrial
bodies.

FISH AND WILDLIFE SERVICE

Micron

Figure 312. — Transverse section of the tails of oyster sperm of C. virginica slightly below the level of the middle piece.

Electron micrograph.

EGG, SPERM, FERTILIZATION. AND CLEAVAGE

339

Centimeters

Figure 313. — Sperm suspension of C. virginica on a slide
in sea water (left) and in sea water containing a small
quantity of egg water (right). Drawn from life.
Natural size.

Agglutination also occurs in the sperm of C.
gigas and in 0. circumpicta Pilsbry. Terao (1927)
experimented with the latter species using egg
water made by mixing 0.55 ml. of ripe eggs in 9 ml.
of sea water and removing the eggs by centrifug-
ing and filtering after they had stood for 20
minutes. The filtrate caused the isoagglutination
of sperm even in a dilution of 1 to 10 millions.

Heteroagglutination

by the

water of 0.

circumpicta has been observed in the suspensions
of sperm of the bivalve Area, sea urchin Toxo-
cidaris tuberculatus, and starfish Luidia quinaria.
LUlie (1919) regarded the sperm agglutinating
factor he discovered in Arbacia eggs as an essential
to fertilization, and to the active substance of egg
water he gave the name fertilizin. Tyler (1948)
identified fertilizin with the jelly substance of the
egg and on the basis of experimental data con-
cluded that the presence of the jelly coat has a
favorable effect on fertilization. B_v biochemical
analysis of sea urchin eggs, Vasseur (194Sa, 1948b)
determined the composition of the jelly coat and
found that 80 percent of it consists of polysac-
charide and 20 percent of amino acids. The sub-
stance was found to exert a heparinlike action in a
blood-clotting system (Immers and Vasseur, 1949).
After removing the jelly coat with acidified water,
Hagstrom (1956a, 1956b, 1956c, 1956d) found
that the rate of fertilization was higher than in the
presence of the coat. It is, therefore, apparent
that the jelly coat is not essential for fertilization.

° Microns ^^ if.

Fjgube 314. — Photomicrograph of sperm of C. virginica agglutinated by egg water of the same species. Phase contrast

oil immersion.

340

FISH AND WILDLIFE SERVICE

This view agrees with the conditions found in
oyster eggs which have no jelly coat.

According to the modern view discussed in the
re%new of the problem by Runnstrom, Hagstrom,
and Perlmann (1959), the jelly coat not only fails
to improve fertilization but impedes it by acting
as a sieve. Its action may be considered as an
elimination process by which the number of
spermatozoa capable of attaching to the cytoplas-
mic surface is substantially reduced.

Fertilizin of sea urchin eggs has two distinct
properties: it agglutinates sperm suspension and
activates the motility of free, single spermatozoa.
Both of these properties are present in the fertilizin
of an oyster egg.

ACROSOMAL REACTION

Spermatozoa of various invertebrates have been
found to carry a substance of protein character,
probably a lysine, capable of dissolving the
vitellme membrane of the egg. Such lysine is
present in the sperm of the giant keyhole limpet,
Megafhura crenulata (Tyler, 1939), in the sperm of
Mytilus, where it is probably located in the
acrosome (Berg, 1950; Wada, Collier, and Dan,
1956), and in other marine animals (Tyler, 1948,
1949).

Upon contact with the surface of an egg, the
spermatozoon undergoes a so-called acrosomal
reaction, which is described as the deterioration
of the surface of the acrosomal region of the head
followed bj' a projection of a stalklike filament.
The acrosomal reaction and the discharge of the
filament have been observed in starfish, holo-
thurians, mollusks, and annelids. The reaction
was studied by Col win, A. L., and L. H. Col win
(1955) and Colwin, L. H., and A. L. Colwin (1956)
in the annelid Hydroides hexagonus and enterop-
neust Saccoglossus kowalewskii. Using electron
microscopy, the Colwins revealed many interesting
details of the penetration of the spermatozoon
into egg cytoplasm. In pelecypod mollusks the
discharge of the acrosomal filament was observed
in Mytilus and in the three species of oysters, C.
echinata, C. nipjxnm, and C. gigas (Wada, Collier,
and Dan, 1956; Dan and Wada, 1955). The
reaction can be induced by egg water as well as
by the contact of a spermatozoon with the egg
surface. The first sign of acrosomal reaction in
oyster sperm is the flattening of the anterior
surface of the spermatozoon. At the same time
the head becomes extremely adhesive, the acro-

EGG, SPERM, FERTILIZATION, AND CLEAVAGE

some membrane bursts, and the filament is
discharged. The reaction can be observed when
a small drop of live sperm suspension is placed on
a cover slip, a minute quantity of egg water is
added, and the cover slip then inverted on a slide.
The preparation is examined with phase contrast
oil immersion lens using anisol (Crown oil) of
refractive index 1.515 instead of cedar oU.

The acrosome reaction of C inrginica is similar
to that observed by Dan and Wada in three other
species of oysters. Under the effect of egg water
the head becomes swollen and rounded and the
filament is ejected from the acrosome. The dis-
charged filament is wider than the tail and is
about three to four times longer than the length of the
head (fig. 315 ). In my observations only a small
number of oyster spermatozoa suspended in egg
water discharged acrosomal filaments.

The exact role of the filament in the fertilization
of oyster eggs is still unknown. Investigations

Microns

Figure 31.5. — Diagrammatic drawing of acrosomal reaction
in the spermatozoon of C. virginica produced by egg
water. Onlj' a part of the sperm tail is shown in the
drawing. Drawn from Uve preparation.

341

with eggs of other invertebrates suggest that the
acrosome region of a spermatozoon is active during
the first stages of fertihzation and that it carries
a lysine which facilitates the attachment of the
spermatozoon to the egg membrane and its pene-
tration into the cytoplasm.

The old view that spermatozoa penetrate the
egg by a mechanical action of screw-borer move-
ments of the pointed end (the perforatorium) has
been abandoned. It is now generally accepted
that the action of the sperm head is primarily
chemical and that probably several enzymes are
carried by the acrosome. Readers interested in
the problem of fertilization are referred to compre-
hensive reviews of this subject by Runnstrom,
Hagstrom, and Perlmann (1959), Colwin, A. L.,
and L. H. Colwin (1961a, 1961b), and Colwin,
L. H., and A. L. Colwin (1961).

FERTILIZATION OF EGG

Eggs for fertilization experiments may be ob-
tained in the laboratory by stimulating a single
female spawn as described in chapter XIV. A
suspension of eggs pipetted off the bottom of a
laboratory tank is free of blood and other body
fluids. Eggs may also be taken directly from the
ovary by cutting off smsxll slices from the surface
of the gonad and mincing or shaking them in sea
water. Cutting into the underlying layer of
digestive diverticula should be avoided to prevent
contamination with body fluids. The eggs must
be washed se^ eral times in filtered seawater by de-
canting or by filtration through a fine sieve until
the suspension is free of tissue cells and debris.
After being in sea water for a short time, the eggs
change their shape and become globular but their
large germinal vesicles remain clearly visible
(fig. 316).

A sperm suspension may be obtained by any
one of three methods. Male spawning can be
induced by raising the water temper atiu-e or by
adding a small amount of thyroid suspension, and
live spermatozoa collected as they are discharged
through the cloaca; small pieces of ripe spermary
can be e.xcised and the spermatozoa liberated in
sea water by shaking; or a very ripe spermary
can be pressed gently with the fingertip and the
spermatic fluid pipetted as it comes from the
gonoduct. Concentrated sperm suspension must
be diluted for fertilization. I found it convenient
to make a standard suspension using 0.2 g. of
gonad material in 50 ml. of sea water and then

Microns

30

Figure 316. — Camera lucida drawing of naturally spawned
but unfertilized egg of C. virginica.

diluting it, using 0.5 ml. for 100 or 150 ml. of
water containing eggs.

Although several spermatozoa may attach
themselves to an egg, (fig. 317), only one pene-
trates the cytoplasm. The others, called super-
numeraries, eventually are cast off when cleavage

Microns

25

Figure 317. — Photomicrograph of a fertilized egg of C.
virginica a few minutes after the formation of the
fertilization membrane. Several spermatozoa are at-
tached to the egg membrane but only one will penetrate
it. The germinal vesicle is intact. Contrast phase oil
immersion lens.

342

FISH AND WILDLIFE SERVICE

begins. If the sperm suspension is too concen-
trated, many spermatozoa enter one egg and
cause polyspermy, a condition which may inter-
fere with normal development of the egg.

The spermatozoon which succeeds in pene-
trating the egg's surface midergoes great changes.
Its acrosome region becomes swollen and dis-
rupted and the tail loses its motility; the head
gradually penetrates the egg membrane as the
sperm moves deeper into the cytoplasm. At the
same time the fertilized egg contracts and assumes
a globular shape if it was not round before; the
cytoplasm becomes so dense that the germinal
vesicle is no longer visible under the layer of yolk
granules. A few seconds after the sperm head
touches the egg's surface a thin, transparent
fertilization membrane is elevated and imder the
hght microscope appears to be homogeneous.
This membrane apparently is formed from the
pre-existing vitelline membrane and is under-
lined by a layer of subcortical particles (fig. 318).
The two layers are optically separated. It is
generally accepted (Runnstrom, 1952) that in
Arhacia and many other species the fertilization
membrane originates from the vitelline membrane
because it fails to form after the vitelline mem-
brane has been removed with potassium chloride,
trypsin, or urea. No experimental work of this
type has been done on oyster eggs.

AGING OF EGGS AND SPERM

The longevity of eggs of marine invertebrates,
i.e., their ability to form fertilization membrane
and undergo cleavage, was observed in the sea
urchin {Arhacia) and in other common species
(Harvey, 1956). Oyster eggs also undergo aging
changes and lose their ability to be fertilized.
This has been demonstrated in a number of tests
made in the Bureau's shellfish laboratory at Woods
Hole. Because of wide individual variability in
fertilization capacities only one female and one
male were used in each series of tests. The
following technique was used : Suspension of egga
was made by shaking 0.5 g. of ripe ovary tissue
in about 200 ml. of sea water: eggs released by
this action were permitted to settle on the bottom
and the supernatant water was decanted; the
remaining eggs were rinsed twice in sea water and
transferred to a beaker filled with 500 ml. of filtered
sea water. The beaker was kept half submerged
in running sea water to prevent heating to room
temperature. Samples of eggs were taken for
fertilization every hour during the first 4 hours,
then at 2-hour intervals for the next 6 hours, and
finally one sample was taken each time after 12
and 24 hours. Eggs were collected at random
from the bottom of the beaker and placed in a
finger bowl in 100 ml. of filtered sea water. To
fertilize them 0.5 ml. of dilute stock suspension
of sperm was used; the water was gently stirred

• %

S.p. (>

0

Microns

8

Figure 318. — Photomicrograph of a portion of fertihzed egg of C. virginka shorly after the attachment of sperm. Fer-
tilization membrane (f.m.) (outside layer) is underlined by the vitelline membrane (v.m.) with a dense row of subcor-
tical particles (s.p.). Live preparation. Oil immersion phase contrast lens.

EGG, SPERM, FERTILIZATION, AND CLEAVAGE

343

to obtain uniform distribution of sperm, and the
finger bowl set aside for 5 hours, half submerged
in running sea water. At the expiration of this
period a sample was taken for examination and
cleaved and uncleaved eggs were counted. In
each case 300 eggs of the sample wei-e examined.
All tests were made in water of 31 to 32°/oo
salinity and 20.8° to 21.4° C. During the first 4
hours of aging the percentages of cleaved eggs
declined from 90 to 70. After 5 or 6 hours the
percentages dropped to 60. Then the fertilizabil-
ity decreased to about 20 percent in 10 hours, and
only a few eggs cleaved normally after 12 and 24
hom's of aging.

It is common knowledge among embryologists
that the fertilizing power of spermatozoa is not
decreased if sperm is kept at 10° to 12° C. in a
concentrated suspension in a tightly closed con-
tainer. This is also true for oyster sperm. Its
fertilizing power is affected by dilution and in-
creased temperature. At room temperature in a
dilute suspension, the spermatozoa lose their
fertilizing ability within 4 to 5 hours. However,
in a concentrated suspension, protected from
evaporation, and stored in a refrigerator at about
10° C. the sperm remains active and retains its
full fertilizing power for 24 hours and possibly
longer.

The effect of cold storage on the fertilizability
of eggs is not known. On several occasions ripe
females with intact shells were kept for 3 to 4
days in a refrigerator (about 10° C) and after
that were successfully used in spawning experi-
ments. The effect of cold storage on eggs of the
excised ovary has not been studied.

POLARITY OF EGG

The polarity of all moUuscan eggs apparently
is determined while they are still attached to the
wall of the ovarian follicle. Presumably the side
on which the food reaches the growing ovocyte
becomes the vegetative pole of the mature egg
(Raven, 1958).

The metabolic gradient along the egg axis is
indicated by a concentration of cytochrome oxidase
which Kobayashi (1959) detected with M-Nadi
reaction ; the activity of the enzyme was observed
using Graff's modification of this method. (The
reader not familiar with the reaction and its sig-
nificance in cytochemical research is referred to
the publications of Danielli (1958), Deane, Barr-
nett, and Seligman (1960), and to a review by

NovikoflF (1961a, p. 308).) In brief, the localiza-
tion of oxidative enzymes and their presence in
mitochondria can be determined by the staining
reactions. The rate of respiration of eggs of C.
mrginica increases with fertilization by a factor of
1.4 (Ballentine, 1940) but in the eggs of the
Sydney rock oyster, C. commercialis, the rate of
respiration increases only at the onset of the first
cleavage (Cleland, 1950).

CLEAVAGE

The spermatozoon may enter the oyster egg
anywhere. Its path inside the egg cytoplasm
toward the nucleus has not been described, and
cytological details of the process leading to the
fusion of the female and male pronuclei have not
been studied. It is probable that the major fea-
tures of these events are not different from those
found in other mollusks. Maturation divisions
occur in the oyster egg after the elevation of the
fertilization membrane. The germinal vesicle
(ovocyte nucleus) breaks down and moves toward
the egg's periphery. At temperatures of 22° to
24° C. the first polar body is formed within 25 to
50 minutes after the addition of sperm. The re-
duction of the number of chi'omosomes probably
takes place during the first meiotic division. This
is to a certain extent corroborated by an exami-
nation of fertilized eggs of C. mrginica stained in
toto with Feulgen reagent or with acetic orcein.
Unfortunately the results are not consistent enough
to draw a final conclusion and the question remains
unanswered, awaiting a complete cytological study.

The second polar body is formed shortly after
the first, within 45 to 70 minutes after fertilization
(at 22.5° to 24° C). The two polar bodies remain
attached to the surface of the egg (fig. 319) until
the completion of cleavage and emergence of the
trochophore.

The fu'st cleavage following the formation of
the second polar body divides the egg meridionally
into two unecjual cells designated as AB and CD
(fig. 320). The inequality of the blastomeres is
due to the occurrence of a polar lobe. Because
the egg appears to consist of three cells this stage
received the name trefoil.

The plane of the second division, also meridional,
is at a right angle to the first. Both blastomeres
divide synchronously and separate into the four
quadrants. In fig. 321, drawn from a photograph
of a cleaving egg taken from the animal pole, the
position of the spindles indicates the plane of new

344

FISH AND WILDLIFE SERVICE

Microns

Figure 319. — Photomicrograph of a live fertilized egg of
C. virginica after the formation of two polar bodies (top
of egg). High-phase oil immersion lens.

0

Microns

40

Figure 320. — First cleavage division of the egg of
virginica 70 minutes after fertilization. Blastomere
(left) and CD (right). Polar body — ^'-

C-

ion. Blastomere AB

on top.

furrows which will intersect the egg irito four cells,
A, B, C, and D. At this stage the mitotic figures
are fairlj- large and the ckromosomes are in a
favorable position for examination. In the best
sectioned preparations of the cleaving egg, eight
daughter chromosomes were counted at the begin-
ning of anaphase (fig. 322). It would be, however,
premature to state that the diploid number of
chi'omosomes in C. virginica is 8 because on other
preparations 7, 9, and 10 were counted.

The third division of each quadrant cuts the
cells in the equatorial plane and separates the

Microns

10

Figure 321. — Beginning of second cleavage of the egg.
Viewed from the animal pole. Whole mount, Kahl^
Feulgen stain.

Microns

20

Figure 322. — Section of an egg of C. virginica at the second
cleavage. Beginning of anaphase. Kahle, Heidenhain
iron-hematoxylin.

first quartet of micromeres, small ceUs at the
animal pole, from the macromeres, or larger cells
at the vegetal pole (fig. 323).

At the fourth and fifth cleavages, resulting in
16- and 32-cell stages, the micromeres overgrow
the macromeres. Only one of the macromeres is
visible in the figure 324 and two in 325, which show
a side view of an oyster egg at these two stages of
development.

Cell lineage, or tracing the developmental
history of the cleavage blastomeres through to

EGG, SPERM, FERTILIZATION, AND CLEAVAGE

345

Microns

Figure 323. — Third division of fertilized egg of C. vir-
ginica; side view. Separation of mieromeres (on top)
from macromeres (bottom). Only one macroraere is
seen in the plane of view. Live egg.

0

Microns

20

Figure 324. — Fourth cleavage of egg of C. virginica and
the formation of 8 mieromeres (2d quartet). Side view.
Drawn from photomicrograph of live cell.

their ultimate fates as parts of the larva or adult,
was first described by Wliitiuan (1878) for tlie
egg of C'kpsine, and followed by Wilson (1892) for
the egg of Nereis. The works of Lillie (1895)
on the development of Unionidae, C'onklin (1897,
1908) on Crepidula and Fulgur, Meisenheimer

Microns

20

Figure 325. — Fifth cleavage of egg of C. nirginica and
the formation of the third quartet of mieromeres (32-
cell stage). Side view. Drawn from photomicrograph
of live cell.

(1901a, 1901b) on Dreissensia and Cydas, and
Wilson (1904a, 1904b) on Dentalium and Patella
constitute major contributions to the embryology
of mollusks.

The nomenclature of the cleavage blastomeres,
as developed by Wilson (1892), was progressively
modified by Conklin (1897), Mead (1897), and
Child (1900); the present system is based largely
on the work of Robert (1902) on the development
of the Trochti^ egg. The system is a combination
of letters and numbers by which the blastomeres
are identified.

The fu'st four cells or macromeres are designated
as A, B, C, and D; in the majority of cases studied
D is the largest of the four and is situated at the
side which will develop into the posterior portion
of the embryo. When the first four blastomeres
divide, their daughter cells are denominated la and
lA, lb and IB, Ic and IC, and Id and ID, the
small letters in each case referring to the micromere
and the capital letter to the macromere.

In successive dixisions lA divides into 2a and
2A, IB into 2b and 2B, and so on. When the
mieromeres divide, la is divided into la' and la^,
tlie superscript 1 denoting the daughter cell which
is nearest to the animal pole and superscript 2 the
one nearer to the vegetal pole. The nomenclature
is capable of indefinite expansion, but certain con-
fusion arises when the two daughter cells resulting

346

FISH AND WILDLIFE SERVICE

from the di\asion of one cell lie at an equal distance
from the pole. In this case the letters r for right
and 1 for left are used. The practice is, however,
not generally followed.

Descriptions of various types of cleavage can be
found in volume 1 of MacBride (1914). The equal
and unequal cleavages in the spu-ally cleaving eggs
of annelids and molluscs are discussed by Costello
(1955) in chapter 2 of Willier, Weiss, and Ham-
burger (1955).

During division the micromeres of a quartet,
viewed from the animal pole, become slightly dis-
placed because the spindles of the dividing cells
(not shown in fig. 324 or 325) occupy an oblique
position with respect to the egg's axis. At the
following di\T[sions the plane of separation of
daughter cells is oriented approximately at a right
angle to the preceding divisions. The pattern of
such cleavage is called spiral. It gives rise to an
irregular morula (sterroblastula according to Kor-
schelt and Heider, 1895, from the Greek "sterros"
meaning firm) found in annelids (Nereis), in some
bivalves (Ostreidae, Teredo), and in gastropods
(Crepidula, Fulgur, Nassa), and others. In all
cases the sterroblastula arises from an unequal
cleavage dm-ing which the micromeres overlie the
macromeres, and at each division are slightly dis-
placed to the right (dexiotropic cleavage) or to the
left (laeotropic cleavage). Sometimes, as in the
case of Dreissensia, the second dexiotropic cleavage
is followed by a third dexiotropic cleavage after
which the normal alternating coiKse is established
(Meisenheimer, 1901a). In the case of oyster
eggs, as shown by Fujita (1929) for C. gigas, the
cleavage is laeotropic.

The multicellular stages of a C. virginica egg are
reached in the course of the sixth and ensuing
cleavages (figs. 326 and 327) during which the
micromeres divide much more rapidly than the
macromeres, become progressively smaller and
overgi-ow the vegetal pole. Approximately at this
stage the sterroblastula of an oyster Is formed.

Gastrulation begins with epibolic extension of
the micromeres. At 22° to 24° C. the stage
shown in fig. 328 is reached within 4 to 6 hours.

The cell lineage of C. virginica has not been
studied; the stages of development of eggs of the
species shown in figm'es 320 to 328 are similar
to those previously described by Brooks (1898);
Horst (1882) for O. ediilis; Seno (1929) for 0.
denselamellosa; Hori (1933) for 0. lurida; Yasugi
(1938) for 0. spinosa, and C. gigas. Yasugi

Microns

Figure 326. — Formation of sterroblastula in C. virginica
egg; of the four macromeres only two are visible from
the side. Micromeres begin to overgrow the vegetal
pole. Drawn from photomicrograph of live egg.

0

M icrons

20

Figure 327. — Advanced sterroblastula stage of C. vir-
ginica. One of the macromeres has divided into two
daughter cells of equal size. Drawn from photomicro-
graph of live egg.

EGG; SPERM, FERTILIZATION, AND CLEAVAGE

347

Microns

20

Figure 328. — Early stage of gastrulation in the egg of
C. virginica. Seen at a section of an egg cut along its
axis. Heidenhain, iron-hematoxvlin.

found that equal cleavage can be induced arti-
ficially in eggs of the Japanese oyster by cen-
trifuging for 2 minutes at 1,500 r.p.m. and at the
centrifuge radius of 14 cm.

Fujita (1929) gives a brief account of the cell
lineage of the eggs of C. gigas and states that the
mode of cleavage of this species is identical to
that of C. mrgmica. The main features described
by him are as follows. The first polar body in
the fertilized egg of C. gigas appears 15 minutes
after insemination. At the two-cell stage (fig.
329) the two blastomeres of unequal size, AB and
CD, are separated along the meridional plane.
Their position corresponds to the anterior (Ant.)
and posterior (Pst.) ends of the embryo. The
second division, also meridional, separates the
four blastomeres A, B, C, and D (fig. 329b).

A and B represent the anterior, and C and D
the posterior hahes of the embryo, while B and
C form its left and A and D its right halves
(fig. 329b) . The ensuing cleavage starts with the
blastomere B and proceeds in laeotropic order to
C, D, and A; tlie resulting daughter cells, the
micromeres a,, b,, c,, and di, retain the shape of
the mother cells but are smaller. The macromere
D and micromere d, are respectively the largest.
The four daughter cells aj through d, form the

first quartet of micromeres located between the
macromeres on the dorsal side of the embryo.

The 12-cell stage is initiated by the division of
the macromere D; the ensuing larger cell da
(fig. 329c) is generally known as the first somato-
blast X. (In the system of nomenclature used
by American and European embryologists (see
p. 346) the ID cell gives rise to 2d and 2D and the
2d is the X cell.).

The cleavage is continued laeotropically, and
the second generations of micromeres as, bj,
and C2 are smaller than the first macromeres.
They lie on the outside of the macromeres. The
third cleavage of macromeres A, B, and C con-
tinues in laeotropic order and results in the
micromeres aj, bj, and Cj; they are larger than
other micromeres. After the third cleavage the
macromeres make no further contribution to the
formation of micromeres and in the course of
development become the entoderm. The first
somatoblast (X cell) gives rise to many organs of
ectodermal origin. At the 18-cell stage of the
embryo the position of cell X and its first divisions
mark the beginning of the transition from spiral
to bilateral symmetry (fig. 329d).

The mesoderm begins to form at about the
32-cell stage with the appearance of cell 4d,
the second somatoblast, also designated as cell
M. In bivalves the cell M remains at the surface
for a long time, then divides into the two meso-
dermal teloblasts which sink into blastocoel
(Raven, 1958, p. 117). The formation of meso-
derm in C. gigas has not been followed in detail,
but as a rule the mesoderm bands in bivalves
remain rather rudimentary (Raven, 1958). Fujita
states that the establishment of the three germinal
layers in C. gigas is completed at the 30-cell
stage (fig. 329 e and f).

The gastrula stage is reached in 4 to 6 hours.
The cell lineage of C. gigas is generally comparable
to that described for other bivalves (see: Raven,
195S, p. 70), but for details the reader should
consult Fujita's (1929) original text and his
drawings.

In about 4 to 6 lioui-s after fertilization, an egg
of C. virginica reaches the stage (fig. 330) when
a few large cilia become nsible at the vegetal
pole, the oval-shaped body is covered with very

348

FISH AND WILDLIFE SERVICE

M

M, P

z.t

Microns

40

Figure 329. — Several stages of development of the egg of C. gigas. Redrawn from Fujita, 1929. a — Two-cell stage,
Ant. — anterior, Pst. — posterior ends; b — Four-cell stage, formation of blastomeres A, B, C, and D; c — 12-cell stage and
the formation of the first somatoblast (cell X), viewed from the animal pole; d — embryo viewed from vegetative
pole after the formation of the mesomere M; e — cleavage of mesomere M and the first somatoblast X, posterior
view optical section; f — advanced stage of development showing the arrangement of the mesomeres M, M, and the
somatoblasts, X, X, X,, X,, posterior view optical section. Cleavage nomenclature as given by Fujita.

Microns

20

Figure 330. — Larva of C virginica ready to hatch.
Drawn from photomicrograph of live larva.

fine ciliation, and two polar bodies still remain
attached to the animal pole. The beating of the
cilia is not coordinated at this stage, and the
movements of the larva are irregular and spas-
modic. A few minutes later a girdle of powerful
cilia is formed, the polar bodies are lost, and the
larva begins to swim upward (fig. 331). In a
finger bowl containing cleaving eggs, the newly
hatched larvae appear as white columns rising from
the layer of fertilized eggs on the bottom of the
container (fig. 332). The larvae can be pipetted
off easily, transferred into larger containers and
provided with suitable food.

The time requu-ed to complete the development
of an oyster egg varies, depending on condition
of eggs, temperature, salinity, oxygenation of
water, and other environmental factors. Records
of three sets of observations made in the Woods

EGG, SPERM, FERTILIZ.\TIOX, AND CLEAVAGE
733-851 O— 64 23

349

Microns

20

Figure 331. — Larva of C virginica at the time of its
emergence 6 to 6}^ hours after fertilization. Drawn
from a photomicrograph of a live larva.

Hole laboratory at room temperatures varjdng
from 22. .5° to 24.5° C. and salinity of water of
32.2 °/oo are given in table 37. To obtain re-
cords of rates of development at different tem-
peratm-es, several hundred artificially fertilized
eggs were placed in each Syracuse dish filled with
fresh sea water and coveretl to prevent evaporation.
The debris was removed, and the water contained
no unfertilized or cytolyzed eggs.

Table 37. — Observations on the time required for artificially
fertilized eggs of C. virginica to reach trochophore stage

All observations were made at Woods Hole in July at room temperatures
varying from 23° to 25" C. Tlie time required to reach different stages
varied in different groups of eggs. Tlie observations are arranged in two
groups; A and B. wliich differ primarily in the duration of time required
to reach rotating blastula and trochopliore stages.

STAGES

Stage of development

Fertilization membrane-
First polar body _..

Second polar body

First cleavage

Second cleavage

Third cleavage

Morula stage

Rotating blastula

Trochophore

5 min

40 min

1 hr. 10 min.
1 hr. 12 min.

2 hr. 10 min.

4hr.
5hr.

10 to 25 min.
25 to 62 min.
40 to 65 min.
45 min.
62 to 120 min.
55 to 195 min.
135 min.
fi hr. 30 min.
8 to9hr.

0

Centimeters

Figure 332. — The emergence of larvae of C. virginica from fertilized eggs? kept in a finger bowl. The free-swimming
larvae form columns, which tend to disper.se at the surface. Drawn from life.

350

FISH AND WILDLIFE SERVICE

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MuLNARD, Jacques, Walter Auclair. and Douglas
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1960. Aster-associated particles in the cleavage of
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354

FISH AND WILDLIFE SERVICE

CHAPTER XVI
LARVAL DEVELOPMENT AND METAMORPHOSIS

Page

Anatomy of trochophore and veliger 355

Morphology of larval shell - 363

Attachment and metamorphosis 3(i5

Dispersal of larvae 369

Reaction of larvae 371

To the external environment 371

To angle of surface 372

To the properties of surface 373

Gregariousness 373

Artificial rearing of oyster larvae 374

Bibliography 376

The anatomical structure of an oyster larva is
known primarily from works on the development
of 0. edulis by Horst (1883), Huxley (1883),
Dantan (1917), and Erdmann (1935). Fragmen-
tary information regarding other species is found
in publications of Stafford (1913) on 0. lurida;
Prytherch (1934) on C. virgmka and Fujita
(1934); on C. gigas. Larval histology is described
in a comprehensive paper by Erdmann (1935),
and fate of larval organs in the metamorphosis
of 0. edulis is discussed by Cole (1938b).

The voluminous literature on the ecology and
biology of oyster larvae of 0. edulis and other
species has been reviewed by Korringa (1941) in a
lengthy publication which places emphasis on
spawning and the setting of oysters. An abun-
dance of ecological data found in the reports of
Federal, State, and private organizations con-
cerned with the conservation and management
of oj'ster bottoms, deals mainly with the time of
appearance and setting of oj^ster larvae. Rela-
tively little is known about the factors which
control the life and behavior of the larvae, and
only a few studies have been made in recent
years on larval physiology, nutritive requu-ements,
and metabolism. However, advances in the tech-
nique of artificial rearing of oyster larvae from
fertilized eggs (Loosanoff and Davis, 1963a, 1963b)
now make it possible to obtain a continuous
supply of larvae of known age regardless of the
season of the j^ear. This advantage may stimulate
future studies of larval physiology.

FISHERY bulletin: VOLUME 6 4, CHAPTER XVI

ANATOMY OF TROCHOPHORE AND
VELIGER

The sHghtly flattened embryo which forms at
the completion of cleavage does not increase in
bulk during embryonic development and is about
40ju to 50iLi along its dorso-ventral dimension, about
the same size as the egg. The two polar bodies may
still be attached to some of the embryos and a
tuft of robust cilia marks their anterior ends. The
larva, which at this moment begins to swim, is
called trochophore from the Greek "trochos," a
wheel: and "phero," to bear.

The formation of the trochophore results from
the epiboly, i.e., the midtiplication of small ecto-
dermic cells, their arrangement around the single
and much larger macromere, and invagination of
the endoderm. At the early stage of larval de-
velopment, described by Horst (1883) for 0. edulis,
the invagination of endodermic cells (fig. 333, en.)
marks the position of the blastopore, bl., (from
the Greek "blastos," bud, and "poros," passage),
a channel which leads to the archenteron (the
cavity of the gastrula). A small invagination of
the ectodermic cells at the animal pole of the
larva indicates the location of a saddlelike shell
gland (sh.g.), which at the later stages gives rise
to the larval shell called prodissoconch (from the
Greek "pro," before, "dissos," double, and
"kongche," conch or shell).

The invagination of the blastopore (fig. 334,
bl.) becomes deeper and narrower; the mesoderm,
me., is formed; and the shell gland, sh., increases
in size. At the trochophore stage (fig. 335) the
blastopore is closed and the mouth, m., is formed
aliove it; the ectodermic cells, ec, develop cilia
and are now called the trochoblasts. They form
a cihated ring or prototroch, which functions as
an organ of swimming. The position of the pro-
totroch is indicated in fig. 335 by two ectodermic
cells, c, with cilia.

As the development of the trochophore ad-
vances, the prototroch forms a ciliated crown at

355

sh.g

en.

0

Microns

30

Figure 333. — Optical section of an early stage of devel-
opment of the larva of 0. edulis according to Horst.
Reproduced from Pelseneer, 1906. bl. — blastopore;
en. — endoderm; ec. — ectoderm; sh.g. — rudimentary shell
gland.

the ventral side of the larva (fig. 1^36, pr.). The
digestive system consists of tlie mouth (m.) sur-
roimded by ciliated lobes; large stomach (st.);
relatively short mtestine (int.); and anus (a.).
The thickened central ])art surrounded by the
prototroch is considered to be a rudiment of the
cephalic ganglion. The larval shell (sh.) covers a
considerable part of the body and is formed into
right and left oval valves t)f equal size and shape
joined at the dorsal side of the larva. At the be-
ginning of larval life tlie beating of the cilia of
the prototrocli is sporadic and disorganized.
Within the ne.xt 15 to 20 minutes the larva first

me

Microns

30

Figure 334. — Optical section of the gastrula stage of de-
velopment of the larva of 0. edulis according to Horst.
Reproduced from Pelseneer, IttOG. bl. — blastopore;
me. — mesodermic cells; sh. — rudiment of shell.

rotates around its dorsoventral axis and swinas
with the ciliated crown directed forward and up
toward the surface of the water. The trochophore
stage of C. virginica is short; in the laboratory at
22° to 24° C. it lasts no longer than 48 hours and
in some instances only 24 hours.

The ne.Kt stage is Icnown as veliger (from the
Latin "veliuii", veil; and "gerere", to carrjO- A
detailed account of the structure and development
of bivalve veliger was made by Meisenheuner
(1901) for Dreissensia polymorpha. MacBride
(1914) stated that the development of larvae of
Pecten, Teredo, Pholas, Cardium and Ostrea (in-
cluding Crassostrea) is virtually identical with that
of Drciiisensia. The early larval stages of these
forms are so similar that their recognition in
])lankton sam])les cannot be made with confidence
until their larval shells have been developed. The
structm'e of an early veliger of 0. edulis, described
by Yonge (1926, 1960), is similar to that of C.
nrginica and C. (jigas. The description given
below is based primarily on publications by Yonge
(1960) and Erdmann (1935) on 0. edulis.

The veliger (fig. 337) is a highly complex orga-
nism containing several larval organs which dis-
appear with the end of free-swimming life. The
most conspicuous among the larval structures is
the velum, v., which is formed by an outgrowth
of the lateral parts of the prototroch area in two
semicircular folds or lobes bearing large cilia along
their margins. The prototroch thus develops at

mes

0

Microns

30

Figure 335. — Optical section of the trochophore larva of
O. ediilis according to Horst, 1883. Reproduced from
Pelseneer, 1906. c. — cilia; ec. — ectoderm; en. — endo-
derm; m. — mouth; mes. — mesodermic cells; sh. — shell;
St. — stomach.

356

FISH AND WILDLIFE SERVICE

0

30

Microns

Figure 336. — Trochopliore of O. edulis according to Horst,
1S83. Reproduced from Pelseneer, 1906. a. — anus; e. —
esophagus; int. — intestine; m. — mouth; st. — stomach;
sh. — shell; pr. — prototroch.

the veliger stage into a powerful organ for swim-
ming. During swimmmg tlie velum projects be-
tween tlie valves of the shell. It is highly con-
tractile and at the slightest disturbance is with-
drawn between the valves by several velar
retractor muscles (r.v.), which are attached to
the velum and are anchored at the opposite end
to the sheU.

For examination of the velum, the larvae should
be narcotized with menthol, chloral hydrate, or
other narcotics, and made transparent with
glycerol. The larvae can be satisfactorily narco-
tized in a small dish l)y placing tiny crystals of
menthol on the sm'face of the water and allowing
them to relax before giving additional crystals.
When narcosis appears to be complete, glycerol
should be added slowly, drop by ch'op, to avoid
disturbing the larvae and causing th^m to con-
tract. The method is tedious, time-consuming,
and requires a great deal of patience.

liarge cilia around the margin of tlie velum are
for swuiiming; small cilia (not shown in fig. 337),
covering tlie base of the velum carry food particles
toward the mouth (m.). A relatively long esopha-
gus (e.) leads to a barreUike .stomach (st.), which
is in close contact with the glandular structure of
digestive diverticula (dig. d.). The crystalline
style sac (cr. s.) is at the lower part of the stomacli.
The intestine (int.) emerging from the stomach

makes a single loop and continues into the rectum
(r.); the anus (a.) opens into the mantle cavity
fm.c). The foot rudiment (f.) appears as a cili-
ated outgrowth of the body under the mouth and
reaches its full development toward the end of
larval life. The anterior adductor muscle (ant.
ad.), destined to disappear in older larvae, is con-
spicuous; the posterior adductor has not yet
developed.

The early larvae of C. virginica found in plank-
ton samples or developed in the laboratory are
oval-shaped and shghtly as3Tiimetrical. Because
the hinge side of their shells is straight, they are
called straight-hinge larvae or D-shaped larvae.
Rees (1950) refers to this stage as Prodissoconch I.
Dimensions of the larvae vary from 70^ to 75^ in
length, i.e., parallel to the hinge side, and from
60m to 68m in height, with the greatest distance
at a right angle to the hinge side. The prodis-
soconchs of C. virginica are shown in the photo-
micrographs in fig. 338.

Major changes take place in the appearance and
structure of the larva as it grows, reaches its full
development, and becomes ready to set. The ad-
vanced stages of larval development are called by
various descriptive names referring to the most
conspicuous morphological change of each stage:
umboned larva, eyed larva, adult, and mature
larva. The latter expression is frequently used

dig. d

ant. ad

m.c.

Microns

Figure 337. — Early free-swimming veliger of 0. edulis.
From Yonge, 1926. a. — anus; ant. ad. — anterior ad-
ductor muscle; cr.s. — crystalline style sac; dig.d. —
digestive diverticula; e. — esophagus; f. — rudiment of
foot; int. — intestine; m. — mouth; m.c. — mantle cavity;
r. — rectum; r.v. — velar retractor muscles; sh. — shell;
St. — stomach; v. — velum.

LAR\^AL DE\'ELOPMENT AND MET.-UVIORPHOSIS

357

B.

0 ^,■ 100

Microns

0

Microns

90

Figure 338. — Photomicrographs of early straight-hinge live larvae of C. virginica. A — larvae resting on bottom, shells
closed; B — the slightly narcotized larva (upper part) has its velum protruding from the shell; the lower larva has
closed its shell and withdrawn its velum.

by English-speaking oyster biologists in spite of
the obvious contradiction in applying the adjec-
tives adult and matiu'e to larval stages. The
term "velichoncha" proposed by Werner (1940)
and adopted by Rees (1950) refers to the advanced
stages of development of bivalve larvae, but the
expression is not generally used in malacological
literature. The name "pediveliger" was proposed
bj" Carriker ('1961) to designate the "swunming-
creeping" stage of clam larva, Merce/naria (Venun)
mercenaria. The term deserves to be accepted in
malacological literature because it indicates the
major character, i.e., the presence of a foot, and
is applicable to many bivalve species, including

oysters, in which a larval foot appears during the
planktonic period.

As the larva grows its valves become deeper and
almost circular. The liinge develops two bulgings
or umb(uies, the one on the left side larger than
its opposite number. At these stages the umbones
bend toward the posterior end of the shell, which
at this tune has pronounced concentric rings, is
heavy, and obscures the organs under it. In
swuiiming the umbo larva protrudes its large cili-
ated foot forward. The larvae of C. virginica
now have diameters of more than 300 /i in both
length and height. The photomicrograph in
figm-e 339 shows the side view of the advanced

358

FISH AND WILDLIFE SERVICE

Figure 339. — Photomicrograph of live, slightly narcotized umbo larva of C. virginica.

,

umbo larva of this species, slightly narcotized to
reduce its movements.

The anatomy of fully developed oyster larva is
known primarily from the work of Erdmann
(1935) on 0. edulis. Figure 340, reproduced from
his publication, shows the velum (v.) with a crown
of powerful ciHa arranged in a preoral ciliated
circle, and a ciliated aboral belt or zone covei'ed
with small cilia (ab.c).

Four pairs of velar retractors (r.v.) withdraw
the velum. The muscle bands consist of bundles
of cross-striated fibers along the dorsal side of the
body. The cross striation of the velar retractors
of oyster larva is typical for rapidly contracting
muscles. In swimming the veliger rapidly changes
the degree of expansion and the position of the
velmn, and withdraws the organ with great rapid-
ity when the valves begin to close. The striated
muscles in the larva indicate the high degree of
specialization of larval organs needed for the
organism to function effectively. The muscles of
an adult oyster are nonstriated. Tlieir contrac-
tions are relatively slow and do not require tlie
mechanism typical for the rapid movements of
the free-swimming organism. The apical sense
organ (a.p.o.) ("Scheitelorgan", according to

LARVAL DEVELOPMENT AND METAMORPHOSIS

Erdmann) and cerebral ganglion occupy a central
position in the crown of the velum. The function
of the apical organ is not known.

A new feature in the larval anatomy, not pres-
ent at earlier stages, is a well-developed foot (f.)
covered with strong cilia. The foot is highly con-
tractile and can be withdrawn by its retractor
muscle (f.r.). A byssus gland (b.g.) with a small
duct opening into the mantle cavity (m.c.) is
located at the base of the foot. Both the foot and
the gland are typical larval structm-es which dis-
appear after performing their function during the
attachment. Two muscles, the anterior and pos-
terior adductors (ant. ad. and post. ad.), close the
valves. The mouth (m.) is surrounded by a cili-
ated ridge which develops into the labial palps.
The esophagus (e.) leads to the stomach (st.),
part of which is covered with the gastric
shield (g.sh.). The crystalline style sac (cr.s.)
and digestive tubules have greatly increased in
size, and the ciliary motion inside them is acceler-
ated. The intestinal tract (int.) forms a loop
and ends in the rectmn (r.), which has an anal (a.)
opening into the mantle cavity (m.c). The rudi-
ment of heart and kidney is represented by a
gi'oup of cells (h.r.) shown in figm-e 340 above the

359

a.po

Figure 340. — Fully developed larva of 0. edulis viewed from the right side with velum and foot at the ventral side in
the uppermost position, typical for swimming. According to Erdmann, 1935. a. — anus; ab.c. — aboral belt of cilia;
ant. ad. — anterior adductor muscle; a.p.o. — apical sense organ and ganglion; b.g. — byssus gland; cr.s. — crystalline
style sac; d.div. — digestive diverticula; e. — esophagus; ey. — eye; f. — foot; f.r. — foot retractor muscles; g. — gill
rudiment; g.sh. — gastric shield; h.r. — heart and kidney rudiment; int. — intestine; m. — mouth; m.c. — mantle cavity;
p.g. — pedal ganglion; post. ad. — posterior adductor muscle; r. — rectum; r.v. — velar retractor muscles; st. — stomach;
stc. — statooyst; v. — velum; v.g. — visceral ganglion.

rectum. The gill ruduiient (g.), located between
the base of tlie foot and heart rudmient, consists
of a series of short, tubular channels. The pedal
ganglia (p.g.), a round structure at the base of
the foot, disappear with the dissolution of the
foot. The visceral ganglion (,v.g.) appears in its
permanent position at the ventral side of the
posterior adductor. The larval sense organs com-
prise a pair of statocysts (stc.) in the foot tissue
and a pair of dark pigmented eyes (ey.) which
develop toward the end of larval life. Their pres-
ence in the free-swimming larvae indicates the
approaching of setting and metamorphosis.

The nervous system of the larva, shown dia-

grammatically in figure 341, is more complex than
that of the adult oyster. It contains the pedal
ganglia (ped.g.), which are absent in the adult;
the pleural ganglia are present as a separate
structure and are connected to the statocysts
(syc.) and eyes, which disappear without a trace
during metajnorphosis. The visceral ganglia
(visc.g.) of the larvae are less conspicuous than in
the adult. All these organs are obviously neces-
sary to a free-swimming organism, and some of
them disappear with the loss of locomotion and
the change to a sedentary mode of living.

The anatomy of the fully developed larva of
C. viryinica is smiilar to that of 0. edulis. Figure

360

FISH AND WILDLIFE SERVICE

<S?. -S, o.

'H-

vise.

Microns

100

Figure 341. — Diagram of the nervous S}'stcm and sense
organs of fully developed larva of 0. edulh. According
to Erdmann, 1935. a.s.o. — apical sense organ; e.g. —
cerebral ganglion; ey. — eye; ped.g. — pedal ganglia;
pl.g. — pleural ganglion; stc. — statocyst; visc.g. — visceral
ganglia.

342 shows the structure of the larva as it appears
in the narcotized live specimen. The drawing is
a composite from a number of photographs of
live larvae taken with the microscope magnifica-
tion of about 100 X, and from examination under
higher power of specimens mounted in glycerin
jelly. Only the organs visible under these con-
ditions are shown in this illustration. The larvae
were at the last stage of development, over 300/u
in height, with eyes (ey.) and a well-developed
foot (f.). The velum was large with long cilia at
the top and a row of shorter ones forming an
aboral circle (ab.c.) at the base. The apical
organ could not be seen in the whole mount prep-
arations. The retractors of the velum (r.v.)
were well developed. As in 0. edulis they con-
sisted of rapidly contracting bands of striated
fibers. When the velum is completely withdrawn
within the shell cavity, the valves close and the larva
drops to the bottom. In a contracted state the
different organs become undistinguishable. The
well-developed foot (f.) contains a large byssus
gland (b.g.). During swimming it protrudes be-
tween tlie valves and is kept in the direction of
swimming. The tip of the foot frequently turns

right or left and up and down while the larva is
swimming. This behavior suggests that it serves
to orient the movements. At the last phase of
larval life the foot is used for crawling over the
hard surface where the young oyster will finally
attach itself. The funnel-shaped mouth (m.)
leads to a narrow and long esophagus (e.), which
opens into a barrellike stomach (st.) partially
surrounded with massive and dark digestive diver-
ticula (d.div.). The intestinal loop (int.) and
rectum (r.) are similar to those described for
0. edulis. Both adductor muscles (ant.ad. and
post. ad.) are well developed. The gill rudiment
(g.) appears as a strand of cells in the mantle
cavity, and the beating of the heart (h.), located
between the stomach and the posterior adductor,
can be seen in live specimens. At 24° C. the
beating of the heart is rapid, varying from 80 to
100 pulsations per minute.

Food apparently is gathered by the ciliary
mechanism of the velum, and small food particles
can be observed entering the esophagus and
moving inside tlie stomach wliere they are rotated
by the ciliary epithelium. The ciliated apparatus
of the gills has not yet fully developed, and food
is gathered only by the aboral circle of the velum
(ab.c.) and by the labial palps around the mouth.
The statocysts (stc.) and the eye (ey.) are well
formed. In a tangential section the eye of C.
tirtjimca appears as a transparent lens surrounded
by a circle of darkly pigmented cells (fig. 343).
The dark liand is a short branch of a nerve leading
to the eye.

The highly developed ciliary mechanism of the
velum and the rapidly contracting velar retractors
are essential to the life of a free-swimming larva.
Their structure appears to be better developed
than those of the muscles and ciliary epithelium
of adult oysters. Electron microscopy reveals
that the ciliated cells of the velum ha\'e a highly
complex system of basal bodies and rootlets with
distinct periodicity (fig. 344). Intercommunica-
tion between adjacent cilia through the basal
bodies and their branches pro\-ides a system for
tlie coordination of ciliary motion. The com-
plexity of the ultrastructure conforms to the com-
plexity of the ciliary activity of the velum, making
it possible for the larva to swim in any direction,
to turn around, or instantaneously to stop ciliary
activity. The ciliated cells of the velum are very
large; their surface is covered with microvilli, and

LARVAL DEVELOPMENT AND METAMORPHOSIS

361

post, ad

Figure 342. — Optical section of fully dpvelopcd larva (pediveligcr) of C. virginica viewed from the left side, m swimming
position. Composite drawing from a numlier of photomicrographs of live, slightly narcotized larvae and whole
mounts m glycerol, a. — anus; ab.c. — aboral circle of cilia; ant. ad. — anterior adductor muscle; b.g. — byssus gland;
d.div. — digestive diverticula; e. — esophagus; ey. — eye; f. — foot; f.r. — foot retractor muscles; g. — gill rudiment; h. —
heart; int. — intestine; m. — mouth; m.c. — mantle cavity; post.ad. — posterior adductor muscle; r. — rectum; r.v.
velar retractor muscles; st. — stomach; stc. — statocysts; v. — velum.

they contain lartce oval mitochondria close to the
rootlets.

The high degree of specialization of lar\al organs
may he regarded as an adaptive organization of a
free-swunming organism to its environment and
may have no phylogenetic significance. The
pelagic larva of a hivalve has a douhle task: to
distribute the species and grow into an adnlt.

The performance of these tasks requires the main-
tenance of an equilibrium between the locomotive
efficiency and the weight to be carried; this main-
tenance is accomplished by the development of
the velum. As the shell grows and becomes
tiiicker and heavier, the task of swimming becomes
more difhcult, and the fully grown larva sinks to
tiie bottom more rapidly and possibly more often

362

FISH AND WILDLIFE SERVICE

f^

Figure 343. — Photomicrograph of a tangential, slightly
slanted section of the larval eye of C. virginica preserved
in osmie acid.

than it does at the straight-hinge stage. When
the larva attaches to the substratum, the velum
and the foot are no longer needed. Their disap-
pearance marks the transition from free-swimming
to a sedentary mode of life. Garstang (1929)
expresses the correct opinion that larval organs
should be regarded as an adaptation to the con-
dition of life during development and need not
affect the organization of the adult. His charming
book on larval forms (Garstang, 1951) summarizes
in a somewhat unorthodox way the ideas and
theories concerning the significance of various
larval forms in the evolution of aquatic animals.

MORPHOLOGY OF LARVAL SHELL

The morphology of the larval shell differs from
that of an adult oyster primarily in the greater

t

#

|l

t "i^.

m

\

V.

