i;

NATIONAL

PROCEEDINGS
of the
SHELLFISHERIES

ASSOCIATION

Official Publication of the National
Shellf isheries Association; an Annual
Journal Devoted to Shellf ishery Biology

Volume k&
August 1957

Published at the University of North Carolina,
Chapel Hill, North Carolina, for the National
Shellfisheries Assoc

Chapel Hill, N. C, 1958

TABLE OF CONTENTS

INTRODUCTION OF HONORARY MEMBERS.

REVIEW OF SHELLFISH BIOLOGY

The past and future of oyster research Paul S. Galtsoff 8

SANITATION OF SHELLFISH

Sanitary criteria for shellfish by

species and by area Robert L. Dow 23

Bacteriological studies of harvest- R. H. Gregory
ing and processing oysters in R. T. Hill
Virginia and J. A. Hope 30

ECOLOGY OF SHELLFISH

Unused oyster shell in South Caro-
lina suitable for seed oyster
production G. Robert Lunz hk

Survival of some juvenile bivalves

in water of low salinity Paul E. Chanley 52

Causes of depletion of oysters in R. Winston Menzel
St. Vincent Bay, Apalachicola Bay, Neil C. Hulings
Florida and Ralph R. Hathaway 66

The seasonal significance of total
solids of oysters in commercial
exploitation James B. Engle 72

An automatic water sampler for

marine shore stations Ronald E. Westley 79

Burial as a method for control of the

common oyster drill, Urosalpinx V. L. Loosanoff

cinerea of Long Island Sound". . and C. A. Nomejko 83

Vertical distribution of oyster
larvae in Delaware Bay
(Summary) Donald E. Kunkle 90

-in-

ECOLOGY OF SHELLFISH (Continued)

A. K. Sparks
Studies on the comparative utilization J. L. Boswell

of oxygen by living and dead oysters and J. G. Mackin 92

Management of the abalone fishery in

California K. Cox 103

The Maryland soft clam industry, its

potentials and problems , J. H. Manning 110

PHYSIOLOGY OF SHELLFISH

Filtering efficiency of hard clams in
mixed suspensions of radioactive
phytoplankton Rebecca Joyce Smith 115

Histophysiology of the oyster Milton Fingerman

kidney and Laurence D. Fairbanks 125

Some factors in the use of

nannoplankton cultures as food for

larval and juvenile bivalves Robert R. Guillard 13^

Disposal by oysters of intracardially

injected red blood cells of vertebrates M. R. Tripp 1^3

Some observations on blood circulation

in the oyster A. Eble lk&

MORPHOLOGY OF SHELLFISH

An abnormal Virginia oyster with a

bifurcated muscle Gordon Gunter 152

Observations on muscle attachments,
ciliary motion and the pallial
organ of oysters Paul S . Galtsof f 15k

PARASITES AND PREDATORS OF SHELLFISH

Observations on the distribution and
elimination of spores of Nematopsis
ostrearum in oysters Sung Yen Feng 162

Laboratory and field studies of an

oyster drill parasite Nelson R. Cooley 17^

-iv-

PARASITES AND PREDATORS OF SHELLFISH (Continued)

The crown conch Melongena corona
Graelin; its habits, sex ratios,
and possible relations to the oyster.... Ralph R. Hathaway 189

ASSOCIATION AFFAIRS

Annual convention 195

Officers and committees 195

Brief history of the National Shellf isheries Association 196

Membership 197

Editors ' notes 198

Information for contributors 198

Directory of members 200

TITLES OF OTHER PAPERS GIVEN AT THE 1957 CONVENTION

Some effects of storms on spatfall P. A. Butler

Bacteriological studies of growing and harvesting

oysters on the Gulf Coast M. W. Presnell & C. B. Kelly

Cooling time and its influence on the bacterio-
logical quality of oysters during processing
and shipment C. B. Kelly, M. W. Presnell & A. C. Sexton >

Survival and growth of clam and oyster larvae

at different salinities (Biol. Bull. 112: ) ,H. C. Davis

Some aspects of behavior of oysters at different

temperatures (Biol. Bull. 112:57-70) V. L. Loosanoff

Epidemic oyster disease in New Brunswick and

Nova Scotia R. R. Logie

Design and function of a suction drill dredge

for small boats James B. Engle

Oyster drill predation in Mississippi Sound C. R. Chapman

The life history of the oyster pea crab,

Pinnotheres ostrearum A. M. Christensen & J. J. McDermott

Field and laboratory studies of the crown conch,

Melongena corona Gmelin K. D. Woodburn

Some practical problems of the Delaware Bay seed

beds H. H. Raskin

The natural beds of Delaware C . N . Shuster

Oyster drills and eel grass W. J. Hargis, Jr.

Anatomical studies of the drilling mechanism of

the Gulf oyster borer, Thais haemastoma W. J. Demoran

Movement of migration of Japanese drills, Peine bra

japonica C . E . Woelke

-VI-

INTRODUCTION OF HONORARY MEMBERS

Oyster biology has benefited greatly from long continuous
service from several famous scientists. At the 1957 Convention,
three biologists, all retired from positions of authority, but all
very actively engaged in research, were introduced as Honorary Mem-
bers. Through teaching, research and students, these scientists
have made a profound impression on shellf isheries activities in this
country. Together, these three men have given approximately 157
years of service to shellf isheries.

It is our pleasure to honor these men in this volume of the
Proceedings and to acknowledge their good works. At the Convention
banquet, Dr. Trevor Kincaid was introduced by Mr. George D. Esveldt,
Dr. Paul Simon Galtsoff by Victor L. Loosanoff, and Dr. Thurlow
Christian Nelson by Dr. Melbourne R. Carriker . A brief biography of
each follows.

TREVOR KINCAID

Professor Emeritus
University of Washington, Seattle

For 69 years Dr. Trevor Kincaid has been observer, investi-
gator and councilor to the oyster industry of the West Coast of North
America. Born in Ontario in 1872, he is the son of a physician who
moved in 1889 to Olympia, the center of the native oyster industry.
Since that date Dr. Kincaid has seen native Olympia oysters decline,
eastern oysters imported but also fail, and finally Japanese oysters
become the basis for a prospering industry.

Although a professor at the University of Washington since
1901 with many other duties, Dr. Kincaid has always maintained a
keen interest in oysters. For several years he was employed by the
U. S. Bureau of Fisheries as a consultant to Olympia oysterraen. He
made surveys of state oyster reserves for the State Department of
Fisheries and for eleven years directed the fisheries laboratory of
the Department on Willapa Bay. He was intimately associated with
the men who established the culture of Japanese oysters, on the West
Coast.

Dr. Kincaid was "fully" retired by the University in 19^2 at
the age of 70 with the title Professor Emeritus in Zoology and Re-
search Consultant, but he is still active and maintains an office
and laboratory at the University. Each summer he returns to Nahcotta
on Willapa Bay where he has a series of covered tanks for experiments
with larvae and breeding of Olympia oysters. As a hobby, he has
installed a printing press in his basement and now prints his own
research papers. The latest paper, on races and clines in the gastro-
pod Thais lamellosa, contains 75 printed pages and 65 plates.

Dr. Kincaid organized the School of Fisheries of the Univer-
sity of Washington and graduated the first class before other hands
began building it into the present extensive organization entitled
the College of Fisheries. As founder and past director of the Puget
Sound Marine Biological Laboratories at Friday Harbor, Dr. Kincaid 's
reputation is worldwide and his influence reaches far beyond the realm
of oyster culture . We are proud to honor him as the father of oyster
science on the West Coast and one of the "Grand Old Men" of biological
science. Sir, we salute you!

--Jay D. Andrews

•3-

PAUL SIMON GALTSOFF

U. S. Fish and Wildlife Service
Woods Hole, Massachusetts

I consider it a distinct privilege to have the opportunity to
talk to you on this occasion about a man who is an outstanding biologist
and whose scientific efforts and contributions in the field of applied
biology will constitute a remarkable chapter in the hsitory of shell-
fisheries. This man is Dr. Paul S. Galtsoff.