X

\«

\

Micron

Figure 344. — Electron micrograph of a tangential section of a portion of a ciliated cell of the velum of the larva of C.

virginica.

LARVAL DEVELOPMENT AND METAMORPHOSIS

363

complexity of the hinge apparatus of the prodis-
soconch. The hinge ensures the exact closui-e of
the valves and prevents them from sliding on each
other under uneven pressure. Consequently, as
the larva grows the hinge apparatus increases in
strength and complexity. According to Bernard
(1898), who made an extensive study of the
ontogeny and morphology of larval shells of
bivalves, the straight part of the dorsal shell
margin thickens to form a provinculum (from the
Latin "pro", before, and "vinculum", bond or
band) or primitive hinge. The provinculum (by
definition) always bears teeth or is shaped into
toothlike projections which fit into the correspond-
ing gaps of the opposite valve.

On the basis of the liinge structure Rees (1950)
proposed a system of classification of bivalve
larvae that greatly facilitates their recognition
in plankton samples (fig. 345). He postulated
that each superfamOy of bivalves has a distinct
type of larval hinge; that the shape of the hinge
is typical as a generic and species characteristic;
and that the texture of the larval shell can be used
in certain cases in the recognition of a species.
In the families Pteriacea and Ostreacea the hinge
apparatus consists of a series of small, uniform
teeth (taxodont teeth) in the central portion of
the strip and a few larger rectangidar teeth with
clear gaps between them (fig. 346) at the posterior
section. The distinguisliing features of the species
of these families is the absence of lateral and
special teeth and of flanges, i.e., the thick edges
of the valves on both sides of the provinculum.
The hgament lies between the posterior rectangular
teeth and the taxodont strip (Bernard, 1898;
Borisiak, 1909). In 0. edulis there are some large

RIGHT

Figure 34.5. — Type of hinge of the families Ostreacea and
Pteriacea. Redrawn from Rees, 1950.

364

Figure 346. — ^Drawing of a 5- to 6-day-old prodissoconch
of C. virginica, 70 m long, examined from the dorsal
side.

corrugations anterior to the taxodont teeth (Rees,
1950), but their taxonomic value is doubtful.

Differing arrangements and numbers of taxodont
teeth in the shells of various species of oyster
larvae are used for their identification. The
straight-hinge line of a 5- to 6-day-old larva of
C. mrginica grown in laboratory culture has two
groups of rectangular teetli that can be clearly
seen by examining the shell from the dorsal side
(fig. 346). At this stage there is only a slight
difi"erence between the upper (riglit) and lower
(left) valves. The difl^erence becomes more
pronounced as the larva reaches the umbo stage.
In a series of papers Ranson (1943, pp. 52-58)
attempted to establish the classification of all
adult Ostreacea on the basis of the fully developed
prodissoconchs. Essentially this work was based
on the investigations pubhshed long ago by Bernard
(1898) and Borisiak (1909). Ranson (1960)
separates the oysters into three genera: Pycno-
(lonte, Crassostrea, and Ostrea. Each genus, accord-
ing to his data, is determuied by the character of
the final prodissoconch hinge and tlie position of
the ligament in relation to the hinge. He con-
cludes his paper with the statement that "as far
as the Ostreidae are concerned, the species can now
be established on a firm basis, which so far had
never been done by studying the adult." The list
publislied by Ranson includes 5 species of Pycno-
donte, 12 species of Crassostrea, and 19 species of
Ostrea. Unfortunately the diagnosis is given only
for each genus without descriptions of taxonomic
cliaracters which are shown by the illustrations.
The drawings referring to the five species found in
the waters of the United States are reproduced in
figures 347 through 351. Ranson's text does not
include the larvae of C. gigas or 0. equestris.

Ranson states also that the oysters can be cor-
rectly identified by the structure of the larval

FISH AND WILDLIFE SERVICE

0

Microns

300

Figure 347.— Prodissoconch of C. virginica (Gmelin). Inner view of the valves. Left valve on the left and right valve
on the right. Knoblike structure indicates the location of the ligament. From Ranson, 1960.

shells still visible on tiie shells of adults. Examina-
tion of the many shells of adult C. virginica, C.
gigas, C. rhizophorae, and 0. equestris in my collec-
tion did not reveal the structure of their larval
shells, which in many instances appeared to he
eroded or were missing. It is doubtful tiiat
Ranson's method of identification of adults by
tlieir larval shells will gain acceptance by ta.vono-
mists. Comparison of liis illustrations of the
closely related species, such as C. virginica and
C. rhizophorae, indicates no significant differences
between the two. On the otiier hand, his set of
drawings of pelagic prodissoconclis may be useful
for planktonologists, at least for separating the
tlu'ee genera of ovster larvae.

ATTACHMENT AND METAMORPHOSIS

I-iarval life ends when the oyster attaches itself
to a substratum. This event is called setting,
settlement, or spatfall; the different expressions
are used interchangeably and are synonymous.
The word setting is commonty used bj^ American
biologists and oyster growers; the expressions
settlement and spatfall are more frequently found
in Canadian and British publications. The term
setting will be used tlu-oughout this text except in
quotations from other authors.

The fully developed larva of C. virginica swims
with its foot projecting between the valves.
When the foot touches a solid sm'face, the larva

0

300

Microns

Figure 348.— Prodissoconch of C. rhizophorae (Guilding). Arrangement as in figure 347. From Ranson, 1960.

LARVAL DE\ELOPMEXT AND METAMORPHOSIS 365

733-851 O — 64 24

0

300

Microns
FiQiiRE 349. — Prodissoconch of 0. edulis Liiine. Arrangement as in figure 347. From Ranson, 1960.

stops swimming, the velum is partially withdrawn,
and the larva begins to crawl on its foot. This
behavior may be changed suddenly by the re-
sumption of swinnning; the foot may be with-
drawn, tlie velum expands again, and the larva
swims away. When it is ready to set, the larva
crawls until it encounters suitable condition for
final attachment.

Phases of setting of C. rinjinica were recorded
by a motion picture camera nearly 30 years ago
(Prytherch, 1934) and the photogi'aphs were
recently reproduced bj^ Medcof (1961, p. 19).
To facilitate photography, the larvae were ce-
mented with marine glue on their left valves to a
glass slide whicli was tilted at a 45° angle. Under
such conditions the larvae had no free choice in
selecting the place for attachment, and the records
obtained in this manner do not represent normal
behavior. The attachment of fully developed
larvae can be observed, however, by placing them

0

Microns

300

Figure 350. — Prodissoconch of 0. lurida Carpenter.
Arrangement as in figure 347. From Ranson, 1960.

in sea water in a petri dish and observing their
behavior with a binocular microscope.

The foot of the larva extends forward, its tip
attaches temporarily to the substratum, and the
whole body is pulled over by the contraction of the
foot. The direction of crawling changes and
occasionally reverses as the foot extends at dif-
ferent angles. The movement continues for some
time, gradually becoming shorter and slower.
Finally the foot extends far beyond the edges of the
shell, the larva turns sideways with its left valve
touching the substratum, and comes to a standstill.
The attachment is made permanent when the
byssus gland discharges a cementing fluid, which
sets within a few minutes (Nelson, 1924). A
similar process takes place in tlie setting of 0.
eduJis and is probably common to other species of
oysters.

The change from larva to juvenile oyster (spat)
then begins innnediately. The process of this
metamorphosis is better known for O. edulis than
for other species of oysters, for it has been studied by
Davaine (1853), Huxley (1883), and more recently
by Cole (1938b). The work of early European
zoologists influenced the study of the American
oyster to such an extent that in several instances
the description of the metamorphosis of C.
virginica has been repeated almost verbatim from
studies on 0. edulis with only slight changes
(Ryder, 1883; Jackson, 1888, 1890). A somewhat
more detailed account of the transformation of
larva into spat of C. virginica and 0. lurida was
given by Stafford (1913).

During the metamorphosis the larval organs

366

FISH AND WILDLIFE SERVICE

0

300

Microns
Figure 351. — Pro disso conch of Pycnodonte hyolis (L.). Arrangement as in figure 347. From Ranson, 1960.

disappear and there is an anatomical reorganiza-
tion of the permanent organs. At this time the
rehitive size of the organs and their orientation
are changed. The extent of topographical changes
in the relative position of organs during the transi-
tion from larva to spat can be appraised by com-
paring the position of some of the larval organs
with that in the adult oyster. In figure 352 the
principal organs of the early larval (1), fully
grown larva (2), and of the juvenile oyster or
spat (3) of 0. edulis are shown diagrammatically
in three drawings oriented along the dorso-
ventral axis. The mouth (m.), nearly ventral
in the larvae (1, 2), has shifted counterclockwise
(when viewed from the right side of the oyster)

about half the periphery of the larva and in the
spat occupies an area in the anterio-dorsal part
near the hinge. The position of the anus (a.)
changes in the same direction, from the dorso-
posterior part in the larva to dorso-ventral in
the adult. The retractor muscles of the velum
(r. V.) disappear by the end of the larval period
and in the spat and adult are replaced by the
radiating and marginal pallial muscles.

The most conspicuous and rapid changes take
place in the velum. Davaine (1853) suggested
that in 0. edulis the velum is cast off about the
end of the larval period, a conclusion not con-
firmed by Ryder (1883) and Stafford (1913).
Illustrations by Meisenheimer (1901) of the larva

DORSAL

DORSAL

ant add Q
post ad

d.div

VENTRAL

I.

DORSAL

post add

VENTRAL

Figure 3.52. — Diagram showing the changes in the topographical relation of various organs of 0. edulis during the transi-
tion from free-swimming larva (1) to fully developed larva ready to set (2) and juvenile oyster (3). From Erdmann
(1935). a. — anus; ant. ad. — anterior adductor; ey. — eye; f. — foot; g. — gills; int. — intestine; l.p. — labial palps;
post. ad. — posterior adductor; r.v. — retractors of velum; v. — velum.

LARVAL DEVELOPMENT AND METAMORPHOSIS

367

post, add.

Figure 353. — Sagittal section of spat of 0. edulis about 24 hours after attachment, ant. add. — disintegrating anterior
adductor muscle; ap. — apical area of the velum; e.g. — cerebro-pleural ganglion; f. — foot; mn. — mantle; m. — mouth;
post. add. — posterior adductor muscle; s. — stomach; v. — velum; v.g. — visceral ganglion. After Cole, 1938b.

of Dreissensia polymorpha and unknown to
Stafford showed very clearly that the velum
of this bivalve disintegrates and is absorbed,
and that the apical area comes to lie outside the
esophagus and later is fused with the upper lip
of tiie moutii to form the basis of the labial palps.
Cole (1938b) sliowed in a series of sections of
0. ediilift that as the velum collapses almost
immediately after setting, its entire structure
is moved upward and forward. Most of it is
either cast off or disintegrates, and parts of it
probably are swallowed. The apical area or
apical plate of the velum becomes detached from
siuTounding tissues and sinks to a position dorsal
to the esophagus below the surface of the body
(fig. 353, ap.) where it fuses with the upper lip
of the mouth. Subsequently the thickened upper
lip extends laterally to form tlie upper labial
palps. The cerebro-pleural gangial (e.g.) can
be seen underlying the apical plate. In 4S hours
all traces of the velum disappear.

Reabsorption of the foot begins after the dis-
charge of the contents of the byssus gland during
attaciiment. Tlie foot gradually shrinks and pro-
jects behind the mouth as an irregular mass of

tissue covered with ciliated cells. Phagocytes
invade the interior of the foot and digest the tissue.
The disentegration of the foot of 0. edulis is
completed in about 3 days.

The fully developed oyster larva has two
adductor muscles. The posterior muscle, dis-
covered by Jackson (ISSS, 1890), is not found in
the early veliger but appears in the umbo larva.
Botli muscles are of approximately equal dimen-
sions. Following attachment the anterior muscle
degenerates while the posterior moves counter-
clockwise in the same direction as the mouth and
anus.

The eyespots of O. edulis break down and dis-
appear after the first 24 hours of attached life.
Tlu' outlines of the epithelial cup become irregular
t)orause it is invaded by phagocytes that ingest
tlie pigmented eye cells, thus causing the liberated
])igment to lie in irregular clumps.

Many pliases of larval-metamorphosis, especially
of the ( 'raxxostred group of oysters, are inadequately
known and need to be more critically studied.
With advances in the technitiue of artificial
rearing of oyster larvae this gap in tiie knowledge
of oyster biology may soon be filled.

368

FISH AND WILDLIFE SERVICE

DISPERSAL OF LARVAE

During the 2 or 3 weeks of free-swiniming life
the hirvae of C. virginica are more or less passivelj-
carried by currents and are \videl.y distributed in
coastal waters. Biologists who have studied the dis-
tribution of planktonic bivalve larvae (Thoreon, 1946)
agree that their swimming is not strong enough to
overcome the water movements which transport
them far from the spawning grounds. To a
certain e.xtent larvae combat the currents by
closing their valves and sinking to the lower level
of the water colunm or to the bottom. However,
observations of the swTmming habits of artificially
raised larvae of C. virgimca kept in tall containers
in the laboratory show that most of them remain
swimming nearly all the time, and only those
that appear to be too weak or are infected by
fungi settle to the bottom.

Various methods are used in oyster research to
study the distribution of larvae by taking quanti-
tative samples, but none are satisfactory, and the re-
sults obtained by the different methods are not
comparable. A pump for pumping measured
volumes of water from different depths, plankton
tow samplers of various designs, plankton
traps, and bottle collectors of the type described
by Thorson are the devices used in the study of
vertical distribution of oyster larvae. The plank-
ton tow net is most frequently employed. Larvae
may be filtered out through screens, or a pre-
served sample of water may be placed in a glass
cylinder with the bottom drawn into a funnel with
a drain cock. The water may be centrifuged at
high speed using the Foerst type electric centrifuge
designed primarily for the collection of minute
organisms that ordinarily pass through the finest
mesh of the plankton net.

Many observers have found that newly attached
young oysters far outnumber the free-swmiming
hirvae, particularly of the umbo stage, found in
plankton samples (Prj'therch, 1924; Galtsoff,
Prytherch, and McMillin, 1930; Loosanoff and
Engle, 1940). Similar observations concerning the
scarcity of larvae of 0. eduHs were reported by
Sparck (1925) for Limfjord waters and by Gaarder
(1933) for two Norwegian oyster ponds where the
oyster larvae were present only in the deeper and
saltier layers of water. Observations on the
abundance and distrilnition of oyster larvae made
m this country and abroad have been adequately
reviewed by Korringa (1941).

The problem of adequacy of plankton sampling

LAinAL DEVELOPIIEXT AND METAMORPHOSIS

in relation to the physical and chemical hydrology
of the James River, Va., oyster seed bed area was
mvestigated by Pritchard (1952, 1953). His
calculations show "that the concentration of late
stage larvae in the overlying water sufficient to
produce the large observed set needs to be, on the
average, only about one larva for 100 liters."
Since the basic sampling employed in these
studies of distribution of oyster larvae was 100 1.,
the inadequacy of such a sampling technique is
obvious and some better automatic sampling
methods should be used to clarify these obscure
points of larval behavior.

In estuaries the vertical distribution of larvae
seems to depend on changes in the velocity and
dn-ection of tidal cuiTents and the vertical salinity
gradients. The oyster larvae have a more or less
uniform vertical distribution in rearing tanks
(Cole and Knight -Jones, 1939) and in the estuaries
and bays wherever water mixing has prevented the
formation of vertical gradients of temperature and
salinity.

Several observers have attempted to correlate
the distribution of larvae with different stages of
tides. Julius Nelson, one of the pioneer students
of the biology of the larva of C. virginica m New
Jersey waters, believed that the larvae could
migrate toward land by rismg at the beginning of
flood tide and settling to the bottom before the
tm-n to the ebb. By this reaction to tidal changes
their dispersion in tidal estuaries is avoided.
This idea influenced the research of his son,
Thm-low Nelson, and his students, who modified
and elaborated the original concept (Nelson,
1917, 1921; Nelson and Perkins, 1931; Carriker,
1951).

According to these observations, which were
made in New Jersey estuaries, the swarms of
larvae are distributed along definite lanes up and
down stream from the spawning grounds. If the
salinity of water is uniform from bottom to surface,
the gi-eatest number of larvae is found at the level
of the highest current velocity. In bodies of
water with distinct salinity stratification the
larvae congregate just above the zone of greatest
salinity change. Nelson beUeved that the ad-
vanced larval stages drop to the bottom and
remain near it during slack water, and that the
increased salinity of early flood tide stimulates
their swimming upward. This performance re-
peated at each change of tide enables the larvae
to move upstream by progressive stages. This

369

mechanism, if true, would explain the location
of many setting areas in tidal rivers above the
principal oyster gi-ounds. The theory has stimu-
lated a great deal of field observation, but, un-
fortunately, the experimental evidence upon
which it rests has not been fully documented.
Only a few laboratory observations have been
made on the effects of changes in current velocities
and salinity on the behavior of oyster larvae, and
the experiments reported by Nelson and Perkins
were performed luider the most primitive con-
ditions. Great experimental difficulties were in-
volved in conducting this type of study, and the
elaborate equipment necessary for recording larval
behavior was not available to the investigators.

Observations on larval distribution in waters
other than New Jersey differ from those described
by Nelson and his associates. Prytherch (1929)
states that in MUford Harbor, Conn., "the oyster
larvae were found to be most abundant at the
time of low slack water and gradually disappeared
as the tide began to run flood." He further states
that no larvae could be found swimming in the
water when the flood current had reached a veloc-
ity of 0.6 foot per second, and supports this state-
ment by observations on oyster larvae kept in a
tank. Oyster larwie remained swimming in the
tank while the water was at a standstill, but
dropped to the bottom wlien the current velocity
produced by artificial circulation was from 0.3 to
0.5 foot per second. The experimental technique
was very primitive, and the results cannot be
considered convincing. Observations made by
Loosanoff (1949) in Long Island Sound do not
confirm Prytherch's interpretations. No evidence
was found that early and late umbo larvae were
common near the bottom. On the contrary, in
several instances "their number was greatest mid-
way between the high and low water when the
tidal current was near the maximum velocity."
A similar conclusion that larvae do not descend
during periods of ra])id tidal flow was reached by
Carriker (1959) from studies of conditions in a
salt-water pond on Gardiners Island at the eastern
end of Long Island, N.Y.

Current views of tlic movement of oyster larvae
up estuaries were summarized by Carriker (1961).
The consensus of opinions of those who studied
the problem in typical estuaries indicates that
fully developed larvae (pediveligers) luive a tend-
ency to remain in lower, more saline strata and
are passively convej'ed toward the upper reaches

370

of an estuary by the net, nontidal flow of deeper
and denser layers of water (circulation in the
estuaries is discussed in Chapter XVIII, p. 402).
The discrepancy between the observations made
in New Jersey waters and in Long Island Sound
may be explained by differences in hydrography.
Long Island Sound is not an estuary in the strict
meaning of the term, but can be regarded as an
embayment with several true estuaries, as for
instance, the mouth of the Housatonic River,
Milford Harbor, New Haven Harbor, and many
others. The distribution of the larvae in the
vSound is not, therefore, comparable to that ob-
served in New Jersey waters. Further, salinity
change from surface to bottom is small, rarely
exceeding 27oo,and there is considerable exchange
of sea water between the Sound and the outside
waters. Under these conditions one may expect
substantial losses of larvae during a tidal cycle.
It is known that abundance of fully grown larvae
in the Sound area is so low that quantitative
sampling is not reliable.

The evidence that oyster larvae are actually
conveyed by tidal current to the upper part of a C
tidal river is provided by the investigations of
Dimick, Egland, and Long (1941) on 0. lurida in
Yaquina Bay, Oreg. Yaquina Bay and River is
a short estuary, about 12 miles, on the coast of
Oregon. The natural oyster beds cover only 101.9
acres. Plankton samples taken systematically at
known distances from the mouth of tlie bay showed
that "up-river limit of the free-swimming larvae
was . . . approxinuitely 4 miles above the upper
limits of the natural oyster beds." No larvae
were found in this area at near low tide. There
is no doubt that these larvae were carried up-
stream by flood tide.

Lack of agreement on the results of field ob-
servations on the relation of larvae to tidal stages
is the result of inadequacy of sampling techniques
and a lack of understanding the responses of the
larva to environmental changes. Changes in
temperature, salinity, current velocities, oxygen,
and food content of water vary in each estuary, so
the occurrence or absence of larvae camiot be
related to a given tidal phase unless the major
conditions during this stage of tide are fully
understootl and their effects on larvae are known.
The volume of water transported by ebb flow
in estuaries usually exceeds the volume of water
le-cntciing at flood, the difference being equal to
tiie volume of river discharge at the head. If

FISH AND WILDLIFE SERVICE

the larvae are unifonnl}- distributed in the water
and swiin most of the time, a certain percentage
of them will be carried away and lost in the sea.
Manj- more are lost as prey to enemies, disease,
and other causes.

In the light of present knowledge only two
general assumptions regarding the larval behavior
can be made: oyster larvae are able to move by
their own power within only a very limited area,
and they are dispersed by tidal currents beyond
the immediate vicinity of spawning grounds.

A survival relationship exists between the age
of the larvae and tidal cycles. After analyzing
daily counts of larvae of O. ediilis in plankton
samples taken in Oostersheld, Holland, Korringa
(1941) concluded that the longer the duration of
the pelagic period the greater is the loss of larvae
and the lower is the percentage reaching maturity:
In 6 to 7 days, equal to 13 tides, 10 percent reach
maturity; in 10 days, equal to 19 tides, 5 percent
reacli maturity; and in 12 days, equal to 23 tides,
2.5 percent reach maturity. If the original num-
ber of larvae is A and the rate of dispersal and
other losses of larvae are equal during their free-
swimming period, the number of larvae at the
completion of pelagic life is A(l-l/p)" where 1/p
is the decrease during one tidal cycle and n is
the number of tides. The loss dm-ing one tidal
cycle is estimated by Korringa at between 13 to
15 percent. About 10 percent of the losses he
attributed to predators and only about 4 percent
to tides. Because of the greater duration of the
pelagic life of the oviparous f. inrritiuca, it is
reasonable to expect that losses of larval popula-
tions of this species probably exceed those deter-
mined by Korringa for the larviparous 0. edulis.

It is generally known that mortality among the
planktotrophic larvae during their pelagic life is
tremendous and that only an insignificant per-
centage of them reach metamorphosis. Korringa
made an interesting computation which shows
tliat out of one milUon 0. edulis larvae produced
in Oostershelde only about 250 attach themselves
and metamorphose, and of this newly set spat 95
percent die before the onset of winter.

The rate of survival of larvae of C. rirginica
and the percentage reaching attachment are not
known, but the principles of Korringa 's method
can be applied to the American species. His
studies show that the success of oyster setting
depends on prolific and shnultaneous spawning of
oysters in an estuary. By determining the abun-

LARVAL DEVELOPMENT AND METAMORPHOSIS

dance of larval population and the rate of exchange
of water during a tidal cycle, an estimate can be
made of the intensity of the forthcoming setting,
barring, of course, unforeseen circumstances which
may destroy the larvae.

REACTION OF LARVAE
TO EXTERNAL ENVIRONMENT

Little is known about the reactions of larvae to
changes in temperature and salinity of water.
Temperature fluctuations dm-ing the reproductive
season apparently have no direct effect on the
beha^^or of larvae of C. virginica, 0. edulis, and
C. gigas. Davis (1958) has demonstrated in a
series of laboratory tests that the reduction of
sahnity from the normal (for Long Island Sound
oysters) level of 267oo to 277oo to 157oo has no ef-
fect on the growth of larvae and that inhibition of
wrowth became noticeable in salinities of 12.5°/oo
and lower (Davis and Ansell, 1962). In water of
107oo salinity 90 to 95 percent of the larvae died
by the 14th day, and at a salinity of 5°/oo they
appeared to be moribund within 48 hours. In
these experunents the behavior of larvae was not
recorded. It would be interesting to repeat these
studies and determine the reactions of larvae to
sudden and to gradual changes of salinities.

Vertical distribution of larvae of 0. edulis ap-
parently is not affected by light (Korrmga, 1941).
This is probably true also for the larvae of C.
virginica, but because no experiments have been
made under controlled laboratory conditions, it is
prematm-e to assume that larvae of the American
oyster are not sensitive to light. The phototactic
responses of larvae to light intensity and color
have not been explored, but the presence of the
eye in the fully developed larva suggests that this
oi-gan is somehow used during the last days of
lanal life. Before attachment the larva crawls
over the surface exploring the substratum with
its foot, whicli acts as a tactile organ. It has not
been established that the eye participates in this
exploration. Nelson (1926) beUeves, however,
that the "eyed" larvae of C. virginica are stimu-
lated by light and continue to move until they
reach a shaded place where they become quiescent.
Hopkins (1937) expresses the opposite view and
states that in settmg of 0. lurida light is not an
orienting factor. He inclines toward Prytherch's
(1934) view that the larval eye has an entirely
different function. Since neither of the quoted
authors can corroborate theu- impressions by ex-

371

perimental evidence, the whole question of the
factors influencing the behavior of oyster larvae
at the time of setting needs to be examined.

TO THE ANGLE OF SURFACE

French oyster growers take advantage of the
preference of oyster larvae for the under surfaces
of submerged objects and use special spat collec-
tors made of tiers of tiles set one upon the other
with their concave surfaces underneath. New
spat is always found in larger numbers on the
lower surfaces. According to Cole and Kjiight-
Jones (1939) the larvae of 0. edulis reared in large
tanks in Conway, Wales, set more mtensely on
the under surfaces of test shells. A study of the
effect of the angle of a flat surface on the attach-
ment of larvae was made by Hopkins (1935) in
his work on 0. birida of the Pacific Coast. He
used glass plates, each 2,400 scj. in., placed at
different angles over the oyster grounds. Tlie
under hox-izontal surface was designated as 0° and
the upper horizontal as 1S0°. Other plates were
set at 45° intervals between the two extremes.
The average number of larvae attached to each
surface were:

0° (under horizontal) 1, 195

45° 181

90° 11

135° 3

180° (upper horizontal) I

Similar observations were made by Schaefer
(1937) with the larvae of C. gigas. He set 150
glass plates in positions varjang by 45°; some of
the plates were parallel to the du'ection of the
tidal current while others were transverse to it.
The plates were left for 5 days before the spat
were counted. There is a functional relationship
between the intensity of setting and the angle of
the surface on which the larvae set, the number
of larvae being greatest on the under horizontal
surface (0° angle) and lowest as the angle ap-
proaches 180°. The curve shown in figru-e 354
is drawn between the points taken as the average
numbers of larvae attaching during 5 days on a
glass surface held at different angles. The curve
is hyperbolic. Schaefer attributes the setting be-
havior of C. gigas to the upward position of the
foot of the swimming larvae and possibly to nega-
tive geotaxis. No experimental evidence is given
to substantiate either point.

The behavior of oyster larvae does not differ
from that of many other fouling organisms which

372

2

CO

li-
o

cr
iij
m

2

300

200

100
n

\

\

\

V

' ^^*--*--^

45 90 135

ANGLE OF SURFACE

180

Figure 354. — Effect of angle of the surface of plate glass
on the number of spat of C. virginica which attach to
it within 5 days, (llass area 2,400 sq. in.; 0° is under
horizontal surface. (Figure 1 from Schaefer, 1937).

were found by Pomerat and Reiner (1942) to
attach in greatest abundance to the imder sm'-
faces of plates held in a horizontal position.

Contradictory residts were obtamed, however,
in experiments with cement covered boards held
at several angles either suspended in water or
placed near the bottom. These tests made by
Butler (1955) on oyster bottoms near Pensacola,
Fla., showed a preponderence of spat on the upper
surfaces of the boards. Setting on upper sur-
faces (135° and 180°) comprised 78 percent of
the total number set, and only 18 percent were
counted on the lower surfaces. The remaining 4
percent were found on vertical boards. Butler
was not able to confirm the results of Pomerat's
and Riener's tests made earlier at Pensacola in
which frosted glass plates suspended in water
attracted the greater percentage of oysters (and
barnacles) to the lower surfaces. Bonnot (1937,
1940) built a spat collector from plywood strips
dipped in a mixture of cement and sand. He
fastened five or six strips of plywood one above the
other so tliat the horizontal surfaces were sepa-
rated by three-fom-ths of an inch. Setting of 0.
Iiin'da averaged 98 larva per square inch on the
upper surfaces and 67 per square inch on the
lower ones. The turbulent cm-rent caused by
narrow spaces between the strips possibly was

FISH AND WILDLIFE SERVICE

responsible for the greater number of larvae setting
on the upper siu-faces.

A lack of consistency in observations of various
investigators in different environments indicates
that it is impossible to ascertain the effects of a
single factor of the environment while testing
under complex and variable natural conditions.
Real progress in the study of the reaction of
oyster larvae may be achieved if further observa-
tions are made under controlled conditions.

Larvae of C. virginica that are grown artificially
in culture jars and not disturbed by stirrmg or
aeration are more or less uniformly distributed.
Eyed larvae frequently congregate on the sm-face,
swimming with their vela uppermost and touching
one onother with the tips of the cilia. They
form groups or "rafts" visible to the naked eye.
Some of them close their valves, fall rapidly to the
bottom, and after a short time resume swhirming.
Falling to the bottom should not be confused with
negative geotaxis, which has not been demon-
strated for oyster larva. In the laboratory, larvae
often attach themselves to the sides of plastic or
glass containers and apparently do not discrmii-
nate between light and dark surfaces.

TO THE PROPERTIES OF SURFACE

Oyster larvae attach themselves to many kinds
of hard and semihard surfaces. They are found
on rocks, gravel, cement, wood, shells of other
mollusks, on stems and leaves of marsh grass, and
on a great variety of miscellaneous objects such
as tin cans, rubber boots and tires, glass, tar paper,
and pieces of plastic that may be accidentally
thrown on the bottom or deliberately used as spat
collectors. There is no evidence that the larvae
are selective in finding a suitable place to set,
provided the surface is not covered with a slimy
film, detritus, or soft mud. Under natural con-
ditions they are never found on shifting sand or
on a bottom covered with loose sediment. Success
of setting always depends primarily on the avail-
ability of clean surfaces rather than on other
factors. Shells covered with oil and greasy sub-
stances in polluted areas are not suitable for the
attachment of larvae. Cole and Knight-Jones
(1939) found that it is difficult to induce 0. edulis
to set on smooth glass, but the larvae of C.
virginica raised in the laboratory readily attach
to polished glass. In fact live preparations of
spat may be obtamed for microscopic examination

of small oysters by suspending glass slides in a
tank with fuUy grown larvae.

GREGARIOUSNESS

An interesting gregarious tendency has been
observed by Cole and Knight-Jones (1949) among
the larvae of 0. edulis. During experunents m
large rearing tanks they found that larvae set
more readily on shells akeady bearmg 50 to 100
spat than on shells bearing fewer spat. They
suogest that a substance secreted into the siir-
rounding water by the spat, and possibly by the
fully developed larvae, encourages the setting.
No attempts were made to isolate the substance
and test its effect. The authors make another
observation which may throw some doubt on the
validity of their interpretation. They state (p. 36)
that "Larvae set more readUy on shells which
had remained uncleaned in the tanks for 2 or
more weeks, and which bore a \'isible film of
bacteria or diatoms, than on similar shells which
were cleaned daily." In their study of gregarious-
ness they placed shells of uniform size and shape
in pairs in a tank containing fully developed
larvae, and the number of spat attached to them
was counted daily. One shell of the pair was con-
sidered a control and was cleaned every day, and
the other (experimental) remained uncleaned. By
tlie end of the setting period the total spat settled
on the experimental shells significantly exceeded
the total spat settled on the controls by a ratio of
2.5 to 1. The figures suggest that the observed
differences may be due to the attraction of larvae
by those which had already settled on the shell,
but the conclusion cannot be accepted without
further verification. The possibility is not ex-
cluded that some other unknown factor, such as
tlie position of the controls in relation to the ex-
perimentals, affected the results or that handling
and removal of spat from the control shells caused
changes to the surface which made them less
attractive to the larvae. It would be profitable
to conduct a series of tests designed to eliminate
bias by placing experimental and control shells at
random and making a statistical analysis of the
significance of the differences.

Yonge (1960) expresses no doubt "that larvae
(of O. edulis) settle more readily on surfaces to
which others are already attached," and points
out that this tendency aids in reproductive effi-
ciency and is, therefore, a major benefit to attached
animals. In view of the fact that a single oyster

L.'iRV.'iL DE\ELOPMEXT AND METAMORPHOSIS

373

shell has sufficient space for only a few spat to
grow to maturity, heavy concentrations of spat
are of doubtful value for reproduction and may
even be liarmful by creating overcrowded
conditions.

ARTIFICIAL REARING OF OYSTER
LARVAE

Early attempts to rear o.yster lar\ae under
artificial conditions produced uncertain results.
Sometimes a small number of spat were obtained,
but the experiments could not be repeated under
similar conditions. At that time oyster larvae
were placed in 5-gallon carboys, and the water
was aerated and circulated. At 2-day intervals
the larvae were concentrated by centrifuging and
transferred into fresh sea water (Wells, 1920). In
another method, tried with only partial success,
the larvae were reared in slowly running sea water
which was filtered through a 2-inch layer of white
sand or porous stone (filtrose) placed on the
bottom of a container. The rates of filtration
and of addition of new water were regulated by a
valve placed below the filtering layer (Prytherch,
1924). In both types of experiments no food was
added to the containers under the assumption
that enougli was present in the water. Then
Gaarder and Sparck (1933) and Gaarder (1933)
studied the food of the larva of 0. ecbiUs in Nor-
wegian oyster ponds and made what may be con-
sidered the first significant step toward solving the
problem of rearing larvae under artificial cotuH-
tions. Sparck observed that the water of the
ponds contained considerable numbers of a small
green unicellular alga which later on was isolated
and cultured in the laboratory. It appeared to
l)e a species of Chlorella which was consumed by
tlie larvae. Studies by these investigators re-
vealed also that nannoplankton of tlie ponds con-
sisted principally of small green algae and flagel-
lates measuring from 2m to 3m. Fertilization of
experimental tanks by the addition of liquid
maimre greatly increased the production not only
of Chlorella but also of various diatoms, chiefly
Nitzschia, flagelhites, various large unicellular
green algae, and bacteria. In this enriched water
a few larvae grew to a size of 300m but failed to
attach (Sparck, 1927). After it was found lliat
( 'hlonlla is present in the Norwegian oyster ponds,
in experimental tanks in Conway, Wales, and in
certain experimental basuis m Denmark, Kandler
(1933) attempted to grow oyster larvae on a diet

374

of this alga alone but had little success. This led
liim to conclude that oyster larvae are unable to
digest Chlorella, which left the intestine apparently
unchanged. Feeding experiments with Carteria
and Chlamydomonas were also unsuccessful. More
critical experiments conducted at Conway, Wales,
showed that the larvae are unable to utilize
nonmotile green algae such as Chlorella and
Collomyra but that yellow-brown chrysomonads
(not identified but designated as flagellate C)
gave satisfactory results (Cole, 1937).

The Conway experiments demonstrated that
organic enrichment of the water of the large
tanks was consistently successful in giving rise to
a good crop of flagellates with the resulting good
growth and setting of larvae (Cole, 1939). The
most satisfactory fertilizer was the meat of the
shore crab Carcinus ground with sand and heated
to the boiling point. The suspension of meat was
added to a 90,000-gallon tank at the average rate
corresponding to 12.5 medium-sized crabs per day
for a period of 3 to 4 weeks. Production of nan-
noplankton was judged by pH readings, and as
soon as the readings reached 8.3 to 8.4 and the
tank had a distinct slight cloudiness, no more
crab meat was added.

Evidence presented by Cole showed that
growth and attachment of 0. edulis larvae in
tanks were significantly increased by organic en-
riclnnent which stimulated the development of
the nannoplankton. Under laboratory conditions
the oyster larvae grew and set satisfactorily in
the water containing cultures of Platymonas
tetrahele. The larvae of oysters and other bivalves
apparently are not able to swallow microorganisms
which exceed 8m, but according to Thorson (1950)
the size of nannoplankton normally devoured by
larval forms is smaller (2m to 3m).

Difficulties in obtaining reproducible results
from using organic enrichment for rearing larvae
suggested that variations in the composition and
quantity of nannoplaidvton may be responsible.
To determine the food requirements of 0. edulis
larvae, Bruce, Knight, and Parke (1940) isolated
from sea water six flagellate organisms ranging in
size from 1.5m t" ~m i" diameter. A known num-
l)er of ovstor larvae were introduced into glass
vessels filled witii 10 1. of uncontaniinated, sterile
sea water whicii was stirred and aerated. The
water was changed continuously by a drop feed;
the loss of larvae was prevented by covering the
outflow tubes with bolting silk. Tiie larvae were

FISH AND WILDLIFE SERVICE

fed pure cultures of flagellates grown in the so-
called "Erdschreiber" medium of the following
composition (Gross, 1937):

Sodium nitrate (XaNOs) 0.1 g.

Sodium orthophosphate (NazHPOj) 0.02 g.

Soil extract 50 ml.

Sea water 1.000 ml.

Soil extract is made b,y boiling 1 kg. of good
potting or garden soil with 1 1. of distilled water
in an autoclave for 1 hour. The flask is set aside
for 2 or 3 days, and the muddy dark fluid is de-
canted and sterilized by heating to the boiling
point. After standing 3 to 4 weeks the suspended
particles settle on the bottom, and the trans-
parent brown or red fluid is poured into another
container and boiled for a short time. Boiling of
the medium sliould be avoided once the required
quantities of nitrates and phosphate have been
added.

Since the six flagellates used in these experi-
ments (Bruce, Knight, and Parke, 1940) were not
identihed and were labeled only by letters, incon-
sistencies in the results reported may be attributed
to the appearance in the culture of other species,
or, as the authors state, "to the supervention of
factors outside experimental control." The au-
thors suggest that one of these conditions may be
the fact that larvae from dift'erent oysters are not
equally viable.

The feeding of oyster larvae (O. edulis) with
pure cultures of nannoplankton was repeated by
Walne (1956). In this case the larvae were kept
in vessels of 1 1. capacity without change of sea
water, and the species of flagellates grown in
cultui'es were identified. Among the Chloro-
phyceae, only Pyramimonaft grossii Parke gave
consistently good results. Tests made with Chlo-
rella stiijmaiophora Butcher seemed to indicate
that those clilorococcales which have a thick cell
wall are poor food for oyster larvae. The best
results were obtained with Isochrysis galbana
Parke, a chrysophycean of about 5ai to 6m in
length. Prymnesium parvum Carter was found
to be toxic to larvae. So far there is no proof
that the species of flagellates used in these experi-
ments form a significant component of the natural
population of nannoplankton and that their pres-
ence in estuaries is necessary for larvae living
under natural conditions.

Imai and Hatanaka (1949, 1950) reported that
the larvae of C. gigas can be reared on a culture
of colorless flageflate, Monas sp., which abounds
in brackish waters of Japan. The authors believe

LARVAL DEVELOPMENT AND METAMORPHOSIS

that the flagellate of the Monas type plays an
important role in the production of oysters in
Japan. The possibility remains, however, that in
their experiments other flagellates were present in
the culture of Monas enriched with glucose, cane
sugar, nitrates, and phosphates.

The pelagic life of C. virginica and C. gigas, and
probably of all oviparous oysters, is longer than
that of larviparous 0. edulis and 0. lurida. Con-
sequently, the rearing of these oviparous larvae
under artificial conditions presents additional dif-
ficulties. Considerable advances in the rearing of
larvae of various bivalve species were made by
Loosanoff, Davis, and their collaborators at the
Bmeau of Commercial Fisheries Biological Lab-
oratory, Milford, Conn. Phases of the work are
summarized by Loosanofl' (1954) and LoosanofT
and Davis (1963a, 1963b). Oysters were induced
to spawn by increasing the temperature and by
adding sperm suspension (seep. 30S, Chapter XIV).
The fertilized eggs were freed from debris by
passing the water through a series of fine screens
and placed in 5-gallon earthenware jars until free-
swimming larvae emerged. Then the water was
changed every 24 to 48 hours by straining it
through fine sieves which retained the larvae.
The sea water in which the larvae lived was filtered
thi-ough cotton to remove detritus and zooplank-
ton. Aeration and mechanical agitation were
considered unnecessary if the water was changed
every other day. The larvae were given measured
amounts of cultm-es of various micro-organisms.
In general the results obtained in Milford cor-
roborate the findings of British investigators.
Davis (1953) established that oyster larvae can
utilize as food the following species of flagellates:
Dicrateria inornata, Chromulina pleiades, Isochrysis
galbana, Hemiselmis rufescens, and Pyramimonas
grossii. Chlorella sp. was used only by advanced
larval stages and not by young veligers.

The utilizable flagellates were added to the
rearing tanks at the rates of 15,000 and 25,000
cells per ml. per day but no toxic effects were
noticed in these heavy concentrations, and the
larval oyster population of approximately 5,000
per 1. showed satisfactory growth. The actual
number of flagellates ingested by the larvae was
not determined, but the inference was made that
"the rate of growth of oyster larvae had an inverse
relation to the number of larvae per unit volume"
(Davis, 1953). Cole (1939) states that a popula-
tion of 20,000 to 30,000 small flagellates per 1 ml.

375

is adequate to promote growth of larvae of 0. edidis
but is insufficient for the spat.

With tlie exception of the toxic Prymnesium
parmim, the naked flagellates provided better food
for young oyster larvae than the organisms with
hea\y cell walls, which can be utilized only by
older larvae. The best single foods were found
to be Isochrysis galbana, ^fonochrysis Intheri,
Chrom.idina ■pleiades, Dicraferia inornata, and
some other unidentified species of Dicrateria.
Since the cultures used in these experiments were
not free of bacteria, the question naturallj' arises
whether the marine bacteria ace utilized as food.
Davis (1953) states that none of the 13 species of
marine bacteria tested by him were used by the
larvae. The species of bacteria have not been
identified. However, the probability that, larvae
nuiy derive a certain amount of food from some
bacteria is strengthened by the observation re-
ported by Davis (1953) and Loosanoff (1954) that
larvae kept in cotton-filtered sea water without
algal food continued to grow for as long as 14 to
IS days. The role of marine bacteria in tlie feed-
ing of oj'ster larvae needs further experimental
study.

Apparently the best results in rearing larvae
under artificial conditions are obtained with a
mixed food of Isochrysis galbana, Monochrysis
lutheri, Chromulina pleiades, and Dicrateria sp.
With such a diet and at 30° C, the larvae of C.
virginica begin setting between the 10th and 12th
days after fertilization; at 24° C, the sibling
larvae are ready to set on the 24th to 26th day;
at 20° C, only a few of the larvae set by the 3Sth
day. Setting of larvae of 0. lurida at a tempera-
ture of 22° C. takes place on the 7th day after
release of larvae from the brood chamber (Loosan-
off and Davis, 1963a).

Under laboratory conditions in Woods Hole the
young larvae of C. virginica are often found on the
bottom of vessels entangled in lumps of several
individuals. These larvae never recover and
usually die within the next 24 hours. Sometimes
the larvae of oysters and clams are attacked and
killed by a fungus which has been tentatively
identified by the workers at the Bureau of Com-
mercial Fisheries Biological Laboratory at MiKord,
Conn., as belonging to the genus Sirolpidium
zoophtorum Vishniac (Davis, Loosanoff, Weston,
and Martin, 19-54; Johnson and Sparrow, 1961).
There are undoubtedly other bacteria and possibly
viruses which inflict epizootic mortality on larval

populations in the laboratory and in natural
waters.

The technique of rearing oyster larvae has pro-
gressed sufficiently to be applicable to practical
purposes of oyster cidture. Details of techniques,
organization, and operation of a mollusk hatchery
are summarized by Loosanoff and Davis (1963b).

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1930. Paper and glass collectors for oyster spat.
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1932. Observations on propagation of oysters in
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LARVAL DEVELOPMENT AND METAMORPHOSIS

379

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380

FISH AND WILDLIFE SERVICE

CHAPTER XVII
CHEMICAL COMPOSITION

Page

Proxiinate composition of oyster meat 381

Seasonal and local variations.. 381

Yield and quality of meat 382

Inorganic constituents 383

Iodine 383

Heavy metals 383

Observations on New England oysters 384

Analytical procedures 384

Variations in glycogen content 386

Iron, copper, zinc, and manganese 387

Variations in thecontentofproteins, amino acids, and vitamins 390

Condition index 392

Antibacterial agents 393

Bibliography 393

Regardless of the zoological group to whicli an
animal belongs the greatest mass of materials
which form the tissues and organs, exclusive of
skeleton or shells, consists of three major groups
of organic compounds: proteins, carbohydrates,
and lipids (fats). Many analyses reported in the
literature show that, in spite of great variability
in tlie composition of meat of several species of
Ostrea and Crassostrea, the order of magnitude of
the three components is common to all the species
studied. The proteins make up 50 percent or
more of tlie solids, carbohydrates are less than 25
percent, lipids constitute less than 20 percent.

PROXIMATE COMPOSITION OF
OYSTER MEAT

A general idea of the pro.ximate composition of
tiie meat of C. rirtjinica can be deduced from
tallies published by the U.S. Department of
Agriculture for dietitians, nutritionists, physicians,
and otiiers engaged in planning diets or in cal-
culating the nutritive value of foods^ (Watt and
Merrill, 1950, p. 36). The material used for such
analyses represents the average sample available
for purchase at the market or delicatessen store.
The figures do not refer, therefore, to oysters of
any particular locality or to time of the year.
For convenience in making a comparison all tlie
values originallv given for 1 cup (240 g.) of raw
oysters were recomputed for 100 g., which corre-
sponds to five to eight medium-size oysters.
The sample contained 9.8 g. of protein, 5.6 g. of
carboliydrates, 2.1 g. of fat, 94.1 mg. of calcium,

FISHERY bulletin: VOLUME 64, CHAPTER XVII
733-851 O— 64 25

143 mg. of phosphorus, and 5.6 mg. of iron, and
80.5 g. of water.

When oysters are prepared for the market the
meats are shucked and washed, either in fresh
water or sea water. During this process the water
is stirred and air is blown through it to remove
grit, pieces of broken shell, and mud. The pro-
cedure affects the chemical composition because
some of the soluble salts present in the body are
lost, and the less soluble constituents, the proteins
and fats, then make up the greater proportion of
solids. Consequently the values for these two
components quoted above are somewhat higher
than for unwashed oysters. Correspondingly the
values of mineral salts in Watt and Merrill's data
are lower.

SEASONAL AND LOCAL VARIATIONS

Variations in the chemical composition of oys-
ters foUow distinct patterns related to environ-
ment and season of the year. The major environ-
mental factor affecting chemical composition is the
salinity of water. C. virginica is an estuarine
species which may be found in waters ranging
from almost 40°/oo, as in the sheltered bayous of
the Gulf Coast, to less than 3°/oo at the upper
reaches of bays after heavy rainfall (upper Chesa-
peake Bay, MobOe Bay, Apalachicola Bay, and
others). A change from wet to dry spells produces
a pattern of fluctuations in the contents of mineral
salts in oysters growing in waters of fluctuating
salinity. Such conditions prevail in the waters of
the south Atlantic and Gulf states where the
annual range of changes from maximum to mini-
mum ash content was reported to be 5.3 to 31.1
percent on a moistm-e-free basis. The solids, for
the same period of time, varied between 7.5 and
18.4 percent of the wet weight of oysters (Lee,
Kurtzman, and Pepper, 1960). Fluctuations in
the moisture content due to absorption of water
and loss of solids are the most significant features
of changes in the chemical composition of oyster
meat which affect their commercial quality. Good
oysters contain two and one-half times more solids

381

per unit of volume or weight, and obviously have
higher nutritive value than the poor ones contain-
ing over 92.5 percent water.

Oysters living under marginal conditions (see
chapter XVIII) are usually low in solids through-
out the year. A comparison of the mean annual
composition of meat based on a series of regular
observations discloses these differences. Table 38
summarizes the chemical studies made for 2 con-
secutive years on oysters from six southern states
(Lee and Pepper, 1956; Lee, Kiu-tzman, and
Pepper, 1960). The lowest values of total solids,
and of proteins, carbohydrates, and fat were found
in Georgia and the highest in Louisiana and
Alabama oysters. Data on seasonal variation in
the composition of meat for the southern oysters
were analyzed by Lee and Pepper (1956). Solids
increase steadily from 9.5 percent in October to
about 13.5 percent in March; in the middle of
May they begin to decline and reach the lowest
value of 9.2 percent in September. The fat con-
tent followed the trend appro.ximately. The
changes are associated with the gonad develop-
ment and spawning, which in the southern oysters
begins earlier and continues longer than in oysters
of the northern waters.

T.\BLE 38. — Proximate mean composition of meat of C.
virgiiiica from southern icaters for 2 consecutive years,
from October 1954 to October 1956 inclusive, in -percent
of their net weight

[Recalculated from the data published by C. F. Lee and L. Pe
and C. F. Lee, C. H. Kurtzman, and L. Pepper (1960

r

1956)

State

Solids

Protein

Carbo-
hydrate

Fat

Ash

1st
year

2d
year

1st
year

2d
year

1st
year

2d
year

1st
year

2d
year

1st
year

2d
year

Louisiana

Mississippi-

11.5
11.2
11.8
10.8
10.7
13.5

12.2
12.5
13.8
11.7
10.0
13.9

5.8
.5.1
5.8
5.0
5.0
6.2

6,3
6.1
6.1
5.6
4.4
6.6

2.7
3.0
3.3
2.5
2.3
3.7

3.0
3.3
4.6
2.9
2.1
4.0

1.1
1.1
1.3
0.8
0.7
1.0

1.3
L6
1.6
1.1
1.0
1.1

1.9
2.0
1.4
2.5
2.7
2.6

1.6
1.5
1,4

Florida

2.1

2,5

South CaroUna. .

2.9

YIELD AND QUALITY OF MEAT

Some idea of geographical differences in the
productiveness of oyster bottoms may be gained
by comparing the yield of oysters in pounds of
meat per bushel. It can be seen from table 39
copied from Power (1962) that the recorded yield
of market oysters in the waters of Delaware and
further north varies from 6.6 to 7.5 pounds per
standard bushel and is significantly higher than
in the southern states, from Maryland to Texas, in
which the yield is from 3.15 to 5.07.