It was in the early '20s that Dr. Galtsoff directed his chief
attention towards shellf isheries. Soon after, a series of excellent
articles offering comprehensive descriptions of oyster communities in
different parts of the country began to appear. Parallel with that,
numerous treatises on the ecology and physiology of oysters, represent-
ing original research, were published. Biologists soon noticed that
in his experiments, Dr. Galtsoff stressed precise, quantitative
approaches, skillfully separating scientifically established facts from
certain old conceptions sometimes founded only on superstitions or even
emotions. Studies of behavior of oysters in relation to various fac-
tors of environment became more and more stressed in Dr. Galtsoff ' s
contributions and, later on, in those of his associates. In general,
under his leadership oyster research ceased to resemble, in some
respects, the fine arts and began to approach an exact science.

The results of Dr. Galtsoff s work, could not remain unnoticed
and soon his name became familiar in every school, where the principles
of aquatic biology were taught. His contributions were also eagerly
studied by research workers. Because of this I consider that Dr.
Galtsoff, indirectly, has had probably more students and has influenced
more research workers than any of his contemporaries working in the
same field.

In the past Dr. Galtsoff has held several offices in the
National Shellf isheries Association. Here again he worked hard to
attract the best men to work with us. During this period, as well as
during his entire career as a biologist, Dr. Galtsoff was especially
concerned with the preservation and development of oyster industries.
From his earliest days as an aquatic biologist he accepted an uncompro-
mising attitude towards pollution. In several cases, by precise
scientific approaches, he and his associates demonstrated the many-
sided effects of some pollutants. In some of these situations he stood
virtually alone defending his principles. Broadly speaking, he be-
lieves that "there is no pollution that is good pollution" and that a
means should be found to protect our rich aquatic resources from
destruction. Most of us are familiar with his extensive studies on
effect of various industrial pollutants directly on oysters and in-
directly on their environments. These studies conducted in Louisiana
and the York River will long be considered and, as a matter of fact,
are at present used as standards in pollution studies.

--Victor L. Loosanoff

-4-

-5-

THURLOW CHRISTIAN NELSON

Research Specialist, The Julius Nelson Professor of Zoology, Rutgers,
State University of New Jersey

Professor Nelson's warm personality, keen fertile mind, broad
training and interests in biological sciences, and his enviable gift
of vigorous oral and written expression are well known. Over the
years these rich assets have given direction, vitality, and character-
istic "Nelsonian" color to his stimulating teaching, forceful speaking,
lucid writing, productive research, and devoted public service.

This able scientist has energetically and effectively advanced
research and training in the field of shellfish biology. Early in his
professional career he became affiliated with our Association and soon
assumed a leading role. Almost every Association Convention has been
enriched by his scientific papers and discussions. He is quick to
capture significance of scientific discoveries and to underscore poten-
tial practical applications. He is characterized by a unique capacity
to ferret out profitable new lines of research and is generous in
transmitting these discoveries.

Dr. Nelson has contributed much to popularization of technical
reports in shellfishery biology by writing biologist's language in
prose understandable and stimulating to general readers. As a member
of the Department of Zoology at Rutgers University for 37 years and
chairman for 28 years; as Biologist for the New Jersey Board and
Council of Shell Fisheries since 1928; as a member of the New Jersey
State Water Policy Commission and of its successor the Water Policy
and Supply Council since 1929> and for the past 13 years its chair-
man; and as a member of the U. S. Public Health Service Committee on
Shellfish Sanitation, he has rendered services in the fields of water
supply and shellfishery biology to a degree which it is not yet possi-
ble to evaluate .

In American Men of Science his specialties are listed as
biology of the oyster, estuarine ecology, marine biology, and limnology.
Advancement of these disciplines has provided background for more than
125 contributions to knowledge of parasitology, water supply, and
anatomy, physiology, and ecology of the oyster and associated organ-
isms. On July 1, 1956 Professor Nelson was granted permanent leave
from the Rutgers campus with the title of "The Julius Nelson Professor
of Zoology". He retains active membership in the Graduate Faculty.
With full devotion to scientific writing, he aims to revive voluminous
unpublished researches --affectionately nicknamed, by him "my morgue".
We wish him every success!

--Melbourne R. Carriker

THE PAST AND FUTURE OF OYSTER RESEARCH

Paul S. G-altsoff

Shellfish Laboratory, Bureau of Commercial Fisheries
U. S. Fish and Wildlife Service, Woods Hole, Mass.

I wish to express my thanks to the officers of your organiza-
tion for the invitation to address your opening session. This in-
vitation gives me an opportunity to present some of the ideas which
have developed in my mind during the 3-l/2 decades of my participa-
tion in oyster research in this country. As the title of my address
indicates, I intend to speak of the past and future of oyster research.
The omission of the present is deliberate because I am certain that
all the members of this convention are fully aware of the current
developments and achievements in oyster biology. Only occasionally,
I shall refer to current research in relation to my discussion of
future trends .

The beginning of the study of the oyster is shrouded in
ancient history. We all know that oyster culture was practiced many
thousands of years ago and that the oyster culturists of ancient
times succeeded in developing the fundamental techniques of oyster
farming which are used now. The Middle Ages hardly contributed any-
thing worthwhile to the progress of zoology. The development of
natural sciences during the Renaissance and the following period
brought to light several problems in mollusk anatomy and physiology
which are vitally important today. One example may suffice. In
1686 Anton de Heide described a gelatinous structure found in the
stomachs of mussels and other shellfish. He called it "stylus
cristallinus", a name retained ever since. Heide hazarded two
guesses: that the crystalline style has something to do with repro-
duction, and that the structure is involved in the process of diges-
tion. How prophetic was the latter surmise!

For more than two centuries the strange worm-like structure
continued to puzzle biologists until in 1900 Coupin in France and
Mitra in England, working independently, showed that the style is a
protein rod saturated with digestive enzyme. During the intervening
period at least seven different groups of theories were advanced to
explain the nature and function of the style. The story of the
style is given in detail by T. C. Nelson in his paper of 1918 in
which he discusses the significance of this organ in lamellibranch
physiology. This valuable contribution contains also the proof that
the crystalline style is rotated inside its sac and in this mechani-
cal way expedites the separation of food from sand in the stomach,
while the dissolving style digests carbohydrates.

The last quarter of the 19th Century was the time of rapid
advances in invertebrate anatomy and embryology. As a part of this
general progress many facts regarding the structure of oysters and
related mollusks were reported. At the beginning of the present
century biologists began to pay more attention to life histories and
mode of living of creatures of the sea. Aquatic biology, a new branch
of science, was born and investigators began to emphasize the relation-
ship between organism and environment, a new trend which found its
full expression in modern ecological studies.

I want to limit my discussion today to the main phases of
oyster research carried on in this country and abroad during the
30 years from 1921 to 1951 • I know, of course, that breaking the
continuity of historical events is arbitrary, but my reason for
selecting this particular period is two-fold. First, during this
time oyster research rapidly expanded and produced thousands of
technical and popular papers. The second reason is a personal one.
My association with oyster investigations in this country began in
the twenties and has continued without interruption until the present.
I believe, therefore, that I am in a position to add a little color
to my technical presentation.

In 1921, when I joined the U. S. Bureau of Fisheries, the
post of the Commissioner of Fisheries was occupied by Dr. Hugh Smith,
a man of extraordinary scientific ability and broad intellectual
interests. He was an inspiration to the young members of the Bureau
and his collecting trips to the waters of Cape Cod, which he made
regularly during the summers he spent at Woods Hole, gave us an
opportunity to acquire a wealth of information about the local fishes
and their habits. In his office he seemed to be reserved and rather
formal. But we soon realized that under this appearance was a human
heart of great kindness and understanding. An M. D. by education, he
forsook the medical profession for ichthyology and became a world
authority on fishes.

The position of Deputy Commissioner of Fisheries was occupied
by Dr. Henry Frank Moore, whose name is familiar to many of us attend-
ing this convention. Dr. Moore's extensive work on the biology of
oysters, his surveys of oyster grounds in Texas and Louisiana, and
his penetrating study of the problems related to oyster feeding and
oyster culture form a chapter in oyster studies of this period. Many
investigations conducted after he finished his work started from the
point where Dr. Moore left them. I specifically refer to his method
of collecting the material discarded by the oyster and his quantita-
tive studies of the food and feeding habits of this mollusk. The
application of Moore's technique made it possible, for Dr. Thurlow
Nelson to develop the method for accurate determination of the amount
of water filtered through the oyster gills. This technique has
greatly advanced our knowledge of the physiology of the oyster.