APR MAY

Figure 355. — Average protein and carbohydrate content
in monthly samples of southern oysters in percent of dry
weight. Oysters for analysis were collected at the
shucking plant but were not subjected to the routine
washing and air bubbhng procedure which causes large
salt and fluid lo.sscs. From Lee and Pepper, 1956.

The quality of oyster meat is related primarily
to the amounts of protein and carbohydrates.
The ratio between the two components changes
with the season and reproductive cycle. The
percentage of protein sharply decreases in May to
less than 40 percent of the dry weight while at the
same time the cai'bohydrates reach their maxi-
mum of about 60 percent (fig. 355). The actual
changes in the protein content are less pronounced
because of the increase in solids due to storage
of glycogen.

Decline in the ash (mineral matter) content of
oyster meat from the highest value of almost 25
percent (dry weight basis) in October to about 5
percent in May (fig. 356) and a gradual increase
during May to September are probably related to
changes in the salinity of water from which the

Figure 356. — Average mineral matter and salt content in
the montlily samples of southern oysters (unwashed) in
percent of dry weight. From Lee and Pepper, 1956.

382

FISH AND WILDLIFE SERVICE

Table 39. — Yield of market orjsiers 1960, pounds of meat

in U.S. standard bushel

[From Power, 1962]

Maine -- 7.50

Massachusetts 6.50

Rhode Island __ 7.00

Connecticut.. 7. 70

New York 7.50

New Jersey 7.01

Delaware 6.60

Maryland 4.58

Virginia 4. 19

North Carolina 4.21

South CaroUna 2.93

Georgia 3. 15

Florida, east coast 4. 20

Florida, west coast 4.22

Alabama 4. 17

Mississippi... 3.86

Louisiana 4.54

Tc.tas. 5.07

oysters were taken, but the problem requires
further study.

Variations in the chemical composition of the
meat of 0. eduiis are similar to those which take
place in C. virginica. Gaarder and Alvasker
(1941) give a detailed account of these changes in
the oysters of Norwegian waters. The e.xtent of
annual fluctuations that took place in 1936 are
given in table 40.

Table 40. — Extent of changes in the chemical composition

of Ostrea eduiis in A^orway in percentage of ivet weight

[According to Gaarder and Alvasker, 1941]

Weight

of
meat

Mois-
ture

Pro-
tein

Carbo-
hy-
drate

Gly-
cogen

Fat

Ash

Maximum

14.6
7.6
11.3

81.9
76.0

78.7

11.2

8.8
10.0

9.6
6.3

7.8

7.9
5.1
6.8

2.5
1.6
2. 2

1.5

1.2

Average for 1936....

1.3

In a comparison with the analysis of C. virginica,
the 0. eduiis has a relatively higher carbohydrate-
protein ratio and higher fat content. This may
be due primarily to the fact that European oysters
are grown on oyster farms while the sample of
southern American oysters was taken from wild
populations. Like\vise, the higher yields of ('.
virginica in the waters of northern latitudes is
primarily the result of skill in cultivation by
private oyster growers of New England and the
North Atlantic states rather than geography.

INORGANIC CONSTITUENTS

The mineral content of the edible portion of the
oyster consists primarily of sodium chloride (fig.
356), but it also contains almost every chemical
element present in sea water. Spectrographic
analysis of 22 samples of oyster ash (exclusive of
shell) made by the U.S. Bureau of Mines in 1940
at the request of the Bm-eau of Fisheries (data
on file in the library of the Bureau of Commercial
Fisheries Biological Laboratory, Woods Hole,
Massachusetts) showed that the samples consisted
mainly of sodium, potassium, calcium, magnesium,
and phosphorus; and low concentrations of the

following elements: copper, iron, sihcone, alumi-
num, strontium, lithium, rubidium, nickel, silver,
titanium, zinc, vanadium, platinum, manganese,
gold, and zirconium. The results are predictable
since sea water which contains these elements
enters the composition of the oyster's body fluids.

IODINE

The presence of iodine in various sea food
animals has been generally knowm for a long
time and has been studied primarily from the
point of view of dietitians and nutritionists.
Coulson (1934) found that one average serving
of (\ virginica (110 g.) would furnish to the diet
54 /ig. of iodine, an amount higher than that
found in one serving of red salmon, milk, various
vegetables, and beef. The iodine content of fresh
oysters handled in the usual commercial manner
varied from 1,000 to 11,530 parts per billion on
the dry basis, or from 194 to 1,652 parts per biUion
on tlie original wet weight basis. When the
means of individual variation are considered
statistically, there appears to be no significant
variation in the iodine content of oysters from
different Atlantic and Gulf states nor any signif-
icant variation with season. There is, however,
a significant difference between the Atlantic and
Pacific Coast oysters, {C. drginica and 0. lurida):
the Pacific species have a lower iodine content
than the Atlantic species. The mode of accumu-
lation of iodine in the oyster tissue and the role
it plays in the physiology of the oyster are not
known.

The iodine content in oyster meat can be arti-
ficially increased by placing live oysters in sea
water to which free iodine has been added. In
e.xperiments with C. angulata at Arcachon, France,
Loubatie (1931) showed that the concentration
of iodine in the tissues of oysters increased 700
times over its normal value after live oysters
were kept for 4 days in water containing up to
3 mg./l. of free iodine. In 1932 a commercial
concern at Bordeaux, France, artificially produced
such "super-iodized" oysters and advertised
their beneficial effect in cases of anemia and other
maladies attributed to iodine deficiency. When
I visited Arcachon in 1932 there was apparently
a good demand for these oysters, which had a
strong iodine flavor.

HEAVY METALS

The ability to accumulate various elements
present in sea water at very low concentrations

CHEMICAL COMPOSITION

383

is common to many marine invertebrates. Of
particular interest is tlie ability of many
bivalves to accumulate various heavy metals,
such as zinc, copper, iron, manganese, lead, and
arsenic. Tlie problem is of importance because
in polluted coastal waters shellfish may store
substances that may be dangerous to human
health. Hunter and Harrison (192s) showed
that oysters affected by industrial pollution in
certain coastal areas in Connecticut, New York, and
New Jersey contained traces of lead (determined
as Pb) and arsenic (AsaOs), the concentration of
the arsenic varying, depending on locality, from
0.6 mg./kg. to 3.0 mg./kg. of dry weight.

The accumulation of copper causes green dis-
coloration of the mantle and gills of oysters and
gives them an unpleasant coppery flavor. The
problem of greening has attracted many investi-
gators, especially since Lankester (1886) demon-
strated that green color in some oysters may
be due to an excess of copper, while in the green-
gilled European oysters of the west coast of France
the bluish-green coloration was caused by ab-
sorption of a pigment from a diatom, Nitzschia
ostrearia, called marenin (Ranson, 1927). Green
oysters similar to those of Marennes, France,
occur occasionally along the Atlantic coast in
Virginia (Mitchell and Barney, 1917) and in
North Carolina (personal observation). Accu-
mulation of iron, zinc, and manganese does not
change the color of oyster meat. .

The degree of concentration of heavy metals in
the oyster body is related to the environment.
Oysters from the North Atlantic States are poorer
in iron and riclier in copper than oysters of the
South Atlantic and Gulf States in which the rela-
tion is reversed. This has been shown by Coulson,
Levine, and Remington (1932), who analyzed a
number of samples collected from various states
in April and again in November-December, 1931.
Their observations are summarized in table 41.
The data show that the iron content of oyster meat

significantly increases from north to south while
the copper content decreases. The samples show
no significant variations in the manganese content.
The increase in iron content is associated with a
greater percentage of iron (as FcoOj) in the river
water of the South Atlantic States discharged into
the estuaries than is present in the runoff waters
of the North Atlantic States. High copper con-
tent in the oysters of New Jersey, New York,
Connecticut, and Rhode Island is possibly asso-
ciated with the discharge of chemical wastes from
shore installations of these highly industrialized
states.

OBSERVATIONS ON NEW ENGLAND
OYSTERS

Seasonal changes in the composition of oysters
can best be studied by regularly taking samples
from a single bed containing a population of
oysters of known age. Such an investigation was
made by taking samples of oysters from a com-
mercial bed in Long Island Sound, off Charles
Island, and simultaneously recording the temper-
ature, salinity, and pH of the water. The work
was conducted from the Bureau of Commercial
Fisheries Biological Laboratories at Woods Hole
and Milford. For experimental purposes and for
checking analytical methods a large number of
4- to 5-year-old oysters were kept in the outdoor
tanks near the laboratories. Samples of 25 oys-
ters were taken once or twice a month for a period
of 22 months from July 1933 to August 1935.
Ten of the oysters were used for a chemical
analysis of ash, 10 for the extraction of glycogen,
and 5 for biological studies.

ANALYTICAL PROCEDURES

Oyster meats being prepared for chemical
analyses are easily contaminated with iron while
they are being removed from the shell. We found
that the following analytical procedure was most
satisfactory. The surface of the shells was cleaned
with a stiff nonmetal brush, and the whole oysters

Table 41. — Iron, copper, and manganese content of oysters from the Atlantic and Gulf coasts
[Results are expressed in mg./kg. (wet basis). From Coulson, Levine, and Remington, 1932]

Localitj

Spring samples

Winter samples

Iron

Copper

Manganese

Iron

Copper

Manganese

Nortli .\tlantic States

South Atlantic States _

Gulf States

fRange

lAverage

/Range..

"" 1.\verage

fRange -.

"LAverage

24.9- 32.1
28. 9± 1.1
50.0-104.5
66. 0± 8. 0
37.5- 74.8
59. 8± 3.4

41.2-122.9
71. 8± 6.6

4.0- 38.0
16. 1± 3.8

5.9- 26.8
16. 0± 1.9

1.05-3.0O
2.09±0.20
1.26-4.16
2. 49±0. 25
2.93-4.09
3.60±0.15

31.5- 47.1
40. 7± 1.1
45.1-135.3
70. 3± 8. 1
65.2-113.8
82. 5d= 5. 0

34.4-137.2
86. 2± 9. 1
3.4- 36.9
17. 0± 3.0
19.1- 48.2
26. 7± 3.0

1.66-2.82

2. 47±0. 1

2.0-2.87

2. 57+0. 08
3.07-4.40

3. 77+0. 13

384

FISH AND WILDLIFE SERVICE

were put in glass containers and placed in an oven
at 50° C. for about 1 hour. The meats were then
removed witli a glass spatula from the gaping
valves. The shell liquor remaining in the con-
tainers was added to the meats, and a sample of
10 oysters was weighed and placed in a porcelain
dish for drying at 90° C. to a constant weight.

The dried samples were pulverized in a glass
mortar. Then several grams of the powdered and
well-mixed sample were weighed into a silica dish,
charred over a low flame, and ashed in an electric
muffle at a temperature of 500° C. for 3 to 4 hom-s.
After cooling the sample was moistened with water
and 1 ml. of concentrated nitric acid was added.
The sample was evaporated to dryness on a hot
plate and returned to the muffle, this time at a
temperature of 400° C. One application of nitric
acid was usually sufficient to complete ashing.
Ash was dissolved by heating in a 1:1 solution of
hydrochloric acid, 10 drops of hydrogen peroxide
were added, and heat was applied imtil the libera-
tion of oxygen ceased. Finally the sample was
weighed, transferred to a 100-ml. volumetric flask
and diluted to the 100 ml. mark.

For iron determination Kennedy's colorimetric
method of potassium thiocyanate was emploj'ed
(Kennedy, 1927), using ferrous ammonium sulfate
(dried to constant weight) as standard. Copper
was determined by Biazzo's method as described
by Elvehjem and Lindow (1929) and Elvehjem
and Hart (1931). Zinc was determined by
Birclviier's method, using a nephelometer for
comparison of the turbidity of samples (Bh-ckner,
1919) and zinc oxide solution in hydrocliloric acid
as the standard. Manganese was found by
Richards' method (Richards, 1930). To determine
the reliability of analytical procedures, several
analyses were made in duplicate and occasionally
known cjuantities of metal salts were added to the
samples and recovered. In this way error due to
analytical procediues was found to vary between
0.5 and 2.5 percent.

Differences in the results of analyses of oyster
meat often are due to the method of obtaining
samples. Tlie percentage of solids in a sample and
the corresponding figure of moisture content de-
pend on the method of drying. Sometimes the
sample is dried on a steam bath at a temperature
of 90° C; in otlier cases the oyster meat is kept in
an electric oven at 95° or 97° C. The results will
also differ if the sample is first homogenized or if
the whole oyster is used for tlrying.

The main source of inconsistency in the analyses
results from methods of discarding the fluid
retained in the mantle cavity and in the water
tubes and chambers of the gills. This fluid con-
sists primarily of sea water with some blood cells
and excretion from the kidney. Oysters removed
from the shell with no injury to the mantle and
pericardium nevertheless continue to lose blood
from the severed ends of the muscle and from
blood sinuses in the body proper. The loss of
body fluid is very rapid during the first half horn*
after removal from the shell. For as long as 2
hours after shucking the oyster may lose a quantity
of fluids equivalent to 26 percent of the original
body weight (Fingerman and Fairbanks, 1956a,
1956b). Puncturing the mantle and pericardium
results in up to 50-percent loss of body weight.

To minimize losses of weight caused by pro-
longed bleeding, oyster meats may be placed on a
screen and drained for 5 minutes. More consistent
results are obtained if the water captured between
the organs is discarded. If the valves are forced
apart slightly and jammed open by a small
wooden wedge, sliaking the oyster with 10 sharp
jerks is sufficient to dislodge the water from the
gills. This method gives a lower percent of solid
content than those obtained with other procedures,
because bleeding is minimized.

The percent of moisture in the meat is usually
determined by the difference between the total
and dry weight of the sample. Direct determina-
tion of water content can be made by distillation
in xylene in a flask with a reflux condenser. The
sample is boiled continuously for 1 hour at a rate
of approximately 5 ml. of reflux per minute and
for 3 hours at double that rate. Without interrupt-
ing the boiling, two drops of 95 percent ethanol
are added through the top of the condenser. After
the violent ebullations have ceased, boiling is
continued for 5 minutes (Calderwood and Piechow-
ski, 1937), then the volume of water accumulated
in the side arm of the condenser is measured.

The glycogen content of oyster tissues is deter-
mined by digesting them in 30 percent sodium
hydro .xide for 1 hour at 80° C. Glycogen is
precipitated by 95 percent ethanol, washed, dis-
solved in hot water, hydrolyzed with hydrochloric
acid for at least 4 hours at 92° C. and the dextrose
present determined in aliquot sample by use of the
Hagedorn-Jensen procedure. Details modifying
the method to make it suitable for obtaining
glycogen in a high state of piuity from oyster

CHEMICAL COMPOSITION

385

tissues are given by Calderwood and Armstrong
(1941).

VARIATIONS IN GLYCOGEN CONTENT

Glycogen is the reserve material of the oyster.
It is stored primarily in the connective tissue of
the mantle and labial palps. During the rapid
proliferation of se.x cells the reserve supply is used,
and by the end of the reproductive cycle the
amount of glycogen is at a minimum and the
mantle is reduced from a thick heavy layer to a
thin transparent membrane. Soon after spawning
the oysters begin to form and store glycogen and,
in the parlance of oyster growers, become fat.
The expression fatness as it is used in trade is a
misnomer because it does not refer to an increase
in lipids. In New England waters the accumu-
lation of glycogen reaches its maximum during
late autumn but sometimes continues even in
winter. As a rule the glycogen remams at a high
level until the beginning of rapid proliferation of
sex cells in May. Seasonal fluctuations in glycogen
content are common to all the species of oysters
that have been studied. The pattern of changes
varies in different localities and in difi'erent species
depending on local conditions — temperature and
abnormal salinity of water, abundance and type
of food available, and intensity of feeding.

Seasonal changes in the glycogen content of
New England oysters show a definite cycle related
to gonad development and spawning. The rapid
increase in the number of sex cells in the gonad
e.xhausts the reserve materials and brings the
glycogen content to its minimum, which usually
occurs immediately after spawning. After a short
period of relative inactivity during which the
unspawned sex cells are reabsorbed the oysters
begin to accumulate and store glycogen in their
tissues. The process may be rapid, as for instance
in September to December 1933 (fig. 357) or
gradual as in the same period in 1934. The
glycogen count of oysters of the same population
varies from year to year. It can be seen in fig.
357 that in 1934 the content of glycogen after
spawning was significantly higher than in the
preceding year. Microscopic examination of these
and Cape Cod oysters showed that sometimes the
glycogen reserve is not depleted during the growth
of the gonad and remains at a relatively higli
level throughout the spawning season. Another
interesting fact noticeable in the annual glycogen
curve is the continuing increase in glycogen during
the cold months of winter when feeding ceases.

J I I I I \ I I I I I I I I I 1 I I I L_

Figure 357. — Glycogen and water content of adult
oysters (5-years-okl in 1933) from commercial oyster
bed off Charles Island, Long Island Sound.

The amount of glycogen stored in the tissues at a
given moment is the balance resulting from the
glycogen formed (glycogenesis) and that broken
down (glycolysis) . Biochemistry of both processes
known in great detail in mammals, has not been
adequately investigated in bivalves. It appears,
however, reasonable to postulate that the tissue
glycogen continues to be synthesized by the
oyster from the carbohydrates accumulated with
food during the period of active feeding or from
indigenous sources of intermediary metabolism.

Increase in glycogen content is usually associ-
ated with an increase in solids and a corresponding
decrease in water. There are, however, unusual
instances as in the oysters found in November and
December 1933 (fig. 357) which had a higli glyco-
gen content in spite of an increase of water to 88
percent and corresponding loss of solids.

The annual glycogen cycle in oysters of the
York and Piankatank Rivers, Va. (GaltsofF, Chip-
man, Engle, and Calderwood, 1947) follows the
general pattern simihir to that of Long Island
with the only diff'erence that the lowest concen-
trations were observed in July to September and
the highest in November to February. In Loui-
siana the period of low glycogen was found by
Hopkins, Mackin, and Menzel (1954) to extend
from April to the end of November. All the dif-
ferences mentioned above are associated with the
longer reproductive periods in warmer climates.

The cyclic change in glycogen content has been
described for 0. edulis and C. angulaia by Bierry,
Gouzon, and Magnan (1937); Bargeton (1945);

386

FISH AND WILDLIFE SERVICE

Figure 358. — Seasonal changes in iron content in adult
oysters from Long Island Sound in mg./kg. of dry
weight adjusted to weight of total solids less glycogen
(broken line), July 1933 to March 1935.

Gaarder and Alvsaker (1941), and many others.
In general the changes are similar to those ob-
served in C. virginica, the lowest content occm-ring
during the smnmer.

The cycle of fat has not been studied for C.
virginica. According to Watt and Merrill (1950)
the average content of fat of raw oyster meat sold
in U..S. markets is equal to 2.1 percent. Gaarder
and Alvsaker (1941) found that the fat content
of 0. edulis in Norwegian ponds varied from 2.52
to 1.56 percent with the annual average of 2.17
percent. The observed fluctuations were not
seasonal.

IRON, COPPER, ZINC, AND MANGANESE

The four metals present in the meat of Long
Island Sound oysters were found primarily in the
giUs and mantle; lesser quantities were in the
muscle and gonads. Only the ovaries had man-
ganese in quantities greatly exceeding the content
of this metal in other organs. These findings are
based on the series of chemical analyses of different
organs and on histochemical reactions used for the
localization of various metals. The curves in
figures 358 to 361 showing the seasonal changes in
the contents of metals expressed in mg./kg. of dry
weight have a common pattern despite large dif-
ferences in the levels of concentration. The
amounts of metals increase during summer and
decline in the following fall and winter. The in-
crease in metals during the warm feeding season
cannot be associated with the possible presence of

Figure 359. — Seasonal changes in copper content in adult
oysters from Long Island Sound in mg./kg. of dry
weight adjusted to weight of total solids less glycogen
(broken line), July 1933 to March 1935.

food particles in the intestinal tract, since the
total weight of food and fecal masses inside the
intestines constitutes only a minute fraction of the
body weight, and because the mantle and gills are
the principal storage places for iron, copper, and
zinc. Likewise the increase in metal content is not
caused by the loss of glj'cogen since the general
trend of the cm'ves is not affected by adjusting the
values of concentrations to the weight of solids
less glycogen (dotted lines in figures 358 to 361).
With minor exceptions the two types of ciu-ves
(adjusted and nonadjusted) run parallel. It ap-
pears, therefore, a firmly established fact that the
content of the fom' metals increases during the

Figure 360. — Seasonal changes in zinc content in adult
oysters from Long Island Sound in mg./kg. of dry
weight adjusted to weight of total solids less glycogen
(broken line), July 1933 to March 1935.

CHEMICAL COMPOSITION

387

NOT ADJUSTED

I I I I I I I I I 1 I I 1 III

lASONOJFUiaMJJ a S 0 N B J F M «

Figure 361. — Seasonal changes in manganese content in
adult oysters from Long Island Sound in nig./kg. of dry
weight adjusted to weight of total solids less glycogen
(broken line), July 1933 to March 1935.

summer and decreases in winter. Heavy metals
are accumulated in the oyster tissues by direct
absorption from sea water, ingestion in the in-
testinal tract with food, and dispersal by blood
cells throughout the visceral mass.

Individual variations in iron, copper, and zinc
contents are large, and oysters living side by side
frequently were found to vary m the contents of
these metals. This is particularly easy to observe
in green oysters, for the color varies in intensity in
direct relation to the copper content. In the case
of pronounced green discoloration the presence of
metallic copper may be demonstrated by inserting
in the tissues a well-polished steel knife; the surface
becomes copper plated in a short time. This sim-
ple method can be used profitably for a qualitative
demonstration of the presence of copper. Tlie
green pigment of the oyster can be isolated by
grinding the meats with pure sand previously
treated with strong hydroclijoric acid and carefully
washed. The proteins in the extract are precipi-
tated with ammonium sulfate (NH4)2S04, but the
pigment remains in solution. It was shown by
S. Lepkofsky (quoted in Galtsoff and Whipple,
1931) that the green compound is not even re-
motely related to hemocyanin and that it exists in
the oyster as a readily diffusable material. The
green extract is readily soluble in metliyl alcohol,
less so in ethyl alcohol, and quite insoluble in butyl
or amyl alcohol. It is insoluble in chloroform,
ether, acetone, or benzene, but is soluble in
pyridine.

Wlien the extract is left standing for 4 months
or longer in sealed glass tubes it turns to a reddish-
chocolate color, but the green color I'eturns if it is

shaken with methyl alcohol, ethyl alcohol, or pyri-
dine. Bubbling air or oxygen fails to bring back
the green color.

The content of copper in the tissues can be arti-
ficially increased by placing the mollusks in sea
water containing an excess of this metal. Green
discoloration develops in the oysters kept in sea
water which is in contact with copper pipes or
valves. Within about 6 summer weeks the copper
content in oysters kept under such conditions in-
creased up to 20 times and the meats became deep
green. Analyses of samples of Woods Hole water
taken in the harbor and from the laboratory supply
pipe sliowed that the copper content in the labora-
tory water sometimes exceeded 20 to 40 times its
concentration in the harbor near the intake pipe.

The iron content of oyster meat may be arti-
ficially augmented by adding ferric salts to the
water in which the oysters are kept. The iron in
sea water was enriched by suspending several
pounds of iron nails in the large outdoor tank with
the oysters or by adding ferrous iron sulphate
(copperas). Although large quantities of iron
oxide particles were formed and remained in
suspension, the concentration of iron dissolved in
sea water did not change significantly in 28 days
but the content of iron in suspension increased
about five tunes. Particles of iron oxide were
noticeable in the feces, which contained as high
as 13,000 mg. of iron per kg. (dry basis). Oysters
being prepared for chemical analysis were placed
for several days in running sea water containing
no iron particles in suspension so that all loose
sediment in the mantle cavity and the gills would
be discarded. The removed meats were thor-
oughly mspected and rinsed in sea water. Micro-
scopic examination of sections of the gills and other
organs was made at intervals varying from 20
minutes to several days following the initial
feeding with iron oxide suspension. The oysters
treated with potassium ferrocyanide and hydro-
chloric acid (Prussian blue reaction) show that
leucocytes on the surface of the gills actively ingest
iron particles, migrate throughout the body, and
aggregate near the wall of the intestines and in
blood vessels (fig. 362). No iron was detected in
the digestive diverticula, sex cells, or in the ad-
ductor muscle. wSome iron is eliminated through
the epithelial cells of the mantle (fig. 363).

Histological localization of copper is not
entirely reliable. According to Mallory (Lillie,
1948; Click, 1949) copper compounds produce a

388

FISH AND WILDLIFE SERVICE

■•« •■

Sfe ®^p

;.V«S

0

(?-

a 5:

<©■■

:.;t*A-

(5>.

: -. :>^>' I

M:

w^m

Microns

80

Figure 362. — Blood cells of C. virginlca containing iron in the connective tissue under the digestive tract. Drawing of
a section of oyster fed iron particles and treated with potassium ferrocyanide and hydrochloric acid.

■■:■©

0

Microns

100

Figure 363. — Iron particles in the mantle epithelium of ('. virginica fed iron oxide. Treated with ferrocyanide and

hydrochloric acid.

CHEMICAL COMPOSITION

389

light to dark blue color with an unoxidized fresh
aqueous solution of hematoxylin made by dis-
solving from 5 to 10 mg. of pure hematoxylin in
0.5 to 1 ml. of 100 percent ethyl alcohol and 10
ml. of distilled water boiled 5 minutes to drive off
carbon dioxide. Sections of celloidin-embedded
tissues were stained for 1 hour or longer. Copper
compounds appeared as a light to clear blue color.
The reaction is to a certain extent obscured by
a mass of yellow to brown colors produced by the
iron in the tissues. The surface of the mantle and
gills of green oysters usually contains large masses
of blood cells loaded with dark granules which
react strongly with Mallory reagent. It is obvious
that a large proportion of the copper in the oyster
is found in the blood cells.

For histological localization of zinc, the nitro-
prusside reaction proposed by Mendel and Bradley
(1905) can be used. The reaction is considered by
Lison as specific (Lillie, 1948). The method
involves treatment of the paraffin section of
tissues for 15 minutes at 50° C. in 10 percent
sodium nitroprusside solution. The section is
washed for 15 minutes in gently running water.
Then a drop of sodium or potassium sulfide solu-
tion is introduced under one side of the cover
glass. The reagent elicits an intense piu-ple
color in the zinc precipitated by the nitroprusside.
In many preparations of green oysters treated
by this method a diffuse purple coloration of
varying degrees of intensity was produced in
different organs, the mantle and gills staining
conspicuously deeper than the rest of the body.
The concentration of zinc within the blood cells
could not be demonstrated by this method. It
appears probable that zinc is present in a soluble
state and is more universally distributed through
the tissues than iron, copper, and manganese.
Observations on the uptake and accmnulation of
radioactive zinc Zn''^ confirm tiiis view. Chipman,
Rice, and Price (1958) demonstrated that zinc
in surrounding water is rapidly taken up in great
amounts by the bodies of oysters, clams, and
scallops. The gills of oysters were found to
accumulate almost twice the concentration of
radioactive zinc, as did the organs and tissues.
The digestive diverticula and body mass contained
a considerable amount of Zn"^, The zinc content
of sea water along the Atlantic and Gulf of Mexico
inshore waters averages 10.6 Mg-/1-

Several histochemical reactions for the localiza-
tion of manganese in the oyster tissue have been

tried without success. So far as I know there is
no satisfactory method for demonstrating this
element in the cells and tissues.

The distribution of manganese in the oyster
body is related to the female reproductive cycle,
because the concentration of this element in fully
developed ovaries (see fourth column of table 42),
is 15 times that of the spermary (Galtsoff, 1943)
and its total concentration materially decreases
after the discharge of eggs. No such relationship
is apparent for the other three metals.

The role of heavy metals in the physiology of
the oyster is not clear. It is reasonable to as-
sume that manganese performs some function
during the rapid propagation of ovocytes, possibly
as a catalyst.

Iron, copper, and zinc may be stored in the
tissues and in some blood cells as excess materials
which are slowly eliminated. Observations on
excretion of iron by the mantle epithelium (fig.
363) and accumulation of iron, copper, and zinc
in the mantle and gills support this view. The
distribution of the four metals in different organs
of Woods Hole oysters was studied analytically.
The organs were excised by fine scissors, weighed,
and analyzed separately. The results of the
analyses are shown in table 42 as means of
10 samples taken from natural environment.
The lower part of the table summarizes the results
obtained after keeping the oysters in a tank with
an excess of copperas. It appears significant
that both mantle and gills have absorbed relatively
large quantities of the metals.

Table 42.- — Distribution of metals in the body of adult C.

virginiea in Cape Cod waters {mg.jkg., dry weight)

[Mean of 10 samples. Early August and October, 1936]

Body portion*

Iron

Copper

Zinc

Man-
ganese

Remarks

Gills

382
136
151
136

252

178
65
63
65

163

4,480
1.420
1,710
1,420
4,630

39
4

60
4
9

Summer samples from

Ovary

Spermary

Residue

ment.

Mantle

184

194

75

401

1.840

1,920

172

1.490

22,000

19.400

1,590

14,400

14
25
4
9

Autumn samples from

Gills

Muscle

tanks with e.xcess of
copperas after 26
days of exposure.

Residue

*In summer the mantle could not be separated witliout contaminating
the sample with underlying gonads; in the autumn, after spawning, the
gonads contain only few imdifferentiated cells of germinal epithelium.

VARIATIONS IN THE CONTENT OF PRO-
TEINS, AMINO ACIDS, AND VITAMINS

The protein content in oyster meat of C. vir-
giniea, determined by the Kjeldahl method as
N X 6.25, fluctuates between 5.1 and 9.8 percent

390

FISH AND WILDLIFE SERVICE

of a fresh wet sample and between 42 and 57
percent of a dry sample. The figures quoted
above from the paper by Wentworth and Lewis
(1958) refer to the oysters of Apalachicola Bay,
Fla., the extent of fluctuations is probably common
to all oysters of the Atlantic and Gulf states since
occasional observations on oysters from various
states fall within this range (Jones, 1926). Ac-
cording to monthly observations by Gaarder and
Alvsaker (1941), the protein content in the meat
of 0. edulis from Norwegian ponds ranks somewhat
higher, varying from 8.8 to 11.2 percent (fresh,
wet basis) with an annual average of 10.5 percent.

Interesting biological observations were made
by Duchateau, Sarlet, Camien, and Florkin (1952)
on free amino acids in the muscles of marine bi-
valves, 0. edulis and Mytilus edulis, and the fresh-
water mussel, Anodonta cygnea. The muscles of
these mollusks were isolated after bleeding, boiled
for 5 minutes to inactivate proteolytic enzymes,
homogenized, and treated wdth tungstic acid.
Protein-free samples were hydrolyzed and ana-
lyzed. The results (table 43) show that the
amino acid contents differ greatly between the
marine and fresh-water species. Generally higher
concentrations of amino acids in the muscles of
marine forms is related to the osmotic equilibrium
with the blood, which in these anunals has nearly the
same concentration as that of sea water. Because
the concentration of inorganic ions in the tissues
is lower than in the blood, a relatively high con-
centration of free amino acids in the tissues is
necessary for maintaining osmotic equilibrium.

The concentration of protein in blood plasma
in 0. edidis, Pecten maximus, j\Iya arenaria, and
Mytilus edulis is about 0.1 percent (Florkin and
Blum, 1934). Samples of blood were collected

T.ABLE 43. — Free amino acids {mg./lOO g. of ivater) in the
muscles of marine and fresh-water bivalves*

Alanine.--

Arginine

Aspartic acid-.
Glutamic acid.

Glycine

Histidine

Isoleuciue

Leucine

Lysine

Methionine

Phenylalanine

Proline

Threonine

Tyrosine

Valine

Ostrea
edulis

646.0
66.6
26. 1

264.0

248.0
22.9
19.2
12.9
22.0
8.4
8.5

166 0
9.7
10.3
10.8

Mytilus
edalis

340

415.5

200.4

317.0

399.0

12.1

24.8

15.4

39.4

9.8

9.6

29.0

30.5

12.7

14.4

Anodonta
cygnea

8.8
36 5
4.4
29.4
13.2
2.5
6.3
3.6
8.2
0.4
1.6
1.0
3.6
2.2
3.3

after tearing off the gills, and the cells were
removed by centrifugation.

Nutritional studies have been made by feeding
)'aw and frozen oysters to albino rats suffering
from artificially induced vitamin deficiency (Ran-
doin and Portier, 1923; Jones, Murphy, and
Nelson, 1928; Whipple, 1935). E.xperimental
results showed that oysters are a good source
of vitamins A, B, and D. Daily feeding of 2 g.
of fresh Chesapeake Bay oysters (0.32 g. on a dry
basis) furnished sufficient vitamin A to cure rats
of xerophthalmia (chronic inflammation and
thickening of the conjunctiva of the eye) in 18 to
20 days. According to Whipple's data the
vitamin content of oysters taken in October from
Great South Bay, Long Island, N.Y., was ap-
proximately three LT.S.P. units/g. The vitamin
D content of oysters harvested from the same
bay in the faU was approximately 0.05 U.S. P.
units/g. and the vitamin B (B') content was
found by Whipple to be approximately 1.5
Sherman units/g. Oysters are a very modest
source of vitamin D and their antiricketic value
is low.

In more recent work Wentworth and Lewis
(1958) determined by chemical analyses the
contents of niacin, riboflavin, and thiamine
(table 44). None of these vitamins was found to
have a distinct pattern of seasonal fluctuation.

Table 44. — Range of vitamin contents in Apalachicola,

Fla., oysters in mg./lOO g. wet weight
[From February 1955 to August 1956 From Wentworth and Lewis, 1958]

February

August

1.41

0.06
0.08

2.52

Riboflavin

0.28

0. 13

•From: Duchateau, Sarlet. Camien, and Florkin, 1952. The French
investigators express the concentration in a rather iinique manner as mg./
100 g. of "d'cau de fibre."

Thiamine content of raw shucked oysters
studied by Goldbeck (194/) varied by region.
Oysters collected in the waters of Connecticut
and New York contained more thiamine per unit
of fresh weight than those from Louisiana, Georgia,
Virginia, and Maryland (table 45). The determi-
nation of thiamine was made by chemical method
(using thiochrome) and by rat growth method,
which gave values about 9 percent smaller than
the chemical tests.

The sterol mixtures of bivalves are of particular
interest, because in certain species they are the rich-
est natural sources of provitamin D. In Modiolus
demissus of the Atlantic coast of America the
content of provitamin D was found to be suffi-

CHEMICAL COMPOSITION

391

ciently rich to warrant commercial exploitation
for the manufacture of vitamin D preparations
(Bergmann, 1962).

The cholesterol in bivalves constitutes but a
small portion of the sterol mixtures in comparison
with those obtainable from gastropods. In C.
virginica and C. gigas Bergmann (1934) found a
new sterol which he named ostreasterol. Similar
compounds found in the sponge Chalina and in
Japanese oyster (C. gigas) which were named
chalinasterol and conchasterol. Reinvestigation
of bivalve sterols proved the identity of ostreas-
terol, chalinasterol, and conchasterol with 24-
methylenecholasterol (Bergmann, 1962).

The conditions and type of food which favor the
enrichment of the bivalve body with sterols and
vitamins are not known.

Table 45. — Thiamine contents per 100 g. of raw oysters

from different states

(From Goldbeck, 1947]

State

Thiamine
In^g.

State

Thiamine

Connpp.tlniit

170
170-180
100-103

Virginia

lOO-llO

New York

Georgia

98-106

Maryland.

110-130

CONDITION INDEX

Oysters of good quality have relatively large
amounts of meat in relation to their total volume.
Their glycogen content is high, and the meat
has a creamy color and pleasant flavor. De-
termination of glycogen and of the total solids
is a time-consuming procedure which cannot be
regularly used in the oyster trade. To Caswell
Grave (1912) belongs the credit of expressing the
quality or fatness of oysters as the percentage
of the volume of space enclosed between the two
valves occupied by the oyster body. Hopkins
(quoted from Higgins, 1938, p. 49-50) developed
this idea further and suggested that the ratio

dry weight of meat in g. X 100
volume of cavity in ml.

is a useful index of quality. Since then the ratio
between the dry or wet weight of meat to the
volume of the cavity has been used by many
investigators in determining the condition index
of oysters. The volume of the cavity can be
measured by displacement. The oyster shells
are thoroughly scrubbed with a wire brush, and
each oyster is placed in a glass container provided

Figure 364. — Glass container used for determining the
volumes of whole oysters and oyster shells by
displacement.

with a side arm set at an angle to the side wall of
the container (fig. 364). First the zero level of
water is marked; then the oyster is introduced,
and the level is brought back to zero position by
draining the water through a drain pipe at the
bottom. The water is collected, and its volume
measured. The oysters are then taken from the
container, opened carefully, and the meats re-
moved. The volume of shells without meat is
measured, and the volume of shell cavity is found
by the dift'erence between the volume of the whole
oyster and the volume of its shell.

The methods used in determining condition
index have not been standardized and, therefore,
the values given by different investigators vary.
Baird (1958) applied statistical analysis in evaluat-
ing the significance of variation of the index.
He also demonstrated that little accm'acy is
gained by using dry weights as an index measure-
ment and that even with fau'ly large samples the
fluctuations may be considerable. He concludes
that 50 oysters per sample is the largest practicable
number.

392

FISH AND WILDLIFE SERVICE

The condition index has a practical use for
oyster growers. It gives an objective method
of comparing the quahty of oysters talcen from
difl'erent commercially exploited oyster beds,
but this comparison is valid only if oysters of the
same species and of approximately the same age
are used. Oysters from overcrowded natural
reefs and young oysters are usually Hat, witli very
little inner space between the valves; consequently
their condition factor will be relatively high
because the bodies occupy almost the enthe shell
cavity.

Westley (1961) found that the condition index
of samples of C. gigas in Oakland Bay, Wash., in
1956 varied between 6.2 and 8.1. The condition
index of the oysters in North Bay, Wash., in
August 1957 was 12.3 to 15.0.

Observations were made on 0. lurida in Oyster
Bay on July 11 and repeated on August S, 1957.
On the first date the range of condition index
varied from 6.6 to 7.2, while a month later it had
increased to 16.0 to 17.3. The improvement was
probably associated with an accumulation of
glycogen after discharge of the larvae. In this
case the volume of the oysters was measui'ed by
weighing them first in air and then in water, and
computing the volimie from the difference between
the weights. The removed meats were oven-
dried to constant weight (at 100° C).

Although the condition index may be useful to
oyster growers as a measure of quahty of oyster
meats, it provides no advantages for physiological
studies, and cannot be used for the study of
growth.

ANTIBACTERIAL AGENTS

Antibacterial and antiviral agents were found
in the meat of the oyster {C. virginica), in abalone
(Haliotis rufescens), and in a number of other
mollusks. These substances have been isolated
in the laboratory of the U.S. Pubhc Health Service
and their activity tested in vitro on a number of
pathogenic bacteria (Li, Prescott, Jahnes, Martino,
1962), and on various strains of influenza and polio
viruses. In vitro tests were made by using cul-
tures of monkey kidney tissue; tests in vivo were
conducted by feeding white mice with the extracts
and recording the death rate after infection.
There are two differe'nt extracts which the authors
call Paolin 1 and Paolin 2 (according to Li, Paolin
is a Chinese word which means "abalone extract").
Paolin 1 was found to inhibit the growth of

Staphylococcus aureus, Streptococcus j)yogenes, Sal-
monella typhosa, Shizella dysenteriae, and others.
Paohn 2 fed to white mice decreased the death
rate of animals experimentally infected by virus.
The decrease was from 36 percent in the controls,
which received no extracts, to 10 percent in the
animals fed with oyster or abalone extracts before
infection (Li and Prescott, 1963). The discovery
by Li and his coworkers is of great practical sig-
nificance and opens a new chapter of research into
the role of antibacterial and antiviral agents in
the tissues of mollusks.

BIBLIOGRAPHY

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CHEMICAL COMPOSITION

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1949. Techniques of histo- and cytochemistry.
Interscience Publishers, Inc., New York, 531 pp.
Goldbeck, Clar.a Gale.

1947. Some studies on the content of thiamine and
anti-thiamine factor in fishery products. [U.S.]
Fish and Wildlife Service, Commercial Fisheries
Review, vol. 9, No. 8, pp. 13-21.
Grave, Caswell (editor).

1912. A manual of oyster culture in Maryland.
Fourth Report of the Shell Fish Commission of
Maryland, 1912, pp. 279-348. (Reprinted in 1944
by King Brothers, Inc., Baltimore, Md.]
Gunter, Gordon.

1942. Seasonal condition of Texas oysters. Proceed-
ings and Transactions of the Texas Academy of
Science, vol. 25, pp. 89-93.

/^

394

FISH AND WILDLIFE SERVICE

HiGGiNS, Elmer.

1938. Progress in biological inquiries, 1937. [U.S.]
Bureau of Fisheries, Report of the Commissioner
of Fisheries for the fiscal year 1938, appendix 1
(Administrative Report Xo. 30), pp. 1-70.
HiLTNER, R. S., and H. J, Wichmann.

1919. Zinc in oysters. Journal of Biological Chem-
istry, vol. 38, No. 2, pp. 205-221.
Hopkins, A. E.

1949. Determination of condition of oysters. Sci-
ence, vol. 110, No. 2865, pp. 567-568.
Hopkins, Sewell H., J. G. Mackin, and R. Winston
Menzel.

1954. The annual cycle of reproduction, growi^h, and
fattening in Louisiana oysters. National Shell-
fisheries Association, 1953 Convention Papers, pp.
39-50.
Hunter, Albert C, and Channing W. Harrison.

1928. Bacteriology and chemistry of oysters, with
special reference to regulatory control of produc-
tion, handling, and shipment. U.S. Department
of Agriculture, Technical Bulletin No. 64, 75 pp.
Jones, D. Breese.

1926. The nutritional value of oysters and other sea
food. American Journal of Public Health, vol. 16,
No. 12, pp. 1177-1182.

Jones, D. Breese, J. C. Murphy, and E. M. Nelson.
1928. Biological values of certain types of sea food.
II. Vitamins in oysters {Ostrea virginica). Indus-
trial and Engineering Chemistry, vol. 20, No. 2,
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Kennedy, R. P.

1927. The quantitative determination of iron in tis-
sues. Journal of Biological Chemistry, vol. 74,
No. 2, pp. 385-391.

Lankester, E. Ray.
'^ 1886. On green oysters. Quarterly Journal of Mi-
croscopical Science, vol. 26, pp. 71-94.
Lee, Charles F., Caroline H. Kurtzman, and Leonard
Pepper.

1960. Proximate composition of southern oysters —
factors affecting variability. U.S. Fish and Wild-
life Service, Commercial Fisheries Review, vol. 22,
No. 7, pp. 1-8.
Lee, Charles F., and Leonard Pepper.

1956. Composition of southern oysters. U.S. Fish
and Wildlife Service, Commercial Fisheries Review,
vol. 18, No. 7, pp. 1-6.
Li, C. p.

1960. Antimicrobial activity of certain marine fauna.
Proceedings of the Society for Experimental Biology
and Medicine, vol. 104, pp. 366-368.
Li, C. p., and B. Prescott.

1963. Antimicrobial agents from shellfish. [Ab-
stract.] Abstracts 16th annual Gulf and Caribbean
Fisheries Institute, November 12-16, 1963, 2 pp.
[Mimeographed.]
Li, C. p., B. Prescott, W. G. Jahnes, and E. C. Martino.
1962. Antimicrobial agents from moUusks. Trans-
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LiLLIB, R. D.

1948. Histopathologic technic. The Blakiston Com-
pany, Philadelphia, Pa., 300 pp.
LoosANOFF, V. L., and James B. Engle.

1947. Effect of different concentrations of micro-
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U.S. Fish and Wildlife Service, Fishery Bulletin 42,
vol. 51, pp. 31-57.
Loubatie, Ren^.

1931. La fixation de I'iode a I'etat organise dans le
corps de I'hultre vivante. Journal de Medecine de
Bordeaux et de la Region du Sud-Ouest, vol. 108,
No. 4, pp. 99-110.
./Marks, Graham Wallace.

1938. The copper content and copper tolerance of
some species of mollusks of the southern California
coast. Biological Bulletin, vol. 75, No. 2, pp. 224-
237.

McCollum, E. V.

1926. V. Vitamins in fish and shellfish. In Harry
R. Beard (compiler). Nutritive value of fish and
shellfish, pp. 546-548. [U.S.] Bureau of Fisheries,
Report of the Commissioner of Fisheries for the
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McHugh, J. L., and J. D. Andrews.

1957. Computation of oyster yields in Virginia.
Virginia Fisheries Laboratory Contribution No.
55. Virginia Fisheries Laboratory of the College
of William and Mary and the Commission of Fish-
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Medcof, J. C, and A. W. H. Needler.

1941. The influence of temperature and salinity on
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Mendel, Lafayette B., and Harold C. Bradley.

1905. Experimental studies on the physiology of the
mollusks. — second paper. American Journal of
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Mitchell, Philip H., and Raymond L. Barney.

1917. The occurrence in Virginia of green-gilled
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pp. 135-150. (Document 850.)

Nilson, Hugo W., and E. J. Coulson.

1939. The mineral content of the edible portions of
some American fishery products. [L^.S.] Bureau of
Fisheries, Investigational Report No. 41, vol. 2
(1950), pp. 1-7.

Power, E. A.

1962. Fishery statistics of the United States, 1960.

U.S. Fish and Wildlife Service, Statistical Digest

53, 529 pp.
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1923. Etude des vitamines des moUusques. Presence

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CHEMICAL COMPOSITION

395

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1930. The colorimetric determination of manganese

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1923. The occurrence of copper and zinc in certain
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1926. II. Fish and shellfish as a source of protein.
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Tressler, Donald K., and Arthur W. Wells.

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U.S. Department of Agriculture.

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Wells, Arthur W.

1925. Iodine content of preserved sea foods. [U.S.]
Bureau of Fisheries, Report of the Commissioner of
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(Document 979), pp. 441-444.

Wentworth, Jane, and Harvye Lewls.

1958. Riboflavin, niacin, and thiamine in Apalachi-
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Westley, Ronald E.

1959. Olympia and Pacific oyster condition factor
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Washington, Department of Fisheries, Shellfish
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[Mimeographed.]

1961. Selection and evaluation of a method for
quantitative measurement of oyster condition.
Proceedings of the National Shellfisheries Associa-
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Whipple, Dorotht V.

1935. Vitamins A, D, and B in oysters — effect of
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396

FISH AND WILDLIFE SERVICE

CHAPTER XVIIl
ENVIRONMENTAL FACTORS AFFECTING OYSTER POPULATIONS

Page

Positive factors of environment _ 399

Character of bottom -. _. 399

Water movements. 400

Salinity -.- - --. 404

Temperature 407

Food - 408

Negative factors of environment _ 409

Sedimentation. 410

Disease 415

Malpeque Bay disease - - 415

DeTmocystidium marinum 415

Disease associated with HaplospoTidium 417

Shell disease... 417

Foot disease 418

Hexamita. 419

Nematopsis 419

Trematodes and parasitic copepods 419

Commensals and competitors... 420

Boring sponges 420

Boring clam 421

Mud worms 421

Oyster crab 425

Splrochaetes 426

Perforating algae 426

Fouling organisms 426

Predators 430

Carnivorous gastropods. 430

Starfish 437

Flatworms 438

Crabs 439

Mud prawns and flsh. 439

Birds 439

Man 440

Pollution 441

Domestic sewage 442

Industrial waste 443

Radioactive waste-.. 445

Combined effect of environmental factors 445

Bibliography 446

The various species of the family Ostreidae
inhabit the coastal waters within the broad belt
of the sea, limited by the latitudes 64° N. and 44°
S. Some large aggregations cover many square
miles of bottom of littoral and intertidal zones;
they also thrive above the bottom attached to
rocks and underwater structures, branches and
trunks of fallen trees, and miscellaneous objects.
These aggregations of live oysters and empty shells
are called oyster bottoms, oyster beds, oyster
banks, or oyster reefs. The expressions are not
well defuied either biologically or in the legal
sense and are used interchangeably. Only those
species of oysters which form large and dense
populations are important to man as a source of

FISHERY bulletin: VOLUME 64, CHAPTER XVIII
T33-851 O — 64 26

food. Those living singly and widely dispersed
are of no commercial value.

Descriptions of oyster bottoms found in the
world literature combined with personal observa-
tions over the course of years in the United States,
France, Italy, the West Indies, Cuba, Venezuela,
Panama, Hawaii, and some of the South Pacific
islands have convinced me that, regardless of the
species of oysters present, certain major factors
are common to all oyster bottoms.

It is a matter of historical interest that more
than SO years ago Mobius (1883) established the
concept of a biocenosis or a social community
using an oyster bank as an example. According
to his definition every oyster bed is to a certain
degree —

... a communitv of living beings, a collection of species,
and a massing of individuals, which find here everything
necessary for their growth and continuance, such as suitable
soil, sufficient food, the requisite percentage of salt, and
a temperature favorable to their development. Each
species which lives here is represented by the greatest
number of individuals which can grow to maturity subject
to the conditions which surround them, for among all
species the number of individuals which arrive at maturity
at each breeding period is much smaller than the number
of germs produced at that time. The total number of
mature individuals of all the species living together in
any region is the sum of the survivors of all the germs
which have been produced at all past breeding or brood
periods; and this sum of matured germs represents a
certain quantum of life which enters into a certain number
of individuals, and which, as does all life, gains permanence
by means of transmission.

Mobius further commented that a change in one
factor of a biocenosis affects other factors of the
environment and eventually changes the com-
munity character. Relative abundance of various
species constituting a bottom community is
affected by changes in estuarine environment
and by man's activities which alter the environ-
ment. Patterns of currents, salinity gradients,
and turbidity of water may be changed by dredg-
ing operations, construction of inshore installa-
tions, and other harbor and waterway improve-

397

ments. Commercial dredging removes substantial
portions of natural oyster grounds; planting of
seed oysters for growing increases artificially the
population densities. Inadvertent introduction
of foreign species, competitors, and predators
disturbs the established biological balance. Final-
ly, excessive discharge of domestic sewage and
trade wastes causes irreparable damage to pro-
ductive oyster bottoms.

The productivity of a sea bottom may be
measured by determining the sum of weights of
all animals and plants in a unit of area. The
value, called community biomass, is of consider-
able theoretical interest to the marine ecologist
engaged in the study of oceanic productivity. It
has, however, no practical application in deter-
minations of the productive capabilities of a
community dominated by a single species such
as oyster, clam, or scallop. The species produc-
ti\'ity of any bottom may be materially reduced
by competitors, predators, and other conditions
that may suppress the reproduction and gi-owth
of commercially utilizable organisms while not
affecting or even sometimes encom'aging the
growth of noncommercial forms.

Descriptions of oyster bottoms usually provide
information regarding their location, type of
bottom, depth and salinity of water, the principal
species associated with oysters, and the abundance
or absence of predators. This type of description
is found in the papers of Dean (1892) on .South
Carolina grounds; Moore on the condition and
e.xtent of oyster gi-ounds in Texas (1907), Louisi-
ana (1899), Mississippi Sound (1913), James
River, Virginia (1910), Delaware Bay (1911);
Pearse and Wharton (19.38) on oysters of Apala-
chicola Ba}^; Frey (1946) on oyster bars in the
Potomac River; Hagmeier and Kandler (1927)
on oyster banks in North Freisland shoals,
Germany; Joubin on the coast of France (1906,
1908), and many othei's.'

Because of the great diversity in the kind
and number of species forming an oyster com-
nmnity only a few generalizations can be drawn
from descriptive data: 1) in common with other
bottom communities, oyster grounds of tlie warm
southern waters support a gi-eater \ariety nf
species than do the colder waters of the nortliern
latitudes, and 2) the variety of plant and animal
species is less in waters of low salinity than in
adjacent areas of higher salt concentrations.

The inferences are in accord with observations
made by European ecologists and summarized
by Hedgpeth (1953). In the Elbe estuary the
weight of all invertebrates per square meter of
bottom decreases from 6,068 g. in the area of full
oceanic salinity to only 37 g. in brackish water.
A similar decrease in the weight of community
biomass is found along the northern coast of
Germany, although the difference is much smaller
ranging from 304 g. per square meter of sea
bottom to 16 g. in the inshore areas. The decrease
in the biomass cannot be attributed to a single
factor of the envu"onment since other conditions
such as rate of water movements, sedimentation,
and food content are associated with the salinity
changes.

There are many well-documented cases of
destruction of productive oyster bottoms by
human activities. Mobius (1883) cites formerly
rich oyster beds of Cancale, Rochefort, Marennes,
and Oleron on the West Coast of Europe in which
the oyster populations were replaced by cockles
and mussels. The newcomers were present in
small numbers while the oysters floui'ished but
greatly increased in abundance when the removal
of oysters left more space for them to settle.

Many well-documented examples may be cited
of the destruction of oyster bottoms by sand and
mud stirred up by dredging operations in nearby
areas. One incidence of this nature occurred in
1935 to 1938 near the Buzzards Bay entrance to
the Cape Cod canal, Mass., where valuable oyster
grounds were bm-ied under 8 to 12 inches of
material that was disturbed by dredging and then
settled on the oyster grounds. Three to four
years later the area was repopulated by quahogs
and continues to remain highly productive,
although the species composition has been
completely changed.

Discounting minor local variations, the basic
requirements of the oyster are identical regardless
of the location of the oyster bottoms. The
suitability of a bottom area for the development
of a productive oyster community can, therefore,
be evaluated if the effects of different environ-
mental factors are estinaated.

Principal factors favorable for the propagation,
growth, and general welfare of an oyster com-
munity are character of bottom, water movements,
salinity of water, temperatm'e, and food. The
unfavorable or destructive factors that tend to
inhibit the growth and productivity of a com-

398

PISH AND WILDLIFE SERVICE

munity are sedimentation, pollution, competition,
disease, and predation. The interaction of these
five positive and five negative factors acting
simultaneously on a community determines its
utiUzable productivity.

POSITIVE FACTORS OF
ENVIRONMENT

CHARACTER OF BOTTOM

Oysters may grow equally well on a hard, rocky
bottom or on semihard mud firm enough to sup-
port their weight. Shifting sand and soft mud
are the only types of bottoms which are totally
unsuitable for oyster communities. With the
exception of these extreme conditions oysters
adapt themselves to a great variety of bottoms.
They thrive well on shore rocks and underwater
structures which are left exposed at low tide. The
controlling factor in this situation is the climate,
since no oyster can survive several hours exposure
to below freezing temperature, and, therefore,
none are found growing near the surface in the
latitudes where in winter they may be killed at
low tide, or frozen in the ice and carried away by
tidal currents. The degree of softness and insta-
bility of the bottom can be quantitatively meas-
ured by penetrometers, and by determining the
amount of bottom material transported by cur-
rents of different velocities. The depth of sinking
into a mud layer of a probe of known dunensions
imder constant weight can be used as a measure
of the relative softness of sediment. The deter-
mination must be made in situ because the removal
of a sample and handling in the laboratoxy changes
the consistency of mud. A penetrometer to eval-
uate certain physical properties of marine sedi-
ments in situ has been designed by Miller (1961).
The instrument is a conical probe which is driven
at low, constant speed into the bottom; resistance
to penetration as the prol^e sinks mto the bottom
is recorded graphically. An instrument answering
these specifications and constructed at the Uni-
versity of Rhode Island has been used in research
at the Narragansett Marine Laboratory at Kings-
ton, R.I., and has proved reliable in providing
information about compactness, degree of plas-
ticity, allowable bearing loads, and other proper-
ties of sediments. No reliable method has yet
been developed for measuring the resistance of
sediment to water current in the sea.

A soft muddy bottom may be artificially im-
proved by planting oyster or clam shells to attain
the desired firmness. Other materials, such as
gravel and slag from blast iron furnaces, have
been tried experimentally but are less satisfactory,
primarily because of the greater weight and higher
cost. At present the reinforcement of oyster
bottoms by shells remains the principal practical
method used on a large scale for the improvement
of oyster bottoms or for the establishment of new
ones.

Soft nmddy bottoms may be gradually con-
verted by the oysters themselves into oyster
banks or reefs because of an innate ability of
larvae to choose a substratum upon which to
settle. This ability is probably common to most
species of bottom invertebrates having free-
swimming larvae (Verwey, 1949). The process
begins with the attachment of several larvae to
a single shell or other hard object lying on the
surface of the mud. Other larvae attach to
those that have already settled, and soon a cluster
of oysters is formed on the surface of the mud
(fig. 365).

Dead oyster shells dropping from clusters pro-
vide additional surfaces, and the reef begins to
grow horizontally and vertically. The process
is t3rpical for the tidal flats of South Carolina
and Georgia where successive phases can be
easily observed. Oysters grown on mud have
long, slender shells.

The suitabihty of bottom to an oyster com-
munity may be expressed by an arbitrary scale
from 0 to 10, according to the relative softness
and stability. Bottom conditions fully unsuit-
able for the formation of any oyster community
may be designated as zero. The zero value of
any positive factor denotes conditions under
which the community cannot e.xist, regardless
of the values of all other factors of the environ-
ment. The zero value of bottom factor refers
either to extremely soft mud not capable of sup-
porting the weight of an empty shell or to shifting
sand; both conditions are unsuitable for oysters.
Marginal conditions are indicated by 1 , and opti-
mum conditions by 10. The highest value of
bottom factor may be assigned to firm and stable
bottoms such as rocks and hard or sticky mud.
The value of 1 is assigned to the soft muddy
bottoms of the South Atlantic States and Texas.

FACTORS AFFECTING OYSTEH POPULATIONS

399

'^t

Figure 365.— Initial stage in the formation of an oyster bank on very soft mud of a tidal flat. Photographed at low

tide near Brunswick, Ga.

WATER MOVEMENTS

Free exchange of water is essential for the
growth, fattening, and reproduction of oysters.
An ideal condition is represented by a steady,
nonturbulent flow of water over an oyster bed,
strong enough to carry away the liquid and gaseous
metabohtes and feces and to provide oxygen and
food. Furthermore, an oyster bed can expand
only if the larvae are carried by the currents and
at the time of setting are brought in contact
with clean, hard sm-faces. Estuaries seem par-
ticularly suitable for the expansion of oyster
communities and for the annual rehabihtation of
oyster populations reduced by harvesting because
some larvae, carried back and forth by the oscil-
lating movements of tidal waters, eventually
settle beyond the place of their origin.

In large embayments, such as Long Island
Sound, the difference between the surface and
bottom sahnities is small, about l°/oo or 2%©.
In tidal rivers and true estuaries the differences
between the salinities of the lower strata and those
at the surface are considerable. Salinity strati-

fication, as will be shown later, complicates the
pattern of circulation.

The great variety of conditions found in the
bodies of water within the tidal zone nuikes it
difficult to define the term "estuary" in a few
precise words. A Latin dictionary (Andrews,
1907) defines the word "Aestuarium" or "Aestus"
as a part of the seacoast overflowed at flood tide
Init at ebb tide left covered with mud and slime.
Some authors extend the concept of an estuary to
include such large bodies of water as the Mediter-
ranean Sea and the Gulf of Mexico, while others
restrict the use of the term to relatively small
coastal indentures in which the hydrogi-aphic
regime is influenced by the river discharge at the
head and the intrusion of sea water at the mouth.
Cameron and Pritchard (1963) define an estuary
as "a semienclosed coastal body of water having
free connection with the open sea and within
which the sea water is measurably diluted with
fresh water deriving from land drainage." The
essential features of a true estuary are the inflow
of river water at the head and the periodical
intrusion of sea water at its mouth. Stommel
(1951) classifies estuaries by the predominant

400

FISH AND WILDLIFE SERVICE

causes of movement and mixing of water, which
may be due either to tide, wind, or river flow.
Rochford (1951) points out the significant differ-
ences between brackish water and estuarine en-
vironment. According to his ideas, brackish water
refers to a dynamically stable environment of
lakes, lagoons, etc., in which sea water, diluted
by fresh water, is not necessarily influenced by
tidal movements. On the other hand, the estua-
rine environment, influenced by tidal rise and
fall, is dynamically unstable.

The persisting factors of a typical estuarine
envh'onment are seasonal salinity variations and
circidation exchange between the river and sea
water. The intruding salt water forms a wedge
or prism with its base at the mouth and the tip
at the upper part of the estuary. The position of
the wedge along the bottom and its dimensions
depend to a gi-eat extent on the flow of the river
water.

Circulation and mixing of water is a highly
complex problem adecjuately discussed in the
papers of Rochford (1951), Ketchum (1951a,
1951b), Pritchard (1951, 1952a, 1952b), and in
the textbook. The Sea, vol. II, edited by Hill
(1963).

It is important for a biologist to understand
that the type of circulation that prevails in a
specific estuary depends on physical structure,
i.e., size, depth, bottom configuration, etc., river
flow, and vertical salinity gxadients along the
enth'e length from head to mouth. Circulation
pattern and mixing have important biological
implications in the study of the distribution and
transport of sediments, pollutants, and plank-
ton, including free-swimming larvae of sedentary
invertebrates.

The volume of fresh water entering at the head
of an estuary occupies the upper layer and
exceeds the volume of the up-estuary flow in the
lower and more saline layer by an amount sufficient
to move the fresh water toward the sea (Pritchard,
1951). As one moves seaward, the volume of
saline water contributed by the ocean increases
while the river water, entered at the head of the
estuary is being removed. The process of removal
of river water, called flushing, continues ttu-oughout
the estuary from its mouth to the so-called "inner
end" which is defined by Ketchum (1951b) as
"the section (of an estuary) above which the
volume required to raise the level of the water
from low to high water mark is equal to the

volume contributed by the river dm-ing a tidal
cycle." Consequently, there will be no net
exchange of water through this section dm-ing a
flood tide and the water above the section should
be completely fresh.

The sahnity at any location below the inner
end varies with tide but retm-ns to substantially
the same level on successive tidal stages. Assum-
ing that a net seaward transport of fresh water
during any tidal cycle is equal to the volume
introduced by the river in the same period of time,
and that there is no net exchange of salt water
through the cross section during the same tidal
cycle, Ketchum (1951b, 1954) advanced a simpli-
fied theory which permits an easy calculation of
the proportion of water removed by the ebb tide.
This theory is based on the assumption that in
each of the volume segments, limited by the
average excm'sion of water on the flood tide, the
water is completely mixed at high tide. Accepting
this assumption, which is obviously a simplification
of the actual conditions, the rate of exchange in a

given segment (r^) has the value rp=p ^ in

which Pn is the intertidal volume and Vn the
low tide volume of the segment n. The average
length of time required for the river water with
a particle suspended in it to move thi-ough a
segment of an estuary is called flushing time,
which is defined as a ratio obtained by dividing
the volume of river water, calculated from the
salinity data, by river flow. The ratio is expressed
in number of tides. In Raritan Bay, N.J., a
sm'vey made by the Woods Hole Oceanographic
Institution indicates the flushing time for the
entire estuary was 60 tides.

If a stable pollutant is discharged at a constant
rate at the head of an estuary and is uniformly
mixed as it is transported downstream, its propor-
tion in the water can be calculated by using the
formula of Hotelling which was applied in deter-
mining the concentration of poUutant over
Olympia oyster beds (Hopkins, Galtsoff, and
McMiUin, 1931). According to this formula the
proportion p of a contaminant in a basin is:

a+bl

l-{l-a+b)

V

]

where a is the rate of discharge of contaminant
in acre-feet per day, b the rate of influx of water
into the basin in acre-feet per day, V the total

FACTORS AFFECTING OYSTER POPULATIONS

401

'^ which are usually encountered in estu-

volume of the basin in acre feet, and t time in
days since the pollution started.

In this formula it is assumed that the influx
and efflux occur discontinuously once a day. By
assuming that the influx of contaminated water
and the efflux are contmuous, Tuckerman (see:
Galtsoff, Chipman, Engle, and Calderwood, 1947,
p. 100) arrives at the foHowing formula:

p^po.[l-{l-K)']

where poo is the hmit which the proportion of
contaminated water approaches after a long time

and X=^^^- Computations made by using the

two formulas indicate the same value for the

nm=—^! but differ in the rate at which this
' a-\-b

limit is approached. For the small values of

aries, the two rates are practically identical and
the simpler Hotteling formula may be applicable.

In many estuaries the water is stratified. With
relation to stratification and circulation patterns
Pritchard (1955) distinguishes four types of
estuaries — highly stratified (type A), moderately
stratified (type B), and vdrtually homogeneous
(types C or D). The reader interested in the
dynamics and flushing in different types of es-
tuaries should consult the original contributions
of Pritchard (1952a, 1952b, 1955) and Pritchard
and Kent (1953) in which the complex hydro-
dynamical problem is analyzed. It is sufficient
for a student of oyster ecology to realize that
vertical and horizontal distributions of oyster
larvae will be different in each of the four principal
types of estuaries.

A free-swimming organism such as bivalve larva
cannot be considered in the same manner as a ma-
terial dissolved in water or as an inanimate body
passively transported by water movements. Lar-
vae of bivalves, barnacles, and other invertebrates
may have a tendency to swarm and, therefore,
their distribution may not be uniform even in a
homogeneous environment. Oyster larvae react
to changes in the environment by periodically
closing their valves and dropping to the bottom or
by swimming actively upward or in a horizontal
plane. Consequently, they may be carried up-
stream or downstream according to their position
in the water column. Field observations in the

estuaries of New Jersey and Chesapeake Bay
(Carriker, 1951; Manning and Whaley, 1955;
Nelson, 1952) in which the salinity of water in-
creases from surface to bottom indicate that ver-
tical distribution of larvae is not uniform. The
late umbo larvae of C. virginica have a tendency
to remain within the lower and more saline strata,
and are probably stimulated to swim by the change
in salmity at flood tide. A brood of larvae swim-
mmg withm a given salinity layer will be passively
moved in the direction of the current. Nelson
observed that in Barnegat Bay, N.J., the brood of
larvae of setting size was carried about 3 miles up
the bay in a single evening spring tide. In
Yaquina River, a small tidal stream along the
ocean shore of Oregon, swimming larvae were
transferred by tidal currents and set several miles
above the natural beds (Fasten, 1931; Dimick,
Egland, and Long, 1941). There are many other
places where setting grounds are far above the
spawning grounds. Since the seaward discharge
of water in an estuary usuaUy exceeds the land-
ward movement, it was difficult to visualize a
mechanism by which the larvae can be transported
up an estuary and left there. The existence of
such a mechanism became apparent, however,
from the hydrographic studies by Pritchard (1951).
He found that estuaries may be considered as being
composed of two distinct layers: an upper layer
in which the net movement is toward the mouth
(positive movement), and the lower layer in which
the net movement is toward the head of the estuary
(negative movement). There is a boundary be-
tween the two layers which may be called "the
level of no net motion" (fig. 366). Since the net
movement seaward does not result in a net dis-
placement of the fines of constant safinity in the
upper layer, there must be a progressive transfer
of the deeper, higher safinity water of the lower
layer upward, across the boundary level, to be
included in the seaward transfer. The role of the
strongest tidal currents is primarily that of pro-
viding energy for the mixing processes. Compu-
tations made by Pritchard show that superimposed
on tidal oscillation there is a residual or nontidal
seaward drift on the surface and a net landward
drift along the bottom. He appfied his theory to
a study of the seed oyster area of the James River,
Va., and found that below a depth of about 10 feet
there is a net (or residual) upstream movement of
water at an average speed of slightly more than
one-tenth of a knot. This is the type of estuary

402

FISH AND WILDLIFE SERVICE

— RIVER

OCEAN

Figure 366. — Estuarine structure and net (i.e., nontidal or residual motion of water). Dotted line marks the boundary
between the upper and lower net movement; the boundary is the level of "no net motion." From Pritchard, 1951,
fig. 3.

in which larvae move from the spawning grounds
at the mouth of the river up into the seed oyster
bed area. Bousfield (1955) applied Pritchard's
ideas to an analysis of distribution of barnacle
larvae in the Miramichi estuary, New Brunswick,
Canada. The retention of larval populations in
this estuary was found to be due to a combination
of two factors: changes in the average vertical
distribution of successive larval stages, and the
strength and direction of transport by residual
drift at different depths. This theory of larval
retention and the mechanism of transport is not
apphcable to bodies of water in which there is no
saUnity stratification or where the residual upriver
drift is insignificant.

Least favorable in the life of an oyster com-
munity are occasional turbulent currents of high
velocities which may dislodge and cany away
young and even adult oysters not attached to the
bottom. Oysters attached to rocks and other
structures are not destroyed by strong currents,
but their valves are injured by small pebbles and
sand acting as an abrasive material. Live
oysters with shells damaged by abrasion can be
found in the Sheepscot and other tidal rivers
along the coast of Maine.

Continuous renewal of sea water running over
a bottom in a nonturbulent flow is the most
desirable condition for a flourishing oyster com-
munity. On the basis of experimental studies
discussed in chapter IX, p. 195, it may be assumed
that under optimal conditions of temperature
and salinity an average adult C. virginica trans-
ports water at the rate of 15 1. per hour. With
250 large oysters to a bushel and 1,000 bushels
per acre, an oyster bed of that size would require
3.75 million liters of water per hour. My
observations show that under the best of condi-

tions oysters can take in water only from a
distance not exceeding 2 inches from the shell.
It is, therefore, necessary to know the rate of
water exchange within the narrow layer adjacent
to the oyster bottom. The situation may be
dift'erent in the case of vertical mixing of water
due to turbulent flow.

The amount of water available to an oyster
population can be calculated if the number of
individuals on the bottom and the rate of water
movements are known. In the case of a turbulent
flow, vertical mixmg of water depends on the
degree of turbulency. If the mixing extends to
the height of 1 foot above the mud line, the total
volume of water in which the oyster population
lives in our example is 1 acre-foot or 325,851
gallons (1.25 million 1.). It follows that the
current velocity must be strong enough to renew
the volume of the layer above three times (3.75-^
1.25) every hour or 72 times in 24 hours. In
cases of greater population density the current
requirements are proportionally higher. Due
allowance should be made, of course, for the
presence of other water-filtering animals which
compete with the oyster for food. It is clear,
therefore, that great concentrations of water-
filtering organisms are possible only where there
is sufficient renewal of water. The oyster reef
in the Altamaha Sound, Ga., (fig. 367) is a good
example of this condition. Such concentrations
of live oysters crowded over a limited space
cannot exist in sluggish water and are found only
in rapid tidal streams.

The water movement factor can be evaluated
by determining whether the rate of renewal of
water over the bottom is sufficient for the needs
of the population and whether the pattern of
circulation is such that a certain percentage of

FACTORS AFFECTING OYSTER POPULATIONS

403

Figure 367. — Oyster reef in Altamaha Sound, Ga., at low tide, March 1925. The highest point was about 6 feet above
the bottom; water at the foot of the reef was 8 inches deep. The reef consists of live oysters growing on the side
and upper surfaces and attached to empty shells.

oyster larvae will be retained in the estuary by
the end of the larval period. It is obvious that
the requirements of water movements for the
growing of oysters are different from those nec-
essary for the settlement of larvae.

SALINITY

The general rule that the composition of sea
water is constant and varies only in the degree
of dilution by fresh water is applicable to the
estuaries and other basins which have direct
communication with the sea. Only in exceptional
cases is the circulation in an estuary so impeded
that stagnation and oxygen deficiency develop
and render the area unsuitable for oyster growth
and reproduction.

Oysters like many other euryhaline organisms
are able to live in sea water of very wide range
of salinity. According to the so-called Venice
system of classification of saline waters adopted
at the symposium organized by the International

Association of Limnology and the International
Union of Biological Sciences at Venice, Italy, in
April 1958, the range of salinity favorable for
C. virginica falls within two zones, the polyhaline,
from 307oo to lS7oo, and the mesohahne, from
l<S%oto 57oo (Symposium for the Classification of
Brackish Waters, 1958). Populations of oysters
found beyond the upper or lower limits of the
range exist under marginal conditions. Their
growth and gonad formation are inhibited, and
they are often decmiated either by floods, in the
lower zone of the range, or by predators which
usually remain in more saline waters. The non-
commercial oyster of the Atlantic and Gulf
coasts, 0. equestris, prefers more saline waters
and has been found on buoys as far as 20 miles
offshore where surface salmities ranged from 33%o
to 36 7oo (Galtsoff and Merrill, 1962). In the
subtidal regions of the coastal waters this species
is fomid in salinities of 207oo to 257oo-

404

FISH AND WILDLIFE SERVICE

An unstable salinity regime is an important
ecological factor in the tidal rivers and streams
inhabited by C. tdrginica; diurnal, seasonal, and
annual fluctuations are the normal features of
such an environment. Their effect on C. virginica
depends on the range of fluctuations and the
suddenness of the changes. For instance, oysters
that were replanted in September from a low
salinity area of the upper Chesapeake Bay (10%o
to 12 7oo) to the high salinity water of Sinepuxent
Bay (32%o to 33 7oo) all perished within 3 to 4
weeks after planting. Examination of the new
grounds disclosed that the sudden change in
salinity during hot weather was the primary
cause of mortality. On the other hand, a sunilar
transfer made by the same grower succeeded in
October and November when the air and water
temperatures were much lower.

The mean values of diurnal, seasonal, and
annual salinities are of little significance for
evaluating their effect on an oyster population.
The oyster can isolate itself from the outside
environment by closing its valves tightly and
survive adverse conditions, provided they do not
last indefinitely (see: ch. VIII). Since changes in
salinity are commonly associated with tempera-
ture changes, an attempt was made by Hedgpeth
(1953) to combine the two factors to express
what he calls "hydrographic climate." He plotted
ranges of means and extremes or monthly means of
salinity against temperature and obtained poly-
gons which provide a graphic representation of
the conditions existing in a given area for the
indicated period. The method appears to be
useful and may be profitably applied to oyster
research.

Oysters inhabiting the parts of estuaries in
which salinity is below 10 %o are seriously af-
fected by fresh water and could be destroyed by
floods lasting for several weeks. Mobile Bay,
Ala., investigated in 1929, may be cited as an
example of this condition. Oysters in Mobile
Bay grew on reefs which extended from the upper
to the lower parts of the bay. The river discharge
into the bay normally resulted in a salinity
gradient from 5%o to 30%o. However, in the 36
years from 1893 to 1929 the two tributaries, tlie
Tombigbee and Selma Rivers, rose 27 times to
flood stage with the flood conditions lasting from-
4 to 31 days. The height of the rivers at flood
stage in Februarj' and April 1929 was 65.4 and
56.2 feet, respectively, and lasted for 32 days

(GaltsofT, 1930) with the result that fresh water
prevailed over almost the entire bay and the
mortality at different parts of the bay varied from
100 percent in the upper parts to 85 and 54 percent
at the passes to Mississippi Sound.

Oysters in Mississippi Sound often suffer from
long-continued low salinities. Mortality of oys- .
ters in the Sound occurs when the local precipita-
tion in the Sound area, in the Pearl River basin,
or at some more distant point in the Mississippi
River basin occurs more or less simultaneously
and lowers the salinity to a harmful level (Butler,
1949b, 1952).

Freshets sometimes kill oysters in the James
River, Va. During a 6-week period from May 1 to
June 15, 1958, many of the native oysters died,
and as many as 90 percent perished on some
grounds where salinity did not become suitable
until July 1 (Andrews, Haven, and Quayle, 1959).
In a test made at the Virginia Fisheries Labora-
tory at Gloucester Point, oysters held in trays in
low salmity areas were "conditioned" to a low
physiological state (absence of heart beat and
ciliary motion and loss of mantle sensitivity).
The investigation lead to the conclusion that
oysters conditioned slowly at low temperatures
and low salinities can endure a prolonged situation
of unsuitable environment. Andrews, Haven,
and Quayle infer that "the mechanism of condi-
tioning appears to be a type of narcotization,"
an interesting idea which, however, needs veri-
fication.

The first symptoms displayed by an oyster
affected by water of lowered salinity are partial
or complete contraction of the adductor muscle
and slowing or cessation of water current through
the gills. With the drop counting technique
described in chapter IX it can be shown that the
ciUary activity of the gifl epithelium immediately
decreases when it comes in contact wdth water of
lowered salinity. The effect may be brief or
prolonged, depending on the degree of change
from the salinity level to which the oyster had
been adapted. When the salinity change is
about 10°/oo and continues for several hours,
both the rate of water transport and the time the
oyster remains open are decreased, and under
extreme conditions the feeding and respiration
cease. Experimental studies on the adaptation
of oysters to salinity changes were first made by
Hopkins (1936) on C. gigas of the Pacific coast.
He recorded the changes in the opening and closing

FACTORS AFFECTING OYSTER POPULATIONS

405

of the valves, and registered the deflection of a
small plate placed in front of the cloacal current.
As indicated in chapter IX this method is not
reliable for a quantitative determination of the
volume of water transported through the gills but
is adequate for determining the relative strength
of the cloacal current. The results show that the
adaptation to new conditions depends upon the
degree of change. Recovery was more rapid when
the salinity was increased than when the same
degree of change was made in the opposite direc-
tion. At a sahnity of about 13°/oo very little
water was transported even after several days
were allowed for adaptation, but recovery to
normal activity followed rapidly after the return
of the oyster to a normal environment in water of
267ooto297oo. Increased salinity, from 257oo to
39°/oo, produced no significant changes in the
water transport by C. gigas. An unfavorable
effect was recorded at 567oo, which is considerably
above the normal range of the oyster's habitat.

In experiments at the Bureau of Commercial
Fisheries Biological Laboratory, Milford, Conn.
(Loosanoff, 1952), C. virginica from Long Island
Sound accustomed to water of a stable salinity of
about 27 %o were placed directly in water of 20,
15, 10, and 5 °/oo made by the addition of a cor-
responding amount of fresh water. The loss of
food caused by the addition of plankton-free water
was compensated by providing measured amounts
of phytoplankton culture. The decrease in the
rate of water transport was proportionate to the
degree of change and varied from 24 to 99.6 per-
cent of the normal rate. Six hours of exposure to
the lowest salinities tested resulted in no perma-
nent injuries, and within a few hours after transfer
to the salinity of their natural habitat the oysters
fed, reformed the crystalline styles, and dis-
charged true feces and pseudo feces. Other ex-
periments at Milford at the same time demon-
strated that oysters conditioned to live in low
salinities can tolerate lower concentrations of salts
than oysters living in more saline waters. Al-
though the oysters were observed to feed in water
of 5 %o salinity their shell movement and water
transport were abnormal and growth was inhibited.

The reproductive capability of oysters is in-
liibited by low salinity. Butler (1949a) showed
that this is due primarily to the failure of gonad
development in oysters of the marginal area of
upper Chesapeake Bay; his findings were con-
firmed by experiments with Long Island Sound

oysters (Loosanoff, 1952). These experiments
have not demonstrated whether the failure of
gonad development is the direct result of lowered
salinity or is due to inadequate feeding.

Long-continued exposure to salinities above the
32 %o level also has an unfavorable effect on oyster
populations. This can be seen from the conditions
of Texas oyster beds. During the 6-year drought
from 1948 to 1953, the salinity in the bays of the
central Texas coast generally rose well over 36%o
and at times reached the 40%o level without an
appreciable decrease in tlie winter (Parker, 1955).
Previous records, from 1922 until 1948, show that
during most of the year salinity in this area ranged
from 5%o to 25%o with somewhat higher sal-
inities in the summer. With the increase in salin-
ity there was a gradual replacement of C. virginica
populations by 0. equestris. In 1952 over half of
the young oysters (spat) were 0. equestris, whereas
in years of low salinity the reefs were comprised
primarily of C. virginica. It is not known whether
the observed change was due to the inhibition of
gonad formation or to the failure of oyster larvae
to reach setting stage. From an ecological point
of view it is, however, significant that the replace-
ment of one species by another took place at the
time of the increase in salinity of water. The
surviving C. virginica were observed to develop
different shell characteristics: the valves became
crenulated, thin, sharp, and higlily pigmented.

Under certsiin circumstances the influx of fresh
water into estuaries may be beneficial. Some of
the carnivorous gastropods, flatworms, and star-
fishes, which are highly destructive to oysters, are
killed by brackish water that constitutes a barrier
through which they cannot penetrate. Decrease
in the salinity of water protects the populations of
oysters at the heads of the bays. Periodical flush-
ing wipes out the predators and restores the pro-
ductivity of beds. The population of oysters in
areas highly infested by their enemies, as in the
Apalachicola Bay, tlie upper half of the Delaware
Bay, and many others, cannot exist if the access
of fresh water to oyster bottoms is diminished and
the salinity increases above the 15%o level.

The evaluation of the salinity factor can be esti-
mated by determining the total percentage of
time the brackish water of less than 10%o
or water of salinity exceeding 34 %o remains on
an oyster bottom. The zero value is assigned to
conditions unsuitable for the oyster's existence;
marginal conditions are indicated by 1, and opti-

406

FISH AND WILDLIFE SERVICE

mum conditions by 10. Tlie values between are
based on the percentage of time the oyster popula-
tion is effected by unfavorable salinities.

TEMPERATURE

A great difference in climatic conditions exists
within the range of distribution of C. virginica.
The water temperature under which the species
lives varies from a minimum of about 1 ° C. during
the winter in northern states to a maximum of
about 36° C, which occasionally has been ob-
served in Texas, Florida, and Louisiana. The
temperature of oysters exposed to the sun at low
tide on the flat registers 46° to 49° C. measured
by inserting a small thermometer between the
slightly opened valves. Normally oysters of the
tidal zone remain exposed for 2 to 3 hours at the
maximum. Occasionally strong offshore winds
drive the waters away and oysters beds in shallow
places remain out of water for several days. Such
instances occur along the coast of Texas where as
a rule the low stage of water is caused by strong
northern winds. The exposed population may
perish either from excessive warming or from
freezing temperatures brought from the north by
cold fronts.

The temperature regime affects the life of the
oyster by controlling the rate of water transport,
feeding, respiration, gonad formation, and spawn-
ing. C. virginica ceases feeding at a temperature
of 6° to 7° C The maximum rate of ciliary
activity responsible for the transport of water is
at about 25° to 26° C; above 32° C. ciliary
movement rapidly declines. Nearly all functions
of the body cease or are reduced to a minimum at
about 42° C Using the seasonal fluctuations of
temperature, it is easy to determine the percentage
of time dm'ing which oysters in any given locality
continue to feed and reproduce. Similar observa-
tions may be made on the growth of shells and
calcification. Two curves in figure 368 show the
seasonal changes in mean monthly temperatures
in two localities separated by about 11.5 degrees
of latitude. The northern location of oyster
grounds of Long Island Sound is at about lat. 41 °
30' N.; the southern location is that of Apalachi-
cola Bay, Fla., at about lat. 30° N. The two
cui-ves, upper for Apalachicola and lower for Long
Island Sound, parallel each other but are at two
distinct levels. The difference is greatest during
the winter and early spring and is smallest during
the fall. The two temperature levels indicated

a:

3

<
a.

a.

5

<

5

2
O

32
30
28

-

_r-

\

APALACHICOLA
BAY

\
\

26
24

- /

A

\
\

\

h

1 2

22

-

/

/ ^

\ \

/ g

1 O

20

r

—

\ \

/ 2
/

18

-

1

\ "^

/
/

16

~

/

\ \
\ \

/

14

-

/

\ ^

/

12

.^

>

\

10

\

8

—

—

V

6

\

/ 3)

/ Z

A

-

LONG ISLAND \

/ >

y °

2

-

SOUND V

y^ H

0

1

1

1 1 1 1

1 1 1

Figure 368. — Mean monthly temperature in °C. of water
in Apalachicola Bay (upper curve) and Long Island
Sound (lower curve).

by broken lines mark the periods of successful
mass spawning and setting of oysters at tempera-
tures of 20° C. and above and the inhibition of
feeding and growth of the oysters below 8° C,
often called hibernation. Apalachicola oysters
continue to feed and grow throughout the year,
and then- reproductive season may last for 7
months or 58 percent of the year, whereas the
period of feeding and growth of the northern
oysters is limited to about 6K months, or 56 per-
cent of the time and the reproductive season is
restricted to 2 summer months, or about 16
percent of the time. The use of monthly means
based on several years of observations gives a
pictm-e of a general condition not unduly in-
fluenced by short-term fluctuations which may
differ from year to year.

Little is known about the prolonged effect of
temperatures above 32° to 34° C. on oyster popu-
lations. From a few physiological observations
it may be inferred that long continued exposure
to high temperature is unfavorable and impedes
the normal rate of water transport by the gUls.

FACTORS AFFECTING OYSTER POPULATIONS

407

The percentage of time available to an oyster
population for growth and "fattening", or for re-
production, can be used in evaluating the effect
of the temperature factor on the productivity of
an oyster bottom. A distinction should be made
between the reproductive capability of the popu-
lation and its growth and "fattening". In the
practice of oyster culture the areas of bottom
most suitable for setting are not considered de-
sirable for the rapid growth and conditioning of
oysters for market and vice versa.

FOOD

The quantities of food available to water-
filtering animals may be determined by taking
plankton and nannoplankton samples and by
noting the food requirements of a given species.
It has been shown by J0rgensen (1952) and
J0rgensen and Goldberg (1953) that the oyster
(C. virginica) and the ascidians {Ciona intestinalis
and Molgula manhattensis) filter about 10 to 20 1.
of water for each ml. of oxygen consumed, and
that about two-thirds of the energy absorbed by
them can be used for growth. The actual food
requirements of the animals studied by J0rgensen
probably do not exceed 0.15 mg. of utiUzable or-
ganic matter per liter of water used. Determina-
tions of phytoplankton in American coastal waters
made by Riley (1941), Riley, Stommel, and
Bumpus (1949), and Riley and Gorgy (1948),
show that the organic matter of the phytoplankton
in their samples ranged from 0.17 to 2.8 mg. per
liter. These waters contain enough material to
supply the energy requirements of C. virginica
which, according to my determinations, differ
from those made by J0rgensen (see p. 210 in ch. IX) ;
under normal conditions at 24° to 25° C. C. vir-
ginica uses from 3 to 4 mg. of oxygen per hour.

Quantitative samples of plankton and micro-
plankton taken throughout the year from the
water over a thriving oyster population can be
compared with samples collected in the plankton-
poor waters of the tropics. The water should be
pumped from the bottom zone, with care being
taken not to stir the sediment. Vertical hauls are
useless since the water a few inches above the
oysters does not come in contact with them ex-
cept in the case of strong vertical mixing.

Seasonal changes in the volume of plankton and
microplankton of water over a commercially pro-
ductive oyster bed in Long Ishind Sound are shown
in figure 369. Both types of samples were collected

S 0 N D

Figure 369. — Seasonal changes in the volume of plankton
(upper curve) in cm.' per 50 1. and microplankton
(nannoplankton) in cu. cm. per 1 m.' (lower curve) in
Long Island Sound in 1932 and 1933.

at the same time. For the plankton study 50 1.
of water were filtered through No. 20 bolting silk.
The collected material was preserved in 2 percent
formalin and transferred into tall glass cylinders,
and its volume read 24 hours later after the mate-
rial had settled at the bottom; the results are ex-
pressed in cm.'. For microplankton determina-
tion a 1 liter sample was taken from the bottom
and the water passed through a high-speed Foerst-
Juday type centrifuge rotating at 20,000 r.p.m.
The centrifugate was transferred to a 15 mm.
diameter tube and centrifuged for 5 minutes in a
clinical centrifuge at 14,000 r.p.m. and its volume
measured. Since the waters of Long Island Sound
are relatively free of silt, the amounts of detritus

408

FISH AND WILDLIFE SERVICE

in the samples were insignificant. Seasonal fluc-
tuations of plankton and nannoplankton follow a
typical pattern of maximum development from
about the middle of January to the end of April,
followed by a decrease during May to September
and a second maximum, smaller than the first, in
September to November. The latter period is of
greater significance for the northern oysters be-
cause they do not feed during the winter months of
January and February.

Another method of determining plankton pro-
ductivity of water over an oyster bottom was used
in a study of the York River, Va. (Galtsoff, Chip-
man, Engle, and Calderwood, 1947). In this case
the water sample was obtained by means of a
Birge-Juday plankton trap. Two liters of the
sample were passed through the Foerst-Juday
centrifuge, and the centrifugate extracted for 15
minutes in 10 ml. of 80 percent acetone. A com-
parison of the color was then made with standards
of nickel sulfate and potassium dichromate (Har-
vey, 1934), and the results expressed in pigment
units per liter of original sample. Each unit was
found to be equivalent to approximately 10,000
diatoms and dinoflagellates present in York River
water. So far as seasonal changes in plankton are
concerned, the results were similar to those ob-
served in Long Island Sound. Although the total
energy requu'ements of certain filter-feeders are
known, no information is available about the
specific food that is needed for their growth and
reproduction. Certain types of phytoplankton,
such as Chlorella, have antibiotic properties and
are harmful to some bivalves. Too little is knowTi
about the specific food requirements of various
organisms. The total bulk of phytoplankton may
be made of the materials of low nutritive value or
may consist of such organisms as Rhizosolenia,
Chaetoceras, and others which cannot be ingested
by the oyster because of their size and shape.

In evaluating the food factor the abundance of
plankton should be compared in different areas
during the period when the oyster is actively
feeding and accumulating glycogen. This period
usually occurs shortly after spawning (ch. XVII).
The amount of phytoplankton per unit of volume
of water on oyster bottoms of highest commercial
yield and consisting primarily of algae that can be
utilized bj^ the oyster represents the optimum
value of food factor (score 10); while the phyto-
plankton content of water from the marginal
areas is assigned the score 1.

Another and more accurate method may be
used. It is based on the determination of meta-
bolic rate of oysters during the period of feeding.
The nutritive value (food energy) of the sample
of phytoplankton can be determined by using a
bomb calorimeter and measuring the heat of
combustion in calories. Knowing the rate of
water transport by the oyster at a given tempera-
ture and salinity, it is easy to calculate whether
the food supply on oyster grounds is adequate.
Unfortunately, the method widely used in nutri-
tional studies has not yet been applied in oyster
research.

The high concentrations of phytoplankton
which occur during blooms are not desirable
features and can be harmful. Experimental work
has clearly shown that at a certain high concen-
tration of several forms {Nitzschia closterium,
Prorocentrum triangulatum, Euglena viridis, and
Chlorella sp.) the rate of water transport of oysters
is reduced and feeding ceases (Loosanoff and
Engle, 1947). The deleterious effect is caused by
the cells themselves and by their metabolites.
These laboratory findings are in accord with field
observations in Great South Bay, N.Y., where a
mass development of a Chlorella like organism
adversely affected valuable oyster beds. Another
example of a danger of excessive development of a
single microorganism is the so-called red tide
(Galtsoff, 1948, 1949) along the western coast of
Florida. Sudden development of the dinoflagel-
late, Gymnodiniuin breve, causes extensive mor-
tality of fishes and kills many oysters growing
along the shores of the affected area.

Conditions are ideal for the feeding of oysters
when water free of pollution and containing a low
concentration of small diatoms and dinoflagellates
runs over a bottom in a nonturbuleut flow.

NEGATIVE FACTORS OF ENVIRONMENT

The environment itself may interfere with the
welfare of oyster populations. Negative factors
decrease or inhibit reproductive capabilities;
destroy the population by causing extreme adverse
conditions; increase the incidence of disease;
inhibit the fattening and the growth of oyster
body, thus decreasmg the productiveness of an
oyster bed; and interfere with the formation of
shell and so deprive the oysters of their principal
means of protection against adverse situations
and attacks of enemies. All negative factors are
evaluated by determining the degree of their

FACTORS AFFECTING OYSTER POPULATIONS

409

harmf ulness and assigning them scores from 1 , for
10 percent effectiveness, to 9, for 90 percent. The
score 10, indicating 100 percent destructiveness, is
omitted because no oyster population can exist
under such a condition. Zero score means
complete absence of a negative factor.

SEDIMENTATION

Rapid settling of suspended material may be
highly destructive to an oyster community. All
coastal waters contain a certain amount of solids
in suspension of either organic or inorganic origin.
The particles settle on the bottom, depending on
their weight and shape, chemical composition,
temperature, viscosity of water, and character of
water movements. The velocity of the fall of a
particle through a liquid is a function of the radius
of the particle governed by Stoke's law:

_2{Po-P)9r'

gn

where v is the velocity of sinking, ?„ and P are the
densities of the particle and of the liquid respec-
tively, r the radius of the particle, n the absolute
viscosity of the liquid, and g the acceleration of
gravity. It is assumed in the equation that the
particles are spherical, that they are small enough
so that the viscosity of the water is the only
resistance to their fall, and that their sinking is not
impeded by adjacent particles. The equation is
appHcable to spheres varying in size from 0.2 to
200ju and suspended in quiet water. Obxdously
such conditions represent the "ideal" situation
which cannot be found in an estuarine environ-
ment. Here the water is in almost constant
motion with rapid changes in the du-ection and
velocity, and carries sediments consisting of parti-
cles of differing sizes, shapes, and densities. The
discussion of the physical aspects of sedimentation
problems is beyond the scope of this book. The
reader interested in these problems is referred to
comprehensive textbooks on the subject
(Twenhofel, 1961 ; Linsley, Kohler, and Paulhus,
1949). For an understanding of the ecological
effects of sedimentation it is enough to describe
in general terms the conditions under which silt
particles are transported and deposited on es-
tuarine bottoms.

Observations made on waterflow in a tube show
that at low velocities the particles of a liquid move

in parallel lines and the resistance to their motion
is due to viscosity. This condition is called
streamline or laminar flow. High velocity of the
water and roughness in the walls of the tube make
the stream break into a turbulent flow charac-
terized by iiTegular, eddying, and rolling move-
ments. The formation of the eddies counteracts
the gravitational settling of particles, and more
material is moved upward than sinks toward the
bottom. When the amount of sediment picked
up by a turbulent flow exceeds the amount
deposited, the liottom is eroded. If the gravita-
tional force predominates, more material is being
deposited than carried away and the bottom is
rapidly covered by the sediment. In an estuary
both processes alternate following the rhythmic
changes in the direction and velocity of tidal
cm-rents. In many instances an equilibrium is
established and continues for a long period of time
unless the balance between the two forces is upset
by \'iolent water movements from severe storms
or floods. Depending on the configuration of the
bottom, certain areas of an estuary are scoured,
while vast quantities of sediments are deposited
on others. This is typical on oyster grounds in
many tidal streams.

The material suspended in coastal water is a
very complex mixture of particles of differing size,
shape, specific gravity, and mineralogical compo-
sition. The particles are sorted out as they are
moved by the water. According to Trask (1950,
p. 10), slowly moving water seems to make for
poorer sorting than a fast moving current. To a
sedimentation geologist the resulting pattern of

SILT FLOCCULATION / , A /• /

l9%o ■ ^ ^I0%«

SILT RUNOFF

FiGi'RE 370. — Diagrammatic representation of the sorting
out and deposition of coarse sand, fine sand, and silt in
an estuary. The lower part of the diagram indicates
the complex system of current and counter current.
From Rochford, 1951.

410

FISH AND WILDLIFE SERVICE

the size distribution of sediment may serve as an
indication of the mode of its deposition. A
schematic representation of sorting of the material
suspended in water and its deposition in the four
zones of an estuarine system according to the
distance from the mouth of the river is shown in
figure 370, redrawn from Rochford (1951). At
the head of an estuary silt is transported by tidal
streams further than sand and because of floccula-
tion, caused by the influx of saltier water, smothers
the beds near the mouth (middle and left parts of
fig. 370). The complexity of the horizontal
pattern of currents is shown on the lower part of
the diagram.

Particles immediately above the bottom move
by rolling, sliding, and jxunping. Outside of this
narrow "bed layer" (Einstein, 1950) the particles
in water constitute the suspended load; their
weight is continuously supported by the fluid
until they reach the lower part of the estuary
where they are deposited over the bottom and on
tidal flats.

The problem of transport and settling of sedi-
ments in the sea is not well understood (Trask,
1950, p. 9) and is being intensively studied by
many oceanographic institutions of the world.
In tidal waters where oyster beds are located the
problem becomes more complex because the rate
of transport and settling is greatly influenced by
periodic changes in cmrent velocities, tm'bulence,
salinity, temperatiu'e, density and viscosity of
water, and size, shape, roughness, and specific
gravity of transportable particles. In the tidal
regime where salty oceanic water mixes with fresh
water another important factor called floccula-
tion enters into this already complex picture.
Small dispersed particles of clay and of organic
detritus have a tendency to aggregate in small
lumps. In the pm'ification of drinking water
flocculation is produced artificially by adding a
coagulant such as aluminum sulphate (Al2(S04)3- 18
HoO). In estuaries where sea and fresh water
mix the flocculation is caused by the change in the
electric charges of the particles which occur with
the change in the hydi'ogen ion concentrations.

Laboratory experiments with kaolin suspension
show that flocculation of particles about 4^ in
diameter may occm- at constant pH (8.5) by in-
creased concentrations of Na+ at constant S04-_

concentrations (Whitehouse, 951, 11952).' The
results indicate the tendency of positive ions to
cause the flocculations of the negatively charged
particles.

Flocculation in coastal waters may be observed
from the deck of a ship at the time of freshets
along the coastline of the Carolinas and Georgia.
Large patches of aggregated silt particles show
clearl}' in the sahy offshore water while the brown-
ish color of less saline water at the mouth of a
river remains uniform.

Rochford (1951) states that flocculation of silt
takes place in the salinity zone of less than 19°/oo
and higher than 10°/oo (fig. 370). Since floccula-
tion depends on several factors including tempera-
ture, pH, and the type of sediments in suspension,
one may expect a great variation in the rates of
settling in the various estuaries of the Atlantic
and Gulf coasts.

The gross effect of sedimentation may be meas-
ured by determining the depth of deposition of
silt over an oyster bottom per unit of time and
area. Less noticeable, but highly significant, is
the deposition of a thin layer of sediment over
the hard surfaces to which many organisms, in-
cluding oyster larvae, attach. A deposit of loose
sediment only 1 or 2 mm. thick is enough to make
the sm'face of shells and rocks unsuitable for the
attachment of larvae and to cause failure of
setting. I have observed such conditions many
times in certain sections of Oyster River, Chatham,
Mass.; in the Wiweantic River of the Cape Cod
area; in the small rivers and creeks emptying into
Delaware Bay; and in the Rappahannock and
York rivers, Va. U^ndoubtedly similar conditions
may be found in many other places where silt is
transported by estuarine currents. Light sedi-
mentation is not harmful to populations of adult
oysters, but may be hea\^ enough to interfere
with their reproduction.

' Whitehouse, U. Grant.

1951. A study of chemical sedimentation and of physical oceanography
sponsored at Texas A. and M. College by the American Petroleum
Institute through the Scripps Institution of Oceanography of the
University of California (A.P.I. Project 51). Progress report tor the
quarter ending December 31, 1951. The Te.Nas A. and M. Research
Foimdation, Project 34, The Agricultural and Mechanical College of
Texas, Department of Oceanography, College Station, Texas, 0 pp.
(Preliminary report).

1952. A study of the chemistry of oceanic sedimentation sponsored at
Texas A. and M. College through the Scripps Institution of Oceanog-
raphy of the University of California (A.P.I. Project 51). Progress
report for quarter ending December 31, 1952. The Texas A. and M.
Research Foundation, Project 34. The Agricultural and Mechanical
College of Texas, Department of Oceanography, College Station,
Texas, 11 pp. (Preliminary report).