-9-

In the early twenties scientific circles in the United
States showed very little interest in marine sciences in general.
Persons trained in this discipline were scarce. Wo courses in ocean-
ography and fishery biology were given at any college, and consequently
the Government positions which required training in this field remained
vacant. My appointment to the staff of the Bureau of Fisheries as a
naturalist of the Albatross was made on the basis of Section 10, Rule
2, of the Civil Service Commission which gives a Commissioner authority
to fill existing vacancies without competitive examination. The posi-
tion of naturalist of the Albatross remained vacant for nearly two
years before my appointment on December 20, 1921. '

Oyster research at this time centered in New Jersey where Dr.
Thurlow C. Nelson continued the work started by his father, with
special attention to behavior of oyster larvae, feeding of oysters,
and shell movements. He made the first attempts to record behavior
of the mollusk in its natural habitat; by using a kymograph he
observed the peculiar contractions of the adductor muscle during
spawning.

The importance of filtration of water by oysters and other
pelecypods was already understood at that time, especially in its
sanitary aspects. The question of obtaining pure oysters, devoid of
contamination by pathogenic bacteria, became an acute and vital issue
to the industry. The consuming public had no assurance that shell-
fish sold on the market came from unpolluted water and therefore was
not dangerous to eat. A disastrous crisis in the oyster industry
caused by the epidemic in Chicago in 1924-25 left the leaders of the
industry disorganized and in great doubt concerning the future of
their business. I clearly remember the great tension felt by every-
body present at the meeting called in 1925 by the Commissioner of
Fisheries to find a practical solution for a difficult situation.
Sales dropped almost to zero; hundreds of letters were received every
week by the Bureau asking if all oysters were contaminated, and
several housewives had written us that they discontinued buying
chicken because poultry was fed on oyster shells. The Government
officials, members of the state and federal public health services,
two or three biologists, and many oyster growers were present at the
meeting. The main scientific questions under discussion were the
rate of water filtration and the effect of temperature on feeding
and so-called hibernation of oysters. I was asked how temperature
affects the rate of feeding of the oyster--and was not able to give
an answer because the problem had not been studied. From bacterio-
logical data it became evident that the Bacterium coli (now
Escherischia coli ) score in oysters is lowest in winter and highest
in summer. Bacteriologists (Gorham, Gage and others) concluded from
these observations that the difference must be due to suppression of
vital activities by low temperature and called it hibernation. As a
consequence of the conference I started a study of the ciliary motion
of the gills and found that this motion is suppressed at temperatures

-10-

below kQ F. In the course of this study two methods were developed
for measuring the volume of water passed through the gills. In the
first method rubber tubing was introduced into the cloaca and made
fast by cotton paddings. The free end of the tubing was attached to

lorizontal glass tube of known diameter and graduated in centimeters.
The velocity of the current inside the tube was measured by adding,
through an inverted T tube, small quantities of carmine suspension

lich formed a clearly visible cone moving along the entire length of
the tube. The so-called "carmine cone" method proved to be useful
for rapid bioassays in cases when it was necessary to demonstrate the
effects of toxic substances on ciliary mechanisms.

The other method involved the use of one large and one small
connecting vessel in which the level of running water was kept con-
stant. An oyster with the rubber tubing inserted into the cloaca
was placed into the large vessel; the free end of the tubing was con-
nected to a glass tube inserted in the wall between the two vessels.
Water pumped by the oyster into the small vessel was collected through
an overflow, measured and analyzed. With modifications added by
various investigators the method became generally used in studies of
feeding and respiration of oysters.

Determination of the rate of water pumping was much improved
by Thurlow C. Nelson's apron technique which consisted of wrapping the
oyster in plastic in such a way as to intercept the excurrent water.
At the same time Nelson called our attention to the existence of the
promyal chamber in the species of oysters which are now grouped under
the generic name Crassostrea. The promyal chamber was depicted in
earlier work of J. L. Kellogg (1892) but the significance of this
asymmetrical area on the right side of the body was overlooked. The
importance of the promyal chamber fully justifies the splitting of
the genus Qstrea into two genera, Crassostrea with the chamber and
Ostrea without it.

Almost every oyster biologist of the last quarter of a century
was more or less involved in a study of sex, spawning, and setting of
oysters. In a series of carefully documented papers W. R. Coe (193 6,
1938) showed that along the coast of the United States from New Eng-
land to the Gulf of Mexico young oysters, less than one year old are
predominantly males. The sex ratios are, however, highly variable.
In northern localities there may be only 8 females to 100 males while
in southern waters the ratio increases to an average of kO females to
100 males. The reasons for the diverse sex ratios are unknown. The
differences may be attributed according to Coe to local races with
differing genetic factors and to environmental conditions. During
the second year of their life some of the males change sex and become
females. In this way a normal 1:1 sex ratio is restored.

A. B. Needier (1932) found that adult C. virginica also change
their sex. Her observations were fully confirmed by my experiments

-11-

with about 200 five -year -old oysters which were kept in the hay near

the laboratory for six years. Every summer each of these oysters was

induced to spawn and its sex ascertained. About 10 percent of them

changed sex annually either from male to female or vice versa. One

oyster changed its sex five times. Sex changes in adult Japanese

oysters, C. gigas, were described by I. Ameniya (1929). Rapid sex

changes in the hermaphroditic 0. edulis and 0. lurida were reported

in considerable detail by J. H. Orton (1927-33, 1936), R. Sparck

(1925) and W. R. Coe (1932).

>

Hundreds of papers in oyster literature deal with the effect
of temperature of water on spawning. From purely descriptive data it
is difficult to determine the nature of this relationship. My experi-
mental studies of the physiology of reproduction of C. virginica
(Galtsoff, 1938) showed the fundamental differences between male and
female spawning. The discharge of ripe eggs by the female is accom-
plished by a complex process in which eggs are first discharged into
the suprabranchial chamber, then forced through the gills into the
mantle cavity, and finally are thrown off by violent movements of
the valves. Spawning of the male is much simpler; sperm is discharged
into the cloaca and is dispersed in water by the outgoing current. No
definite temperature of spawning can be established for a given popu-
lation of oysters. In my experiments specimens at different degrees
of ripeness spawned at any temperature between 21 and 32°C. Some of
them were able to spawn only under the combined influence of tempera-
ture and chemical stimulation by sperm. I would prefer to think of
a critical condition of a fully ripe organism ready to discharge its
sexual products rather than the existence of critical temperatures.
Experimental evidence is against the latter view. The trigger-like
effect in releasing sex products from ripe gonads may be produced by
slight changes in temperature at different levels, by chemical stimu-
lation, and by a combined effect of both agents. An attempt to
explain the differences in the temperatures of spawning of C. virginica
living at different latitudes was made by L. A. Stauber (19^7) by
assuming the existence of "physiological races". This view is shared
by some biologists (V. L. Loosanoff and C. A. Nomejko, 1951) but in
my opinion the final test should come from study of the genetics of
the oyster. The existence of a "physiological race" can be proved
only by breeding experiments and by testing under controlled condi-
tions the spawning reaction of specimens of known genetic origin.

Spawning of oysters has been associated frequently with lunar
phases. The work of the Milford Laboratory under Loosanoff demon-
strated that such relationship does not exist in C. virginica in Long
Island Sound, although according to P. Korringa (19^-7) it is present
in 0. edulis.

Practical considerations demanded that oyster biologists
predict time of spawning and setting. Based on many years of obser-
vations the laboratory at Milford arrived at a general empirical

-12-

formula which lias proved to be useful. A slightly different but also
empirical method is being used by the oyster biologists of the Pacific
Coist in predicting spawning and setting of C. gigas. There are so
many factors affecting the life and survival of oyster larvae that
probably many years of intensive research will pass before the pre-
diction of time and intensity of setting can be placed on a sound
scientific basis.