FACTORS AFFECTING OYSTER POPULATIONS

411

Many formerly productive oyster bottoms along
the Atlantic Coast of the United States have been
destroyed by a high rate of sedimentation. Dead
oyster reefs buried below a surface of mud in the
waters of Louisiana and Texas are good examples
of this process. The silting of estuaries may be
studied by the simple method of comparing the
depth of water shown in navigation charts of 25
or 50 years ago with present soundings. From
these dift'erences the total amount of deposit
accumulated over the given period of time can be
computed. The reduction of volume of water in
a basin due to sedimentation can be determined
from these data and from the computations of
the capacities of reservoirs (Dobson, 1936).
Brown, Seavy, and Rittenhouse (1939) success-
fully used this method in determining the rate of
silting over a distance of 19.4 miles of the York
River, Va. In 1857 the water volume of this
sector of the river, estimated at mean low tide,
was 227,780 acre-feet. By 1911 it was reduced
to 222,189 acre-feet, and in 1938 was only 206,896
acre-feet. The cumulative volume of sediment
deposited during the period 1857-1911 was 5,591
acre-feet, and from 1911 to 1938 reached 20,884
acre-feet. The annual deposition for the first
period of 55 years was 104 acre-feet, which repre-
sented 0.05 percent loss of water volume; during
the second period of 27 years (1911 to 1938) the
annual deposition increased to 566 acre-feet,
which corresponded to an annual 0.25 percent
loss of water. The increased rate of silting during
the later period was explained by an increased
erosion of soil over the watershed resulting from
faulty agricultural practice, deforestation, and
an increase in population.

The filling of bays and estuaries with sediments
is a general phenomenon along the Texas coast,
and is particularly pronounced in Laguna Madre
and near the mouth of the Colorado River in
Matagorda Bay where some of the buried oyster
reefs are found under 14 feet of mud (Norris,
1953). Duj-ing the last 36 years silting has de-
stroyed 6,000 to 7,000 acres of productive oyster
reefs near Matagorda. These beds were described
in detail by Moore (1907) and resurveyed in 1926
by Galtsolf (1931a). In 1926 the principal reefs
opposite the mouth of the Colorado River were
sm-rounded by very soft mud but were still pro-
ductive. Now the nmd of the Colorado River has
completely buried these reefs and pushed the head
of fresh water seaward. For the Neuces River,

a small stream emptying into Corpus Christi Bay,
the accumulation of silt recorded by comparing
the depths given on 1880 charts with those issued
in 1937 varied from 4 feet near the ship channel to
less than 1 foot at the south shore (Price and
Gunter, 1943). The average annual accumulation
of sediment near the ship channel was about
0.8 inches, only slightly less than the annual in-
crease of oyster shell in height. Under such
conditions an individual oyster, even if it grew in
the vertical position that oysters usually assume
in soft mud, in 6 to 7 years would have sunk in
mud for about three-quarters of its height and
perished.

The soft, muddy tidal flats typical for the in-
shore waters of the South Carolina and Georgia
coast are usually devoid of oysters. U.S. Bm-eau
of Fisheries experiments on oyster farming in 1939
and 1940 in the vicinity of Beaufort, S.C., demon-
strated the complete unsuitability of these areas
for oyster culture. Reinforcement of these flats
by shells planted in a layer about 1-foot thick and
strong enough to support a man's weight was a
complete failm-e. The shells acted as baffles,
with the result that in a short time mud filled
all the crevices between them and in about 6
weeks completely covered everything with a
smooth layer of silt (Smith, 1949). Similar results
were obtained with brush and other materials
placed on the surface of the mud; in a few months
not a trace of them could be seen on the surface.
On the other hand, oysters grew well along the
opposite side of the river where a swift current
kept the bottom scoured.

Accumulation of silt over an oyster bottom is
sometimes caused by the activity of various mud-
gathering and nmd-feeding invertebrates. Chief
among them are several species of the mud worm,
Polydora. The two long antennae of these worms
protrude from the tube in which the animal lives
and sweep the surrounding water. Mud particles
suspended in the water are caught by the epithe-
lium of the antennae and by ciHary motion are
transported toward the head to accunmlate around
the worm's body and thus making the tube.
Some of the mud is ingested and passes through
the intestime. P. websteri invades the shell cavity
of the oyster, settles on the inner surface at a riglit
angle to the edge, and builds a U-shaped nmd tube
with both orifices external. The structiu-e is soon
covered by a layer of conchiolin deposited by
oyster and becomes a semitransparent blister.

412

FISH AND WILDLIFE SERVICE

As the worm grows the cavity it occupies is
enlarged by boring to provide for its increased size.
Several shell layers are deposited by the oyster
over the bUster. The mechanism of boring is not
well understood; probably erosion of the shell is
accomplished by a combined chemical and me-
chanical action. P. ligni is found living in mud
tubes on tidal flats or attached to shells and rocks.
On several occasions the reproduction of P. ligni
on the oyster bottoms of Delaware Bay was so
rapid that nearly every live oyster of the aft'ected
area was killed by a deposit of mud several inches
thick consisting of numberless hve worms and their
tubes. The process of gathering mud by Polydora
is shown in a photograph (fig. 371) taken of a live
worm, which was placed in a glass tubing for
observation in the laboratory.

Oysters themselves accumulate large quantities
of organic sediment, which is discarded with the
feces. During feeding the oyster discharges fecal
ribbons at the ratp of several centimeters per
hour. In a sluggish current large cfuantities of

fecal masses settle in the crevices between the
oysters and contaminate the bottom. The situa-
tion may become serious enough to cause a decline
in the productivity of oyster beds, as has been
demonstrated by Japanese biologists (Ito and
Imai, 1955).

Organic material constitutes a major portion
of marine muds. The physical properties of a
sediment may be of lesser importance to oyster
ecology than the comple.x biochemical changes
associated with the bacterial decomposition of
its organic components that result in the forma-
tion of carbon dioxide, ammonia, phosphates,
sulfates, and various organic acids. In the case
of anaerobic oxidation, methane and hydrogen
sulfide are formed (Waksman, 1942; Waksman
and Hotchkiss, 1937). The effect of these prod-
ucts of decomposition on bottom populations
probably is the mam reason for the slower rate
of growth for oysters on the bottom than for
those which are kept above the bottom on trays
or are suspended from rafts and floats (Shaw,

/

Figure 371. — Photograph of live P. ligni collecting mud. The worm i.s contained in a glass tube placed in sea water
containing silt in suspension. i\Iud particles along the grooved antennae are transported toward the mouth and
have begun to accumulate at the edge of the glass tubing.

FACTORS AFFECTING OYSTER POPULATIONS
733-851 O— 64 27

413

1962). It appears paradoxical but true that the
conditions of a natural environment do not add
up to the ideal situation for the life of a bottom
inhabiting mollusk such as the oyster. Location
on rocks and undenvater structures above the
mud line appears to offer a more favorable environ-
ment.

Determination of the amount of silt setthng
on the bottom can be made with a mud trap of
the type shown in fig. 372. The trap consists
of a metal funnel riveted to a metal frame and a
quart size container (fruit jar) screwed to the
funnel. The trap is set on the bottom for the
desired length of time, then carefully lifted, and
the amount of sediment settled over the area of
the funnel and inside the jar is measured.

Data obtained with a mud trap of the type
shown above measure the rate of settling of sedi-
ments on the bottom. One should bear in mind,
however, that a certain portion of the sediment
deposited during slack tide may be washed away
as the current velocity increases with tidal
changes. Furthermore, mud already settled on
the bottom may be stirred by wave action and
resettle on an adjacent area. The actual accumu-
lation of sediment can be measured with a mud
board of the type shown in fig. 373. A wooden
stake 36 inches high supports a flat board 18
inches long and 6 inches wide, mounted hori-
zontally, and the stake is forced into mud so
that the board is level with the bottom. This
type of trap can be used conveniently on tidal
fiats.

A trap to be used below low water can be made
of a flat board ji square yard in area. To prevent

CONE FUNNEL

'2 STRAP IRONS FORMING 4 LEGS

Figure 372. — Mud trap for collecting sediments settling
from water.

10"

36"

-18"-

<-Z"-^

3/4"

Figure 373. — Mud board for measuring the accumulation
of sediment on tidal flats.

the loss of sediment the board has slanted borders
about 4 inches wide, projected above its upper
surface. The trap is set on four short legs; its
surface is ruled in squares to facilitate the measure-
ments of the area covered with sedunent. Tiie
thickness of the sediment is measured with a
ruler. Since the board cannot be lifted without
disturbing and losing the accumulated sediment,
observations must be made by a diver.

An indirect estimate of the amount of material
in suspension can be made by using a Secchi
disc and recording the depth of extinction of white
color. The results are affected by the visual
acuity of the observer, illumination, and the
condition of the sea surface.

The amount of suspended material in a sample
of water can be determined with a simple tur-

414

FISH AND WILDLIFE SERVICE

bidimeter of the type used primarily in fresh-water
studies; more accurate results are obtained with an
electrophotometer and spectrophotometer. The
turbidity of the collected samples is compared with
standards made of known dilutions of a suspension
of 1 g. of kaolin (or silica clay) in 1 1. of water.

Evaluation of the effect of sedimentation on
oyster bottoms can be made by considering the
location of oyster bottom in the estuary; the
amount and character of sediment in suspension;
the type of estuarine circulation; and the rate of
accumulation of sediment on oysters. A score
of zero is assigned to the ideal conditions under
which no deposits settle on live oysters. The
opposite extreme, valued at 10, is found in the
areas of heavy sedimentation, not suitable to
oysters. All intermediate conditions scored from
1 to 9 can be evaluated on the basis of field
observations.

DISEASE

Oysters suffer from both noncontagious and
infectious diseases. The first category is associated
with the malfunction of physiological systems of
organs and deficiencies in the environment, such
as lack of food, unsuitable salinity and water
temperature, and pollution by domestic sewage
and various trade wastes. The second category,
infectious diseases, is caused by pathogens and
parasites. Clear distinction between the two
types of pathological conditions is not always
possible because resistance to infection is lowered
by an unfavorable environment, and an oyster
weakened by adverse conditions more easily
succumbs to infection.

With few exceptions the outward symptoms of
a disease are nonspecific. The more common
symptoms are slow growth, failure to fatten and
develop gonads, recession of the mantle, and
valves that remain slightly agape. There is often
a corresponding abnormal deposition of shell
material that in a chronic condition causes the
formation of short and thick shells ("huitre
boudeuse" of French biologists). The valves do
not close tightly because the adductor muscle is
weakened. The body of a sick oyster is watery,
often discolored (dirty green and brown), and
bloody with blood cells accumulating on the
mantle and on the surface of the gills.

The etiology of oyster diseases is not well
known. A few microorganisms infecting tlie
oyster have been definitely identified as pathogens;
the taxonomic position of others is not known.

and some are called by code numbers. Oyster
populations throughout the world suffer from
periodic widespread mortalities which may be
associated with infections, but since the life
cycles of some of the pathogens have not been
described, the evidence remains circumstantial.

The widespread mortality of oysters rarely can
be attributed to a single factor of the environ-
ment; in most cases it occiu's as a result of the
combination of several adverse conditions in-
cluding infection.

Malpeque Bay disease

One of the most persistent and mysterious ail-
ments of oysters is the Malpeque Bay disease,
which in 1915 and 1916 struck the populations of
C. virginica in the bays of Prince Edward Island,
Canada, causing the death of 90 percent of the
oysters, and in later years appeared along the
Canadian mainland. The most distinctive symp-
tom associated with the disease was the occurrence
of yellow-green pustules, up to 0.5 cm. in diame-
ter, on the surface of the visceral mass, along the
edges of the mantle, and on the adductor muscle
and the heart. Despite lengthy field and experi-
mental studies conducted from the epidemic year
to the present, the causative agent has not been
found, although there is no doubt that the mortal-
ity of Malpeque Bay oysters was due to an in-
fection (Needier and I^ogie, 1947). With the
expectation that the survived oysters were of
disease resistant stock, the Department of Fish-
eries of Canada and the Fisheries Research Board
organized ua 1957 a rehabilitation project and
transferred oysters from Prince Edward Island
to devastated mainland areas. Unfortunately the
hopes did not materialize fully since a high propor-
tion of the spat that settled on the rehabilitated
area did not show the expected level of resistance.
It is hoped, however, that a resistant stock will
develop from a small number of survivors over a
period of several years (Drinnan and Medcof,
1961).

Dermocystidium marinutn

Dermocystidium marinum Mackin, Owen, and
Collier, a fungus of uncertain taxonomic position
infecting C. fdrginica, is probably the most danger-
ous pathogen associated with periodic mortalities
of oysters in the waters of southern States. The
microorganism infects oj^ster tissues producing
single, spherical, vacuolate cells which reproduce
by endogenous free cell formation and subse-

PACTORS AFFECTING OYSTER POPULATIONS

415

quently liberate uninucleate sporelike bodies.
The following detailed description of the species is
reproduced verbatim from Johnson and Sparrow
(1961, p. 539):

Mature thallus a hyaline, spherical, spore-like body,
2-30m, averaging about 10m in diameter; each cell con-
taining a large, slightly eccentric vacuole in which a
polymorphic, refractive vacuoplast usually occurs; nucleus
oval, eccentric; cleaving internally to form a short hypha
terminated l)y an apical, conidium-like swelling.

Dermocystidium can be identified on cross-sections
of an oyster stained with hematoxylin, Giemsa or
other histological stains, or in teased preparations
stained with Lugol solution (fig. 374).

Identification of Dermocystidium by microscopic
examination of tissues is time consuming and
difficult. The diagnostic technique developed by
Ray, Mackin, and Boswell (1953) facihtates the
examination of large numbers of samples. Small
pieces of tissues are removed from the gaping
oysters and placed in Carrel tissue culture flasks
containing a small amount of sterile water to which
streptomycin and penicillin have been added to
prevent bacterial growth. Prior to excision the

A.

B.

V

Figure 374. — ^Drawings of Dermocystidium marinuni
stained with Heidenhain's iron hematoxylin and eosin.
A — A mature spore with markedly irregular vacuoplast,
cytoplasmic inclusions, and very large vacuole. B —
Multiple fission resulting in several daughter cells. C —
A binucleate stage with chromatin in diffuse condition,
and showing beginning vacuolation of the cytoplasm.
D — An immature .spore with small vacuole and vesicular
nucleus. Figure 1 from Mackin, Owen, and Collier,
Science, vol. Ill, No. 2883, 1950, p. 329.

tissues are washed in sterile sea water, then placed
for 10 minutes in a 10 percent solution of sodium
merthiolate (1:10,000), waslied again in sea water,
and allowed to remain for several hours in sterile
sea water fortified with 1 ,000 units eacli of strepto-
mycin and penicillin. Tissues parasitized with
Dermocystidium disintegrate completely in about
a week, while in the controls they remain intact.
The debris of disintegrated tissues consists mainly
of minute spheres of Dermocystidium cells. Unfor-
tunately the contamination of samples with molds,
yeast, and ciliate protozoans could not be entirely
prevented and failures were "much more frequent
than were successes, and most of the experiments
were discarded." The data presented by Ray,
Mackin, and Boswell show that the major effect of
Dermocystidium infection is marked loss of weight,
averaging 33 percent.

The infection may combine with other factors to
produce a mortality of oysters which, according to
IMackin (1961a), can virtually destroy seed oysters
planted in Louisiana in a single summer. Dermo-
cystidium studies in southern waters established
the significant fact that the effect of the parasite
"is not only a matter of disease but of season,
summer losses accruing from disease being signif-
icantly greater than those of early spring months"
(Ray, Mackin, and Boswell, 1953). The impor-
tance of environmental factors (temperature and
salinity) is clearly demonstrated by these findings.

According to Mackm (1961b), the disease
caused by Dermocystidium affects the oysters from
Delaware Bay to Mexico but in the more northerly
part of the range is not apparent in winter. It is
not clear, however, if Dermocystidium remains in a
dormant stage or if it disappears from oysters.
In the Gulf States winter temperatures are prob-
ably not low enough to eliminate the parasite, and
consequently considerable mortality may occur in
mild winters.

Dermocystidium marinum and possibly other
species of the genus have been reported from
0. frons, 0. eguestris, 0. lurida, Mya arenaria,
Mulina lateralis, Macoma baltica, Mercenaria
(Venv^) mercenaria, Anadare transversa, Anomia
simplex, Emis minor, Laevicardium mortoni, and
Lyonsia hyaliria (Johnson and Sparrow, 1961, p.
540).

Many phases of tlie life history and biology of
Dermocystidium. require elucidation, particularly
the transport of spores by water and their pene-
tration into the tissues, the details of reproductive

416

FISH AND WILDLIFE SERVICE

cycles, the relationship between environmental
conditions and degree of infection.
Disease associated with Haplosporidiuni

Excessive mortality of oysters in Delaware
Bay in a 6-week period of April and May 1957
wiped out from 35 to 85 percent of planted oysters
and almost completely ruined the oyster industry
of the State. A microorganism consistently
found in tissues of infected oysters was designated
by the code name MSX and later on was tenta-
tively identified by Mackin as one of the Haplo-
sporidia. The organism invades the connective
tissue surrounding the intestine and digestive
diverticula. Early plasmodial stages and en-
suing stages of development are shown in two
illustrations (figs. 375 and 376) made in the
laboratory from a preparation kindly supplied
by Haskin.

Mortality of oysters on the eastern shore of
Virginia near Seaside was investigated from
1959 to 1961 by the Virginia Institute of Marine

Science. The microorganism causing the disease
and fii'st designated as SSO was described by
Wood and Andrews (1962) as a sporozoan,
Haplosporidmm costale, n. sp., infecting connective
tissues of oysters and producing a truncate spore
encased in an operculum with a lid. An early
Plasmodium with 6 to 12 nuclei is from 6 to 8/i
in size (fig. 3~7). Haj^losporidium has been
found in live oysters as early as February, and in
mid-May to June the infection may cause high
mortality. How the parasite infects the oysters
is not known, and its life history is not fully
understood (Andrews, Wood, and Hoese, 1962).

Shell disease

This disease, which is probably associated with
an unidentified fungal infection of oyster shell, is
not particularly serious in C. virginica, but has
been reported to cause catastrophic mortalities in
the population of 0. eduHs in Oosterschelde, Hol-
land. The disease can be recognized by bottle-
green or orange-brown rubberlike warts and spots

0 30

Microns

Figure 375. — Plasmodial stage of MSX in the connective tissue of heavily infected C. virginica from Delaware Bay.

Bouin, hematoxylin -eosin.

FACTORS AFFECTING OYSTER POPULATIONS

417

"■.:^5

"^^^^.■■;

0 20

Microns

Figure 376. — Later stage of development of MSX in
the connective tissue of heavily infected C. virginica
from Delaware Bay. Formalin 10 percent, iron
hematoxylin.

Figure 377. — Haplosporidium costale. A— mature spore ;
B — early Plasmodium. From Wood and Andrews,
fig. 1, Science, vol. 136, 1962, p. 711.

on the inner surfaces of the shell and, in more
advanced cases, by deformation of the shell edges
and hinge. Examination of thin slides of shell
show abundantly branching fungus. The shell
disease in Oosterschelde was studied by Korringa
(1951a), who discovered that it spreads at water
temperatures above 19° C. and that the higher the
temperature the more vigorous the attack. The

fungus was not isolated from Dutch oysters and
remains unidentified. Korringa believes that it
survives in the old green cockle shells scattered as
cultch over the bottom and that its spores are
probably carried by the water currents. Whole-
sale cleaning, removal of old shells, and disinfecting
of young infected oysters with a solution of "an
organic salt of mercury" (not fully specified by
Korringa) are recommended as control measures.
Shell disease in Dutch oysters has been known
since 1902, but at that time occurred only in a
limited percentage of oysters. Its rapid spread in
the years following 1930 was probably due to the
enormous quantities of old cockle shells, about
40,000 to 50,000 m.', scattered annually as spat
collectors. This gave the fungus a chance to pro-
liferate more rapidly and infect the oysters. Voi-
sin (1931) describes the disease in oysters imported
from Zeeland, Holland, for planting in the Ma-
rennes area on the west coast of France. He
states that more than 40 percent of these oysters
had shells infected by a fungus, probably belonging
to the genus Monilia. The identification is merely
a guess and cannot be verified.
Foot disease

Foot disease or "maladie du pied" of French
oyster growers occurs in 0. edulis and C. angulata
in the waters of the western and southern coasts of
Europe. Korringa suggests that it is probably
identical with the shell disease. The name is an
obvious misnomer because the foot is lacking in all
adult oysters.

"Foot disease" has existed in the Arcachon re-
gion smce 1877. Giard (1894) described its para-
sitic nature and attributed it to a schizomycete
fungus Myotomus osfrearum Giard, a genus not
listed in Johnson and Sparrow's treatise on fungi
(1961).

The disease afifects the area of the attachment
of the adductor muscle, primarily on the lower,
concave (left) valve, and in certain cases the upper,
flat valve. The surface of the shell under the
muscle is covered with small, rough dark green
spots. In advanced cases the muscle becomes
detached from the valve and forms irregular cysts
of horny and slightly elastic material. Ijater on
when the cyst extends beyond the area of the
muscle attachment, the cyst walls become covered
with calcareous shell deposit. According to
Giard (1894) and Dollfus (1922), the parasitic
fungus grows by utilizing the conchiolin of the
shell and stmiulates its secretion by the mantle.

418

FISH AND WILDLIFE SERVICE

The progress of the disease is slow. During the
advanced stage shell movements are affected and
the oyster has difficulty in closing its valves, thus
becoming an easy prey for its enemies.

Foot disease is found in C. virginica, particularly
in oysters inhabiting muddy waters of the southern
States, but in my experience it never reaches
epizootic proportions. The cysts of an affected
oyster (fig. 378) contain a suspension of blood
cells, debris, and numerous bacteria which prob-
ably represent secondary infection. The disease
does not present a serious menace to the oyster
fishery of the coastal states.
Hexamita

The flagellate Hexamita inflata was first found
in the intestinal tract of 0. edulis (Certes, 1882).
It is present in C. inrginica of Prince Edward
Island, Canada, and southern Louisiana, and in
0. edulis in Dutch waters (Mackin, Korringa, and
Hopkins, 1952). Heavy infection with Hexamita
causes breakdown of connective tissue cells, gen-

FiGUR^ 378. — Large cyst filled with blood cells, bacteria,
and debris of muscle tissue in C. virginica.

eral inflammation, the appearance of many tropho-
zoites in blood vessels, and necrosis of adjacent
tissues. The early stages of the disease appear
to be intraceUular, and are usually found in the
leucocytes of the blood vessels. The trophozoite
is oblong and narrow at the anterior, with si.x
anterior and two posterior flagella. Cysts found
in advanced stages of the disease are small, about
5 (U ill diameter; they contain two or four small
nuclei and have no flagellar structure. The com-
plete life cycle of the parasite has not been
described. The method of infection appears to
be by cysts liberated after the disintegration of
an infected oyster body. Experimental studies
by Stein, Denison, and Mackin (1961), who used
diseased 0. lurida, give no evidence that Hexamita
is a highly pathogenic parasite because there was
no significant difference in the mortality between
the experimental and control specimens.

I^ematopsis

Cysts of the gregarine Nematopsis are frequently
found in the tissues of several European bivalves
includmg 0. edulis, Mytilus, Cardium, Dotmx,
Tellina, Mactra, Solen, and others (DoUfus, 1922).
Observations by Louis Leger (quoted from Dollfus)
showed that vegetative stages of the gregarine
are often found in the kidneys and that the spores
with sporozoites are usually located in the gills.
Leger also showed that the intermediary hosts
are the crabs Corcinus moneas and Portunus
depurator. Nemaiopsis develops in the intestinal
canal of the crab and forms cysts which are
rejected into water and are transmitted with
water currents. There was no evidence that
Nematopsis is pathogenic.

The species A'', ostrearum from 6*. virginica has
been described by Prytherch (1940), who found
the parasite in the oysters of Virginia, North
Carolina, and Louisiana. He expressed the belief
that mortality of oysters in Virginia and Louisiana
was directly caused by this gregarine. Nema-
topsis is widely distributed throughout the waters
from the Chesapeake Bay states to Louisiana.
Its distribution indicates no correlation with
oyster mortalities m that area (Landau and
Galtsoff, 1951).
Tretnatodes and parasitic copepods

The trematode, Bucepthalus ha.imeanus Lac.
Duth., is occasionally foimd in 0. edulis and C.
vir-ginica. According to Tennent (1906), who
studied its life history, the worm thrives in oysters

FACTORS AFFECTING OYSTER POPULATIONS

419

of brackish water and is inhibited by an increased
salinity. In cases of heavy infestation, the
gonads and digestive diverticula are almost
completely replaced by cercariae and by the long
germ tubes of the sporocysts, which after their
liberation infest Menidia, other small fishes, and
lylosurus marinus. Destruction of the gonad is
the most obvious pathological effect caused by
B-ucephalus. So far this trematode has not been
suspected of causing mortalities in oyster
populations.

The parasitic copepod Mytilicola intestinalis is
common among mussels of the Mediterranean.
Another species, M. orientals, infests C. gigas and
Mytilus crassitesta of the Inland Sea of Japan.
The parasitic copepod is found in the intestinal
tract of bivalves and is easily recognized by its
red color and relatively large size which makes it
visible to the naked eye. In the United States
Mytilicola orientalis is widespread in lower Puget
Sound, occurring in 0. lurida and C. gigas, Mytilus
edulis, Paphia staminea, and Crepidula jornicata.
Infection is heaviest in the common mussels,
often reaching 100 percent in some areas (Odlaug,
1946). A single specimen of Mytilicola intestinalis
was found by Pearse and Wharton (1938) in C.
virginica on the Gulf coast of Florida. The
presence of M. orientalis in 0. lurida in the lower
Puget Sound area interferes with their fatness,
but apparently inflicts no serious injuries to
oyster stocks. In C. gigas the copepod produces
metaplastic changes in the gut, completely de-
stroys the cihated epithehum, and penetrates the
underlying connective tissue (Sparks, 1962).

The presence of parasites in adult oysters
makes them unmarketable for esthetic reasons
and, therefore, detracts from the commercial
productivity of oyster bottoms.

Any disease factor, regardless of the identity of
the pathogen, can be evaluated by determining
the percentage of the infected oysters, the intensity
of infection, the loss caused by the mortalities,
and the decrease in yield of marketable oysters.

COMMENSALS AND COMPETITORS

The shell and body of the oyster are the natural
abodes for many plants and sedentary animals
which attach themselves to the shell surface or
bore through it to make for themselves a well-
protected residence; some settle on the soft body
without penetrating its tissues while others invade
the inner organs. The difference between the

420

commensals, i.e., organisms which share the food

gathered by the host, and the parasites, which

live at the expense of their hosts and sometimes

inflict serious injuries, is not very sharp. Some

commensals may cause injury to the host and

become parasites.

Competitors are those organisms which hve in

close pro.ximity to each other and struggle for the

space and food available in the habitat. Some

appear to be innocuous while others by virtue of

their habits and high reproductive capabilities are

harmful.

Boring Sponges

Small round holes on the surface of moUusk
shells indicate the presence of the most common
animal associated with the oyster, the boring
sponge. There are seven species of the genus
Cliona along the Atlantic Coast of the United
States. In a case of heax'y infestation the shell
becomes brittle, breaks under slight pressure, and
reveals conspicuous tunnels and cavities filled with
yellow sponge tissue. Microscopic examination
shows a typical sponge structure with numerous
siliceous spicules from 150 to 250 m long, of the type
called tylostyles, and small skeletal elements of
different shapes and sizes known as microscleres.
Species identification is based on the type of
cavities or galleries made by the sponge and the
shape and sizes of the spicules (Old, 1941). Small
fragments of shell material at the holes by Cliona
may suggest mechanical action of the sponge.
Warburton (1958) found experimentally that
sponge cells in contact with a surface of calcite
form a reticulum of fine pseudopodia and filaments.
A corresponding pattern of lines is etched into the
mineral, and the marked areas are of the same size
and shape as the fragments discharged by the
sponge. Apparently the cytoplasmic filaments
penetrate the calcite by secretion of minute
amounts of acid and undercut fragments which
are carried out by excurrent canals of the sponge.
It is not known whether boring sponges use the
organic component (conchiolin) of the shell, but
it is obvious that they do not draw their nutrients
from the body of the oyster. The sponge touches
the surface of the body only in cases of old, heavy
infestation. In such instances the holes made by
the sponge are rapidly covered by a deposition of
conchiolin. The holes made by the sponge are
clearly visible on the mner surface of the valve
under a newly deposited layer of conchiolin (fig.
379) . The race between the sponge and the oyster

FISH AND WILDLIFE SERVICE

Figure 379. — Left valve of adult Crassostrea virginica
heavily infested by boring sponge, Cliona celata. Woods
Hole.

continues, and in most cases the oyster's protective
measures prevent direct contact between the
sponge and the mantle. However, should the
deposition of shell material be delayed by adverse
conditions, the sponge makes direct contact with
the mantle and produces lysis of the epithelium
and underlying connective tissue. Dark pig-
mented pustules form exactly opposite the holes
in the shell. This extreme case observed in
oysters kept for several months in the laboratory
is shown in fig. .380. The tissue of these oysters
is flabby, and the mantle is easily detached from
the shell sm'face.

All oyster bottoms are, to a certain degree,
infested by boring sponges which are found in
both live oysters and empty shells. There are
certain areas, however, where the infestation is
particularly heavy and the growth of the sponge
is very rapid. After the death of an oyster the
sponge continues to grow on the shell, form hi g
large, irregular masses 2 or more feet wide and
several inches thick. About 30 years ago such
large specimens were common in the bays and
harbors of southern Cape Cod, but now they are

foimd only in deep offshore waters. The effect of
the bormg sponge can be estimated by determining
the percentage of oysters with heavily infested
and brittle shells and by comparing then- solid
and glycogen contents with those of uninfested
oysters.

Boring clatn

Oyster shells m the south Atlantic are often
infested with a bormg clam, Diplothyra smithii
Tryon of the famOy Pholadidae. Many papers
on oyster biology refer to this clam as Martesia
sp., but the taxonomy of the family revised by
Turner (1955) corrects the nomenclature and re-
stricts the name Martesia to wood-boring clams.

The boring clam D. smithii is about one-half
inch long. It is usually found inside the shell
material m a cavity which increases in size with
the growth of the clam. The range of distribution
extends from northern Cape Cod (Provincetown ,
Mass.) south to the east and west coasts of
Florida, Louisiana, and Texas. I have found no
live clams in oyster shells during my long-con-
tinued studies in New England waters, and only
a few live specimens have been recovered from
dead oyster shells aromid Tangier Sound in the
Chesapeake Bay. In southern waters the boring
clam is very common, particularly on some reefs
on the Texas coast. In 1926 oysters from Mata-
gorda Bay, Tex., were found to be so heavily
infested by Diplothyra that over 200 clams of
various sizes were found in a single adult (fig.
381). In order to make this count the shell was
dissolved in hydrochloric acid and the bodies of
the clams were collected.

As the cavity bored by the clam increases and
approaches the inner shell surface, the oyster pro-
tects itself by depositmg layers of conchiolin over
the nearly perforated areas. Very rarely does one
find an oyster in which there is a direct contact
between the clam and oyster mantle. On the
outer surface of the shell the presence of clams is
indicated by small holes. The weakening of the
shell structm-e is the main effect of the boring
clam on the oyster.

Mud worms

Of the several species of Polydora found in the
intertidal zone of the Atlantic and Pacific coasts
of the United States, only two, P. websteri Hart-
man and P. ligni Webster, are important to
oyster ecology. P. websteri is found in oyster
shells and on the inner surfaces near the valve

FACTORS AFFECTING OYSTER POPULATIONS

421

'..VAr

^ '■*

^

^

Figure 380.^Black pustules on the surface of the visceral mass and mantle of C. virginica caused by contact with boring
sponge, Cliona celaia. Photograph of an oyster kept in the laboratory tanks at Woods Hole.

edges. The worm accumulates mud and builds
a U-shaped tube which is covered by semitrans-
parent shell material secreted by the oyster.
The formation is usually called a blister. P.
ligni is abundant on tidal flats where it can be
found living in small mud tubes or in crevices
of waterlogged wood structures and other sub-
merged objects. The mud worm may be in-
directly destructive to oysters, for when many
worms settle on shells they can smother an entire
oyster population with their tubes. P. ciliafa
(Johnston) has been accused of extensive mortali-
ties of oysters in New South Wales, Australia
(Roughly, 1925). Frecjuent reports of finding
this species on the coast of eastern America are
based on erroneous identifications and probably
should be referred to as P. websteri (Hartman,

1945). Korringa (1951b) finds no serious in-
juries by P. ciliata to oysters {0. edulis) in Dutch
waters and thinks that in many areas the damages
were caused by P. websteri and P. hoplura.

Ivnowledge of the life histories of P. websteri
and P. ligni is incomplete. Both species lay
eggs in capsules attached to the inner walls of
the tube in which the animal lives. The egg-
laying was noticed in the Woods Hole laboratory
when P. ligni were placed in small glass tubing
of appropriate lengtii and diameter (fig. 382). The
process of egg laying has never been observed in
spite of frequent examination of several tubes dur-
ing both day and night (Mortensen and Galtsoff,
1944). However, egg capsules were found
attached to the walls of the tubes shortly after
Polydora were left undisturbed in darkness.

422

FISH AND WILDLIFE SERVICE

The eggs develop within the capsule until the
larvae have acquired three pairs of setiferous
segments; then they leave the tube. At a tem-
peratm-e of 21° to 23° C. the development of
P. ligni under laboratory conditions varied from
4 to 8 days. Larvae of P. websteri (fig. 383) also
have three setiferous segments. According to
Hopkins (1958), planktonic larvae, presumably
P. websteri, occm" m Louisiana waters throughout
the year; eggs were found in the tubes when
water temperature ranged between 12° and 18° C.

The duration of the pelagic life of either species
of Polydora is not known. The planktonic larvae
grow and develop additional segments before they
settle on the substratum. Since the largest
P. websteri worm found in plankton had 17 seg-
ments and the smallest found on oysters also had
1 7 segments, it is probable that this species settles
at that age. The appearance of young P. ligni at
an early bottom stage is shown in fig. 384.

The larvae of P. websteri settle on the rough
exterior surface of young oysters and make shoe-
shaped burrows near the extreme edge of the
valves. As the worm grows it enlarges its burrow.
The process of excavation is probably chemical,
apparently similar to that described by Wilson
(1928) for P. hoplura and by Hannerz (1956) for
P. ciliata.

The tubes of P. ligni are made of mud particles
held together by mucus secreted by the antennae

and the body surface. Ciliary motion along the
tentacle grooves serves as an efficient mud-gather-
ing device. Experimental evidence shows that if
the lumps of mud are too large or if particles con-
sist of the finest sand or foreign materials such as
corn starch or powdered glass, the ciliary motion
is reversed and the material is rejected. These
laboratory observati^ms prove that the worm is
capable of selecting the substances needed for the
building of a soft tube.

The tube inhabited by the worm, whether U-
shaped or straight, is lengthened by the worm at
both ends. To accomplish this P. ligni reverses
its position in the tube by folding itself halfway
and sliding over its own ventral side. The
process, frequently observed in the Woods Hole
laboratory, is accomplished with great speed and
remarkable ease.

The amount of mud which P. ligni can accumu-
late in the formation of their tubes is astonishing.
A sample collected on June 8, 1944 from the tidal
flats of Delaware Bay contained about 430 closely
packed worm tubes per square inch of mud area.
They all lay nearly perpendicular to the surface.
A cubic inch of the washed and dried sample
weighed 20 g., of which 12.8 g. consisted of mud
with the balance made up of sand, empty shells,
and organic matter. On this basis it is estimated
that the worms gathered 4.9 pounds of diy mud

Figure 381. — Photograph of an adult C. virginica from Matagorda Bay, Tex., heavily infested with D. smilhii. The

outer layer of the shell was chiseled off to expose the cavities.

FACTORS AFFECTING OYSTER POPULATIONS

423

Figure 382. — Photograph of live P. ligni lying quiescent inside a glass tube. Dorsal view.

per layer of surface 1 square foot in area and 1
inch deep.

Since P. websteri is confined in oysters to mud
blisters and does not come in direct contact with
oyster tissues, it causes no visible injuries. This
view is corroborated by the observations of
LoosanofI and Engle (1943), who found that
oysters heavily infested with P. websteri and grown
in trays above the bottom were in excellent
condition.

However, personal observations made in Seaside,
Va. and in Texas bays convinced me that oysters
heavily infested by mud worms (fig. 385) are
usually in poor condition. This opinion is shared
by Lunz (1940, 1941), who calls the mud worm a
pest in South Carohna oysters. According to his

FiGDEE 383. — Drawing of newly emerged larva of P.
websteri viewed alive from the dorsal side. From
Hopkins, 1958.

0 0.25

Figure 384. — Young "bottom stage" of P. ligni Webster.
Modified bristles from fifth segment at left, and ventral
hooded crochet at right. From Fauvel, 1927.

observations, 20.9 percent of the oysters gi'owing
on the hard sm-face of tidal flats are infected, and
the percentage increases to 51.9 on soft, muddy
bottoms above low-water mark. There is no

424

FISH AND WILDLIFE SERVICE

Figure 385.— Oyster shells with mud blisters made by P. websteri from the groimds at Assateague Island, Va.

evidence, however, that infestation by the mud
worm constitutes a serious menace to the oyster
population.
Oyster Crab

Several species of the large family Pinnotheridae,
commonly called oyster or pea crabs, are associated
with oysters, mussels, and other bivalves. The
adult females have been known since ancient
times and were fh-st described by Ai-istotle. The
males of the American species. Pinnotheres ostreum
Say, are much smaller than the females and are
rarely seen. Usually one or two adult crabs per
oyster can be found, and the percentage of
infestation varies from zero in some N~ew England
waters to about 77 percent in New Jersey. The
latter figure, quoted from Christensen and
McDermott (1958), refers to the "invasion" of the
oyster crab on certain grounds of Delaware Bay.
The oyster crab is also abundant in Virginia
waters, where its life history has been studied by
Sandoz and Hopkins (1947). Some oysters con-
tain a surprisingly large number of these crabs;
the ma.ximum reported in a seed oyster was 262
(Stauber, 1945).

Larvae of the oyster crab are pelagic until late
summer. At this time larval development is
completed, the fu'st crab stage is reached, and
the small crabs invade the mantle cavities of
oysters. At this time the carapace width of the
young crabs ranges from 0.59 to 0.73 mm.

The female crab may be found in various parts
of the water-conducting system of the oyster, but
settles chiefly on the surface of the gills, in the
promyal and suprabranchial chambers, and grows
with the gi-owth of the host. The males are not
permanently attached to their host and may
leave to enter other oysters for copulation.

For many years the oyster crab has been
considered an innocuous commensal; however, the
female crabs which have settled on the oyster
erode its gills and impair their function. More
serious lesions may develop and cause leakage of
water from the water tubes, which fm-ther reduces
the efficiency of the food collecting apparatus and
of the gills. Rapid regeneration of the damaged
gills probably saves many oysters from death,
but interference with the normal gill functions
causes a relatively poor condition in many infested
oysters.

FACTORS AFFECTING OYSTER POPULATIONS

425

Spirochaetes

Tissues of oysters are often infected by spiro-
chaetes which may be found in the stomach,
crystalline style sac and in the gonads after
spawning. Dimitroff (1926) identified 10 species
and found that 91 percent of the oysters sold in
Baltimore, Md. were infected. He reported the
following species: Saprospira grandis Gross; S.
lepta, S. puricfa; Crisiispira balbiani (Certes); (".
anodontae Keysselitz; C. spicuUJera Schellack; (\
modiola Schellack; 0. mina; C. tena; and Spirillum
ostrae Noguchi. The species are harmless to
oysters and man.

Perforating algae

The empty shells of oysters and other mollusks
found on tidal flats and on the bottom are fre-
quently perforated by various algae. Bornet and
Flahault (1889) gave a detailed description and
illustrations of several species, some of them also
found in the carapaces of crabs. Ijive mollusks
do not escape the attacks of perforating algae.
0. edulis of \'arious ages living in the channel of
Saline de Cagliari, Italy, were found to be infested
by three species: Hyella caespitosa Bornet and
Flahault; Mastigocoleuv testarum Lagerheim; and
Gomontia polyrrhiza (Lagerheim) (Agostini, 1929).
The algae penetrate the periostracum, then spread
across the prismatic layer, and form branching
threads in the inner layer of shell. Apparently
the gi-owing tips of the filaments dissolve the
calcium carbonate of the shell and make possible
the expansion of algae which, in severe cases of
infestation, spread through the entu'e valve and
become noticeable by the gi-eenish color of the
valve's inner surface. The color cannot be rubbed
off the surface since the alga is separated from the
oyster and does not come in du-ect contact with
its body. The algal filaments can be studied on
fragments of shell or after decalcification in acid.

Gomontia polyrrhiza, continuously distributed
along the Atlantic coast, has been reported from
North Carolina and Connecticut, to New Bruns-
wick, Canada, growing in empty shells along the
shores and occasionally found in live Spirorbis
and barnacles (Taylor, 19.37).

Live oysters infested \vith perforating algae are
occasionally found in shallow bays and estuaries
of Cape Cod. The inner surfaces of the valves
are bluish-gi-een. At Woods Hole I saw under a
microscope a network of perforating algae re-
sembling Gomontia and probably mi.xed with other

species. The plants have not been positively
identified.

Perforating algae do not appear to be harmful
ro oysters. Continuous growth in empty shells
accelerates the disintegi-ation of the shells and
the return of calcium salts to the sea.
Fouling organisms

Many sedentary marine organisms use oyster
shells as a convenient place to attach, either per-
manently or temporarily. They do not penetrate
the shell nor do they inflict any direct injury on
the oyster, but they do compete with it for food
and space and sometimes smother the oyster by
their accumulated mass. The most conspicuous
among them is the American species of slipper
shell, Crepidula fornicata (L.), which received in-
ternational notoriety because of the havoc it
caused for oyster growers in Europe.

Various species of Crepidula are very conunon
gastropods found attached to hard objects near
or below low water. C. fornicata does not present
a problem to oyster gi'owcrs in the United States,
although sometimes in certain estuaries, as in
Cotuit Bay, Mass., it becomes a nuisance because
of its extraordinary abundance. .Slipper shells
settle on oyster shells and tend to form a spirally
curved chain of individuals, the sexes of which
change from female to male (fig. 386).

The lowest and, therefore, the oldest members
of the chain are always females. The uppermost
are males, and those between the two extremes
are hermaphrodites, which undergo changes from
female to male. To the biologist the species is of
mterest because the alteration of sex which takes
place in this mollusk offers an excellent opportu-
nity for experimentation. Grounds heavily in-
fested with Crepidula are, tlierefore, of great value
as a source of material for marine biological lab-
oratories. Oyster growers do not share this en-
thusiasm because the presence of large numbers
of unwanted slipper shells requires additional
work in cleaning the oysters before delivery to
market.

On many occasions C. fornicata has been intro-
duced to Europe witli the sliipment of live oysters
from the United States. It has established itself
in Essex, Northumberland, Falmouth, England,
and in Soutli Wales. In 1929 the first specimens
of C. fornicata were noticed in the Oosterschelde,
Netherlands, and in 1932 to 1933, according to
Korringa (1950), the situation became alarming.
The mollusk spread to the German and Dutcli

426

FISH AND WILDLIFE SERVICE

Figure 386. — Chain of C. fornicata. Female mollusks,
the oldest of the group, are at the bottom ; the males
occupy the uppermost position with the hermaphrodites
(tr.) between the two groups. From Coe, 1936.

Wadden Zee and the Limfjord in Denmark, where
it successfully competed for space and settled on
scattered shells, makuag it impossible for the
larvae of 0. edulis to set on them. Furthermore,
a large amount of silt and soft mud was deposited
by Crepidula and rendered the bottom unsuitable
for oyster planting.

The story of C. fornicata is an excellent illustra-
tion of the possible danger of introducing a foreign
species, which under new conditions, and in the
absence of natural enemies and diseases, may re-
produce and survive at a rate which upsets the
natural balance of nature.

Other fouling organisms may be of a seasonal
nature. Some of the oyster bottoms along the
Atlantic coast of the United States are often
covered with miUions of tunicates of the species
AloJgida manhattends (De Kay). This ascidian
can be so abundant in a dredged sample that the
oysters are hidden under the gi'ay mass of tunicates.
T observed this condition in the mouth of Chester
River, Md.; undoubtedly it occurs in other places
along the coast. The fouling by I\Iolgula is sea-
sonal; the organism dies and the renuiants are
sloughed off in the fall. Among the 29 species of
invertebrates collected from oysters suspended

from a raft in the water of Oyster River, Mass.,
four species constituted the largest portion of the
biomass: Molgula manhattensis, Botryllus schlosseri,
Amphitrite ornata, and Balanus halanoides. The
worm Amphitrite was found in typical tubes of
mud about one-quarter-inch or more in diameter.

At the height of the fouling season in August,
the weight of the animals and plants and of sedi-
ment accumulated by them comprised 44 percent
of the total weight of a string of oysters. The
death of Molgvla in October and the sloughing off
of its cases reduced the weight to 11 percent.
Later on in November the weight increased to
about 17 percent because of the gi'owth of the
remaining organisms.

The shells of li\ang oysters are frequently
covered with encrusting Bryozoa. In New Eng-
land waters and in Chesapeake Bay the appear-
ance of Bryozoa usually precedes the time of
setting of oyster larvae. When the oysters com-
plete their development, the shell surfaces may be
covered with Bryozoa colonies and unsuitable to
receive the set of spat. There is a possibility, not
fully substantiated, that a great many oyster
larvae are eaten by Bryozoa, and Osburn (1932)
thinks they are detrimental to the oyster beds in
Chesapeake Bay. Marie Lambert made a faunistic
study of the Bryozoa collected during the summer
on live oysters in the Oyster River near Chatham,
Mass. She recorded two species of Endoprocta
and five species of Ectoprocta. The most common
on oyster shells were Bowerhankia imbricata and
Schizoporella unicornis. The latter is an encrust-
ing bryozoan (fig. 387) commonly found on oyster
gi'ounds of Connecticut, part of Long Island
Sound (Hutchins, 1945), and Chesapeake Bay.

Dense setting of barnacles on oyster shells is
very common throughout the range of distribution
of C. virginica. In many instances, the space
that would have other%vise been available to
oyster larvae is already occupied by barnacles, or
the spat becomes covered with barnacles and fails
to grow. Barnacles have no adverse effect on
aduJt oysters.

The assemblage of invertebrate species found
livmg in close association with the oyster reflects
the fauna of the region and naturally differs from
place to place. In some areas oysters may be
almost entu-ely free of fouling organisms, while
in others their shells are hidden under a heavy
mass of siliceous sponges, hydroids, compound
ascidians {Botryllus), and Bryozoa.

FACTORS AFFECTING OYSTER POPULATIONS

427

Figure 387. — Oyster from Oyster River, Chatham, Mass.,
covered in part by tlie colonies of the bryozoan Schizo-
porella unicornis and the compound ascidian (white
spots) Amaroucium constellatum. Fouling is beginning;
witUn a few days tlie surface of the shell may be com-
pletely covered with these two animals.

The fouling is always seasonal, and with the
onset of cold weather many animals and plants die
and slough off. Possibly because of the periodicity
of fouling, the oysters sur\'ive and with few excep-
tions are not affected by the organisms gi-owing on
their shells. An exception is the invasion of
oyster beds by mussels {Mytilus edulis L.), which
in several situations may completely cover an
oyster bed with a thick layer of mud mixed with
excreta.

A number of annelids are commonly associated
with oyster communities, living between clusters
of oysters or in the shells. Sometimes a sur-
prisingly large numl)er of worms crawl out of the
shell crevices when Epsom salt is added to the
water in which oysters were kept. Hartman
(1945) lists seven species of annelids inhabiting
the spaces between clusters of living oysters.
Korringa (1951b) describes more than 30 species
of annelids which in Dutch waters live on or in

the shell of 0. edulis. Except for the boring
Spionidae, the worms apparently cause no direct
harm to oysters, but some of the mud-gathering
species of Nereidae materially increase the deposi-
tion of sediment over an oyster bed. No evidence
has been found of any other adverse effects of
annelids on oyster communities.

Various siliceous sponges are very common
members of the epifauna of oyster bottoms. With
the exception of the boring sponges, they do not
affect oyster populations. The red sponge,
Microciona proliUca, is often found on highly
productive oyster bottoms.

Of the protozoa that live on oyster shells, the
stentorlike infusorian, Folliculina sp., commonly
inhabits brackish water beds. This relatively
large protozoan, measuring from 200 to 800 n,
lives in bottle-shaped cases attached to the leaves
of Elodea, and Potamogeton found in the mouths
of rivers and on shells of other molhisks. During
the warm season it rapidly multiplies and appears
swimming with other plankton. Mass occurrences
of folliculuiids in the Chesapeake Bay were re-
corded by Andrews (1915) and in Oosterschelde,
Netherlands, by Korringa (1951a). Different
species are wdely distributed in the coastal waters
of the United States (Andrews, 1944). The num-
ber of this infusoria found attached to a single
oyster shell has varied from one to several hundred.
In many localities along the eastern shore of
the United States, oyster beds are frequently
overgrown by various algae. Oracillaria confer-
voides (Linnaeus) Greville is one of the species which
sometimes completely covers an oyster bottom
with its thick growth. Huge masses of the plant
wash away from the home grounds and pile on
beaches. Of the many other algae found growing
on oyster shells, several are in some regions as
abundant as Oracillaria: Enteromorpha, Ulva,
Griffitsia, Ceramium, Chondria, Champia, and
Scytosiphon. During experiments on raft culture
in Oyster River near Chatham, Mass. in 1956 to
1959 (Shaw, 1962), the shells of young oysters
suspended in water became covered with a very
dense growth of G. confervoides. Since there was
no noticeable ill effect on the oysters, an exami-
nation was made of the periphyton, the organisms
living loosely attached to the plant's branches.
The prevailmg form was found to be a diatom
Lycosoma sp., which was not present in the river
plankton outside the immediate area occupied by
Gracillaria. The stomach content of the oysters

428

FISH AND WILDLIFE SERVICE

consisted of many Lycosoma, some half-digested,
and of Skeletonema, which was also abundant
among the branches. It is apparent that some
constituents of the periphyton may be mgested
and that the microscopic flora of the envu-onment
provides a substantial amount of food not avail-
able in true phytoplankton.

Some of the seaweeds cause unexpected damage
to commercial oyster grounds. Colpoinenia sinu-
osa (Toth) Derbes and Solier, a common seaweed
in many parts of the word, is one of them. It
grows along the Pacific Coast of North America
from Alaska to southern California, along the
eastern coast of Australia and in France. The
thallus of Colpomenia is of a papery texture and
hollow; it can grow attached to oyster shells to
the size of a hen's egg or tennis ball. On sunny
days at low tide in shallow water photosynthesis
may be so intense that gas bubbles fiU up the
thallus, and on the return of the tide the inflated
balloon floats out to sea carrying with it the young
oysters. In 1906 Colpomenia became such a
nuisance on the western coast of France at Vannes
that the oystermen called it "oyster thief." The
floating oysters carried out by the ebb current

were not returned to shore with flood tide, and
the losses were severe enough for local oystermen
to organize the recapture of oysters with nets and
to tear off the inflated algal ballons by dragging
faggots over the bottom (Church, 1919).

In 1961 the seaweed, Codium Jragile subsp.
tomentosoides (Goor) Silva, was introduced to
Cape Cod waters with oysters brought from
Peconic Bay, Long Island, N.Y. This Pacific
Ocean species, not indigenous to Massachusetts,
occurs in abundance along the western coast of
Europe. It is not known how the alga was
introduced to Long Island where in January 1957
it was found at East Marion attached to dead
Crepidula shells (Bouck and Morgan, 1957). In
Oyster River, near Chatham, Mass., where the
Long Island oysters were planted the shells were
covered with a luxuriant growth and had to be
thoroughly scrubbed before being shipped to
market. The following year the plants were so
large (fig. 388) that on sunny days they acted as
"oyster thieves" by lifting the oysters from the
bottom with gas-fiUed branches and floating them
off with the tide.

Eel grass, Zosfera marina, frequently covers the

Figure 388. — C. fragile introduced into Oyster River, Chatham, Mass., with oysters from Long Island. Two-year-old

plant.

FACTORS AFFECTING OYSTER POPULATIONS 429

733-851 O — 64 28

entire oyster bottom but apparently exerts no ill
effect on oyster populations. This is not the case,
however, with another aquatic plant, the Eurasian
watermilfoil, Myriophyllum spicatum, which by
1933 was established on the Virginia and Maryland
sides of the Potomac River; since 1959 it has
increased rapidly in the Chesapeake Bay area,
including the Potomac River, and is found also
in the fresh and nontidal waters above Washington
D.C. In recent years the growth of the plant
has become spectacular and is a threat to brackish
water oyster grounds, which may become covered
with a heavy layer of decomposing leaves and
stems of the milfoil (Beaven, 1960; Springer,
Beaven, and Stotts, 1961).

The effect of commensals and competitors on the
productivity of an oyster bottom can be evaluated
for each species if the intimate relationship to the
host is clearly understood and the relative abun-
dance of a species is determined. A single species
which appears to be innocuous imder normal
conditions may become destructive and dangerous
because of its mass development. All these
conditions should be evaluated in order to express
their effect in numerical terms. Commensals
such as bryozoans, barnacles, and tunicates so
completely cover the shell sm'face that the
settlement of young oysters upon it is prevented.
Thus the negative effect of fouling may be con-
sidered in relation to the productivity of setting
groimds. On the other hand, in southern waters
where setting continues for the greater part of the
year and oysters become overcrowded with suc-
cessive generations of young, the reduction and
prevention of settlement of spat may be beneficial
because it reduces overcrowding and permits
better growth and fattening of oyster stock. The
struggle for space is an essential factor in the life
of an oyster community.

PREDATORS

The list of many enemies that prey on oysters
includes flatworms, mollusks, echinoderms, crus-
taceans, fishes, birds, and mammals. Not all of
them are equally destructive to oyster popula-
tions. The most dangerous are those which prefer
oyster meat to other types of food, and in search
of it invade the oyster grounds.
Carnivorous gastropods

The deadliest enemies of oysters are various
gastropods inhabiting coastal waters. The most
widely distributed species is the common oyster
drill, Urosaipinx cinerea (Say), which is found

along the entire Atlantic Coast from Canada to
Florida. With a shipment of C. virginica the
common oyster drill was introduced to the Pacific
Coast of the United States (1870 and the following
decades), and to Great Britain (1920 and probably
earlier) where the American oysters were planted
at Brighthngsea and West Mersea. In a short
time the drill became very abundant along the
coast of Essex and across the Thames estuary.
At present Urosaipinx is the most dangerous and
the most widely distributed of all the predators
of oysters in Em-ope.

Oyster planting by shellfish growers is the major
factor in the wide dispersal of Urosaipinx in this
country and its introduction into areas which
formerly were free of the pest. The migration
of drills is rather limited. Wlien hungry, they
may move at an average rate of 15 to 24 feet per
day in the direction of food. To a certain extent
the drills are dispersed by floating objects to which
they may cling and by hermit and horseshoe crabs
wliich have been seen bearing as many as 140
drills per animal (Carriker, 1955).

The drill is particularly destructive to young
oysters. In Cape Cod coastal waters, which are
infested by these snails, the oyster spat has very
little chance of surviving the first year; often small
seed oysters are wiped out before the end of the
second year. Adult oysters with thick shells suffer
less, and the losses sustained during 1 year by the
4 and 5 year classes are insignificant. There are
many localities in Long Island Sound, on the
eastern shore of Virginia between Chincoteague
and Cape Charles, and in other regions where
drills commonly kill 60 to 70 percent of the seed
oysters and sometimes annihilate the entire crop.

Fortunately, brackish water effectively bars the
drills from the upper parts of estuaries and tidal
rivers. Survival of drills in water of low salinity
depends on temperature and on the concentration
of salts to which they were adjusted. It may be
accepted as a general approximation that mini-
mum survival salinities at summer temperatures
vary from 12°/oo to 17%o in different regions.

Extensive literature on the biology and control
of oyster drills has been critically reviewed by
Carriker (1955), who has also made a detailed
study of the structure and function of the proboscis
and drilling apparatus of the drills (Carriker,
1943).

The maximum height of adult drills varies in
different localities between 25 and 29 mm.; a

430

FISH AND WILDLIFE SERVICE

giant form reaching 51.5 mm. in height is found
in the area of Chincoteague Island, Va., and is
considered a subspecies U. cinerea follyensis (fig.
389). As the common name indicates, the Uro-
salpinx attacks oysters and other moUusks by
drilhng a round hole in the shell. The hole,
usually made m the upper (right) valve of the
oyster, tapers toward the inner surface; the shape
of the hole identifies the attacker, and the presence
of drilled empty shells on oyster grounds is reliable
evidence of the mroads made by the snail on an
oyster population.

For a long time bormg was considered an en-
tirely mechanical process. Observations made by
Carriker (1961a) showed that both chemical and
mechanical actions are involved. Secretion from
the accessory boring organ, called ABO for short,
softens the shell, probably by an enzyme acting
on the conchiolin, and the softened material of
the shell is removed by abrasive action of the
radula. Active drilling continues for a few min-
utes and is followed by a long period lasting up
to an hour of chemical action dm'ing which the
ABO gland remains in contact with the shell.

The oyster is not the only victim of drills.
They show preference, in fact, to barnacles, and
usually stop drilling oysters if a rock covered witli
live barnacles is placed near by. A well-developed
chemical sense permits the drills to distinguish
between young and adult oysters. If both kinds
are offered to hungi'y snails kept in a large tank
with running sea water, the majority of active
drills will choose the young oysters. The drills are
positively rheotactic and in running water orient
themselves agamst the current. The orientation
is not, however, precise and the path of a moving
drill is a meandering line only generally directed
against the current.

Light has an effect on the orientation of drills.
They move away from a strong source of light,
but move toward it at lowefr intensities (Carriker,
1955). In dim light, the phototactic response is
lost. In laboratory tests at Woods Hole, I
noticed no orientation of drills toward the window
side of the tank; the drills distributed themselves
at random. They have a tendency to climb
away from the bottom (negative geotaxis) and
congregate on rocks, pilings, and on the wall

Centimeter

1 I I

0 2

Mil I i meters

Figure 389. — [/. cinerea follijensif: from Chincoteague Island region, Va. 1 — aportural view; 2 — abapertural view;

3 — egg cases.

FACTORS AFFECTING OYSTER POPULATIONS
733-851 O— 64 29

431

of tanks. Drills put in the bottom of a vertical
glass tube about 1 inch in diameter and 6 to 8
feet tall, filled with sea water, will climb to the top
of the tube and remain there. Negative geotaxis
is pronounced, particularly during the reproduc-
tive period. At this time drills climb on any
objects above the bottom level and ascend rocks
and various underwater structm'es to lay theu-
eggs, which are deposited in tough, leathery cap-
sules (fig. 389, 3). The egg-laying period depends
on geographical location and local conditions.
Summarizing the data from various sources,
Carriker (1955) estimates that the number of
egg cases deposited by one drill per season ranges
from 0 (in an imnuiture female) to 90 for older
females. In Woods Hole harbor, the breeding
season lasts from the end of June to the middle
of August. The number of egg cases deposited
by a single female kept in laboratory tanks
varied from five to nine; the average nmnber of
eggs in each case was nine. The number of eggs
per egg case varied in different localities from
eastern Canada to Chesapeake Bay from 0 to
22 and from 1 to 29 in British oyster beds. (Cole,
1942).

A second species of drill, Eirpkura caudafa
(Say), (fig. 390), is found in the same waters as
Urosalpinx but is usually less abundant. Various

observers estimate that in different locations it
comprises from 2 to 29 percent of the total diill
population (Carriker, 1955). The behavior of
Eupleura is similar to that of Urosalpinx. Its
food habits have not been studied, but occasional
observations in the laboratory indicate they are
probably not different from those of Urosalpinx.

In the York River, Va., where the growth and
reproduction of Eupleura were studied by Mac-
Kenzie (1961), the snail becomes active as the
temperatm-e rises over 10° C. Spawning begins
late in May at 18° to 20° C, reaches a peak in
June and early July at 21° to 26° C, and ends in
early August. Mature females (kept in cages)
deposit an average of 55 cases, each containing
an average of 14 eggs. In the absence of mortal-
ity, each female Eupleura may produce over 700
young di'ills each season. The leathery egg
capsules are vase-shaped with two distal pro-
jections (fig. 390, 3) and are easy to distinguish
from the egg cases of Urosalpinx (fig. 389, 3).

Conchs of the genus Thais occur on both the
Atlantic and the Pacific coasts. The snails have
strong polymorphic tendencies and form local
races which greatly complicate the taxonomy of
the species. A review of the speciation problem
of T. lamellosa Gmelin made by Kincaid (1957)

I.

0

0

432

Centimeters Centimeter

Figure 390. — Eupleura caudala (Say) from 1 — apertural side; 2 — from abapertural side, 3 — egg cases.

FISH AND WILDLIFE SERVICE

I.

3.

0 2

Centimeters

0

Centimeter

Figure 391. — T. haemastoma floridana Conrad from the shores of Pensacola Bay, Fla. 1 — apertural view; 2 — abapertural

view; 3 — egg cases.

contains interesting material regarding this and
other species of the genus.

T. haemastoma is a common oyster predator in
the waters of the South Atlantic and Gulf states.
There are two subspecies, T. haemastoma floridana
Conrad, which occurs from North Carolina to
Florida and the Caribbean (fig. 391), and T.
haemastoma haysae Clench (fig. 392), common on
oyster grounds of northwest Florida, Louisiana,
and Texas (Clench, 1947). T. haemastoma flori-
dana is a medium-size gastropod with a relatively
smooth shell and a smgle row of low spmes. T.
haemastoma haysae is a large, rugged snail, some-
times measuring 4}^ inches in height. It can be
distinguished from the other subspecies by double
rows of prominent spines around the whorls and
spire.

The behavior of the two varieties apparently is
similar. The conchs feed on oysters and other
mollusks, penetrating their shells from the edge
by using the ABO gland or by drilling holes in the
shell (Burkenroad, 1931; Carriker, 1961a). The
entrance at the edge of the valves is often in-
conspicuous and may be easdy overlooked.

Conchs multiply very rapidly because of their
great fecundity and high survival rate of larvae.
Thais haemastoma lays eggs in groups of about 800
to 975 enclosed in each egg case, with each female

depositing more than 100 cases. These figures
refer to my laboratory observations on conchs
kept in captivity. The eggs are deposited in
horny and transparent egg cases of a creamy
color, which becomes brownish and finally turns
reddish-purple. The breeding season in Louisiana
waters begins by the end of March and reaches its
peak in April and May. There is usually a rapid
decline of egg laying in June and a complete
cessation of reproduction in July. At the begin-
ning of the breeding period the conchs become very
active and develop a strong tendency to climb on
structures and rocks to attach their egg cases
above the bottom. Because of this behavior they
can be trapped during the breeding season on
stakes which the oyster growers erect on the
grounds. Gregariousness is very pronounced, and
many conchs can be trapped in this way in a rela-
tively short time. The number of egg cases
attached to a single stake may be enormous. One
stake which I obtained as a sample was covered
with a solid mass of egg capsules over a 5-foot
length; the estimated number of cases was about
8,000. The incubation period is not known
definitely, but judging from the growth of hydroids
and other fouling animals on the conch cases,
I believe it is not less than 2 weeks.

FACTORS AFFECTING OYSTER POPULATIONS

433

I.

0

Centimeters

Figure 392. — T. haemnstoma haysae Clench from Bastian Bay in the lower part of the Mississippi River delta.

1 — apertural view; 2 — abapertural view. Natural size.

The larvae that escape from the egg capsules are
veligers, which pass through a free-swimming
period of unknown duration, and are widely dis-
persed by tidal currents before they settle on the
bottom and begin attacking small oysters and
other bivalves.

The distribution of Thais is checked by fresh
water. The conch is immobilized by a salinity of
10%o, and a 1- or 2-week exposure to a salinity
of 77oo kills them (Scliechter, 1943).

The effect of sudden changes in water salinity
on the rate of crawling of Thais was corroborated
by my observations at the Bureau of Commercial
Fisheries Biological Laboratory at Gulf Breeze in
northwestern Florida. The crawling of these
snails in the tanks was automatically recorded on
a kymograph. The movements stopped immedi-
ately when the salinity of the water was artificially
reduced from 157oo or 17%o to 87oo or 97oo.

The snails became active again when the salinity
returned to the former level.

Two species of conchs found on oyster grounds
of tiie Pacific coast are Thais lamellosa Gmelin
(fig. 393), a native snail, and Ocenebra (Tritonalia)
Japonica Dunker (fig. 394), introduced from Japan.
T. lamellosa has been considered by some fishery
biologists as a predator on 0. lurida, but Kincaid
(1957) discards this view as not substantiated by
his 50 years of familiarity with the marine fauna
of the region. He states that since T. lamellosa
feeds mainly upon barnacles and mussels, the snail
sliould be classified as "tlie only invertebrate
friend" of the oyster, presumably because it
destroys its competitors. Chapman and Banner
(1949) found that under experimental conditions
T. lamellosa drilled some 0. lurida, but that in a
natural environment it showed a preference for
mussels (Mijtilus edulis).

434

FISH AND WILDLIFE SERVICE

0 2

Centimeters

Figure 393. — T. lamellosa Gmelin, a native species of the Pacific coast of the United States. 1 — apertural view;

2 — abapertural view.

The Japanese species, Ocenebra japonica, is far
more dangerous than the native snail. Mortal-
ities due to devastation by this snail are estimatetl
at 15.4 to 22.6 percent. The first specimens of
0. japonica were introduced into the waters of
Puget Sound with the planting of Japanese seed
oysters, a practice which began in 1902 and 1903
and wliich reached considerable proportions by
1922 when from 1,500 to 4,000 boxes, each con-
taining about 5,000 seed, were planted annually.
In October 1928 while examining the oyster beds
in Samish Bay, Wash., I found a number of
0. japonica Dunker (Galtsoff, 1929, 19.32), and
warned oystermen and state officials of the possible
damages that could result if the practite of bringing
infested seed oysters from Japan was continued.
The warning received no attention. In the late
1940's Ocenebra was well established in the waters
of Puget Sound and became a serious menace to
the native oysters. When given a choice of food,
Ocenebra prefers 0. lurida and Manila clams,
Venerupif! japonica, to C. gigas (Chew, 1960). It
drills holes in the shell by combined cheniical and
mechanical action (Carriker, 1961a). The fertil-
ity of the species is high, the female laying an

average of 25 egg cases, each containing about
1,500 eggs. The egg cases are often found in the
inaccessible crevices of the concrete walls of dikes
surrounding the Olympia oyster beds. Salinity of
18°/oo adversely affects Ocenebra, and brackish
water of less than 12°/oo salinity is lethal.

Large conchs or whelks, Bu..<^ycon carica Gmelin
and B. canaliculatum Linne, are common in the
shallow water of the Atlantic coast and occa-
sionally attack oysters and open them by inserting
the edge of the shell between the valves and
forcing them apart (Colton, 1908). Carriker
(1951) reinvestigated the problem and found that
penetration of shells of oysters and clams is a
purely mechanical process which consists of chip-
ping by the edge of the conch's shell combined with
rasping of the radula. The shell edge of an oyster
destroyed by these conchs bears the marks of the
attack (fig. 395). In the northern part of Cape
Cod, Busycon seems to attack the oyster in prefer-
ence to other mollusks, annelids, or dead fish,
which they are known to constmie. Local depre-
dations on oysters observed in the Cape Cod area
(Shaw, 1960, 1962) may be severe enough to war-
rant trapping of conchs during their reproductive

FACTORS AFFECTING OYSTER POPULATIONS

435

2.

0

Centimeters

0 0,5

Centimeter

Figure 394. — O. (Trilonalia) japonica Dunker, Japanese species from oyster bottom of Piiget Sound, Wash. 1 — apertural

view; 2 — abapertural view; 3 — egg case.

Figure 395. — Edge of the shell of an oyster killed by
Busycon in Oyster River, Chatham, Mass. Straight
line at the lower edge of the shell indicates the place of
rasping by the conch's radula after the valves were
chipped.

cycles. In Cape Cod estuaries, egg cases of conclis
are a familiar sight on tidal flats at low water
(fig. 396). Under experimental conditions the
conchs were found to consume in summer about
three adult oysters per week (Carriker, 1951).

Small parasitizing pyramilid snails of the genus
Odostomia {Meneslho) congi'egate in large numbers
at the very edge of oyster shells. \Vhen the
valves are open, the snails extend their pro-

FiGURE 396. — B. carica depositing egg capsules at low
Tide. Woods Hole.

436

FISH AND WILDLIFE SERVICE

boscides to the edge of the oyster's mantle and feed
on the mucous and tissues. These ectoparasites
are probably a great nuisance to the oyster, but
there is no evidence that they can be regarded as
important enemies. Two species have been found
associated with C. virginica: 0. {Menestho) bisu-
turalis Say which has a range from New England
to Delaware Bay, and 0. (Menestho) impressa
Say which is found from Massachusetts to the
Gulf of Me.xico.

Starfish

The starfish of the Atlantic Coast is also a
highly destructive predator on oysters. The
common species, Asterias jorhesi (Desor), is the
most familiar animal in tidal pools, on rocks, and
beaches of the Eastern Coast of the United States,
often found exposed by the receding tide. Accu-
rate statistics of the destruction caused by this
species are not available, but a few selected
examples emphasize its deadly efficiency. In
1887 the State of Connecticut estimated the loss
caused by starfish at $463,000; the sum repre-
sented the destruction of over 634,246 bushels
of oysters or nearly half of the total harvest for
the year (1,376,000 bushels). The numerical
strength of a starfish population over a relatively
small area can be visualized from the record of
only one company which in 1929 removed over
10 million adult starfish from 11,000 acres of
oyster grounds in Narragansett Bay.

As a rule the starfish populations on various
parts of the coast fluctuate within wide limits
with years of great abundance usually followed by
relative scarcity. These fluctuations cause many
oystermen to believe that starfishes invade their
grounds periodically. Studies of the problem con-
ducted simultaneously in Buzzards Bay, Nar-
ragansett Bay, and Long Island Sound (Galtsoft"
and Loosanoff, 1939) demonstrated that sudden
increases in the abundance of A. jorhesi are due
primarily to the high percentage of survival of
its free-swimming larvae and their successful
setting (fig. 397).

The reproductive season of A. forbesi in New
England waters slightly precedes that of C.
virginica. When oyster larvae reach setting stage,
the space available for their attachment is already
occupied by young starfishes only several mm. in
diameter, hungry, and ready to attack the spat.
The new set of oysters may be completely wiped
out by young starfish.

Millimeters

Figure 397. — Photomicrograph of live larva, brachiolaria,
of A. forbesi from a plankton tow in Buzzards Bay.

The movements of ^-1. forbesi in concrete tanks
are slow, random, and apparently not directed by
tactic reactions. Initially it was difficult to
reconcile this fact with the experience of oyster
growers in Long Island Sound who reported that
oyster bottoms thorouglily cleaned by mopping or
dredging were invaded within the next 24 hours
by swarms of starfish. Underwater observations were
made in Long Island Sound by members of the
Bureau of Commercial Fisheries Biological Lab-
oratory in Woods Hole who used an underwater
television camera. The underwater photographs
showed clearly that starfishes are passively
transported by the tidal currents which in Long
Island Sound are fairly rapid. The animal curls
up the tips of the rays, releases its hold on the
substratum, and floats just above the bottom.

FACTORS AFFECTING OYSTER POPULATIONS

437

Many thousands of starfish are transported in
this way from place to place and settle on new
grounds when tidal currents slacken.

The starfish leaves no identifying marks on its
victim, and only empty shells remain as evidence
of a destructive attack. Tlie recent death of
oysters is indicated by the cleanliness of the valves,
which contain no foreign growth and are still
attached to each other. The method by which
the starfish succeeds in forcing oysters or clams to
relax their muscles and open the valves has
puzzled biologists for a long time. It seemed
doubtful that the starfish could exhaust its
victim and open it by main force, and suggestions
were made, not well corroborated by observations,
that the prey was killed by suffocation or that a
substance secreted by the stomach of the starfisli
produced relaxation of the adductor muscle of the
oyster. Sawano and Mitsugi (1932) reported
that an extract of starfish stomachs poured over
the heart of living molluscs produced tetanus and
often inhibited the heart beat; this seemed to
give some support to the "anesthetic" hypothesis.
Critical experiments made in Woods Hole by
Lavoie (1956) show, however, that the effects of
extracts prepared from digestive organs of starfish
and introduced into the adductor muscle or
poured over the heart of Mytilus were generally
identical with those produced by plain water.
On the other hand, the force exerted by the tube
feet of starfish in opening shellfish was measured
manometrically and was found to exceed 3,000 g.
The measurement was made using mussels in
which the adductor muscles were severed and
replaced by steel springs or plastic cylinders.

Lavoie noticed that a tiny opening of about 0.1
mm. between the valves of the mollusks was
sufficient to permit tlie insertion of starfish
stomach. The pulling of valves apart is probably
repeated at intervals while the stomacii remains
partially compressed. The observations of Feder
(1955) on Pisaster ochraceus show that this starfisli
can open its prey by force alone. Another
Pacific Coast species, Evasferias troschelii, was
found to exert a force in excess of 5,000 g. during
an attack on artificial clams baited with Mytilus
meat (Christensen, 1957). The fact that star-
fishes are able to open mollusks by force alone
does not eliminate tlie possibility of an addi-
tional narcotizing' eflfect produced by starfisli secretion.
The problem of how the starfish opens its prey

has not yet been finally solved, although present
evidence favors the mechanical hypothesis.

Not all starfishes feed by everting their stomachs
and digesting the body of tlie victim without
ingesting it. Many of them are scavengers feeding
on dead animals found on bottoms while others
are capable of catching and consuming live fishes.
Many interesting cases of starfish attacks on
various marine animals including fishes are
described by Gudger (1933).

Starfish are usually found in water of high
salinity and do not invade the oyster grounds in
brackish waters. The salinity level between
16%o and 18°/oo below which A. jorhesi cannot
exist is a natural barrier to the distribution of
this species. This conclusion is based on field
observations along the Atlantic coast and on
experiments at the Bureau of Commercial Fish-
eries Biological Laboratory, Milford, Conn. (Loos-
anoft", 1945). In New England waters, starfish
are controlled by mopping or dredging to remove
them, and by dispersing calcium oxide and other
chemicals to kill tliem or to make a protective
chemical barrier around an oyster bed.

Flatworms

Turbellarians of the genus Stylochus and Pseu-
dostylochiis, commonly known as oyster leeches,
are predators which attack adult and j'oimg mol-
lusks and frequently inflict serious damage to
oyster populations. In 1916 and 1917 attacks of
Stylochus on oysters in Cedar Keys on the west
coast of Florida killed about 30 percent and in
one or two localities 90 percent of the adult
oysters. The mortality of oysters in Apalachicola
Bay, Fla., allegedly caused by the "leech," was
investigated for the LT.S. Bureau of Fisheries by
Pearse and Wharton (1938), who could not state
definitely that the distruction was due to S. ini-
inicus Palombi" and suggested that the oysters
were first weakened by some unknown cause and
that Stylochus invaded those which were unable
to protect themselves. S./rotitalis tolerates water
of low salinity (6 7oo), but according to Pearse
and Wharton does not lay eggs in salinities less
than 157oo.

S. ellipticus (Girard), found in Atlantic coastal
waters and also reported from the Gulf (Hyman,
1939, 1954), lives among oysters, shells, barnacles,
and rocks. The turbellarian was reported to
destroy young oysters on the flats at Milford,

'0 The identification was corrected by Hyman fl9y9) who found that the
Florida leech belongs to the species .S. fro-ntalis Verrill.

438

FISH AND ViaLDLIFE SERVICE

Conn. (Loosanoff, 1956). Apparently it has no
difficulty in entering oyster spat through the
slightly opened valves. On the Pacific coast, the
flatworm Pseudosti/lochus ostreophagus Hyman
(Hyman, 1955) was reported to cause mortalities
of from 6 to 42 percent among the imported
Japanese seed oysters on various grounds. The
worm bores keyhole perforations in the shells of
young oysters (Woelke, 1957).

Crabs

Ryder (1884) was the first to include the blue
crab, Callinectes sapidiis Rathbun, and the com-
mon rock crab. Cancer irroratus Say, in the list
of oyster enemies. He quoted complaints of oys-
termen working in Great South Bay, Long Island,
N.Y., who stated that the crabs eat small oysters
up to the size of a 25-cent com and invade the
the oyster planting grounds.

For many years crabs were not mentioned in
oyster literature as potential enemies, but in the
1930's and 1940's there were reports from the
U.S. Bureau of Fisheries Biological Laboratories
at Milford, Conn., and at Pensacola (Gulf Breeze),
Fla., that under certain conditions the blue crab,
the rock crab, and the green crab, Carcinides
moenas (Linneaus) destroyed oysters kept in
outdoor tanks or placed in baskets with the crabs.
Lunz (1947) reported that at Wadmalaw Island,
S.C., the blue crab was probably the most serious
pest in 1946 and destroyed more than 80 percent
of the young oysters set on collectors. The
crab's diet includes a great variety of food, includ-
ing oysters. There is no evidence that they are
attracted specifically by oysters, but it is apparent
that they may destroy many small oysters in
clusters by cracking their shells.
Mud prawns and fish

Brief mention should be made of the family
Calianassidae (genera Upogebia and Callianassa),
popularly known as "mud prawns" or "burrowing
shrmips", which excavate deep burrows under
oyster bed dikes. This activity drains water
from the grounds, exposes the beds of 0. lurida,
and smothers the young oysters with material
thrown up in burrowing (Stevens, 1928).

In the southern waters of the Atlantic coast,
oyster beds are often invaded by schools of
black drum, Por/oiuas cro7ms (Linnaeus), which
feed on mollusks and occasionally cause extensive
destruction of oysters, leaving behind piles of

crushed shells. The fish uses its powerful pharyn-
geal teeth to crush the shells (fig. 398).

The diamond stingray of the Pacific coast,
Dasyafis dipterurus (Jordan and Gilbert), also
devours oysters, crushing them with powerful
teeth. To ward off attacks by this fish, oyster
grounds in California are surrounded by high
fences, a practice used for the same purpose by
French oystermen.

Birds

Various species of ducks are enemies of small
0. lurida of the Puget Sound area. The extent of
damage to oyster grounds near Olympia, Wash.,
was estimated in the fall of 1928 by the United
States Biological Sm-vey. McAfee, who con-
ducted the field studies, reported (quoted from
GaltsofT, 1929) that at that time 87 percent of
the bluebills {Nyroca marilla and A'', affinis) fed
principally on oysters, which comprised 80.5
percent of the bulk of the food found in their
stomachs. In 38 percent of white-winged scoters

A.

B.

Centimeters

Figure 398. — Pharyngeal teeth of small size black drum
P. cromis, used for crushing oyster shell. A — upper
teeth; B — lower teeth.

FACTORS AFFECTING OYSTER POPULATIONS

439

(Melamita deglandi), about 70 percent of their
stomach contents consisted of oysters. The
number of birds in the Olympia Bay of Puget
Sound during the 2-week period of daily observa-
tions (November 16-29, 1928) averaged 2,000.
Together the three species of ducks were destroy-
ing about 8,000 oysters per day and causing ma-
terial damage to the small oyster industry of the

area.

The effect of predators on an oyster population
can be evaluated by determining the percentage
of oysters killed.
Man

Among the highly destructive predators of
oysters, man occupies the most prominent posi-
tion. Long before our era the stone age dwellers
of the coast of Europe subsisted primarily on
shellfish which they gathered from shallow water
by wading and hand picking. The American
Indians used oysters and clams for food, and
dried and smoked shellfish meat for the food
supplies which they took on their travels. On
both continents numerous shell heaps or so-called
kitchen middens dot the coastline and indicate
the locations of primitive habitations or camp
sites. A famous shell heap on the banks of the
Damariscotta River, Maine, and many others
are evidence of the former productivity of the
oyster beds of past centuries. With the develop-
ment of oyster fishing gear, man became able to
gather oysters much more efficiently and extended
his efforts to deeper water. Oyster dredges of
various designs and dimensions remained for a
long time the principal and very effective gear,
until the appearance in the last quarter of a
century of various mechanical suction pumps and
other harvesters of much greater efficiency.

With tlie improvement of fishing methods, the
oyster bottoms of the northern States became
overfished and many were depleted. This was
the fate of many oj^ster grounds along the shores
of the Gulf of Maine, in New Hampshire, Massa-
chusetts, and Rhode Island. In colonial times
the earliest white settlers of New England feared
the disappearance of their favored seafood and saw
the necessity of protecting their shellfish resources
by such legislative measures as restricting the size
of catch and prohibiting the selling of o_ysters out
of town. The results were ineffective, and many
oyster bottoms, particularly in the northern part
of New England, were destroyed.

The world's richest oyster bottoms in the Chesa-
peake Bay suffered a similar fate, but the deple-
tion was more gradual and not as complete as in
more northern waters. Regulations prohibited
power dredging and set aside certain areas for
the use of tongers only, but they were not sufficient
to maintain the productivity of the oyster bot-
toms. Tlie production of oysters continued to de-
cline because of a general disregard of the basic
conservation principle that the sustained yield of
any renewable natural resource can be maintained
only if the quantity removed does not exceed the
quantity restored annually by reproduction and
growth. Throughout the world the shellfish
resources are depleted when more are taken than
nature is able to replace.

Man must be regarded, therefore, as the most
dangerous predator. On the other hand, through
his action the productivity of an oyster bottom
can be brought to the highest level. Since ancient
times it has been known that oysters can be propa-
gated and cultivated. The development of oyster
culture in this country was particularly successful
in the waters of Long Island Sound where the
depleted shellfish resources were not only restored
by oyster farming, but many thousands of acres
of previously barren bottom were converted into
productive farms under water. Thus, man as an
ecological factor appears in a dual capacity — as
a primitive destructor and as a progressive culti-
vator. LTnfortunately, at present Long Island
Sound is no longer a highly productive oyster
farming area. The decline may be attributed to
poor setting, low survival rate of young oysters,
devastation caused by several hurricanes, and the
high cost of farming operations.

At present the knowledge of oyster biology has
advanced to such a level that effective methods
can be employed both for sound management of
natural, wild populations of oysters, and for de-
velopment of highly productive farms for breeding
selected strains of oj'sters. The continuous decline
of oyster beds is due not to a lack of knowledge
but to failure to apply it.

Aquatic resources of tlic tidal areas along the
Atlantic and Gulf coasts of the United States are
threatened by human activities other than over-
fisliing. Many formerly productive areas of the
coast have been damaged beyond reconstruction
by the filling of marsh lands for industrial sites,
by the construction of thruways, marimis, real
estate developments, and trash and garbage dis-

440

FISH AND WILDLIFE SERVICE

posal areas, by ever-increasing discharge of domes-
tic sewage and trade wastes, and by numerous
contaminants which reach natural waters as a
result of widespread and nonselective use of
insecticides and pesticides. Danger from the
discharge of radioactive materials from nuclear
plants and the disposal of low level radioactive
wastes in the sea not far from shore presents a
new and serious threat to the usefulness of the
renewable aquatic resources of coastal areas.

Some of the changes produced by man such as
improvement of coastal waters for navigation,
construction of liunicane barriers, use of tidal
land for Iniikling of industrial plants are consist-
ent with rapid population gi'owth and industriali-
zation. Other changes, such as pollution, destruc-
tion of natural oyster beds by failure to return
shells and other materials needed for the attacli-
ment of young oysters, and overfisliing are
unnecessary and should be avoided.

A balance between the needs associated with
industrial progi-ess and population pressure on
one side, and effective conservation of natural
aquatic resources on the other can and must be
found.

POLLUTION

The pollution problem is complex. It has
many facets that should be studied from social,
economic, and biological points of view. An in-
vestigation of the biological aspects of pollution,
discussed in this section, deals with the complex
ecological relationship between the life in tlie
tidal areas and the environment affected by the
addition of a number of organic and inorganic
contaminants.

One of the major difficulties encountered in
studies of the biological effects of pollution is the
lack of a generally accepted definition of the
term. Pollution means different things to differ-
ent people: to a Public Health officer pollution
implies a potential health hazard caused by the
dischai'ge of domestic sewage and industrial waste;
an engineer of a manufacturing plant is primarily
concerned with the quality of water needed for
the industry; the conserxationist has in mind
danger to wildlife and means for its protection ;
sport and commercial fishermen fear that foreign
substances discharged into coastal waters will
affect tlie availability of fish; a marine ecologist
tries to find out how the animal and plant life is
affected by changes in the environment; and the
layman, considering that pollution is synonymous

with filthy conditions on beaches and in coastal
waters, raises his voice in protest against unsani-
tary and esthetically objectionable situations.

In court litigations involving damages allegedly
caused by pollution, a biologist appearing as an
expert for either side is handicapped in his testi-
mony either by lack of a legal definition of pollu-
tion or by the generalities used to describe it.
No definition of the term pollution is given in the
Oil Pollution Acts of 1924 and 1961. The Water
Pollution Control Acts of 1948 and 1961 (United
States Congress, 1948, 1961) make frequent refer-
ences to the "abatement of stream pollution" and
declare in the 1948 act that pollution is a public
nuisance "which endangers the health or welfare
of persons in a State other than that in which the
discharge originates." The inclusion of the word
"welfare" puts emphasis on the economic aspects
of pollution and, therefore, increases the scope of
the definition.

After conducting a comprehensive study of all
available State, Federal, and international pollu-
tion laws, the U.S. Public Health Service (1950)
prepared the following broad definition of pollution:

"Pollution" means such contamination, or other altera-
tion of the physical, chemical or biological properties, of
any waters of the State, or such discharge of any liquid,
gaseous or solid substance into any waters of the State as
will or is likely to create a nuisance or render such waters
harmful or detrimental or injurious to public health, safety
or welfare, or to domestic, commercial, industrial, agricul-
tural, recreational, or other legitimate beneficial uses, or
to livestock, wild animals, birds, fish or other aquatic life.

Although this definition is broad and useful, it has
not been incorporated in existing Federal statutes
and, therefore, lacks legal weight.

The amount of waste discharged into coastal
waters of the United States from municipalities
and industrial plants in the last decade has reached
astronomical proportions and is being augmented
by runoff water which carries the numerous or-
ganic phosphorus and hydrocarbon insecticides
used in both control and eradication of agricul-
tural crop-damaging pests. Under present condi-
tions it is probably impossible to find water along
our coast which has not been contaminated.

Some pollutants contain highly toxic substances
and cause mortalities among marine populations.
Others are less toxic and have no lethal effect on
adult organisms but decrease the rate of survival
of their larvae; decrease the rate of growth of
juvenile forms and afl^ect the reproductive capa-

FACTORS AFFECTING OYSTER POPULATIONS

441

bility of an organism. Sublethal concentrations
of such poisons can also destroy one or several
links of the food chain in the sea, and so affect
the food supply for the population of animals or
plants important for human welfare. The normal
ecological environment may be so changed that
some planktonic organisms, most useful to shell-
fish as food, disappear and are replaced by a
luxurious growth of microorganisms not only
useless but even harmful to water-filtering mol-
lusks. Although great advances have been made
in the technique of bioassays, the results of
short-term tests lasting no longer than 72 hours
are of little use in determining the effects of
prolonged exposures of fish or shellfish to low
concentrations of poison. Furthermore, since the
criteria for the welfare of marine populations are
not known, it is impossible to set requirements for
purification of pollutants before they are permitted
to be discharged into the sea. The Federal
Water Pollution Control Act of 1961 authorizes
the Secretary of Health, Education, and Welfare
to organize comprehensive programs of investi-
gation which in the course of years will solve many
of the e.xisting pollution problems.

Detailed descriptions of all types of pollution
that may affect the productivity of oyster bottoms
and methods of their detection and control are
beyond the scope of the present chapter, which is
limited to a discussion of the general principles
apphcable to the majority of situations and to a
description of the most important tj'pes of pollut-
ants encountered on oyster bottoms. Bibho-
graphical references listed at the end of the
chapter are limited to the more pertinent papers.
Discussions of more specialized pollution problems
are listed in a bibliograph}' prepared by Ingram
(1957) and also appear in papers published in
Tarzwell (1957, 1960).

The production of oysters in the United States
is declining at a rapid rate (Galtsoff, 1956). As a
sedentary animal devoid of any means of loco-
motion after setting, the oyster is vulnerable to
environmental changes which weaken it and make
it less resistant to infection. Under natural
conditions unspoiled by human activities, the
oyster is in an equilibrium with its environment;
this adjustment, which may be called a steady
state, is the result of thousands of years of
adaptation and natural selection. It may be
upset by the sudden presence of materials not

normally found in sea water or by excesses or
deficiencies of its normal components.

Two types of pollution are commonly found on
03'ster grounds: domestic sewage and trade wastes.
In natural waters both types of pollutants undergo
gradual changes which lead to a degree of purifi-
cation, but at the same time deposit sediments
that cover oyster beds and change the character
of the bottom. Natural purification is not effec-
tive, however, in the case of detergents and radio-
active waste, which constitute a growing menace
to the safety and purity of our coastal waters.

Domestic sewage

Contamination of water by domestic sewage is
the oldest type of pollution; it probably began
during prehistoric times when man settled on the
shores of the rivers and bays and used natural
waters as the easiest and most convenient way of
disposing of the excrements and unwanted waste.
The problem has reached enormous proportions
with the population growth and the necessitj' of
disposing of quantities of domestic sewage in an
organized manner.

The discharge of untreated domestic waste has
a threefold effect. It covers the bottom with a
sludge which smothers the oyster bed, affects the
normal functions of moUusks by reducing the
oxygen content of the water, and at the same time
greatly increases the bacterial content of the water.
Oysters, in common with other water-filtering
mollusks, retain and accumulate these bacteria in
their bodies. The degree of pollution is deter-
mined by the abundance of Escherichia coH found
in the water. The bacterium itself is not patho-
genic, but is used as an index of pollution. Pro-
cedures for determining the abundance of E. coli,
the so-called MPN (most probable number), are
described in great detail in Jensen (1959). They
are strictly followed by State and Federal Public
Health Officers and other officials responsible for
certifying grounds from which shellfish may be
liarvested for human consumption. Areas in
which the MPN of E. coli exceed the permissible
maximum of 70 per 100 ml. are condemned
and cannot be used for harvesting, but under
certain specified conditions the polluted oysters
and clams can be taken for planting to an unpol-
luted area. The presence of E. coli above the
prescribed MPN eliminates tlie utilization of
grounds for commercial fishery, but does not affect
the survival and growth of the oyster population.

442

FISH AND WILDLIFE SERVICE

Industrial waste

The most common industrial pollutants entering
oyster-producing areas stem primarily from the
following industries: oil; paper; steel; chemicals;
paints; plastics; leather; and food. The character
of industrial waste varies with the product.

Because of the increase in the number of oil
burning ships and the necessity of transporting
crude oil in huge tankers that occasionally break
and spill their cargo, oil pollution of the open sea
has become a difficult international problem.
Although federal and state laws forbid the dis-
charge of oil into coastal waters, many of the bays
and harbors of the United States are heavily
polluted by oil. Through surface tension oil
spilled on the surface of water spreads rapidly
into a thin film or oil slick. In muddy waters
suspended particles of clay and sand absorb oil,
coalesce, and gradually sink to the bottom. In
shallow waters oil laden sediment is disturbed by
waves, and an oil slick reappears on the surface,
sometimes considerable distances from the source
of pollution. The absence of an oil slick is not,
therefore, a reliable sign that water is not polluted.
Crude oil absorbed by sediments retains its toxicity
to oysters and other organisms for a considerable
time (Chipman and GaltsofT, 1949).

With the expansion of the pulp and paper
industry along the Atlantic and Pacific Coasts,
pollution of coastal waters by red and black
liquors, the waste products of this industiy,
became serious. Both types of waste contain
toxic substances which adversely affect oyster
physiology. As in other types of pollution, the
discarded material is usually oxidizable and has
high oxygen demand. It is, however, only in
extreme instances of gross pollution that the
oxygen content of the water is lowered to the
point that it suppresses the principal physiological
functions. Poisons, present in trade waste, are
more dangerous than the high oxygen demand
because they directly affect the function of the
various organs. In spite of great variety in the
composition of trade wastes their toxic effect can
be demonstrated by constructing a toxicity curve
which shows how the pollutant depresses the
function that was selected for testing. An oyster
heart preparation (see ch. XI, p. 247) can be used
conveniently because of the great sensitivity of
the heart muscle to many poisons and drugs.
Another measurable function is the transport of
water by the gills for feeding, respiration, and

discharge of excreta. This function ceases when
the valves are closed. The presence of pulp mill
pollutants reduces the number of hours the valves
are open in proportion to the concentration of
toxic substances in the water. Under normal
conditions and at temperatures of 60° to 70° F.
oysters remam open on an average of 20 to 22
hours a day. If the logarithm of concentration of
black liquor or crude oil extract is plotted against
the number of hours closed, the relationship can
be expressed by a straight line as shown in fig. 399.
Toxic substances of pulp mill effluents and the
extracts of crude oil affect the frequency of ciliary
beat and so interfere with the coordination of
ciliary motion with the result that the pumping
capacity of the gills is reduced. The reduction is
proportional to the concentration of physiologi-
cally active materials Cfig. 400). This type of
relationship was found in studies on the pollution
of oysters by red and black liquor and by water
soluble components of crude oil (Galtsoff, 1931b;
Galtsoff, Chipman, Engle, and Calderwood, 1947;
Chipman and Galtsoff, 1949). The observations
on crude oil are in agreement with data reported

<

UJ

u

z
o

O

0.5

0 5 10 15

HOURS CLOSED

Figure 399. — Effect of concentration of pulp mill effluent
discharged into the York River on the number of hours
oj-sters are closed during every 24-hour period. From
Galtsoff, Chipman, Engle, and Calderwood, 1947.

FACTORS .\FFECTING OYSTER POPULATIONS

443

100

0 ID 20
CONCENTRATION IN

30 40 50
PARTS PER THOUSAND

Figure 400. — Depression of the activity of ciliated
epithelium of oyster gill by increased concentration of
pulp mill effluent (black liquor) of specific gravity
1.0028. From Galtsoflf, Chipman, Engle, and Calder-
wood, 1947.

by other investigators (Seydel, 1913; Veselov,
194S) on the toxicity of crude oil to fishes.

Determination of the toxicity of some pollutants
is difficult because they may be present in such low
concentrations that they are near or below the
threshold of sensitivity to chemical methods.
Their presence in even mmimal quantities should
be considered potentially dangerous to sedentary
animals unable to avoid them. Another detection
problem is that in many industrial plants the dis-
charge of effluents is not contmuous but is fre-
quently interrupted or made during the night and
early hours of the morning. Pollution studies
must include taking composite samples of water
with automatic samplers over a period of several
hours. Some contaminants are unstable; after
being discharged into sea water they are graduallj'
oxidized, precipitated, neutralized, and become
less harmful. The rate of this self-purification of
water depends on many conditions, temperature,
salinity gradient, sedimentation, and currents.
To avoid inconsistencies in results, toxicity tests
with such materials should be carried out only with
stabilized samples (Odlaug, 1949).

Bioassays made within a few days indicate the
presence or absence in water of a physiologically
active substance but do not determine whether the
pollution is lethal to the animal. Long-term field
and laboratory' observations are needed to deter-

mine the lethal effects of a low concentration of
pollutants.

Ecological studies in polluted waters show that
under certain conditions the normal environment
may be modified by the contaminant and become
unsuitable for growth and reproduction of oysters.
Pollution of Shelton Bay, Puget Sound, Wash.,
with red liquor discharged by a local pulp mill
boosted the production of the diatom Melosira sp.
to such an extent that the beds of 0. lurida in the
bay became covered with a thick layer of this
fouling plant. A similar effect occurred in labora-
tory tests with red liquor made by Odlaug (1949).
Oysters affected by red liquor were useless because
of then poor quality and poor taste; their repro-
duction stopped completely. Normal conditions
were restored after discharge of the pollutant was
discontinued (McKernan, Tartar, and Tollefson,
1949).

The biologist who studies pollution of natural
water should remember that there is no harmless
pollution. All types of pollution are harmful to
marine populations; only the degree of their effects
differs. Frequently it is claimed tliat the enrich-
ment of sea water by phosphates, nitrates, carbo-
hydrates, and other organic matter is beneficial
and will tend to increase productivity. In the
case of water pollution by duck farms in Moriches
Bay, Long Island, N.Y., indiscriminate pollution
by duck manure caused an imbalance of nutrient
salts and boosted the outbreak of microorganisms
which had an adverse effect on shellfish (Redfield,
1952). LTseful enrichment of sea water can be
achieved only by controlled and balanced fertiliza-
tion.

Oxidation is important in reducing or destroying
the toxicity of certain contaminants of sea water
(Galtsoff, Chipman, Engle, and Calderwood, 1947).
The efficiency of oxidation is influenced by tem-
perature and by the manner in whicn the pollutant
is added to the water. Preliminary storage in
tanks is helpful in removing objectionable solids,
and cascading the effluent from storage tanks to
tlie place of discharge will expedite its oxidation.
Tlie U.S. Public Health Service found that 10,400
factory outlets in 1950 were pouring their waste
into natural waters of the United States; only 657
of them had waste treatment plants of adequate
capacities. In about 30 percent of tlie plants, the
method of treatment was unsatisfactory. The
number of plants which at present discharge their

444

FISH AND WILDLIFE SERVICE

waste into coastal waters and the amount of
waste are not known.

Radioactive waste

The disposal of radioactive waste in the sea
presents a new threat to shellfish resources; the
concentration of radioactive materials in the bodies
of water-filtering mollusks may render them unsafe
for human consumption. Chipman (1960) showed
that many of the radionuclides added to sea water
become associated with both living and nonliving
particles suspended in water. Experiments at the
Radiobiological Laboratory of the Bureau of
Commercial Fisheries at Beaufort, N.C. (Chipman,
Rice, and Price, 1958; Rice and Willis, 1959)!
indicated that nearly all fission product radio-
nuclides, and also those of the trace metals, that
are added to algal cultures associate with marine
plankton used by shellfish. If continuously
available, radioactive particles may accumulate in
filtering organs, on the body surface, and in the
digestive tract of oysters and other shellfish.

The accumulation of radioactive pollutants in
coastal waters is likely to become higher than it is
at present if the current practice of dumping radio-
active wastes from nuclear plants and many
research institutions close to shore or in the lower
parts of a river (Columbia River) continues
indefinitely. This unwelcome possibility must be
watched carefully, and a great deal of research
remains to be done before a clear picture emerges
of the potential dangers associated with the dis-
posal of low level radioactive waste and the con-
tamination of our fisheries resources.

To evaluate the effect of pollution on the pro-
ductivity of oyster bottoms the following data are
needed: the type and extent of pollution in rela-
tion to the total volume and movements of water
in an estuary; the stability of the pollutant; its
physiological action; the effect of long-continued
exposure of oysters to low concentrations; and the
determination of the lethal concentration of a
pollutant killing 50 percent of a population, the
so-called LD 50.

COMBINED EFFECT OF ENVIRONMENTAL
FACTORS

Known effects of any single factor of tlie environ-
ment do not give a true picture of tlie situation
found in nature. Factors of the environment
always act jointly. One serious weakness of
many ecological studies of marine populations
is the tendency to correlate the results of biological

FACTORS AFFECTING OYSTER POPULATIONS

observations with one or possibly two selected
factors of the environment, such as temperature,
salinity, or hydrography, and to disregard the
effect of others. In reality, any factor can exert
its effect only in conjunction with others. It
is impossible to separate the effect of chemical
changes caused by a pollutant from the move-
ments of water and from the effects of the pollu-
tant on the food chain. Changes in the character
of a bottom brought about by sedimentation can-
not be separated from changes in sea water
chemistry, or the food chain. An increase in the
salinity of water encourages the invasion of
grounds by some competitors and predators,
while lowered salinity forms a barrier to inroads
by starfishes and drills.