The oyster larva still remains a very elusive little creature.
Correct identification of larvae of oysters and other pelecypods pres-
ents great difficulty. Many so-called quantitative records of the
abundance of larvae in various estuaries and sounds are unreliable
because of probable errors of identification. One incident, which I
remember very clearly, illustrates this point. An oyster culturist
employed by the Conservation Commission of one of the coastal states
reported faithfully every year the number of oyster larvae found in
July and August in the waters under the jurisdiction of the state.
One day he showed me his samples which supposedly contained many
thousands of larvae in advanced stages of development. I saw under
a microscope numerous round diatoms of the genus Coscinodiscus but
no pelecypod larvae. The man was quite indignant when I tried to
explain the difference between the two forms; he told me that what
he was doing was the established practice of his office and, "any-
way, " he added, "we do not want to be troubled with scientific
technicalities".

In the last ten years the problem of identification of larvae
was greatly facilitated by the work of the Milford Laboratory which
distributed microscopic preparations of larvae, the identity of which
was beyond any doubt because they were all produced from eggs fertil-
ized and developed in the laboratory.

A classification of laraellibranch larvae, based on the struc-
ture of hinge and detailed descriptions of larvae of several species
of American and European oysters greatly facilitates identification
(G. Ranson, 19^+8; C. B. Rees, 1950).

Although we have at present a fair idea of the time and place
of setting of oysters in various localities, the questions how and
why larvae select definite vertical levels for attachment, and what
causes their aggregation in one place in preference to another, remain
unanswered. Hydrographical studies conducted in Chesapeake Bay,
jointly by D. W. Pritchard (1953) of the Chesapeake Institute, the
Virginia Fisheries Laboratory, and the U. S. Fish and Wildlife Service,
show that the pattern of circulation in a tidal estuary is a major
factor in the transport and retention of oyster larvae. This factor
is greatly influenced by prevailing winds. In this situation the
density of water may play a predominant role in influencing vertical
distribution of larvae, an idea developed long ago by New Jersey
biologists who studied the setting of oysters in Barnegat Bay.

■13-

Correct interpretation of ecological factors which control
setting is difficult because of the insufficiency of experimental data
concerning the physiology and behavior of oyster larvae. So far, the
experimental approach to the problem has been impossible because of
the uncertainty in obtaining sufficient numbers of healthy larvae of
known age for this type of study.

The need for raising oyster larvae in tanks was recognized
long ago. In 1920, Dr. W. F. Wells developed a technique for growing
larvae in large jars in which sea water was changed by centrifuging. >
For several years he conducted an oyster hatchery at Oyster Bay and
later transferred his operation to the plant of the Blue Point Oyster
Company at West Sayville. Although a fairly large number of young
oysters was produced in this way, the method was too complex and un-
certain to be of practical value.

Food and feeding is the main problem in raising oyster larvae.
Great advances in this problem were made in 19^-0 by J. R. Bruce, M.
Knight and M. W. Parker (19^0) in England. They definitely established
the feasibility of rearing larvae of 0. edulis by feeding several
species of flagellates. In 19^9 and 1950 Imai and Hatanaka in Japan
were successful in raising C. gigas and other species by feeding
larvae with naked flagellates which they cultured in tanks. In this
country V. L. Loosanoff (195^) arid H. C. Davis (1953) at the Milford
Laboratory developed an excellent technique of raising pelecypod
larvae by feeding them several species of naked flagellates and by
determining the nutritive value of these microorganisms. The results
open up new and broad possibilities for scientific research on larval
physiology and ecology.

Ecological studies conducted during the past 25-35 years
failed to answer the most important question—why certain grounds
produce an excellent set but are not suitable for rapid growth and
fattening of marketable oysters, while other grounds, not far away
from the first ones, are good for growing marketable stock but can-
not be used to catch spat. Statistical study of associations or
correlations between the setting and various factors of the environ-
ment so far have failed to produce tangible results. I am certain
that the solution of this problem eventually will be found through
a well planned study of the physiology of feeding and food require-
ments of larval and adult oysters.

The knowledge of feeding requirements is equally essential
for understanding the reasons for differences in the productivity of
oyster bottoms. There are many places along the coast where oyster
beds located only a couple of miles apart are markedly different in
their productive capacity and in the flavor of oyster meat. Routine
biological and chemical analyses show no significant differences
between the waters of the adjacent bottoms. Apparently other com-
ponents of oyster environments which are not disclosed by present
methods of study are responsible for differences.

-Ik-

It has been well established by numerous investigations con-
ducted in this country and in Europe that chemical composition of
oyster meat does not remain constant throughout the year. Particu-
larly spectacular is the seasonal cycle of glycogen. The accumula-
tion of this carbohydrate usually takes place in the autumn when
oysters become fat. We still know very little about the carbohydrate
metabolism of lamellibranchs. This consists of complex biochemical
reactions in which several enzymes are involved. Likewise, the
breaking down of carbohydrate in the oyster has not yet been studied.
It is reasonable to assume that an insulin-like substance may be
present in the oyster but so far attempts to extract insulin from
oysters have given negative results. Work along these lines appears
to offer interesting possibilities for further research.

Digestion in the oyster and various enzymes involved in this
process were studied by C . M. Yonge (1926) in England. The continu-
ation of these investigations using modern techniques appears to be
very promising, since the question of the ability of oysters to
digest certain types of food has not yet been settled.

Pathology of the oyster is a relatively new science. Most
parasites and commensals found in the oyster, such as protozoa,
sponges, crustacea and worms, are not pathogens in a true sense of
the word although they may inflict damage to the tissues of the
host. The discovery of the fungus-like microorganism Dermocystidium
marinum a few years ago by J. G. Mackin, H. M. Owen and A. Collier
(1950), and the studies by S. M. Ray (1954), focused the attention
of oyster biologists on this dangerous pathogen as a probable cause
of extensive mortalities among southern oysters. It is reasonable
to expect that further pathological studies may disclose other types
of infection including disease-producing viruses.

Many research studies conducted in the United States were
concerned with the problem of the decline in oyster production. The
question is of great complexity because the productivity of shell-
fish grounds depends on interaction of many physical and biological
factors. The annual production of oysters in the United Stated has
declined from an average level of 165 million pounds of meat during
the decade 1893-1902 to 77 million pounds per annum in 19^3-1952
(P. S. Galtsoff, I956). Several factors may be responsible for the
decrease, but the primary cause is the destruction of public grounds
especially in the Middle Atlantic and South Atlantic States. The
acreage of productive oyster bottoms in these states has been steadily
decreasing through overfishing, dredging of coastal waters for navi-
gation, sedimentation, and pollution. Lack of management or ineffec-
tive management of public grounds has also contributed to the general
trend and may be considered as a major factor in the depletion of
oyster resources. Numerous studies conducted by various states and
federal organizations demonstrated that the production of oyster
bottoms cannot be maintained on a sustained basis without applying

-15-

oyster-farming methods. Several years ago the problem of state
management was investigated in Maryland by J. B. Engle of the U. S.
Fish and Wildlife Service, and by our- present director of the Oyster
Institute. The cost of rehabilitation of the depleted oyster grounds
was determined and several possibilities were found for developing
seed -producing areas of the state. Because of high initial cost of
this type of work, state governments are not in a position to under-
take large scale projects of rehabilitation. This could be accom-
plished by private oyster farmers, but strong opposition against
private leasing prevails in many southern states and usually succeeds
in preventing passage of necessary legislation.

About 25-30 years ago the feeling against private oyster
farming in some communities along the Atlantic seaboard was so strong
that anybody who publicly advocated this course could have met with
physical violence. On one occasion I was threatened with this possi-
bility after a discussion of local oyster problems at a public meet-
ing in a small town.

At present the low level of productivity of public grounds
is maintained by administrative measures designed to restrict the
efficiency of harvesting. The use of inefficient and therefore
expensive methods of fishing is, however, against the general trend
in other industries which try to attain the highest efficiency
through mechanization and automation.

No oyster biologist in the employ of a state or federal
agency can escape being involved in the problem of pollution. In-
creased discharges of domestic sewage from municipalities and of
trade wastes from industrial centers greatly damage shellfish bottoms.
According to the Public Health Service statement in a publication
entitled, "Environment and Health" (I95l)> there were in 1950-51
over 22,000 sources of pollution in the United States, 11,800 munici-
pal sewer systems and 10,1+00 factory waste outlets. Only 9>300 treat-
ment plants were in operation; the remaining sources discharged their
wastes without even a partial treatment. Pollution of inshore waters
became so great that at least 400 shellfish growing areas were con-
demned by state authorities. Damages caused by pollution are exten-
sive. For instance, in New York-New Jersey harbor, water pollution
has caused an estimated 100 million dollars of damage in 20 years to
fisheries, recreation, and property values.