The combined action of several factors pro-
duces a far greater effect than that caused by any
single factor. Findings of what effects combined
factors have on agricultural plants (Riibel, 1935)
are fully applicable to conditions affecting aquatic
animals. So far, however, no adequate studies
have been made on the problem of measm-ing the
joint effect of several factors of aquatic environ-
ment. The relationship of all factors probably
can be expressed by a very complex formula of the
type developed by Riley (1947) for seasonal
fluctuations of phytoplankton populations in
New England coastal waters. The very com-
plexity of a formula of this type precludes its
usefulness for the practical purpose of evaluating
conditions on oyster bottoms. The oyster biolo-
gist is often confronted with the necessity of
expressing his opinion on the quality of the oyster
beds. His impression is given in general, non-
specific terms as adequate, good, very good,
marginal, etc., which do not disclose the reasons
for a particular evaluation.

My proposed method of scoring eliminates the
uncertainty of personal impressions and assigns
to each factor a value which indicates the degree
of its effectiveness on a given- population of
oysters. The method has been applied success-
fully in the evaluation of oyster bottoms in New
England, the south Atlantic coast, and in some
Gulf States (Galtsoff, 1959). It has been already
stated above (p. 399) that the optimal condition
of ex-istence with reference to a single positive
factor can be assigned the numerical value of 10.
Degrees of inadequacies are given numerical
values in descending numbers from nine to one.
Negative factors are treated in much the same

445

way. Complete absence of a negative factor
refers to optimal conditions, and therefore, is
designated zero, wliile the degrees by which the
factor adversely affects an oyster population are
assigned the numbers diminishing from nine,
for 90 percent of negative influence, to one.
which denotes 10 percent or less of harmful effect.
The zero value of a positive and 10 value of a
negative factor are omitted because under the
proposed system such values denote complete
unsuitability of environment for the existence of
an oyster population.

A combination of environmental conditions
which determine the producti\'ity of an oyster
bottom is summarized in simple tabular form by
listing in two separate columns all positive ( + )
and all negative ( — ) factors and assigning to them
their rank. As an example of the method, the
data for one of the highly productive areas in the
northern Cape Cod area, where observations were
made for several years, are presented in table 46.
In this area, which approaches ideal conditions,
the presence of predators is the only serious
problem.

The overall evaluation is inade by summing up
all positive factors, 2 f"^ and all negative factors,
2 f" and by deducting the sum of the negative
factors from the sum of the positive. Under this
system the highest score of 50 refers to a theoreti-
cal situation where all positive factors are optimal
and negative factors are absent. The low score
of 10 and less refers to marginal conditions.
Tabulation of factors is of great practical ad-
vantage because it shows at a glance the causes of
low productivity and how it can be improved.
The following tabulation shows the scores that
in my opinion apply to various degrees of pro-
ductiveness of oyster bottoms:

Excellent 41-50

Good 31-40

Average 21-30

Poor 11-20

Marginal 10 and less.

Table 46. — Evaluation oj the productiveness of an oyster
ground in the northern part of Cape Cod

Positive factors (+)

Score

Negative factors (-)

Score

10
10
5
10
10

2

5

Food

Pollution

Total.

Total

45

7

Note. — Overall score 45-7=38; in negative score indicates absence of

a factor.

In its present form, the method obviously over-
simplifies the problem because it considers all the
factors as equally significant, which may not be
true. The present lack of understanding of the
interaction within a complex ecological system
bars expression of this interrelation in a more
precise form. Growing interest in studies of the
sea and its resources, however, gives promise of
rapid progress in determining the intricate rela-
tionships among the principal factors that govern
the prosperity of marine populations. The result-
ing knowledge will pro\'ide the basic data for
designing effective methods of utilization and
conservation of the renewable resources of the sea.

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1949. The mystery of the red tide. Scientific
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1956. Ecological changes affecting the productivity
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1947. Ecological and physiological studies of the
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1939. Natural history and method of controlling the
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1962. Notes on shell morphology, growth, and dis-
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1937. Natural history and methods of controlling
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1950. Seasonal population changes and di.stribu-
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1956. Larval development of the polychaete families
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1945. The marine annelids of North Carolina.
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1934. Measurement of phytoplankton population.
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1961. Studies on the crown conch Melongena corona
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1953. An introduction to the zoogeography of the
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1958. The planktonic larvae of Polydora websteri
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1945. An annotated check-list of the salt-water

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1957. Handbook of selected biological references on
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1955. Ecology of oyster bed. I. On the decline of
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1959. Manual of recommended practice for sanitary
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1952. On the relation between water transport and
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1953. Particle filtration in some ascidians and
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1906. Notes preliminaires sur les gisements des
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1908. Etudes sur les gisements de mollusques
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1951a. The flushing of tidal estuaries. Sewage and
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1951b. The exchanges of fresh and salt waters in
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1954. Relation between circulation and planktonic
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1957. Local races and clines in the marine gastropod
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1950. A review of the papers on molluscs presented
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1951a. Investigations on shell-disease in the oyster,
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1951b. The shell of Ostrea edulis as a habitat.
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1951c. Crepidula fornicata a.sa.n oyster-pest. Conseil
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1952. General character of plankton organisms in
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1951. Distribution of Nematopsis infection on the
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1956. How sea stars open bivalves. Biological Bul-
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1949. Applied hydrology. 1st ed. McGraw-Hill
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1945. Effects of sea water of reduced salinities upon
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1952. Behavior of oysters in water of low salinities.
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1956. Two obscure oyster enemies in New England
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1943. Polydora in oysters suspended in the water.
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1947. Effect of diiferent concentrations of micro-
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1940. The annelid worm, Polydora, as an oyster pest.
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1941. Polydora, a pest in South Carolina oysters.
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1947. Callinecles versus Ostrea. Journal of the
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1955. Fluctuations of animal populations, and a
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1961. Growth and reproduction of the oyster drill
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Mackin, J. G.

1951. Histopathology of infection of Crassoslrea
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1961a. Mortalities of oysters. Proceedings of the
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1961b. Status of researches on oyster diseases in
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1952. Hexamitiasis of Ostrea edulis L. and Crassoslrea
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1950. Preliminary note on the occurrence of a new
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1955. Distribution of oyster larvae and spat in

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1949. An investigation of the decline of the native
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1962. Seasonal variability of compactness in marine
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1941. The influence of temperature and salinity on the
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1899. Report on the oyster-beds of Louisiana.
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1911. Condition and extent of the natural oy.ster
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1944. Behavior and tube building habits of Polydora
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1947. Serious mortalities in Prince Edward Island
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1952. Some observations on the migrations and
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1938. Oxygen-poor waters of the Chesapeake Bay.
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1939. Studies on the physics and chemistry of
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NoRRis, Robert M.

1953. Buried oyster reefs in some Texas bays.
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1946. The effect of the copepod, Mytilicola orientalis
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1949. Effects of stabilized and unstabilized waste
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1941. The taxonomy and distribution of the boring
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1932. Bryozoa from Chesapeake Bay. Ohio Journal
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1957. Etiological studies on oyster mortality. II.
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1955. Changes in the invertebrate fauna, apparently
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1938. The oyster "leech" Stylochus inimicus Palomlii,
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1943. Certain recent geological and biological
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1951. The physical hydrography of estuaries and
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1952a. A review of our present knowledge of the
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1952b. Salinity distribution and circulation in the
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1953. Distribution of oyster larvae in relation to
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1955. Estuarine circulation patterns. Proceedings,
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1953. The reduction and analysis of data from the
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1940. The life cycle and morphology of A^ematopsis
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1953. The molluscan community of the oyster-reef
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Rasmussen, Erik.

1951. Faunistic and biological notes on marine
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1952. A culture technique for the diagnosis of in-
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1954. Biological studies of Dermocystidium marinum.
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Ray, S. M., and A. C. Chandler.

1955. Parasitological reviews, Dermocystidium mari-
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Ray, Sammy, J. G. Mackin, and James L. Boswell.

1953. Quantitative measurement of the effect on
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Redfield, Alfred C.

1952. Report to the towns of Brookhaven and
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Reid, George K.

1956. Ecological investigations in a disturbed Texas
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1957. Biologic and hydrographic adjustment in a
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Renn, Charles E.

1940. Effects of marme mud upon the aerobic de-
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Rice, T. R., and Virginia M. Willis.

1959. Uptake, accumulation and loss of radioactive
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454

FISH AND WILDLIFE SERVICE

Stevens, Belle A.

1928. Callianassidae from the west coast of Xorth
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1951. Recent developments in the study of tidal
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1949. Habitat selection in marine animals. Folia
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1952. On the ecology of distribution of cockle and
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1948. Miyanie na ryb zagryazneniya vody neftyu.
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1931. Biologic ostreicole. La maladie des huitres de
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1937. On the oxidation of organic matter in marine
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1958. The manner in which the sponge Cliona bores
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FACTORS .\FFECTrSG OYSTER POPUL.\TIOXS

455

Wells, Harry W.

1959. Notes on Odostomia impressa {Sa,y) . Nautilus,
vol. 72, No. 4, pp. 140-144.
Wells, Harry W., and Mary Jane Wells.

1961. Three species of Odoslomia from North Caro-
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1928. The larvae of Polydora ciliata Johnston and
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1957. The flatworm Pseudostylochus osireophagus
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1956, pp. 62-67.

1961. Pacific oyster Crassoslrea gigas mortalities with
notes on common oyster predators in Washington
waters. Proceedings of the National Shellfisheries
Association, vol. 50. August 1959, pp. 53-66.

Wood, John L., and Jay D. Andrews.

1962. Haplosporidium costale (Sporozoa) associated
with a disease of Virginia oysters. Science, vol.
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Yonge, C. M.

1955. Adaptation to rock boring in Botula and Lith-
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456

FISH AND WILDLIFE SERVICE

SUBJECT AND AUTHOR INDEX

Page

ABO (accessory boring organ) 430

absorpt ion t hrough the gills and mantle 23 1

accessory heart 76, 258, 259

connections with pallial artery 259

function of 259

injection of 259

pulsation 259

acetylcholine, content in heart 252

effect on ciliary motion 141

effect on heart 252

sensitivity of bivalve heart 252

actin, in adductor muscle 164

action currents in heart 244

action potentials in heart 250

Acroloius lacuslris, adhesive epithelium 160

acrosomal reaction 341

actomyosin, in adductor muscle 157

adductor muscle 70, 152

anatomy. . 152

attachment of 160

bundles parallel to valve surface 159, 160

chemical composition 161

duration of contraction 165

electrical activity 166

gross analysis 162

innervation in Crassostrea and Mytilus 166

inorganic salts 162

in larva 359,368

microscopic structure 153

power of 175, 180

proteins 164

relative weight 152

role in water transport 185

speed of contraction 165

stretching of 180

tonus 165, 166

translucent part 153, 154

white part 154

adhesive (holding) epithelium, of adductor muscle. 160

ADP (Adenosinediphosphate) 1 160

adrenalin, effect on cilia 168

adrenalin, effect on heart 140

afferent vein, structure 253

agirg of eggs and sperm 254, 257

agglutination of sperm 338, 340

Agostini, A., perforating algae 426

Alectryonia 6

alfalfa, as food 235

algae, on oyster shells 428

perforating 426

alimentary tract, length 227

alizarin, as calcium stain 101

alkaline phosphatase, in mantle 88

Page

Allen, W. R., labial palps in Anodonta 114

rotation of crystalline style 225

Allison, J. B., diantlin in sperm 312

Altamaha Sound, Ga., oyster reef 403

Alvsaker, E., chemical composition 383

glycogen 387

fat content in O. ediilis 387

proteins 391

Amberson, W. R., respiratory quotient 212

ambisexual oysters 324

ambisexual primary gonad 314

Amemiya, I., hermaphroditism in C. gigas 316

sex change 316

amino acids, in conchiolin 41

in excreta 277

in marine and freshwater species 391

in Pinclada shell 41

in Pinna shell 41

Amirthalingham, C 32

ammonia, in kidney excreta 276

ammonia carmine, concentrated in pericardial

glands , 274

Amoebocytes 262

Atnphilrile ornata 427

amylase 229

pH optimum 230

Anadara (^rca), antisera 266

antigens 142

anal sphincter 226

analyses, for heavy metal 385

anatomical peculiarities of oysters 72

anatomy 65

methods 65, 66

Andrews, E. A., estuary, definition 400

FoUiculinids 428

Andrews, J. D., effect of low salinity 405

Haploaporidium 417

anions, effect on ciliary motion 139, 140

annelids, on oyster beds 428

Anodonta cygnea, amino acids 391

calcification 95

oxygen debt 214

Anodonta sp., antiserum of gill epithelium 141

blood filtration with pericardium method — 275

cessation of ciliary motion 146

crystalline style 225

effect of drugs on cilia 140

heart beat 249

heart physiology 242

innervation of adductor muscle 166

labial palps 113

oxygen utilization 214

SUBJECT AND AUTHOR INDEX

457

Page
Anodonia sp. — Continued

pacemaker system of heart 246

subligamental ridge 89

ventilation of the gills 214

Antheunisse, L. J 312

Anthony, R 2

striated muscles 154

anthrone, determination of carbohydrates in sea

water 198

antibacterial agents in mollusks 393

antibiotic properties of Chlorella 409

antigens, effect on ciliary motion 141, 142

antigens, in C. gigas and C. circumpicta, common

and specific 266

antigens, in Crassostrea 266

antigill serum 142

antimuscle serum 142

antiviral agents in mollusks 393

Anton, H.E 2,3

anus 71, 226

Apalachicola Bay, destruction of oysters by

Stylochus frontalis 438

mean monthly temperature 407

apical sense organ 359

arabinose, effect on water transport 199, 200

in sea water 199

stimulating effect on tentacles 294

aragonite 4, 37, 104

affected by strontium and magnesium 103

increased content with temperature 103

mineralogical identification 103

Arbacia, fertilizin 340

Areachon oysters, iodine content of 383

Arcisz, W., retention of coliform bacteria 233

arginine phosphate 167

Arrhenius equation 248

arterial system 253, 254

arteries 84

artery, circumpallial 72

ascorbic acid, effect on water transport 199

ascorl^ic (dehydro-) acid, in sea water 198

ash content 382

Astarlidae 29

Asterias extract, effect on mantle 294

Aslerias forbesi 437

salinity barrier 438

Aslropeden irradians, closing of valves 165

Atkins, D., gill's cila 128-129

ciliary currents 142-143

ATP, adenosinetriphosphate, role in muscular

contraction 167-168

atropine, effect on ciliary motion 141

auricles 240-241

contraction of 244, 250

auriculo-ventricular valves 241

auxocyte 325, 326

Awati, P. B 65

injection of digestive system 219

labial palps 111

nervous system of 0. cucullata 281

pallial organ 289, 291

axes of growth, changes in 27

458

Page

axoneme, in cilia 132

Babak, E 137

Babor, J. F., nervous system 281

bacteria, as food 233

food of larvae 376

retention by gills 233

Bacterium coli, synonym for Escherichia coli 442

Baird, R. H., condition index 392

Bandmann, contraction of muscles in Pinna 159

Bang, F. B., phagocytosis 264

Ballentine, R., respiration of egg 344

Banner, A. H., Thais lamellosa 434

Bargeton, M 82

glycogen 386

barnacles 427

barnacle larvae, transport of 403

Barnes, G. E., adductor of Anodonta 166

Barnes, textbook on physiology 24 8

Barrnett, R. J., cytochemistry 344

Barrois, T., chemical composition of crystalline

style 225

basal body, of cilium 129, 132

role in cilia 132

basal arrangement in ciliated cell 133

Battle, H. I., gastric shield 223

stomach histology 222

Bayliss, L. E., adductor of Pecten 165

Bayliss, W. M., catch mechanism 165

beak 16

Heaven, G. F., milfoil 430

bed layer of sediment 411

Belehradek, J., temperature coefficient 249

temperature, effect of 249

Benson, A. A., inhibition of contraction 166

Benzer, P., calcification 97

Berg, W. E., lytic effect of sperm 341

Bergmann, W., cholesterol in oysters 392

Berkley, C., chemical composition of crystalline

style 225

crystalline style as a reserve of oxygen 229

Bernard, F., larval shell 364

Berthe, C, heart -- 245

Bethel, F. M., effect of drugs on cilia 140

Bevelander, G., calcification 89,97

Bhatia, D 137

Biederman, W 48

Calcium phosphate in mantle 97

Bierrj', H., glycogen- . 386

bioassays 442

biocoenose 397

Birckner, V., zinc determination 385

birds, destructive to oysters 439

bisexual oysters 5

bisexual potency, of gonad 316

Blum, H. F., protein in blood plasma 391

blastopore 355

Blegvad, H., detritus as food 233

blisters, in 0. iridescens 36

on shells 105

blood 259

circulation 257-258

FISH AND WILDLIFE SERVICE

Page

granular cells 262

granulocytes 259

hyaline cells 259,261

specific gravity 265

blood cells, agglutination 262

amoeboid movement 260,262

coalescence 262

blood cells, filamentous pseudopod under electron

microscope 264

enzymes 264

granules 263

heavy metals in cells. _ 264

survival in vitro 260

blood clots, in connective tissues 262

blood plasma, ionic concentration 278

protein content 278

blood vessels 253, 258

histology 84

injection of 253

of mantle 70, 84

blue crab 439

Blue Point oyster 17

Bochenek, M. A., glia cells 285

body fluid, ionic concentration in 278

B0ggild, C. B 37

conohiolin secretion 93

identification of aragonite 103

Bojanus organ, kidney 70, 273

Bonnot, P., setting 372

boring clam 421

boring sponges 420

Borisiak, A., prodissoconchs 364

Bornet, E., perforating algae 426

Borradaile, L. A 154

Boswell, J. L., Dermocystidium 416

Bosworth, M. W., respiratory quotients 212

Botryllus schlosseri 427

bottom, character of muddy 399

Bouck, G. B., Codium in Long Island 429

Bousfield, E. L., transport of larvae 403

Boutan, L., pearl formation 95

Bowden, J., striation in muscle fiber 157

Bowerbank, J. S 48

Bowerbankia imhricala 427

Boyland, R., adductor of Pedere 165

brachiolaria 437

brackish water, classification of 404

Bradley, H. C, zinc in tissues J" 390

branchial nerve 72

microscopic structure 289

Brandes, R., chemical analysis of shell 39

Breder, C. M., blood cells 260

Brezina, M., dropping mercury electrode. _ 214

Brooks, W. K 65

Brown, F. A., Jr., cycles of shell movement 180

Bruce, .J. R., food of larvae 374

Briick, A., muscle attachment in Anodonta 160

Briicke, E. T 136

Bryozoa, encrusting shells 427

Bucephalus haimeanus 419

SUBJECT AND AUTHOR INDEX

Page

Buchholz, chemical analysis of shell 39

Bumpus, D. F., organic matter in phytoplankton.. 408

Bureau of Mines, spectrographic analysis of meat.. 383

Burkenroad, M. D., hermaphroditism. 314

Thais haemastoma 433

burrowing shrimp 439

Busycon, manner of opening bivalve shells 435, 436

number of oysters destroyed per week .. 436

Busycon carica 435

Busycon canaliculatum 435

Butler, P. A., inhibition of reproduction by low-
salinity 406

mortality of oysters, Mississippi Sound.. .. 405

setting 372

water transport and carbohydrates 200

butyrase, in stomach 230

Buzzards Bay, oyster bottoms destroyed by dredg-
ing 398

byssus gland 359

calcification 93-98

difference between right and left valve . _ __ 104

of shell 93

rate of 104

seasonal variation 105

theories 94

calcite 103

mineralogical identification 103

calcite and gypsum crystals 102-103

calcite ostracum, ultrastructure 4,37

calcium 45, use in calcification studies 103

calcium, cytological identification 101

calcium-secreting cells 87

calcium, sources of 103

factors in shell formation 98

use of heavy metal for identification 101

calcium carbonate, minerology 103

use in water filtration studies 193

calcium phosphate, in mantle 97

in new shell 97

calcospherites 93

Calderwood, H. N., glycogen 386

method of glycogen determination 386

moisture determination by xylene distillation. 385

pulp mill pollution 443

York River plankton 409

Callianassa sp 439

Callianassidae, destructive to oyster dikes 439

Callinectes sapidus 439

Cameron, W. M., definition of an estuary 400

Camien, M. N., amino acids 391

Cancer irroratus 439

cane sugar, stimulating effect on tentacles 294

carbohydrates 381-382

determination in salt water 198-199

in sea water 197

in Woods Hole sea water 199

no effect on water transport 199-200

carbon dioxide, fixation during shell formation. 95-96, 99

459

Page

carbonic anhydrase 103

role in calcification 103

Carcinus moenas 439

Carcinus, used as food for larvae 374

Cardium, chemical composition of crystalline style. 225

heart beat 250

labial palps 114

valves 24-25

Cardium corbis 48

Carlson, A. J., heart physiology 242

heart regulation 250

carmine cone method 144

carmineathrocy tes 279

Carriker, M. R., clam larvae 358

conchs 433

distribution of larvae 369

drUls 430

manner of opening shell by Busycon 435

Ocenebra 435

transport of larvae in estuaries 402

Carter, G. S 137

Carteria, food of larvae 374

catch mechanism 165, 166

crystallization hypothesis 167

cells, secreting calcium 87-89

cell lineage 345

in Crassostrea gigas 348

cellulase, absence in oysters 230

cement film, in adductor muscle attachment 161

Ceramiii ni 428

cerebral ganglia, excision provokes spawning 312

microscopic structure 288

cerebro-visceral connectives 285

miscroscopic structure 288

Cerruti, A., fecundity of 0. edulis 313

cessation of ciliary motion 146

Chaetoceras sp 409

chalky deposits 32,37

importance 35

Korringa's theory 34

location of 32, 34, 35

Chama 29

chambering 35

chambers, in shell of C. virginica 36

Champia 428

Chapman, W. Mc, Thais lamellosa 434

chemical composition 381-382

adductor muscle 161-164

mud shells 43

0. edulis 383

proximate 381

seasonal and local variation 381-382

shell of C. virginica 43

shell of 0. edulis 44

Chestnut, A. F., proteolytic action in stomach 231

Child, C. M., cell lineage 346

Chipman, W. A 174

glycogen 386

method of recording water transport 188

oil pollution 443

460

Page

pulp mill pollution 443

radioactive waste 445

removal of plankton cells by the gills 194

water propulsion by scallop 195

York River plankton 409

zinc absorption 390

chitin, color reaction of Zander 222

Chlainydomonas, food of larvae 374

removal by gills 194

Chlamys opercularis, closing of valves 165

chloral hydrate, effect on cilia 141

Chtoretla sp 374, 409

Chlorella stigmatophora, as food of larvae 375

cholesterol, in oysters 392

Chondria 428

Christensen, A. M., oyster crab 425

starfish food used for bait 438

chromosomes, diploid number 345

number of 327

ChromuUna pleiades, as food of larvae 375

chronotropic effect of potassium 251

Church, A. H., Colpomemia 429

Churchill, E. P., Jr 65

ciliation of labial palps 115

cilia, frontal 129

lateral 129

laterofrontal 128

rate of work 145, 146

structure of 132

ultrastructure 132, 133

ciliary beat, after spawning 138

control by temperature 137, 138

frequency 137

variations of 138

ciliary currents, of mantle 90, 91

on gill's surface 142

ciliary motion, crawling method of measuring 142

effect of chemicals 139-141

in genital ducts 300

nervous control of 136, 137

spontaneous cessation 106, 107

ciliary tracts 143

on gill's surface 128

ciliated cell, ultrastructure 134

ciliated epithelium , of labial palps 111

cilium, effective stroke 135

mechanical properties 135

recovery stroke 135

Ciona intestinalis, oxygen uptake 211

circulation 239

circulation in estuary 401

circumpallial artery 76

circum pallial nerve 285

connections 85

microscopic structure 288, 289

citrate, role in calcification 96

clay, nephelenic gray, use in water filtration studies. 193

cleavage 344-346

spiral 347

Cleland, K. W., gametogenesis 325

ovogenesis in C. commercialis 326

respiration of egg 344

FISH AND WILDLIFE SERVICE

Page

Cliona, manner of boring 420

pathological effect of 421

cloaca 71, 123

closing during female spawning 306

Cobb, P. H., labial palps of Anodonta 119

Codium fragile, introduced to New England 429

Coe, W. R., alternation of sex 314-316

gametogenesis 325

gonad development 315

gonad histology 300

number of chromosomes 327

primord'al gonad 324

sex classes at first breeding season 315,316

sex ratio 324

spermatogenesis 326

spermatocytes of 0. lurida 327

synapsis in spermatogonia 327

coelom 271

Cole, H. A., fecundity of 0. edulis 313

gregariousness 373

metamorphosis 366-368

metamorphosis of 0. edulis 355

pallial organ of spat 292

rearing of larvae 369, 374

setting 372

Urosalpinx fertility 432

collagen, in ligament 57

tanning of 58

collagenase, effect on adductor attachment 160

Collier, A., effect of carbohydrates on gill activity. 197-199

Collier, J. R., acrosomal reaction 341

colloidal carbon, for study of ciliary currents 90

colloidal graphite 115

Colorado River, sedimentation at mouth 412

Colpomenia sinuosa (oyster thief) 429

Columbia Southern Corporation, analysis of shell.. 43

Colwin, A., acrosomal reaction 341

Colwin, L. H., acrosomal reaction 341

commensals of oysters 420, 430

commissure, cerebral 72, 284

competitors, effect on productivity 430

competitors, of oysters 420, 430

conchoid curve 23

conchiolin 37, 39

amino acids content 41

chemical composition 39

in ligament 57

in prismatic layer 39, 40

in C. angulata a,nd 0. edulis T 41

percentage in shell 40

role in calcification 40

staining properties 39

test for chitin 39

ultrastructure 39

conchiolin gland 86, 88

conchiolin secreting cells 86

condition index 392

in C. gigas 393

in 0. lurida 393

method of determination 392

significance of 392

SUBJECT AND AUTHOR INDEX

Page

Conklin, E.G., cell Imeage 346

connective, cerebro- visceral 285

microscopic structure 288

connective tissue, of the mantle 79

constant level tank, for measurmg water transport. 186

Conway, experiments in rearing larvae 374

copepods, parasites in bivalves 420

copper 387

accumulation in tissues 384

cause of green color 388

content artificially increased 388

determination, Biazzo method 385

histological localization 388

copperas, use in feeding oysters 388

corn starch, as food 235

Corpus Christi Bay, Tex., sedimentation 412

Costello, D. P., cleavage 347

Coulson, E. J., iodine in tissues 383

heavy metals in tissues 384

Coupin, H., labial palps 114

crab meat, ground, as fertilizer 235

crabs, oyster enemies 439

Crassostrea sp., acrosomal reaction 341

generic definition 7

ostia 127

promyal chamber 123

salinity tolerance 5

striation in muscle fibers 154

Crassostrea angulata, adductor muscle 154, 155

ciliary tracts 143

stomach histology 222

torch bearing cells near gastric shield 222

Crassostrea circumpicta, antigens 266

Crassostrea commercialis, diagnosis of species 8

gametogenesis 325

glycolysis in muscle extracts 168

inorganic salts in adductor muscle 162

male spawning 310

ovogenesis 326

Crassostrea gigas, antigens 266

chemical composition of crystalline style 226

diagnosis of species 7

hermaphroditism 314

male spawning 310, 311

setting 372

spawning 309

Crassostrea rhizophorae, diagnosis of species 7

Crassostrea rivularis 28

diagnosis of species 8

Crassostrea Sacco 3

Crassostrea virginica, cessation of ciliary motion. 146, 147

ciliary tracts 143

diagnosis of species 7

effect of adrenalin on cilia 140, 141

effect of atropine on ciliary motion 141

gametogenes's 325

salinity range 403

setting in jars 373

sorting of food by labial palps 114

Creac'h, P. V., phosphorous in shells of 0. edulis.. 44

creatine phosphate 167

Creighton, C 82

461

Page

Crepidula, sp 426

Crepidula fornicata, introduction to Europe 426

critical temperature of spawning 309

Crofts, D. R., calcification theory 94

cross striated muscles 154

crossing over, in ovocj'tc of C. commercialis 326

Crozier, W. J 30, 248

Crijptomonas, affected by style enzyme 231

crystalline style 71, 223-225

appearance 224

chemical composition 225

dissolution 225

formation 225

in larva 359

pH of 230

role in feeding 225

rotat ion 225

topography 223-224

crystaUine style sac 219-220

secretion 225

ctenidia 121

Cuenot, L., excretion 274, 279

cumarin, detected by tentacles 294

curare, effect on heart 253

currents, nontidal residual 402, 403

currents, turbulent effect of 403

current indicators . 190, 192

Cyclas (Sphaerium), cessation of ciliary motion 146

cycles of shell movement, daily 180

Dahmen, P., nervous system of 0. chilensis 281

pallial organ 291

stomach histology 222

Dakin, W. J., calcification theory 94

mantle muscles 84

Dall, W. H '_ 2,48

Damariscotta River 21

shell heap 440

Dan, J. C, acrosomal reaction 341

Danielli, J. F., cytochemistry 344

Daniels, F., dropping mercury electrodes 214

Dantan, J. L., fecundity of 0. edulis 313

larval development of 0. edulis 355

Dasijbatis dipterus, synonym Atnphovistius 439

Davaine, C, metamorphosis of larva 366

Davis, H. C, food of larvae 375

reactions of larvae 371

rearing of larvae in laboratory 375

Dean, B., oyster bottoms 398

Dean, D., enzyme in style 231

pH of crystalline style 231

Deane, H . W., cytochemistry 344

DeBocr, S., heart 243

pacemaker in Mytilus heart 246

decompression, effect on ciliary beat 142

Delauney, H., excretion 274

uric acid in Mya and C. angulata 277

demibranchs 121

movements of 131

Dendoslrea 6

Denison, J. G., Hcxamila 419

Dennell, R 58

462

Page
Dermocystidium, cause of mortality 415

effect on oysters 4ig

growth in tissue culture 415

occurrence in various bivalves 416

Dermocystidium marinum, diagnosis of species 416

DeRobertis, E. D. P 133^ 1(34

destruction of oyster bottoms by man 440-441

detritus, as food 232

development rate 349-350

De Waele, calcification theory 95

dextrose, effect on R. Q 213

effect on water transport 199

diamond lattice pattern, in dark muscle fibers 154

in muscle fibrillae 157

diantlin 312

diapedesis 83, 185, 239

diastole 244

diatoms, as food 231, 232

on oyster shells 428

diatom growth, stimulated by pulp mill pollution.. 444

Dicrateria, sp., as food of larvae 375

Diederichs, W., nervous control of heart beat 250

digestion 219, 228-23 1

extracellular 231

digestive diverticula 71, 226

ducts 226

histology 226

pH of extracts 231

digestive system 219

digitalin, effect on ciliary motion 141

dimensions of shell 20

Dimitroff, V. T., spirochaetes 426

Dimick, R. E., Yakina Bay oyster beds 402

Diplolhyra, in Texas 421

Diplolhyra smithii, synonym Mariesia 421

direction of growth 25

change in 27-29

direction plane 25

in Pecten 24

disease 415

Divaris, G. A., heart physiology 246

heat beat 251

Dobson, G. C, capacity of reservoirs 412

Dollfus, R. P., foot disease 418

Dosinia discus 30

Dotterweieh, H., calcification 96

Douville, H 39

Dow Chemical Company, analysis of shell 44

DPN (diphosphopyridinc nucleotide) in muscle 168

dredging, effect on oyster bottom 398

Dreissensia (Dreisensia) polymorpha, develop-
ment 346, 368

neurosecretion 312

Drew, G. H., blood cells of bivalves 262

drills, destructiveness 430

dispersal of 430

effect of low salinity 430

Drinnan, R. E., Malpeque Bay disease 4' 5

drop counter, electric 190

drop counting technique 197

drugs, effect on ciliary motion. 140

effect on heart 251, 252

FISH AND WILDLIFE SERVICE

Page

drum fish, destructive to oysters..- 439

dry weight 382-383

Duchateau, G., amino acids.. 391

structure of nacre 40

ducks, destructive to 0. lurida 439

duck farms, pollution by 444

dumping vessel, use in recording water transport.. 188

Duvernoy, M., nervous system 281

Eble, A. F., injection of blood vessels 253

Edmundson, C. H., crystalline style 225

eel grass 429

See: Zostera

Egami, N., transplantation of gonad 317

egg, cytochrome oxidase 344

fertilized 342

mature 327

metabolic gradient 344

polarity of 344

respiration rate 344

egg water 338

Eiger, M., electrocardiogram 244

Einstein, H . A., sedimentation 411

elastic fibrils, in connective tissue 79

electric shock, effect on ciliary motion 147

electrocardiogram 244

electron microscope, in study of cilia 132

Eliassen, E., respiration in 0. edulis 213

Elliptio complanatus, cilia of 133

Elsey, C. R., accessory heart 258

pallial organ 291

promyal chamber 123, 125

spawning induced in oyster bottom 320

tentacles 79

Elssner, E., calcification 96

Elvehjem, C. A., copper determination 385

Emeljanenko, P., excretion of dyes 274

endomysium 153

Engle, J. B., effect of P. websteri on oysters 424

glycogen 386

influence of environment on gonad 299

inhibition of feeding by microorganisms 409

microorganisms undigested by oyster 234

pulp mill pollution 443

York River plankton 409

Engstrom, A 164

Ensis sp., crystalline style 224

Ensis ensix, striation in muscle fibers 157

Enteromorpha . 428

enzymes, in blood cells 264

in stomach 229

proteolytic 230

sucroclastic 230

eosinophilic cells, on labial palps 111

epibranchial chamber 70-71, 122

epithelium, holding (adhesive) in muscle attach-
ment 160

Epstein, S., effect of temperature on aragonite

formation 104

aragonite in shells 104

Erdmann, W., larval development 0. edulis 355

veliger, O. edulis 356

SUBJECT AND AUTHOR INDEX

Page

erosion, of bottom 412

Escherichia coli 442

retention by gills 233

retention by oyster body 190

eserine, effect on ciliary motion 141

effect on heart 252

esophagus 71, 219

Esser, W., innervation of heart 242

pacemaker system of heart 246

estuary, definition 400

distribution of pollutant 401-402

"inner end" 401

nontidal currents 402

silting of 411

structure of 400

types of 402

EulamelUbranchia 121

Eupleura caudala 432

reproduction in York River, Va 432

evaluation of factors, method 445

Evans, C. L 164

Evasierias troschelii 438

excretion of dyes 274

excretory cells 273

excretory system 271

anatomical terminology 273

anatomy 271, 273

histology 273

physiology 274

topography 272

extracardial regulation 250

eye, larval 360, 361

eyespot, in 0. edulis 368

Eysseric-Lafon, M., amino acids in conchiolin 41, 103

factors, negative 399, 409, 446

factors, positive 399

factors of environment, combined effect of 445

Fairbanks . L. D . , excretory system 271

loss of fluids 385

osmoregulation in C. virginica 278

falling drop method, for specific gravity determina-
tion of blood 265

Fasten, N., Yakina River oysters 402

fat, content 387

hydrolysis 230

in vesicular cells 81-82

fattening, due to Nitzschiella 233

fatigue, of muscle 167

Faussek, V., pigmentation 279

Fawcett, D. W 133

fecal ribbon, rate of formation 228

feces 228

fecundity, larviparous oysters 313

oviparous oysters 313

Feder, H. M., starfish opening its prey 438

Federighi, H., heart 248

heart beat 247

feeding, artificial 234

adverse effect of heavy concentration of

microorganisms 234-235

inhibition by Chlorella 234-235

463

feeding, artifical — Continued Page
Inhibition by high concentration of micro-
organisms 409

inhibition by low salinity 405

Feltham, C. B., bacteria as food 233

femaleness 315

female spawning, specificity 309

fences, to ward off sting rays 439

fertilization 338

fertilization membrane 343

fertilizin 340

properties of 341

fibers, white muscle 154

fibrils, arrangement of in white muscles 154

filament, of acrosome 341

filament, of the gills 126

Filibranchia 121

filtration rate, into pericardium 274

Finean, J. B 164

Fingerraan, M., excretion system 271

loss of fluids 385

osmoregulation in C. virginica 278

Fish, P. A., storage of fat in birds and mammals,. 230

filtrose 374

flagellates, as food of larvae 375

culture, as food for larvae 375

naked, as food 233

Flahault, C, perforating algae 426

flatworms 438

destructiveness 438-439

flocculation, of silt 411

flood, Mobile Bay 405

Florliin, M 40,54

amino acids 391

excreta 276

protein in blood plasma 391

fluids, in tissues 385

loss after shucking 385

flushing, of estuary 401, 406

Fol, H., mantle muscles 83

folding, of muscle fibers 158-159

follicle cells 300

Folliculina, on oyster shells 428

foUiculinids, in Chesapeake Bay 428

food 231

availability 408

as factor of environment 408, 409

of O. edulis 232

selection 233

volumetric determination of gut and stomach

content 232

sorting by labial palps 115

food factor, evaluation of 408

foot, larval 361

reabsorption of 368

foot disease, in C. virginica 418

symptoms 418

synonym of shell disease 417

Fosse, R., excretion 274

fouling 426

fouling of shells, Oyster River, Mass 428

Fox, D. L., water propulsion by Mytilus califor-

nianus 193

464

Page

F0yn, E., dropping mercury electrodes 214

Franc, A., excretion 274

excretory system 271

heart 240

pallial organ 291

freezing, effect on oyster community 399

Freidenfelt, T., nervous system 281

glia cells 285

Fremy, E 39

Frey, D. C, oyster bottoms, Potomac River 398

Friza, F sg

fructose, effect on water transport 199

Fujita, T 347

cell lineage 345

larval development, C. gigas 355

fumaric acid, in calcification 99

fungi, effect on oyster larvae 376

Gaarder, T., chemical composition 383

fat content in O. ed!(h's 387

glycogen 387

proteins in O. edulis 391

rearing of larvae 374

respiration in O. edulis 213

setting 369

Gabe, M., neurosecretory cells 293

Gage, S. H., fat storage in birds and mammals 230

Galileo theorem, applied to growth 27

Galli-Valerio, B., antiserum effect on ciliary motion. 141

Galtsoff, P. S 65

carmine cone method 144

constant level tank 186

duration of shell opening 174

effect of pH on oyster cilia 140

effect of temperature on shell movement 174

evaluation of environmental factors 445

flood in Mobile Bay 405

glycogen 386

method of recording water transport 188

Nematopsis distribution 419

Ocenebra, introduction from Japan 435

O. equestris, salinity range 404

oil pollution 443

passage of eggs through gills 304

pollution of Olympia oyster beds 402

pulp mill pollution 443

red tide 409

release of sex cells 303

respiration 201

respiration affected by oxygen tension 214

retention of coliform bacteria 233

setting 369

sex change in adults 316

shell movement, effect of daylight 175

source of calcium 103

spawning 304

spawning of C. gigas and 0. cucullata 306

starfish 437

Texas oyster bottoms 412

water wheel 190

York River plankton 409

Galtsoff, P. S. and A. S. Merrill 2

FISH AND WILDLIFE SERVICE

Page

gametogenesis 324

gamones 338

ganglia, cerebral 72, 284

pedal in larva 360

ganglion cells, in myocardium 242

ganglion, visceral in Ostreidae 71, 281, 283-284, 360

microscopic structure 285-286

Garner, R., nervous system 281

Garstang, W., larval adaptation 363

Gartkiewicz, S. cessation of ciliary motion 146

heart 250

gastric shield 221-222

function 223

resistance to potassium hydroxide 222

gastropods, carnivorous 430, 436

gastrulation 347

Gavard, D., artificial detritus as food 234

gelatin, injection with 219

genera, of living oysters 6

generative curve 24-25

genital canals 300

George, W. C, fat digestion 230

geotaxis, in larvae 372

germ cells, primordial 299, 325

germinal epithelium 300

Giard, A., foot disease 418

Gilchrist, J., nervous system 281

gill axis 121

gill cavity 66

gill filaments 125-127

gill lamellae 121, 125

gills 70, 121

anatomy 121

blood vessels of 71

ciliary tracts 128

currents 123

degenerate in Septibranchia 121

degrees of complexity 121

efficiency in plankton removal 194

function of 72, 185

histology 126-127

interfilamentar junction 125

interlamellar septa 125

muscles 125, 130

muscles used in water transport 185

plica 125

rods 125-126

ventilation of, steady state 195

zinc content 390

Glaister, D., muscle contraction 167

glia cells 285

Glick, D., localization of copper 388

glucose, absorption 231

glycine, in conchiolin 41

glycogen and solids 386

glycogen, in adductor muscle 162

annual cycles 386-387

determination of 385

disappearance of 82

in connective tissue 81-83

in Louisiana oysters 386

SUBJECT AND AUTHOR INDEX
733-851 O— 64 31

Page

in muscular contraction i67

in 0. edulis g2

in Virginia oysters sgg

reagent for 81,385

seasonal changes 357

utilization of 81-82

variations in content 386

glycogen cycle, in 0. edulis and C. migulata 386

glycolysis, in muscle xgg

Gmelin, J. F \

goblet cells 84

Goldberg, E. D. filtration rate and oxygen con-
sumption 408

rate of removal of graphite 194

Gomontia polyrrhiza 426

Gomori method, for localization of alkaline phos-
phate 88

gonads 71

gonad, ambisexual (bisexual) 324

development 315

follicles 300

hermaphroditic 326

histology 299

indifferent stage 302

phases 315

primary 314

primary, transformation of 315

primordial 324

reabsorption 302

thickness. 297

transplantation 317

volume 299

weight 299

gonad development, efifect of environment 298

gonad development seasonal variation 298

suppression by pollution 299

gonia 300

gonoduct 303

Goodalia 29

Gorgy, S., organic matter in phytoplankton 408

GotWin, G. F 137

Gouzon B., glycogen 386

Gracillaria confervoides 428

Graham, A., oxydase in oyster 230

Grant, D. C " 60

granulocytes 259, 262

Grave, C., condition index 392

nervelike apparatus of gills 137

rootlets of ciha 134

Gray, J. E 2, 32

ciliary motion 139

transverse elasticity of cilia 135

Great South Bay, Long Island, N.Y 235

Chlordla bloom 409

green color associated with industrial pollution 384

green oysters 384

green pigment, isolation of 388

Greenberg, M. J., acetylcholine action on heart 252

gregariousness of larvae 373

Gregoire, C 39, 40, 54

Griffitsia 428

465

Page

Grobben, C, excretion of dyes 274

ground substance, connective tissue 79

growth, changes in axes 27

of shell 26

inversion 29

radii 26

rings 26

Gruntzner's model of catch mechanism 165

Gryphaea angulata 2

Gryphaea arcuata 2

Gryphaea, ruling of International Commission 3

Gryphaea vs. Crassostrea 3

Gudger, E. W., attacks of starfish 438

Gunter G 3,6

sedimentation 412

Gutheil, F., crystalline style 225

gastric shield formation 222

gutters, between labial palps 111

Gymnodinium breve, cause of mortality 409

Haas, F 48

excretory system 273

pallial organ 291

habitat 5

Hagmeier, A., oyster bottoms 398

Hagstrom, B. E., jelly coat removal from egg 340

Hall, C. E., paramyosin in oyster 164

Hamburger, V., embryology textbook 347

Hammen, C. S., calcification 99

Hanson, J., helical striation in muscle fibers 155, 157

muscular tension 167

Haplosporidia 417

Haplosporidium costalc 417

Harrison, C. W 43

heavy metals in oysters 384

Hartman, O., annelids on oyster shells 422, 428

Harvey, H. W., pigment units 409

Hatanaka, M., rearing of larvae 375

Haven, D., effect of low saUnity 405

Hayashi, T., hypothesis of catch mechanism 167

Hays, J. T., inhibition in adductor of Pecien 1C6

Hazelhoff, E. H., utilization of oxygen 214

heart _ _ 70, 240-241

accessory 258

automatism 242, 244-245

effect of adrenaline 253

effect of curare 253

effect of drugs 251

effect of hydrostatic pressure 244

fatigue 247

ganglion cells 241

innervation 241

larval 361

nerve fibers 242

peacemaker system 245

physiology 242

rate of beating in larva 361

stimulating nerves 248

use in bioassays 252

heart beat 242

effect of mineral salts 251

effect of pH 251

466

Page

excised 250

frequency 249

i nhibition 249

methods of study 247-248

recorded in situ _ 247

temperature characteristics 248

heart preparation in bioassays 443

heavy metals, analytical procedures 384-385

geographical variation 354

in oyster meat 383-384

locahzation in different organs 390

in New England oysters 334

seasonal differences 390

Hecht, S., sensory stimulation in Mya and Pholas. 293-294

Hedgpeth, J. \V., oyster bottoms 393

sahnity and temperature change 405

Heider, K., sterroblastula 347

Helix, presence of cellulasc 230

Helix pomatia, calcification of epiphragm 95

Hemming, F 2

hermaphrodites 314

hermaphroditism 318

frequency in C virginica 314

Hers, M. J., oxygen debt 214

heteroagglutination of oyster sperm 340

Hexamila 419

Hirata, A. A., calcification 97, 103

Hoagland, H., textbook 248

Hoek, P. P. C., discovery of hermaphroditism in

oysters _ 314

food of oysters 231

genital canals 300

Hopkins, A. E 77

accessory heart 258

adductor muscle 154

cloacal current 405

condition index 392

current indicator 190-193

duration of shell opening 174

fecundity of O lurida 313

function of eye , 371

pollution of Olympia oyster beds 401

sensory stimulation 293-294

shell movement, effect of temperature 175

tentacles 79

Hopkins, H. S 153

oxygen consumption by tissues 200

Hopkins, J. C., water filtration by scallop 194-195

Hopkins, S. H., glycogen in Louisiana oysters 386

oyster crab 425

Polydora larvae 423

Horst, R., larval development, O edulis 355

Hotchkiss, M., decomposition of sediments 413

Hotelliiig's formula, concentration of pollutant in

estuary 401

Houet, R., excreta 276

Hubendick, B., adhesive epithelium 160

Humphrey, G. F., inorganic salts in adductor

muscle 162

chemical changes in oyster adductor 168

Hunter, A. C 43

heavy metals in oysters 384

FISH AND WILDLIFE SERVICE

Page

Hunter, W. R 60

Hutchiiis, L. W., Bryozoa 427

Huxley, J. S 26

Huxley, H. E., sliding theory of muscle contraction . 167

Huxley, T. H., larval development, O edulis 355

metamorphosis ■'OO

hyaline cells, of blood 261

Hydroides hexagonus, acrosomal reaction 341

hydrographic climate 405

Hyella caespitosa 426

Hyman, L. H., flatworms 438

hypostracum 4, 37

Ikemoto, N., cross striated fiber 154

Imai, T., rearing of larvae 375

productivity reduced by feces 413

immunological reactions, method 265

incubatory species 4

index of rejected names 3

India ink, excretion by phagocytes 279

Indians, use of oysters by 440

indifferent (residual) cells 325

indigoathrocy tes 274

indigo-carmin, excreted by kidney 274

industrial waste, number of outlets 443

IngersoU, E 21

Ingram, W. M., pollution bibliography 442

inhibitory axons in Pecten 166

injection, for anatomical studies 66

inorganic constituents of meat 383-384

inorganic salts, in adductor muscle 162

insemination 343

International Commission on Zoological Nomen-
clature 3

internephridial passage 271

intersexuality 324

intestine,. "^^

intracellular fibrillae of gills, existence doubtful... 137

invagination 355

inversion of growth 29

iodine 383

iodine content, artificially increased 383

in 0 hirida 383

iodoacetate, inhibitor of lactic acid formation 167

ions, monovalent, effect on ciliary motion 139

ionic concentration in blood plasma 278

ionic regulation 278

Irisawa, H., heart beat 251

iron content, artificially increased , 388

iron determination, Kennedy's method 385

iron, excretion by epithelial cells 390

histological localization 388

ingestion by leucocytes 388

seasonal changes 387-388

iron saccharatc, use in digestion experiments 228

Isochrysis. not affected by style enzyme 231

Isoehrysis gnlhana, as food of oyster larvae 375

Ito, S , productivity reduced by feces 413

Jackson, R. T 48

adductor muscles of larva 368

meta morphosis 366

Jahnes, W. G., antibacterial agents 393

SUBJECT AND AUTHOR INDEX

Page

Jakubski, A. W., glia cells 286

Jakus, M. A., paramyosin in oyster 164

James River, Va., freshets 405

Jhering, W., nervous system 281

jelly coat of egg 340

Jensen, E. T., pollution manual 442

Jensen, P. B., role of Zostera 233

Jodrey, L. H., calcification 97

Joh- son, W. H., hypothesis of catch mechanusm.. 166

Johnson, T. W., Dermocyslidium 416

marine fungi 376

Jones, D. B., proteins 391

vitamins 391

Jones, E. W. K., gregariousness 373

larvae rearing 369

setting 372

J0rgensen, C. B., filtration rate and oxygen con-
sumption 408

oxygen uptake by oyster 210-211

rate of removal of graphite by oyster 194

Joubin, L., oyster bottoms 398

Julhen, A., heart physiology 242

acetylcholine content of heart 252

adenaline effect on heart 253

curare effect on heart 253

effect of potassium on heart 251

effect of acetylcholine 252

heart 243

pacemaker system of heart 246

veratrine effect on heart 253

Just, E. E., spawning of Nereis 319

Kagawa, K., effect of acetylcholine on cihary motion. 141

Kahlbrock, M 164

electron microscopy of muscle 157

Kahn, J. S., hyopthesis of catch mechanism 166

Kaandler, R., oyster bottoms 398

rearing of larvae 374

Kawaguit, S., cross stirated muscle fibers 154

Kellogg, J. L., adductor muscle 154

ciliary currents 142

hermaphroditism 314

labial palps 114

muscle fiber 154

promyal chamber 123

Kerly, M., muscle contraction 167

Ketchum, B. H., circulation in estuary 401

Keys, A. B., respiration chamber 201

kidney '^0~''l

microscopic structure 272

''72

reservoir "'■'

reservoir lining of 273

Ivineaid, T., bacteria as food 233

Thais lamellosa 432

Kitching, J. A., effect on pressure on ciliary motion. 142

Knight, M., food of larvae 374

Kobayashi, H ., cytochrome oxidase in egg 344

Kobayashi, M., heart beat 251

Kobayashi, S., glycogen in adductor muscle 164

Koch, H. J., oxygen debt 214

Koehring, V., heart beat 247,249

Kohler, M. A., sedimentation 410

467

Page

Koizumi, S., serological differences in vivalves 266

Roller, G., absorption of glucose 231

Korrinsa, P., annelids on oyster shell 428

chalky deposits 32

conchiolin content 39

Crcpidula in Europe 426

Folliculinids 428

larva 0. edulis 355

lunar periodicity in breeding 319

Polydora ciliata 422

setting 369

shell disease in Dutch oysters 418

survival and loss of larvae 371

swarming 306

Korschelt, E., sterroblastula 347

Kowalevski, A., excretion of dyes 274, 279

Kraft, H., velocity of effective stroke of cilium 135

Krijgsman, B. J., heart physiology 242

heart beat 251

Krogh, A., inorganic salts in adductor muscle. 162

organic substances in sea water 197

respiratory quotient 212

Kumano, M., osmoregulation 278

Kurtzman, C. H., chemical composition 381

Kuyper, A. C, calcification theory 94

LD-50 letal concentration killing 50 percent 445

labial palps 70

anatomy 111

Anodonta, autonomous responses 118

blood supply 113

ciliary currents 114

currents on ridged surface 117-118

currents on smooth surface 117-118

direction of currents 114

function of ridges 114

histology 111

innervation 113

method of study of ciliary current 115

muscles of 115

parth of particles across ridges 116-117

reactions of 118

ridges 112

role of mucus 116, 118

surfaces of 111

Lacaze-Duthiers, H., excretory system 272

lactic acid, in muscle 167

Laguna Madre, Tex., sedimentation 412

Lajtha, A., phosphatase activity in muscle 168

Lamarck, J. B. P., species of oysters 1,2,3

Lambert, M. B., Bryozoa 427

laminar flow 410

Lamont, H. C, hypothesis of catch mechanism 167

Lamy, E 2

Landau, H., A''e nioiopsis distribution 419

Landon, W. A., bacteria as food 233

Lang, A 122

Lankester, E. R., green oysters 384

larva, adaptation to pelagic life 362

advanced stage 357-358

anatomy, 0. edulis 359

artificial rearing 374

468

Page

attachment 365

carried upstream 402

classification 364

D-shaped 357

dispersal in sea 369

distribution in relation to tides 369

early shape of 357

effect of salinity on growth 370

emergence from egg 350

eyed 357-359

feeding, 0. edulis 374

food requirements 375

fully developed, C. virginica 362

immobilized 366

loss of during pelagic life 370

mechanism of transport in estuaries 402-403

metamorphosis 365

mortality 370

newly hatched 348-349

number depending on dimensions of adult- _ 313

percent reaching setting 371

rate of loss during tidal cycle 371

reaction to environment 37 1

reaction to surface 373

rearing in laboratory 374

shape of early stage 357

stimulation by light 371

straight-hinge 357

survival 370

transport to up-estuary 402-403

vertical distribution in estuaries 369-370

vertical distribution in rearing tanks 373

larval development 355

larval foot 359

larval organs, fate of 367-368

larval shell 6

latent period, of stimulation of tentacles 294

lateral cilia, mechanical work 143-145

laterofrontal cilia, ultrastructure of 133-134

latex, injection into promyal chamber 123

injection in digestive tract 219

use in anatomical studies 66

Latrigue, A 21

Lavoie, M. E., force exerted by starfish opening

oysters 438

Logic, R. R., Malpeque Bay disease 415

Lee, C. F., chemical composition 381

leech, Stylochus frontalis 438

Leenhardt, H 65

gastric shield 222

labial palps 113

nervous system of C. angulata 281

periostracal groove 86

stomach histology 222

Legendre, R., effect of moon on spawning 319

Leisure, C. E., coordination of ciliary motion 137

Lenhossek, M. V., role of basal bodies in cilia 132

Lenneman, E. E., coordination of ciliary motion — 137

leptonema stage in spermatogenesis 327

Letellier, A., excretion 274

urinary function 277

Levine, H., heavy metals 384

FISH AND WILDLIFE SERVICE

Page
Lewis, G. J., anthrone for carbohydrate determi-
nation 198-199

Lewis, R. N., inhibition of adductor of Pec<en 166

Lewis, S. A., ciliation of hibial palps 115

Lewis, W., proteins 391

Li, C. P., antimicrobial agents 393

ligament 48

alvincular 48

calcification of 58

chemical composition 56

conchiolin content 57

determination of elastic force 59

effect of drying on elasticity 61

elasticity 59

function of 48-49

internal 48

lamellae 49-50

lines of stress 49

moment of thrust (formula) 60

parts of 48

pivotal axis 49

pulling force 59

thrust of 59

ultrastructure of 51

ligamental ridge 66

light, effect on shell movement 175

Lillie, F. R., cell lineage 346

egg water 338

spawning of Nereis 319

Lillie, R. D., histological localization of copper 388

Lillie, R. S., ciliary motion 139

Lima, cross striated muscles 154

Limnaea, cellulase in 230

Lindow, C. W., copper determination 385

Linnaeus, C, taxonomy of oysters 1

Linsley, R. K., sedimentation 410

lipase 230

lipids 381

Lison, L., mathematical analysis of shape of shells. 23-27

List, T., crystalline style rotation 225

labial palps 113

mantle attachment to shell 88

periostracal groove 86

subligamental ridge 89

locking mechanism of muscle 165

Loewenstani, H. A., aragonite in shell 104

logarithmic spiral of shells 22-25

Long, J. B., Yakina River oyster beds 402

Long Island Sound, mean monthly water tem-
perature 407

Loosanoff, V. L., adaption to salinity changes 406

adverse effect of heavy concentrations of

microorganisms on feeding 235

effect of tide on shell movement 175

effect of Polydora websteri on oysters 424

fungi parasities on larvae 376

habitat of Sytlochus ellipiicus 439

influence of environment on gonads 299

inhibition of feeding by microorganisms 409

microorganisms not digested by oyster 234

rearing of larvae in the laboratory 355, 375

selection of food 233

SUBJECT AND AUTHOR INDEX

Page

spawning at low temperatures 307

starfish 437

starfish salinity barrier 438

Lopha 6

Lotsy, J. P., food of the oyster 232

Loubatie, R., iodine in Arcachon oysters 383

low salanity, effect on Virginia oysters 405

Lowy, J., helical striation in muscle fiber 155, 157

muscular tension 167

tonus hypothesis 166

Lubet, P., inhibition of spawning 312

Lucas, A. M 137

Lucina 29

lunar cycle, shell movement 180

lunar periodicity, of spawning 318

Lunz, G. R., crabs, oyster pest 439

mud worm in South Carolina 424

Lycosoma 428

lysin, in sperm 341

McAfee, W. L., destruction of oysters by birds 439

McDermott, J.J 425

McDonald, J. F., coordination of ciliary motion 137

McGrady, J., food of the oyster 232

McMannus, reagent for glycogen 79

McMillin, H. C., pollution of Olympia oyster beds- 401

setting 369

M cell, second somotoblast 348

M-Nadi reaction 344

Macbride, E. W., embryology textbook 347

veliger 356

Mackenzie, C. L., Eupleura 432

Mackin, J. G., Dermocystidium 415

glycogen 386

Hexa mita 419

MSX 417

Mackintosh, N. A., crystalline style formation in

gastropods 225

macroraeres 345-346

Magnan, C, glycogen 386

magnesium, effect on lateral cilia 139

Makino, K., antiserum effect on ciliary motion 141

Makino, M., immunological reactions in moUusks.. 265

male spawning, stimulated by thyroidin 311

maleness 315

Malpeque Bay disease 415

maltose, effect on water transport 199

man, as predator of oyster 440

manganese 387-390

determination by Richard's method 385

localization in tissues 390

Manigault, P., role of phosphatase 98

Manning, J. H., vertical distribution of larvae 402

Mansour-Bek, J. J., proteolytic action in Pinctada.. 231

mantle 66-74

anatomy 74

appearance 74

cavity 66

color of 68

discharge areas 90

edge 66-75

epithelium 84

469

Page
mantle — Continued

folds 76

function of 72, 74, 75

lobes of 76, 77

mantle-shell preparation 99- 1 00

nerve net 86

pigmentation 74

position in spawning female 303-304

reduplication 77

role in formation of shell 91

Manual of recommended practices for sanitary

control 442

manure, use in rearing of larvae 374

Marceau, F 59

mantle muscles 83

muscle fibers 154

power of the adductor muscle 176

relaxation time in white part of adductor _ _ 164-165

Marchal, P., excretion 274

niarenin 384

Marsland's pressure bomb 142

Martin, G. W., feeding experiments 232, 234

fungi parasites of larvae 376

Martin, J., calcification 97

Martino, E. C, antimicrobial agents 393

Mastif/ocoleus leslarum, boring alga 426

Matagorda Bay, sedimentation 412

Matsubayashi, T,, heart beat 251

Matthews, S. A., nerve net in labial palps of Ano-

(lonta 114

Mead, A. D., cell lineage 346

Medcof,J. C 35

Maipeque Bay disease 415

setting, larva 366

Megathura crenulata, acrosomal reaction 341

Meisenlieimer, J., cell lineage 346

metamorphosis 367

Mendel, L. B., zinc in tissues 390

Menestho (See: Odostoniia) 436

Menidia, host of Bucephalus 420

menthol, use in narcosis 357

Menzel, R. W., glycogen 386

Mercenaria mercenaria, catch mechanism 167

oxygen consumption by tissues 200

respiration 205

Meretriz merelrix 31

Merrill, A. L., chemical composition 381

Merrill, A. S., O. equestris, salinity range 404

Merton, N., ciliary motion 137

mesoderm 355

formation of 348

mesohaline zone 404

metabolism chamber 201-202

nietachronic wave 136

metachronism 136

metals, heavy, detection in blood cells 264

metamorphosis 366

Microciona prolifica 428

micromeres 345-346

ci uartet 347

microplankton, seasonal changes in volume 408-409

microvilli, in ciliated cells 131

470

Page
mid-gut 226

milfoil, invasion of 430

Millar, R. H., fecundity of 0. edulis 313

Miller, ('. E., penetrometer 399

Miramachi Estuary, transport of barnacle larvae,. 403

Mironov, f!.\.. turbidity determination 36-37

Mironov's current indicator 191

Mississippi Sound, mortality of oysters due to flood ^ 405

Mitchell, H. 1) ^ 48

mitochondria 326

mitochondrial bodies 325

Mitsugi, K., starfish stomach extract 438

mixing, in estuary 401

Miyazaki, I., male spawning stimulated by Utva

and other algae 311

Mobile Bay, saHnity range 405

Mobius, K., definition of oyster bed 397

oyster beds destroyed by human activity 398

Modiolus demisvs, provitamin D content 391

moisture content, of meat 385

moisture determination, xylene distillation 385

Molyula manhattensis 211,427

Monas, sp., a food for larvae 375

Monilia, suspected in shell disease 418

Monochnjsis luthcri, food of larvae 376

immobilized by style enzyme 231

monomyarian 152

Moore, H. F 65

apron technique 187

food of the oyster 232

oyster bottoms 398

Texas oyster reefs 412

volumetric determination of food 232

Morgan, E., Codium in Long Island Sound 429

Moriches Bay, pollution by duck farms 444

Morin, (!., effect of potassium on heart 251

heart physiology 242

pacemaker system of heart 246

Morton, J. H, osmoregulation 278

Motley, H. L., pacemaker system of heart 246

mouth 70, 219

Moynier de Villepoix, R., periostracal groove 86

MPN, most probable number 442

mucopolysaccharides, in connective tissue 79

mucous cell, in labial palps 111

mud, accumulation by Polydora 412

mud blisters 422

in Virginia oysters 424

mud prawn 439

mud traps 414

mud worms 421-425

Murex, acetylcholine content of heart 252

Murphy, D. B., vitamins 391

muscle attachment, platform 34,41

muscle cells 83

muscles, of gills 130-132

in gill filaments 131

chemical changes during contraction 167-168

rudimentary (in the mantle) 17, 70, 79

muscle fibers, in adductor 153

muscle fibers, striated 84-361

striation 153-1 54

structure 153

FISH AND WILDLIFE SERVICE

Page

translucent (dark) 154

transverse ^3, 160

ultrastructure 157-158

muscle scar 18, 41, 43, 152

ratio of scar and shell areas 43

muscular activity, periodicity 180

mussel, California, water propulsion and size 195

Mya arenaria, absorption of glucose 231

ammonia in excreta 276

ciliary tracts of stomach 220

crystalline style 224

enzymes 229

heart physiology 242

ligament elasticity 59

oxygen debt 214

oxygen utilization 214

pH effect on cilia 246

myogenic heart 1"4

164

myosin "

solubility of 418

Myotomus ostrearum, suspected cause of foot disease- 418
Myriophyllum fpicatum, (milfoil), invasion into

Maryland and Virginia waters 430

Mytilieola orienlalis, parasite in bivalves 420

Mytilus edulis, absorption of glucose 231

amino acids ^"1

aragonite in shell 1"*

crystalline style 224

effect of drugs on ciliary motion 140

gills, effect of chemicals 139

heart beat 250

heart physiology 242

innervation of adductor 106

in v.ision of oyster beds 428

labial palps 114

mechanical properties of cilia 135

pacemaker system of heart 246

sperm 341

subligamental ridge 89

N-ethyl-carbazole, carbohydrate reagent 197

N-ethyl-carbazole method, for determination of

carbohydrates in sea water 198

nacre, ultrastructure of 54-55

nannoplankton, as food 232

in rearing tanks 374

narcosis 65

Navez, A. E., effect of pilocarpine on heart 253

Needham, D. M., chemical changes during mus-
cular contraction 167

Needier, A. B., hermaphroditism 314

sex change 316

Needier, A. W. H., Malpeciue Bay disease 415

negative factors 409, 445-446

Nelson, E. M., vitamins 391

Nelson, T. C 71

apron technique 187

artificial feeding 235

crystalline style rotation 225

diantlin in sperm 312

SUBJECT AND AUTHOR INDEX

Page

distribution of larvae in relation to tides 369

effect of light on shell movement 175

effect of tide on shell movement 175

labial palps of oyster spat 115

mantle currents 91

promyal chamber 123

setting 366

spawning 308

Skeletonema as food 232

stimulation of larvae by light 371

vertical distribution of larvae 402

velum 77

Ne77ialopsis 419

nephridia 271

nephroprokt 272

Nereis, spawning 319

nephrostome 271

nerve cells, of ganglia 285

multipolar 285

under ciliated epithelium 137

unipolar 285

nerve net, in mantle 86

nerve, accelerating heart beat 250-251

branchial, microscopic structure 289

cardiac. 251

circumpallial 285

distribution 281

emerging from visceral ganglion 284

inhibiting heart beat 251

nervous system 71, 281, 289

anatomy 283

larval 360

method of study 281

simplification of 281

neurosecretion, effect on spawning 313

neurosecretory cells 293

Newcombe, C. L., relationship of height and area in

bivalves 31

niacin, in oysters 391

Nicomedes, conchoid curve 23

Nigrelli, R. F., blood cells .--- 260

NissI granules, in nerve cells 286

nitrogen, in excreta of bivalves 278

nitrogen secretion, methods 277

Xitzschia, removal by gills 194

Nomejko, C. A., effect of tides on shell movement.. 175

Nomura, E., growth of Meretrix meretrix 31

Nomura, S., effect of acetylcholine on ciliary mo-
tion 141

effect of adrenaline on cilia 140

nonincubatory species 4

normal axis of shell 25

Novikoff, A. B., cytochrome oxidase in egg 344

novocaine, effect on cilia 140

Nowinski, W. W., paramyosin 164

periodicity in ciliary rootlets 133

Nozawa, A., oxygen consumption in 0. circumpicla. 214

Nueces River, Tex., sedimentation 412

Numachi, Ken-Ichi, local races of C. gigas 266

nymphae 49

O'Brien, H., respiratory quotient 212

471

Page

Ocenebra japonica 434

effect of salinity 435

manner of drilling shell 435

menace to Pacific coast native oysters 435

synonym of Trilonalia 434

Odhner, N., excretory system 271

Odlaug, T. O., toxicity of pulp mill effluent 444

Odostomia, synonym Menestho, ectoparasites of

oyster 436

Odostomia bisuturalis 437

Odostomia impressa 437

oil, crude, toxicity to fishes 443

emulsion, use in digestion experiments 228

oil, pollution 443

Oka, K., heart physiology 242, 251

Oka's method of heart stimulation 248

Okada, K., primordial germ cells 299

Olsson, A. A., tensilia of ligament 49

opening of oysters 66

opening of valves, duration of 172

organic components of adductor muscle 162

organic substances in sea water, effect on water

transport 19'

organs of reproduction, anatomy 297, 299

orthoquinone, in tanning of protein 58

Orton, J. H 16, 32

adductor muscle 154

anatomy of 0. edulis 65

ciliary currents 142

crystalline style formation 225

feeding dependent on stage of tide 175

metabolism and sex change 212

sex changes 314

sex change in 0. edulis 314

Osburn, R. C, bryozoa in Chesapeake Bay 427

osmoregulation 278

in Crassosirea 278

ostia 127

effect on water transport 1 85, 197

Ostrea, diagnosis 1, 6

effect of drugs on ciliary motion 140

taxonomic characters 4

Ostrea angasi, prismatic layer in shell 4

Ostrea chilensis, crystalline style 224

stomach histology 222

Oslrea circumpicta, agglutination of sperm 340

cardial nerves 251

excised heart 243

ganglion cells of the heart 242

glycogen in adductor muscle 162

heart beat 250

respiration 214

specific gravity of blood 265

Ostrea cucullata, hermaphroditism 314

labial palps 111

pallial organ 289

spawning 306

Ostrea dendata, heart beat 250

Ostrea edidis, amino acids 391

chemical analysis of shell 39

ciliary tracts of gills 143

ciliarv tracts in stomach 220

472

Page

crystalline style 224

diagnosis 9

electrocardiogram 244

fecundity 313

gregariousness of larvae 373

hermaphroditism 314

laterofrontal cilia 128

Linnaeus diagnosis 1

spawning 306

sperm balls 302

setting 37 1

sex change 314

stomach histology 222

striation of muscle fibers 157

Ostrea eqnestris 4

diagnosis 12

hermaphroditism 314

on buoys 2

salinity tolerance 5

salinity range 404

Ostrea frons, diagnosis 12

Ostrea haliotidea 2

Ostrea imbrieata, analysis of adductor muscle 162

Ostrea laperousi, heart beat 251

diagnosis 11

gametogenesis 325

hermaphroditic gonad 326

number of larvae in female 313

ostia 127

spawning 306

setting . 372

Ostrea lurida, sex 314

shell movements 175

Ostrea ( A leetryonia) megodon 4, 28

Ostrea mcxicana 28

Ostrea mordax 16

Ostrea permollis, diagnosis 12

Oslrea sandwiehensis 28

Ostrea tuberculata 2

on corals 2

ostrcasterol, identical with 24-methylencholosterol- 392

Ostreidae, taxonomy It 2

Otis, A. B., adrenaline effect on heart 253

effect of pll on heart beat 251

ovary, definite 324

oviparous oysters 5

ovocyte (oocyte) 303, 325

mature, size in C. comjnercialis 326

suppression of development 324

ovogenesis 325

o vogonia 325

ovulation 303-304

Owen, G., normal axis of shell 25

oxalocetic decarbohylase, in calcification 99

oxidase 299

oxygen consumed and weight 208

oxygen debt 208, 214

oxygen tension, effect on respiration 214

oxygen uptake 205

and shell movements 208

and spawning 207

effect of pH 211

FISH AND WILDLIFE SERVICE

Page

effect of salinity 211

seasonal changes 211

under pull of adductor 209

variations 206-207

oxygen utilization 214

coefficient 214

oysters, Blue Points 19

Chincoteagues 19

Cotuits 19

super-iodized 383

trade names 19

use in vitamin deficiency 391

oyster banks 397

oyster beds 397

definition by Mobius 397

oyster bottoms 397, 446

Elbe estuary 398

general description of 398

destruction by human activity 398

oyster community, abundance of species 398

biomass 398

oyster crab 425

effect on oysters 425

oyster culture, success in New England 440

oyster reef 397

Altamaha Sound 403

Oyster River, Mass., sedimentation 411

oyster thief 429

pallial curtain 77

pallial line 75

pallial nerve 72

pallial organ 72,289

function 291

histology 289,290

of spat of 0. eduUs 292

pallial strip (synonym of subligamental ridge) 89

pallium 74

role in water transport 185

Palolo worm 319

paolin 393

palps, labial 67, 70

Paphia staminea, enzymes 229

paramyosin 164

discovery in clam 164

in adductor muscle 167

localization of 164

separation from actomyosin 164

Parke, M. W., food of larva i 374

Parker, R. H., seasonal salinity variation in Texas. 406

parks, for feeding of oysters 234

Patella, calcification 94

pattern of growth 26

uPalhus, J. L. H., sedimentation 410

pea crab, (See: oyster crab) 425

pearl, formation 95

Pearse, A. S., oyster bottom 398

Stylochus 438

Pease, D. C, effect of pressure on cilia 142

Peden, antisera 142

cross striated muscles 154

effect of drugs on ciliary motion 140

SUBJECT AND AUTHOR INDEX

Page

heartbeat 250

heart physiology 242

inhibitory axons 166

mantle muscles 84

Pedersen, E., respiration in O. edulis 213

pediveliger 358

in C. virginica^ 362

Pekelharing, C. A., glycogen used by gonads 82

Pelseneer, P. , chalky deposits 33

excretory system 272

nervous system 281

penetrometer 399

Pepper, L., chemical composition of oysters 381

perforatorium 342

perfusion chamber. Wait's 247

pericardial glands, concentration of ammonia

carmine 274

pericardium 70, 239

pericardium wall 239-240

pcriostracal gland, function 87

periostracal groove 77, 86

periostracum 4, 37

Perkins, E. B., distribution of larva 369

Peter, K. , role of basal body of cilia 132

Petering, H. G., dropping mercury electrodes 214

Petersen, C. G. J., role of Zostera in feeding 233

Petitfrere, J., heart 245

pH, effect on oxygen uptake 211

in gut and stomach 230

phagocytes, ingestion of particles 231

role in excretion 279

phagocytosis 264

in gonad 302

pharyngeal teeth, of drum fish 439

Phiipott, D. E., electron microscopy of muscle. _ 157-158

separation of paramyosin 164

Pholas, labial palps 114

phosphagens 167

phosphatase alkaline in mantle 88

in calcification 88

role in mobilization of calcium 97

phosphatase activity, in bivalve muscles 168

phosphates in shells 44

phosphorous, radioactive, P^-, use in experiments

with gills 194

phosphorylization of ATP 168

phototaxis in larvae 371

Physa, ciliary motion on lip 137

phytoplankton, organic matter content 408

precipitation reaction 266

Picken, L. E. R., osmotic pressure of body fluid — 274

rate of filtration with pericardium, method- . 275
Piechowski, R. J., moisture determination by

xylene distillation 385

Pieron, H., Nissl granules in nerve cells of Mya 286

sensory stimulation in Mya 293

pigment cells, in connective tissue 83

pigment units, in plankton studies 409

pigmentation 279

Pilgrim, R. L. C, effect of acetylcholine on heart — 252

sensitivity of bivalve heart to acetylcholine. - 252

473

Page

pilocarpine, effect on cilia 140-141

effect on heart 253

Pinclada 41

aragonite in shell 104

closing of valves 165

Pinna 41

Pinna nobitis, contraction of muscle 159

Pinnotheres oslreum 425

Pisaster ochraceus 438

plankton, seasonal changes in volume 408

plankton trap, for larvae 369

used in quantitative plankton sampling 409

plaster of paris, injection with 66, 123

Plateau, F., elasticity of ligament 59

power of adductor muscle 176

Plalymonas tetrahele, food of larvae 374

Pogonias crotnis, destructive to oyster 439

Poisseul's formula 145

polar bodies 344

pollen, as food 232

pollution, definition of 441

evaluation of 445

Federal program of investigation 442

from duck farms 235, 444

lack of legal definition 441

oil 443

pulp mill 443

radioactive waste 445

Polydora, sp., mud gathering worms 412

Polydora larvae 423

Polydora ciliala 422

Polydora ligni 413, 422

amount of mud collected by 423

destruction of oysters in Delaware Bay 423

egg laying 422

mud gathering mechanism 423

Polydora wehsleri 422

effect on oysters 424

invasion of oyster shell 412

life history 422

polyhaline zone 404

Pomerat, C. M 372

Porter, K. R., electron micrograph of rootlets in

DeRobertis, etal 133

Portier, P., vitamins in oysters 391

positive factors of environment 399

summation of 445

potassium, effect on frontal cilia of Mytilus 139-140

effect on heart beat 251

Potts, F. A., adductor muscle 154

digestive diverticula 226

Power, E. A., yield of oysters 382

power of adductor muscle 175

in air 177-179

in sea water 178-179

methods of measuring 176

predation, evaluation of 440

predators 430

Prenant, M., calcification in Helii 95

minerals of calcium carbonate 103

Prescott, B 343

pressure, effect on ciliary motion 142

474

Page

pressure, inside the gills 146

pressure bomb, of Marsland 142

Price, T. J., radioactive waste 445

zinc absorption 390

Price, W. A., sedimentation 412

principal axis, instability of 29

prismatic layer 37, 93

Pritchard, D. W., definition of estuary 400

mechanism of transport of larvae 402-403

setting 369

types of estuaries 402

prodissoconch 6, 355, 357, 364, 365

definite 6

C. rhizophorae 365

C. virginica 365

0. edulis 366

0. lurida 366

primitive 6

Pycnodonie hyotis 364

productivity of oyster bottoms, evaluation of 445-446

method of scoring 445-446

productivity of sea bottom 398

promyal chamber 4, 71, 75, 123

cast of 124

importance of 123

in Crassostrea oysters 124

relation to water turbidity 124

relative size of 124

propionic acid, role in calcification 99

Prorocentrum micans, undigested by oyster 234

Prosser, C. L., urine formation 275

proteins 381 , 382

content 390

in adductor muscle 164

in blood plasma 278

prototroch 355-356

pro vinculum 364

provitamin D., in Modiolus demisus 391

Prussian blue reaction 225,264,388

Prym.nesixim parvum, toxic to larvae 375

Pryor, M. G. M 58

Prytherch, H. F., larval development in C. virginica. 355

Nematopsis ostrearum 419

rearing of larvae 374

setting 366, 369

Przylecki, St. J., excretion of nitrogen in Anodonta. 277

Pspudolamellibranchia 121

pseudopodia, bristle, of granulocytes 262

Pseudostylochiis ostreophagus 439

perforation of oyster shell 439

pulp mill effluent, depression of ciliary action 443-444

effect on opening of oyster valves 443

Purdie, A., crystalline style 224

Piitter, A., significance of dissolved organic matter. 198

Puys^gur, M., food of oyster 231

Pycnodonie, definition of gen us 7

rectum, position of 4

shell 4

Pycnodonie hyotis, diagnosis 12

pyloric process 70

Pyramimonas grosii, as food of larvae 375

FISH AND WILDLIFE SERVICE

Page

quartet (quartette) of micro meres 345

Quayle, D. B., effect of low salinity 405

Quenstedt's muscle 17, 43, 70, 104

quinine sulphate, detected by tentacles 294

races of C. gigas 266

radial muscles, contraction of 83

relation to nerve 83

radioactive plankton, use in experiments with gills. _ 194

use in study of filtration by gills 194

radioactive pollutants, in coastal waters 445

radioactive waste 445

Rahl fixative 101

Rai, H. S 65

injection of digestive system 219

nervous system of 0. cucullata 281

pallial organ 289, 291

Randoin, L., vitamins in shellfish 391

Rakestraw, N. W., anthrone method of carbohy-
drate determination 198, 199

Ranson, G., absorption of food 231

chalky deposits 32, 41

classification of larvae 6, 364

green oysters. 384

retention of name of Gryphaea 3

water filtration by oysters 193

Rao, K. P., rate of water transport and size of

California mussel 195

Rappahannock River, Va., sedimentation 411

Rassbach, R., periostracal groove 86

rate of water transport by Black Sea mussels 191

Raven, C. P., egg polarity 344

mesoderm formation 348

Rawitz, B., nerve cells 285

nervous system 281, 283

pallial curtain, function of 77

pathways in ganglia 286

periostracal groove 86

Ray, S., Dermocystidium 416

Reade, J. B., stomach content of oysters 231

rectum 226

red tide 409

Redfield, A. G., adverse effect of unbalanced fer-
tilization 235

pollution by duck farms 444

Rees, C. B., bivalve larvae 357

classification of larvae 364

Reichel, H., contraction of muscle in Pinna 159

Reiner, E. R., setting 372

Reis, O. M., ligament 48

Riesser, O., chemical composition of adductor

muscle 161

rejection reaction 169-170

Remington, R. E., heavy metals 384

renal duct 272

renal opening 272

reno-pericardial canals 239

replacement of species, due to salinity changes 406

reproduction 297

reproduction, See organs of 297

residual cells, in gonad 302

SUBJECT AND AUTHOR INDEX

Page

resihfer ^g

resilium ^g

aragonite in g^

ultrastructure 53-54

respiration 185,200

in Woods Hole oj'sters 206

methods 201-205

in O. edulis 213

in O. edulis and C. angulata 213

related to shell movements 206

respiration chamber of Keys 201

respiratory quotient, R. Q 212

seasonal variation 213

Rhizosolenia 409

riboflavin, in oysters 391

Rice, T. R., zinc absorption 390

Rice, T. R., radioactive waste 445

use of isotopes in removal of plankton cells

by gills 194

Richards, M. B., manganese determination 385

Richardson, H. B., respiratory quotient 212

ridges, on labial palps 114

erection of 115

"right-handed" oysters 29

Riley, G. A., organic matter in phytoplankton 408

relationship of environmental factors 445

Ritchie, A. D., adductor of Peden 164

maintenance of tonus 166

Roliert A., cell lineage 346

Robertson, J. D., criticism of DeWaele's calcifica-
tion theory gg

osmoregulation 278

urine formation 274

Roche, J., amino acids in conchiolin 41

amino acids in shell 41

Rochford, D. J., estuarine environment 401

sorting of sediments 411

roc k crab 439

rootlets of cilia 133

function of 133

periodic striation 133

ultrastructure of 132

Rosenbluth, R., hypothesis of catch mechanism 167

Roughley, T. C, anatomy of O. commercialis 65

Polydora ciliata 422

sex change 316

Rubel, E., combined effect of factors 445

Runnstrom, J., acrosomal reaction 342

jelly coat of egg 341

Russell, P. B., collagen fibers 58

Ryder, J. A., blue cral:) — oyster enemy 439

metamorphosis 366-367

Sabatier, M.A., crystalline style 224

Sacco, F., C. virginica 2,3

Saccoglossus koumlewskii, acrosomal reaction 341

Saez, F. A., paramyosin 133, 164

Saint-Hilaire, C., role of digestive diverticula 229

salinity and temperature changes, hydrographic

cli mate 405

475

Page

salinity, adaptation to 405-406

effect of sudden change on oysters 405

effect on oxygen uptake 211

effect on oyster populations 406

effect on specific gravity of blood 265

effect on water transport 406

inhibition of gonad development by low-
salinity 405

range of tolerance 404

salinity factor, evaluation 406

salinity gradient, effect on distribution of larvae 369

salinity range, Venice system 404

Sandoz, M., oyster crab 425

sarcoplasm 154

Sarlet, H., amino acids 391

Savage, R. E., food of 0. edulis 232

Saville-Kent, W., 0. mordax 16

Sawano, E., but\Tase in crystalline style 230

starfish stomach extract 438

Saxidomus giganteus, crystalline style, chemical

composition 226

enzymes 229

scallop, respiration 205

scallop (Aequipecien), water propulsion by gills 195

Schaefer, M. B., setting at different angles 372

Scheer, B. T., general physiology textbook 167

Schiedt, R. C, pigmentation 279

Schizoporella unicornis 427

Schizolhaerus nutalii, crystalline style, chemical

composition 226

Schlosstjerger, J., analysis of conchiolin 39

Schmidt, W. J., calcite crystals in prism 37

prismatic layer of shell 93

structure of cilium 132

Schmitt, F. C, nervelike apparatus of gills 137

rootlets of cilia 134

Schmitt, F. O., paramyosin in oyster 164

Schwalbe, G., diamond lattice pattern in adductor

muscle fibers 154

Schwaneke, H., blood supply of palps 113

Scytosiphon 428

sea water, diluted, effect on mantle 294

quantity required by oyster community 403

sedimentation. 410

destructive to oyster beds 412

effect on oysters 412

sedimentation factor, evaluation of 415

sediments, biochemical changes 413

sediments in suspension, determination of 414

sediments, sorting of 410-411

suspended- 410-411

self-fertilization 315

Seligman, A. M., cytochemistry 344

Semichon, L., glycogen in vesicular cells. 83

sensory stimulation 293-294

apparatus for study. . 293

Seo, A., nervous control of ciliary motion 137

serological differences in bivalves 266

serological studies, absorption tests 266

serology 265-266

476

Page

setting 365-368

abundance 359

on under and upper surfaces 372

settlement, of larvae 365

sewage, domestic 442

effect on oyster bottom 449

sex, alternation of . . 317

sex change 314-317

effect of excision of gills 3ig

frequency in adults. 317-318

in Crassostrea oysters 316

sex classes at first breeding season 315

sex, recognition of 71

recognition of by inducing spawning 316

sex ratio 314

at first breeding season 314-315

in sex reversed oysters 317

sex cells, release of 303-304

sexual phases 314

Seydel, E., toxicity of crude oil 444

shape of shell, index 29

Shaw, B. L., gastric shield 223

stomach histology .. 222

Shaw, W. N., raft culture 428

Sheepscot River, Me., abrasion of oyster shells 403

shell, analysis by Columbia Southern Corp 43

area and height 30-31

as buffer 212

C. virginica 16

chemical composition 43

circular 27

contour of 23

dimensional relationship 29

gland 355

height 18

larval 363

length 18

morphology 4, 16,363

M\ja arenaria, strontium/calcium ratio 44-45

O. edulis, chemical analysis 39

O. edulis, chemical composition 44

organic material of 39

organic matrix 92-93

phosphates 44

pigmentation 20

prin cipal dimensions 20

rate of secretion 92

right and left 16

shape of 21

structure 36

terminology 36

thickness 18

variability 18

width 18

shell disease 417

shell heap 440

shell liquor 68

shell movements 168

after spawning 174

alleged effect of carbohydrates 199

during male spawning 310

FISH AND WILDLIFE SERVICE

Page

effect of continuous pull 179

effect of light 175

effect of temperature 174

effect of tide 175

female spawning 172

kymograph records 169

major types 169-172

method of recording 16S

of dying oyster 122-173

of spawning female 309-310

percentage of time closed 174

role in water transport 185

Shelton Bay, Wash., pollution 444

Shukow, E. K., muscle tonus 166

Siebert, W., labial palps__ 113

silt, use in determining role of water transport _ 193

sinuses, blood 84

Sirolpidiitm zoophtornm, destructive to larvae _ _ 376

Skelelonema 429

as food of oyster 232, 234

Skramlik, E. V., contraction of auricles 244

"sliaing" hypothesis of muscle contraction 167

Smeltz, H. A., food of oysters 232

Smith, E. A., oysters adhering to Trochns 2

Smith, R. A., shell analysis 44

Smith, R. O., oyster culture in South Carolina 412

sodium potassium ratio in adductor muscle 162

soil extract, used in algal culture 375

solids, in meat 381

local variation 382

seasonal variation 382

somatoblast (X cell) 348

second (M cell) 348

sorting of food, by labial palps 114

sorting mechanism, of labial palps 118

South Carolina, muddy tidal flats 412

Sparck, R., experiments with Zosiera detritus 233

rearing of larvae 374

respiration of 0. ediilis and C. angulata 213

setting 369

sex changes in O. edidis 314

Zostera as food 234

Sparks, A. K., Mytilicola 420

Sparrow, F. K., Dermocyslidium 416

marine fungi 376

spat 366

spatf all 365

spawning 303-313

artificially initiated in oyster population 319

at low temperature 307

biological significance 319-320

critical temperature vs. critical condition 309

eggs discharged through the gills 306

female reaction 303-304

frequency 312

hermaphroditic oyster 318

inhibition 312

initiation by males 319

lunar periodicity 318-319

male reaction 310-312

of Onset oysters 308

sex reversed oysters 318

shell movements 309

SUBJECT AND AUTHOR INDEX

Page

stimulation by temperature 308-309

specific gravity, of blood 265

effect of reduced salinity 265

spectrographic analysis of meat 383

sperm, discharge of 310-311

suspension used in artificial fertilization 342

transport by currents 320

sperm ball 327

sperm extract, stimulation of spawning 312

spermary, definite 324

mature 327-328

ripe 302

spermatocyte 327-328

of 0. lurida 327

secondary 327

spermatogenesis 362

inhibition by ovocytes 315

successive stages 330

spermatogonia, number of divisions 326

primary 325

spermatozoa, in follicles 302

Sphaerium, cessation of ciliary motion 146

heart beat 250

sphaerolithes 103

sphincter, of gonoduct 303

spiral, logarithmic 25

spiral structure, of shell 21

spiremes 325, 327

spirochaetes 426

Spisula solidissima, ligament of 60

catch mechanism 167

Spitzer, J. M., excretory system 271, 274, 276, 277

Spondylus, cross striated muscles 154

sponges, on oyster grounds 428

Sporn, E., analysis of conchoid curve 23

Springer, P. F., Milfoil invasion 430

SSO (Haplosporidium coslale) 417

Stafford, J., eggs of 0. lurida 305

larval development, 0. lurida 355

metamorphosis 367

promyal chamber 123

starfish 437

abundance of 437

destructiveness 437

larvae 437

manner of attacking oysters 438

movements 437

statocyst 360-36 1

Stauber, L. A., excretion of India ink 279

heart beat 247-249

Stein, J. E., Hexamita 419

Steinhardt, B., calcification theory 96

Stella grubi, sponge associated with 0. permollis 12

Stempell, W., ligament 48

Stenzel, H. B., aragonite in resilium 54

aragonite in shells 4

generic names in family Osteridae 6

sterols, in oysters 391-392

sterroblastula 347

Stevens, B. A., Calianassidae 439

stimulation, of nerve by inorganic salts 294

velocity of transmission 293

stimulation of tentacles, latent period 294

477

sting ray 430

Stolkowski, J., role of carbonic anhydrase 104

stomach 71,219-221

ciliary tracts 220

histology 222

lining 220-221

parts of 219-221

Stommel, H., organic matter in phytoplankton 40S

Stoke's law 410

Stotts, V. D., milfoil 430

streamline flow 410

striation of muscle, oblique 154

Striostrea 6

stroboscope, use in study of ciliary motion 137

Strohl, J., excretory system 27 1

excretion of dyes 279

strontium, effect on formation of aragonite 104

in shells 44

strontiuni-calcite ratio in shells 44-45

struggle for space 430

Studnitz, G. V., pallial organ 290

Stylochus ellipticus 438

Stylochus inimicus, synonym S. frontalis 438

Stylochiis frontalis 438

subliga mental ridge 79, 89

histology 89

sulphates, accumulation in Mytilus 278

Suzuki, S., ganglion cells in heart 242

heart innervation 241

swarming of larvae 306

synapsis 327

syringe pipette of Van Dam 204

systole 243

Szent-Gyorgyi, A., theory of muscular contraction, 164, 167
Szent-Gyorgyi, A. G., electron microscopy of

muscle 1 57- 1 58

hypothesis of catch mechanism 166

Tabulae Biologicae 164

Takatsuki, Shun-Ichi, blood cells granules 263

effect of adrenaline on heart 253

enzymes in blood cells 264

excised heart, method 243

heart beat 250

heart physiology 242

Tamura, T., power of adductor muscle 176

Tanaka, K., chalky deposits in shell 35

Tarzwell, C. M., pollution studies 442

taxodont teeth 364

Taylor, I. R., action currents in the heart 244

Taylor, W. R., algae 426

Tellina tenuis, ligament 56,59

teloblasts 348

temperature, as environmental factor 407-408

as factor controlling feeding. 407

as factor controlling reproduction 407

effect on ciliary motion 138-139

effect on rate of work of cilia 145-146

effect on spawning 308-309

mean monthly in Apalachiocola Bay and

Long Island Sound 407

temperature characteristic (m) 249

478

Page

temperature coefficient 249

of ciliary action 146

temperature range 407

Ten Gate, J., heart physiology 242

Tennent, D. H., Bucephalus 419

tensilium 49-50

fibrillae 51, 53

tentacles 66-67, 76

nerves of 84-86

position 78

stimulation of 293

structure of 85

Terao, A., agglutination of sperm 340

Teredo, cellulase in tissues 230

cross striated muscles 154

digestive diverticula 226

terminal groove, of gills 129

Tethys 1

Texas, increase in salinity over oyster beds 406

silting of inshore waters 412

Thais haemasloma, distribution in relation to

salinity 434

fecundity 433

trapping of 433

Thais lamellosa 432, 434

thiamine, in oysters 391

Thiele, J., labial palps 113

pallial organ 289

taxonomy of Oslrea 4

Thompson, d'A. W., mathematical analysis of

growth and form of shells 22, 27

Thorson, G., dispersal of larvae 369

food of larvae 374

thyroidin, as stimulant of male spawning 311

tide, effect on shell movement 175

time marker, for slow motion kymograph 190

Todd, A. R., orthoquinone in arthropod's cuticle.. 58

Tomita, G., serological differences in bivalves 266

tonic activity 166

myogenic vs. neurogenic 166

tonotropic effect of potassium 251

tonus 166

loss of in adductor muscle under continuous

pull 178-179

toxicity, determination in low concentration 444

of pollutants, reduced by oxidation 444

transport of water by the gills 185

automatic recording 187

Trask, P. D., sedimentation 410

settling 411

Trematodes 419

Tridacna, determination of pulling force 59

proteolytic action in stomach 231

trochoblasts 355

trochophore 355

trochophore, anatomy 356

trochophore stage, duration of 356

Trochus marulatus 2

Trueman, E. R., ligament 48,49,56,58,59

role of strontium and magnesium in shell

formation 104

FISH AND WILDLIFE SERVICE

Pag*
Tuekerman's formula, for determining concentra-
tion of pollutant ill estuary 402

Tullberg, T., growth of shell 48

subligamental ridge 89

turbidimeter 193

turbidity determinations 193-194

Turchini, J., excretion 274

Turner, R. D., taxonomy of boring clams_- 421

Twenhofcl, W. H., sedimentation 410

Tyler, A., acrosomal reaction 341

fertilizin 340

Tylosurus marinus, host of Bucephalus 420

typhlosole 224, 225, 226

tyrosin, in conchiolin 103

Uexkull, J., catch mechanism of scallop 165

ultraviolet light, in study of ciliary current 115

Ulva 428

umbo 16

umbo larva 357

Unionidae, excretion rate 274

Upogebia 439

urea, in excreta of Mija and Mytilus 276

urease, in Mijlilus and Helix 277

uric acid, absent in Mytilus, traces in Mya 277

in intestine 277

urine formation 274

urino-genital groove 70

urino-genital vestibule 303

Urosalpinx cinerea 430

dimensions 430-43 1

egg laying 432

fertility 432

manner of drilling 431

rheotaxis 43 1

selection of food.. 431

Urosalpinx extract, effect on mantle 294

U.S. Public Health Service 441

Vaillant, L., Tridacna Ugament 59

valve, equation of 24

Van Dam, L., respiration studies 204

syringe pipette 204

utilization of oxygen 214

Vasseur, E., jelly coat of eggs 340

vaterite 103

veins 84, 254

afferent 254

afferent, structure 257

efferent _" 257

velar retractors 359, 361

veliconcha 358

veliger 356

advanced stages 359

anatomy 357

velum 356, 357

disintegration in metamorphosis 367

venous system 254

ventilation, of gills 185, 214

ventricle of heart 240

peristaltic wave 243

rhythm of 251

SUBJECT AND AUTHOR INDEX

Page

Venus verrucosa, ligament elasticity 59

veratrine, effect on cilia 140

effect on heart 253

Vernon, H. jSI., respiratory quotients 212

Veselov, E. A., toxicity of crude oil 444

vestibule 70

vesicular cell 79

Verwey, J., selection of bottom by larvae 399

Viallanes, H., use of silt in determining water

transport 193

\'incent, D., acetylcholine content in heart 252

\'inogradov, A. P., chemical composition of shell.. 44
vinyl acetate plastic, used for blood venous injec-
tion 253

vinyl resin, used for injection 66

visceral ganglion, extirpation has no effect on ciliary

motion 147

visceral mass 71

viscosity of water, effect on work of cilia 146

vitamin D 391

vitamins in oysters 391

vitelline membrane 343

Voisin, P., shell disease in European oyster 418

Volsella, aragonite in shell 104

Wada, S. K., acrosomal reaction 341

Wait's perfusion chamber 248

Waksman, S. A., decomposition of sediment 413

Wallengreen, H., labial palps 113

ciliary currents 142, 146

Walne, P. R., rearing of larvae 375

Walzl, E. M., action current in heart 244

Wangersky, P. J., dehydroascorbic acid in sea

water 198

Warburton, F. E., Cliona's manner of boring 420

waste, amount discharged into coastal waters 441

importance of sublethal concentrations of — 444

industrial. 443

oxidation of 444

radioactive 445

waste products 276

water content, in oyster meat, seasonal changes 386

water demand, by oyster community 403

water filtration, by oysters. . . 186-192

rate, J0rgensen formula 194

water movements on oyster beds 400-403

water movement factor, evaluation of 403

Water Pollution Control Acts 441

water propulsion, rate of 193

water transport by gills, control of 194, 197

water transport by four species of oysters, deter-
mined by turbidity method 193

direct methods of determination 186

indirect method of determination 186, 193

reduction due to slowing of lateral cilia 197

regulation of I'^S

simultaneous recording of the rate of water

transport and shell movement 187

water transport and shell movement 196

water tubes "1. '^6

water wheel, use in recording water transport 180

Watt, B. K., chemical composition of oysters 381

479

Pag«
Weber, H. H., chemical changes in muscular con-
traction 167

ATP (adenosinetriphosphate) function in

muscular contraction 167-168

weight, of oysters 21

weight of meat, seasonal variation in 0. edulis 383

Weiss, P. A., embryology textbook 347

Wells, W. F., rearing of larvae 374

Wentworth, J., protein in oyster meat 391

Wenyon, C. M., structure of cilia 132

Werner, B., veliconcha, name for oyster larva 358

Westley, C. E., condition index 393

Weston, W. H., fungi, parasites on larvae 376

Wetzel, G., conchiolin in shells 39

Whaley, H. H., vertical distribution of larvae 402

Wharton, G. W., oyster bottoms 398

Stylochus 438

Whipple, D. v., effect of pH on oyster cilia 140

oxygen consumption 201

oxygen tension effect on respiration 214

vitamins in C. virginica 391

Whitman, C. O., cell lineage 346

Wilbur, K. N., calcification 99, 103

shell formation 88

Wilhelmi, R. N., precipitation reaction 266

willeniite, use in study of ciliary currents 90, 115

Willier, B. H., embryology textbook 347

Willis, V. M., radioactive waste 445

Wilson, A. J., water transport by gills and carbo-
hydrates 200

Wilson, E. B., cell lineage 346

Windsor, D. A., acetylcholine action on heart 252

Winton, F. R., tonus in muscle 166

Wiweantic River, Mass., sedimentation 411

Woelke, C. E., Pseudoslylochus 439

Wood, J. L., Haplosporidium costale 417

Woods, F. H., ovogenesis 326

primordial germ cell 299

work of lateral cilia 145

Wright, E. R., shell analysis 44

X cell (first somatoblast) 348

X-ray analysis of shells 103

Page

xerophtalmia 391

Yakina River, Oreg., transport of larvae 402

Yazaki, M., specific gravity of blood 265

yeast, use in feeding experiments 234

yield of oysters 382

local variation 382

yolk nucleus-- 325

Yonge, C. M., absorption of glucose 231, 235

anatomy 65

boring clams 456

cast of digestive system 219

ciliary currents 142

ciliary tracts of stomach 220

crystalline style present in healthy oysters. . 225

digestion __ 229

digestive diverticula 226

enzymes of blood cells 264

gastric shield formation 222

gregariousness of larvae 373

labial palps 115, 118

lipase 230

pll effect on cilia of Mya 140

pH in gut 230

secretion in style sac 225

shell morphology 26

spawning of 0. edidis 305

stomach 219

veliger, O. edulis 356

York River, Va., sedimentation 411

quantitative plankton sampling 409

Zander reaction, for chitiu . 222

zebra mussel, neurosecretion 313

Zinc 65, accumulation in tissues 387, 390

zinc, determination by Birchner's method 385

histological localization 390

ZoBell, C. E., bacteria as food. 233

zoospores of algae, as food 232

Zostera marina 429

role in feeding 233

Zuman, P., dropping mercury electrodes 214

480

FISH AND WILDLIFE SERVICE

U.S. GOVERNMENT PRINTING OFFICE; 1964 O — 733-851

iTHE

AMERICAN

CRASSOSTREA VIRGINICA Gmelin

by PAUL S. GALTSOFF

PIS HE R Y
BULL ETI N

VOLUME 64

UJSTITED STATES

DEPARTMENT OF THE INTERIOR

Fish and Wildlife Service

Bureau of Commercial Fisheries

PUBLICATION BOARD

Carl E. Abegglen Edward A. Power

Robert L. Hacker Osgood R. Smith

Mitchell G. Hanavan Walter H. Stolting

Philip R. Nelson

Leslie W. Scattergood, Chairman

MBL WHOl LIBRARY

li)H nV(3