Biological aspects of water pollution are well understood.
But pollution is primarily a social problem, the solution of which
can be attained through awareness of its great dangers and education
of the general public. As Representative Karl E.. Mundt stated in
his defense of the Ant i -Pollution Bill in I9U5, "Of all the natural
resources, water today suffers from the greatest amount of public
neglect . "

-16-

Numerous investigations conducted in Europe and in this
country have demonstrated the toxicity of various chemical compounds
that are discharged with industrial wastes. The deleterious effect
of pollution is frequently difficult to prove because in a complex
ecological environment the injury caused by poison may be masked.
Pollution frequently weakens organisms, makes them more susceptible
to changes in the environment, and interferes with reproduction and
feeding. Mortality may be due to combined effect of these conditions.

Virtually every state and federal laboratory of this country
has been engaged in the problem of control of predators. Gastropod
snails are undoubtedly the most destructive enemies of the oyster.
Partial success in controlling predators has been achieved by trapping,
dredging, barrier construction, and other physical means. In the
past, careless bedding of native oysters or introduction of foreign
species were the main causes of the wide distribution of carnivorous
drills. These snails have only limited powers of locomotion but are
brought to new territories with shipments of infested oysters. Their
inroads are especially destructive in a new environment in which
natural enemies are absent. Suffice to mention the introduction of
Urosalpinx into European waters and the spread of Tritonalia
japonica in Puget Sound.

The future welfare of the oyster industry calls for the solu-
tion of the following problems: reliable production of seed oysters;
production of rapidly growing oysters with high-glycogen and high-
solid contents; selection of disease-resistant races; improvement of
methods of self -purification of oysters; scientific evaluation or
appraisal of oyster bottoms based on their productive capacity; and
development of oyster pond culture in tidal marshes and other areas
not used at present for oyster farming.

Solution of these problems presents a challenge to young
oyster biologists who are just entering this field of research. From
the background of heretofore accumulated studies they may proceed with
critical and basic research intended to answer the following questions.
How oyster larvae feed and grow, how they react to physical and chemi-
cal changes of the environment and why they set in certain places and
levels and not in others. To answer these questions elaborate physio-
logical studies should be conducted simultaneously with ecological
observations.

Production of oysters of highest quality, judged not by their
size alone, but by flavor and high content of solids and glycogen is
essential for the future of the industry. The problems of carbohydrate
metabolism, feeding, and digestion should be studied and understood
in order to attain this goal. It is futile to enumerate the various
organisms that may be found in the stomachs of the oyster without
knowing their nutritive value. Advance in physiology of nutrition in
shellfish will lead to establishment of feeding or fattening basins
or ponds in which shellfish may be rapidly conditioned for market.

-17-

The progress in methods of raising oyster larvae gives us
assurance that the study of oyster genetics may be undertaken with
reasonable expectation of success. Modern genetics tells us that
variability and adaptability of a species depend on gene combinations
and chromosome changes. The oyster is an extraordinarily adaptable
and plastic organism. It can be assumed on theoretical grounds that
wild populations contain many mutants which can be selected by proper
breeding techniques. Races having desirable characteristics can be
bred and used for establishing breeding stocks of particular qualifi-
cations needed for local conditions. The work along these lines
requires the establishment of oyster breeding centers with adequate
numbers of tanks or ponds, adequate water supply and culture houses
for mass production of microorganisms needed as food for larval and
young oysters. Experiments on hybridization can be conducted in
these places in order to obtain the most desirable hybrids.

Ecological studies in the future should consider the oyster
in relation to its physical and biological environment. Emphasis
should be placed therefore on the factors which control the entire
community of an oyster bottom. It seems erroneous to concentrate on
a study of the biology of a single species without paying attention
to other organisms which form an integral part of an ecological sys-
tem.

The struggle for protection of our inshore waters and oyster
grounds from pollution is one of the major tasks of the conscientious
biologist who feels moral responsibility towards his country. Ample
scientific evidence permits us to state that any pollution of water
is bad and that no harmless pollution ever existed in spite of the
claims made to the contrary by some industrialists. All types of
pollutants which existed in the pre -atomic age have one common
characteristic; after being discharged into natural waters they are
oxidized and undergo other chemical changes which eventually render
them harmless.

At present we are facing a more serious problem of safe
storage and elimination of radioactive wastes which cannot be destroyed
by ordinary methods known to us. These wastes can be only isolated in
special containers or dispersed in water or air in order to reduce the
level of their radioactivity. There are at present more than 4,000
establishments in our country using radioactive isotopes which have to
be disposed of without endangering life. With rapid industrial develop-
ment of nuclear energy the problem will soon reach a critical stage.
The ability of shellfish to accumulate metals in their bodies and
shells makes them particularly susceptible to certain radioactive
elements. This new situation confronting the oys/ter industry should
be given serious consideration. I trust that the realization of
dangers involved in the radioactive pollution of sea water will stimu-
late intensive research and that a satisfactory solution of the prob-
lem will be found in the nearest future.

-18-

It is cleat' from my presentation thai considerable investment
of public and private funds is necessary to implement the outlined
program. No doubt the oyster industry will bear a fair share of this
burden. I believe the industry should do it if it wants to survive.

Centuries ago oysters constituted a cheap food, both in Europe
and in this country. The following quotation from Dickens' "Papers of
the Pickwick Club" expresses this idea. "It is a very remarkable cir-
cumstance, sir," said Sam, "that poverty and oysters always seem to
go together --the poorer a place is, the greater the call there seems
to be for oysters."

Very rapidly the oyster became a luxury in England while in
this country it continued to be inexpensive and available to persons
of low income groups. The situation changed during the last decade
and the American oyster moved into a class of luxury food which poor
people can no longer afford. Being a low energy food, valuable for
its nutrient quality, but providing much less calories than other
types of food, they oyster probably will remain in this category. It
is imperative, therefore, that greater consideration be given by
oyster producers to the attractiveness, the flavor, the content of
solids and the purity of their product. To solve these problems they
have to rely on the scientific resources of modern science and the
skill and imagination of young biologists.

References

Ameniya, I. 1929. On the sex-change of the Japanese common oyster,
Ostrea gigas. Proc. Imp. Acad. Japan (Tokyo) 5(7): 284-286.

Bruce, J. R., M. Knight and M. W. Parke. 1940. The rearing of oyster
larvae on an algal diet. Jour. Mar. Biol. Assoc. 24: 337~374.

Coe, W. R. 1932. Development of the gonad and the sequence of the

sexual phases in the California oyster (Ostrea lurida). Bull.
Scripps Inst. Oceanog. Univ. Calif. Tech. Ser. 3(6): 119-144.

1936. Environment and sex in the oviparous oyster Ostrea
virginica. Biol. Bull. 71: 353-359-

1936. Protandry in the Virginian oyster. Anat . Rec. 67
(Suppl. 1): 89-

1938. Primary sexual phases in the oviparous oyster. Biol.
Bull. 74: 64-75.

Coupin, H. 1900. Sur les fonctions de la tige cristalline des

acephales. Compt. Rend. Acad. Sc . , Paris, 130: 1214-1216.

Davis, H. C. 1953- On food and feeding of larvae of the American
oyster, C. virginica. Biol. Bull. 104: 334-350.

-19-

Gage, S. DeM:, and F. P. Gorham. I925. Self -purification of oysters
during hibernation. Amer. Jour . Pub. Health 15: IO57-IO6I.

Galtsoff, P. S. I926. New methods to measure the rate of flow pro-
duced by the gills of oyster and other molluscs. Science 63:
233-23^.

1928. The effect of temperature on the mechanical activity of
the gills of the oyster (Qstrea virginica Gm). Jour. Gen.
Physiol. 11: 415-431.

1935- The biology of the oyster in relation to sanitation.
Amer. Jour. Pub. Health 26(2): 245-247.

1937- Observations and experiments on sex-change in adult
American oysters, Qstrea virginica. Biol. Bull. 78: 356.

1938. Physiology of reproduction of Qstrea virginica Part I,

II. Biol. Bull. 74: 461-486; 75: 286-307.

1940. Physiology of reproduction of Qstrea virginica, Part

III. Biol. Bull. 78: 117-135.

1956. Ecological changes affecting the productivity of oyster
grounds. Trans. 24th North Amer. Wildlife . Conference, March
5-7, 1956: 408-419-

Gorham, E. P. 1912. Seasonal variations in the bacterial contents
of oysters. Amer. Jour. Pub. Health 2: 23-27.

Heide, A. de. 1686. Experimenta circa sanguinis missionem, fibras
motrices, utricam raarinam, etc. - accedunt auctoris observa-
tions medicoe nee non anatome Mytuli : 35-36. Editio Nova.
Arasterdami .

Iraai, T. and M. Hatanaka. 1950- Studies on marine non-colored

flagellates, Monas sp., favorite food of larvae of various
animals. Sci. Rep. Tohoku Univ. 4th Ser. 18(3): 304-315.

Kellogg, J. L. 1892. A contribution to our knowledge of the mor-
phology of lamellibranchiate molluscs. Bull. U. S. Fish
Coram. 10: 389-436.

Korringa, P. 1947- Relations between the moon and periodicity in
the breeding of marine animals. Ecol. Monogr. 17: 347-381.

Loosanoff, V. L. 1954. New advances in the study of bivalve larvae.
Amer. Scientist 42: 607-624.

-20-

and C. A. Noraejko. 19^6. Spawning and setting of the American

oyster, 0. virginica, in relation to lunar phases. Ecology
32: 113-134.

1951' Existence of physiologically-different races of oysters,
Crassostrea virginica. Biol. Bull. 101: 15I-I56.

Mackin, J. G., H. M. Owen, and A. Collier. 1950. Preliminary note

on the occurrence of a new protistan parasite, Dermocystidium
marinum n. sp., in Crassostrea virginica (Graelinji Science
111: 328-329.

Needier, A. B. 1932. Sex reversal in Qstrea virginica. Contr. Can.
Biol, and Fish. 7(22): 285-29^.

Nelson, T. C. 1918. On the origin, nature, and function of the

crystalline style of lamellibranchs . Jour. Morph. 31: 53-111.

1924. The attachment of oyster larvae. Biol. Bull. 66:
143-151.

1925. Recent contribtuions to the knowledge of the crystalline
style of lamellibranchs. Biol. Bull. k9: 86-99.

1928. Investigations of spawning and setting of oysters in
Barnegat Bay, New Jersey. Rept. Dept. Biol. N. J. Agric.
Exp. St. 1927: 77-83.

1928. On the formation of saliniclines in Barnegat Bay, New
Jersey, with observations of the reactions of oyster larvae
to salinity gradients. Anat . Rec. kl: 8^-85.

1936. Water filtration by the oyster and a new hormone effect
upon the rate of flow. Proc. Soc. Exp. Biol, and Med. 3k:
189-190.

1938. The feeding mechanism of the oyster. I. On the pallium
and the branchial chambers of Qstrea virginica, 0. edulis and
0. angulata, with comparisons with other species of the genus.
Jour. Morph. 63: l-6l.

Orton, J. H. 1927-1933 • Observations and experiments on sex-change
in the European oyster, Parts I, III, IV. Jour. Mar. Biol.
Assoc. Ik: 967-KA5; 19: I-53.

1936. Observations and experiments on sex-change in the
European oyster, Qstrea edulis L. Mem. Mus. Roy. Hist. Nat.
2nd Ser., Fasc. 3: 997-IO55.

-21-

and C. Amirthalingam. 1930- Observations and experiments on

sex-change in the European oyster (0. edulis) Part II. On
the gonad of egg-spawning individuals. Jour. Mar. Biol.
Assoc. 17: 315-324.

Pritchard, D. W. 1953- Distribution of oyster larvae in relation
to hydrographic condition. Proc. Gulf and Carribbean Fish.
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Public Health Service. Federal Security Agency. 1951. Environment
and Health. 152 pp. Washington, D. C.

Ranson, G. 1948. Prodi ssoconches et classification des ostreides
vivants. Bull. Mus. Nat. Belg. 24(42): 1-12.

Ray, S. M. 1954. Biological studies of Dermocystidium marinum a
fungus parasite of oysters. Rice Inst. Pamphlet, Special
Issue, Nov. 1954. 114 pp.

Rees, C. B. 1950- The identification and classification of

lamellibranch larvae. Hull Bull, of Mar. Ecol. 3(19): 73- 104.

Sparck, R. 1925' Studies on the biology of the oyster (Qstrea edulis)
in the Limf jord, with special reference to the influence of
temperature on sex-change. Rept. Dan. Biol. Sta. 30: 1-84.

Stauber, L. A. 1950. The problem of physiological species with

several references to oysters and oyster drills. Ecology 31:
109-118.

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295-386.

-22-

SANITARY CRITERIA FOR SHELLFISH BY SPECIES AND BY AULA

Robert L. Dow

Department of Sea and Shore Fisheries
Augusta, Maine

Abstract

Recommendations to establish shellfish sani-
tary criteria by species and areas were approved by
the National Shellfish Sanitation Conference in 195^
on the basis of laboratory and field observations
and experiments reported by the writer.

To implement these recommendations (l) fur-
ther studies were conducted in Maine to evaluate the
relative importance of hydrographic, geological and
biological factors having actual or potential influ-
ence on the sanitary qualities of shellfish growing
areas and (2) cooperative experiments among the
several northeastern states (Maine, Massachusetts,
Rhode Island, Connecticut and New York) have been
carried on to establish standards for (a) blue mussel
(Mytilus edulis) and (b) (still in progress) soft
clam (My a arenaria) shell stock.

Introduction

Traditionally the problem of sanitary control of shellfish-
growing areas has been the responsibility of chemists, bacteriologists
and sanitarians. With the development of biological knowledge of the
several shellfish species and greater understanding of factors influ-
encing the growing area, it has become apparent that problems of
hydrography, of geology and of biology must also be given considera-
tion in the establishment of criteria for adequate sanitary control
of shellfish-growing areas.

To establish sanitary standards for shellfish-growing areas
without taking into account biological differences among the several
shellfish species, or to conduct sanitary surveys without benefit of
hydrographical and geological study, is to assume that all shellfish
species behave alike physiologically and that all growing areas have
the same hydrographical and geological conditions, These assumptions,
applied indiscriminately, could be disastrous to the industry as well
as to public health.

Laboratory and field observations and experiments of the
Department of Sea and Shore Fisheries indicating the need for a

-23-

review of shellfish sanitary criteria were reported by the writer to
the National Shellfish Sanitation Conference in 1954. As a result of
this report, the Conference amended its resolution on shellfish sani-
tation to include two recommendations:

(1) Establishment of sanitary requirements by shellfish species,
and

(2) Establishment of sanitary requirements on an area basis.

Clarence Sterling and Leo Fox of Massachusetts, Leslie Sherman
of Connecticut, Harold Udell of New York, and the writer arranged for
a northwestern area (New England and New York) meeting to work out a
cooperative research program to implement these proposals.

In addition to the cooperative program, the Department of Sea
and Shore Fisheries undertook an investigation of problems peculiar to
representative shellfish growing areas of Maine.

Factors Influencing the Growing Area

Results of these and previous studies indicate that factors
associated with growing areas and having actual or potential influ-
ence upon sanitary requirements fall into three major categories:
hydrographical, geological, and biological. It was further suggested
that the relative importance of each varies seasonally by area and by
species.

I . Hydrographic factors

A. One of several coves which make up Quahog Bay, Harpswell,
Maine, serves to illustrate the complex influence of inshore hydro-
graphy. This serpentine cove is shallow at its head but slopes to
about sixty feet in depth at its constricted mouth.

1. A survey was made to enumerate sources of pollution and
to estimate the pollution load and its probable distribution. Results
suggested that the cove might be a borderline area as far as satis-
factory sanitary conditions were concerned.

2. scores of bacterial nater sables, ranging fro. zero to

3-6 MPN, compared favorably with those from the most isolated growing
areas .

3. A hydrographic survey of the cove indicated that: (a)
water currents moved independently of wind direction, with large
homogeneous water masses shifting with the direction of the tide;

(b) buoyant material at or near the surface drifted with the direction
of the wind; (c) the volume of fresh sea water moved into the cove
with each eight-to-twelve foot tide could account for low bacterial
scores; and (d) inferences derived from the shoreline survey that the
overlying waters might be excessively contaminated were not supported
by hydrographic studies.

-2k-

B. Similar studies have been made in olher areas. Results
suggest evaluating parts of a sanitary survey in the order: hydro-
graphic studies, bacteriological analysis of water quality, and other
components.

C. Additional experiments during the last several years indi-
cate that complexity of inshore hydrography is seriously underesti-
mated .

1. In one study area it was repeatedly observed that a
water mass moved through another water mass of similar temperature
and salinity. Water pressure created by the tide in combination with
geological features appeared to be the causative factor. Direction
of movement was related to conformation of the bottom.

2. In a second study area, current velocity during the
tidal cycle varied from nearly zero to more than six miles per hour.

3- Even in areas where land mass constrictions or other
obstacles to laminar flow induced turbulence, stratification regularly
occurred with respect to salinity, temperature and bacteriological
water quality.

II. Geological factors

A. Studies of periodic changes in surface hydrography and in
geological modification of intertidal growing areas were carried on
jointly by the Maine Geological Survey and the Department of Sea
and Shore Fisheries from 19^8 through 195^ • Although conclusions
are not final, results of these investigations show that geological
factors with their influence on hydrography must be considered if a
valid appraisal of pollution conditions is to be made.

1. Photographic (both still and motion) records of minor
geological changes were made by the writer while working with Dr.
Joseph M. Trefethen, State Geologist and Dr. Wilraot H. Bradley of the
United States Geological Survey. Redistribution of marine sediments
and other debris by surface tension, sub-surface vortices, tidal run-
off, and ice floes was observed and photographed.

2. Major changes were observed to take place under extra-
ordinary meteorological conditions. Flats in some growing areas were
drastically altered in elevation, conformation and compaction.

3- More permanent geological features, including sub-surface
bedrock, were found to retard distribution.

k. Observations suggest that geological factors including
surface texture, particle size, compaction, permeability and gradient
influence the distribution or redistribution of pollutants.

-25-

III. Biological factors

A. There are several species characteristics which have bearing
upon sanitary criteria.

1. Examination by the Department of Sea and Shore Fisheries
of shell liquors of clams (Mya arenaria) from commercial areas of
Maine indicates that clams did not siphon or feed when salt concentra-
tions of the water dropped below twenty-four parts per thousand. Some
clams have been found to survive in water that, on occasion, dropped >
below one part per thousand. That clams siphon overlying waters of
much lower salinity than twenty parts per thousand in other geographi-
cal areas has been verbally reported to the writer by marine biologists
working in Massachusetts and in Maryland.

2. When the range of salinities is wide, clams siphon only
during the higher concentrations of that range.

3. Grossly-polluted clams repeatedly cleansed themselves at
temperatures as low as kl F., approximately nine degrees lower than
the temperature reported in the "Manual of Recommended Practice for
Sanitary Control of the Shellfish Industry".

h. In shellfish growing areas of Maine where both clams and
quahogs (Mercenaria mercenaria) occur in commercial concentrations,
quahogs invariably will be found in those portions of the area where
the overlying waters are of higher salinity. Since, under normal
conditions, contaminated waters are those of lower salinity, species
that tolerate lower salinity are more likely to be exposed to contami-
nation than are those that require more saline water . Quahogs are less
subject to contamination than clams in the same growing area.

5. Shell form can contribute to contamination. The quahog,
with ability to close its valves completely, is less exposed to con-
tamination from brief periods of polluted fresh water than the clam
which can neither completely retract its siphon nor close its valves.
Although the clam may not feed during these periods, the external
parts of the siphon and the mantle may become contaminated.

6. Differences in viability among shellfish species is an
important health consideration. Experience in holding shellfish
alive under varying conditions indicates that clams will live more
than twice as long as blue mussels, but only from one-half to one-
fifth as long as quahogs. It is apparent that because of biological
differences the same sanitary requirements should not apply to these
three shellfish species.

-26-

Standards for Blue Mussel (Mytilus edulis)

Blue mussel shell stock has for years been a quality problem
for producers, distributors and wholesalers as well as for consumers.
Although the magnitude of production is not comparable to that of
the oyster, clam, and quahog fisheries, the lack of essential informa-
tion on quality control is considered by research personnel of the
northeastern states to be a major deterrent to market development and
production increase. The group selected the blue mussel for its
initial research effort because of industrial considerations.

Minutes of the meeting held in Lawrence, Massachusetts, on
April 18, 1956 contain the results of experiments carried on coopera-
tively by research personnel of New York, Connecticut, Rhode Island,
Massachusetts and Maine. Recommendations on sanitary requirements
covering harvesting, cleansing, shipping, refrigeration, seasonal
harvesting and shipping problems, and bacterial limitations for mussel
shell stock in interstate commerce were prepared from these findings.
After further study by the regulatory and advisory agencies of the
several states, these recommendations were adopted as sanitary
criteria.

Clam (Mya arenaria) Shell Stock

Currently the same group of researchers is cooperating on a
program to establish sanitary requirements for clam shell stock.
Experiments were outlined by the writer and Phillip L. Goggins,
bacteriologist of the Department of Sea and Shore Fisheries.

The purpose of the program is to enhance quality of product
in order that general economy of the industry may be improved. The
primary objective is to evaluate quality of clams from growing areas
to receiving markets and handling and shipping in various containers
at different temperatures. The secondary objective is to relate
these evaluations to biological and conservation problems of the
species.

1. Pre -shipment experiments carried on at Boothbay Harbor by the
Department of Sea and Shore Fisheries include:

a. Harvesting -

(l) From approved areas only, unless otherwise
specified.

b. Handling -

(l) A series of duplicated experiments carried out
under the following conditons:

-27-

(a) washed versus unwashed.

(b) refrigerated versus unrefrigerated.

(c) broken versus unbroken.

(d) stimulated shipping conditions versus
undisturbed storage (room temperature, incubator temperature, and
refrigeration at kO F. )

2. Receiving experiments carried on by agencies of states con-
cerned include :

a. Packaging (three-month trial with each method)

(1) unprotected box and basket (no insulating or
padding material).

(2) protected box and basket.

(l) various degrees of protection using various
materials .

(3) clams from approved versus moderately polluted
areas. Variations of receiving experiments will depend upon results
at Boothbay Harbor.

Summary

For administration and enforcement convenience, it would be
desirable to establish specific sanitary criteria for all species of
shellfish from all growing areas. Biological differences within the
same species from different growing areas and hydrographical and
geological differences for all species from different growing areas
preclude the application of a single index of quality.

Recognizing these problems, researchers from New York and New
England are presently engaged in a cooperative program to develop
applicable standards of quality for the shellfish industry.

Literature Cited

Donohue, John J. March 19^9. Shore studies: Stover's Cove, South
Harpswell, Maine. Rpt . of the state geologist, Augusta, Me.
19^7-19^8, pp. IO3-IO9.

Dow, Robert L. and Dana E. Wallace. Jan. 1950. The Maine clam (My a
arenaria). Bull., Dept. of Sea & Shore Fish., Augusta, Me.

-28-

Dow, Robert L. and Dana E. Wallace. March 1951. Soft shell clam
growth rates in Maine. Bull., Dept. of Sea & Shore Fish.,
Augusta, Me.

Dow, Robert L. 195^. Preliminary experiments in the use of ground
controlled aerial photography in interdial hydrographic sur-
veys. Proc. Natl. Shellfish. Assoc. ^5: 199-208.

Ganaros, Anthony E. Sept. 195^. On the ecology of Mya arenaria L.

and Venus mercenaria L. in Maquoit Bay and Falls Cove, Maine.
Unpub. Ms. Rutgers Univ., New Brunswick, N. J.

Goggins, Phillip L. and John W. Hurst, Jr. 19^9-1957. Bacteriological
surveys; files, Dept. of Sea & Shore Fish., Augusta, Me.

Gustafson, Alton H. 195^- Growth studies in the quahog Venus
mercenaria. Proc. Natl. Shellfish. Assoc, h^: 1^0-150.

Moulton, James M. and Gareth W. Coffin. Feb. 195^- The distribution
of Venus larvae in Orr's Cove plankton over the tide cycle and
during the summer and early fall of 1953- Res. Bull. No. 17>
Dept. of Sea & Shore Fish., Augusta, Me.

Scattergood, Leslie W. and Clyde C Taylor. Jan. 1950. The mussel
resources of the North Atlantic region. Fish. Leaf. 3&+>
U.S.F. & W.L.S., Dept. of the Int., Washington 25, D. C.

Shellfish committee representing northeastern shellfish producing
states, Minutes of. April 18, 1956. Lawrence Experiment
Station, Lawrence, Mass.

Trefethen, Joseph M., John J. Donohue, Philip Stackpole, and William
Fairley. May 1951. Shore studies, Rpt. of the State
Geologist, Augusta, Me. 19^9-1950. pp. 107-158.

U. S. Public Health Service, Sanit. Eng. Div., Fed. Sec. Agency,

Washington 25, D. C. 19^6. Manual of recommended practice
for sanitary control of the shellfish industry.

Zink, Robert M. Dec. 1953- Certain aspects of the ecology of Venus
and Mya at Bunganuc and at Morgan Bay, Maine . Rpt . of the
State Geologist, Augusta, Me. 1951-1952. pp. 71-115.

.29.

BACTERIOLOGICAL STUDIES OF HARVESTING AND
PROCESSING OF OYSTERS IN VIRGINIA

R. H. Gregory, R. T. Hill and J. A. Hope, Jr.

Virginia Department of Health

In 1939 and 19^0 many major improvements were made in the
construction and equipment of oyster shucking plants to meet revised .
standards. The standards were again revised in 19^+6 to include,
among other things, requirements for concrete floors, removable blower
tank pipes, and the present type of shucking pail. During this 1939-
kG period, as now, improved sanitary practices were emphasized. These
changes accomplished some reduction in bacterial content of shucked
oysters examined in the laboratories; however, most samples continued
to show coliform bacteria in excess of the suggested limit of 230 MPN
(most probable number of coliforms per 100 milliliters of sample).
Consequently, it appeared that the shucking plant operation was not
the all-important source of contamination it was previously thought
to be.

It had been known for some time that shell oysters of good
bacteriological quality would show an increase in bacteria when
shucked. There was found to be little difference in this increase
between oysters shucked under controlled commercial conditions known
to be clean and those shucked under regular commercial practice
(Kelly and Arcisz 195^+ )• Preliminary work in the White Stone labora-
tory was conducted from December 195^+ to June 1955 to determine what
steps in oyster handling would be investigated to obtain data on
this increase. Four sampling stations were selected for tracing single
lots of oysters through the plants:

1. "Shell Oysters". A composite sample from shellstock
on shuckers ' benches or in storage bins.

2. "Shell Oyster Washings". Washings of mud and detritus
from the outside surfaces of a composite sample of
"shell oysters".

3. "Meats As Shucked". A composite sample of meats

as they are shucked commercially. Shuckers to shuck
one oyster each directly into the sterile collecting
jar until the quantity of oyster meats required for
examination has been accumulated.

k. "Meats As Packed". Sample of oysters as packed in
plant .

From June 1955 through April 1956, 121 lots of oysters were sampled
and examined in the White Stone and Norfolk laboratories. With few

-30-

exceptions these oysters were taken from beds in the lower Chesapeake
Bay, in Mobjack Bay, and in the York and Rappahannock Rivers near
their mouths; these beds are remote from ship channels and on-shore
pollution. The samples were collected from well-constructed, well-
equipped and well-operated plants.

METHODS OF EXAMINATION

Procedures for the bacteriological examination of shell and
shucked oysters, except for present accepted modifications, followed
those recommended by the American Public Health Association (19^7)-
The program was planned with the cooperation of the Public Health
Service Shellfish Sanitation Laboratory and the Service's Region III
office, and in conjunction with them written procedures were developed;
frequent consultations were held during the project. These procedures
were generally followed by the Service's Shellfish Sanitation Labora-
tory and laboratories in other areas making similar studies.

In preliminary work at the White Stone laboratory need for a
method of examining bacteriologically mud and detritus on the outside
of shell oysters became evident. To fill the need this laboratory
developed uniform procedures for removing this material, submitting
it to examination, and expressing the results in MPN's, as follows:

Sampling. Place in a sterile one-gallon, water-tight oyster-shipping
container at least 12 oysters of uniform size and shape; these should
be representative of the lot to be sampled, and as nearly as possible
between three and four inches in length with deep bowls. If more
than two hours in transit, dry-refrigerate the sample.

Washing. Transfer ten oysters to a sterile one-gallon oyster-shipping
container in good condition with tight-fitting lid. Add to the con-
tainer one liter of sterile phosphate-buffered dilution water. Agitate
the container and contents at least 50 times with a continuous circu-
lar motion. The number of agitations will depend on the thickness of
the mud, but should be limited to that required to free the shells of
clots of mud. Care should be taken to avoid violent motions that
might break the "bills" of the oysters and consequently allow incor-
poration of shell liquor with the washings. Immediately after agita-
tion transfer approximately 100 ml. of the washings to a sterile
bottle .

Examination. Inoculate suitable dilutions into lactose broth. The
sub-sample should be thoroughly shaken prior to each withdrawal of
measured portions. Proceed with incubation and confirmation as in
the examination of samples of water or shellfish, paying due attention
to the necessity of submitting questionable partially-confirmed tubes
to the completed test. This might be indicated more frequently in
muds because of the greater likelihood of spore formers and other
lactose -fermenting non-colif orms .

-31-

Expression of Results. Express results as coliforras per 100 ml. of
washings. If the oysters are of uniform size, and within the standard
size limits, it may be assumed that the MPN is roughly equivalent to
coliforms per shell oyster.

The data were forwarded to the Public Health Service Shellfish
Sanitation Laboratory and statistical analysis was made by Dr. E. K.
Harris, Analytical Statistician, Robert A. Taft Sanitary Engineering
Center. Interpretations and comments were made on the data by the
aforesaid laboratory. Results discussed hereafter developed from
analyses and interpretations.

RESULTS

Changes in Bacterial Quality of Oysters During Processing

Each lot of oysters from shellstock to final pack was followed
through the plant for changes in bacterial quality. The identity of
each lot was maintained. As a base line for comparison, the sum of
MPN's of "shell oysters" and "shell oyster washings" was assumed to
represent unwashed oysters as presented to shuckers. This base line
is an estimate of the total potential coliforra MPN which could be
incorporated in meats during shucking- -exclusive of contamination by
hands, utensils, and equipment in use during the operation.

To show the changes from this base line, "shell oysters +
washings", oysters "as shucked", and oysters "as packed" were assigned
to various groups by MPN's. Group 1 was less than 230, Group 2 from
230 to 2400, Group 3 from 2^01 to 2^,000, Group k from 2^,001 to
160,000, and Group 5 over 160,000 MPN. Oysters in one bacterial range
prior to shucking may fall into another range after shucking and per-
haps still a different range when packed. We assigned zero to samples
remaining in the same group, +1 or -1 to those which rose or fell one
group, and +2 or -2 to samples changing two groups between sampling
points. The changes were averaged at each sampling point.

We find (Table l) that at White Stone the average change in
group units between "shell oysters + washings" and oysters "as shucked"
was in October and November +O.52, in December and January +0.39 and
in February and March +O.39. This shows that shucking increased
bacterial content significantly above the potential from unshucked
oysters. This is a conservative statement for not all detritus and
mud on the outside of shells gets into shuckers' pails. At White Stone,
we found a consistent decrease in the average change from "as shucked"
to "as packed" in all months. From "shell oysters + washings" to "as
packed" there is a slight increase in bacterial content but the rise
is not significant. Interestingly, at Norfolk shucking had little
or no effect on changes in group units.

-32-

Table 1. Average changes in group units during processing

Location & Period

From "shell
oysters +
washings" to
"as shucked"

From as
shucked"
to "as
packed"

From shell
oysters +
washings" to
"as packed"

White Stone

Oct-Nov (23 samples)

Dec-Jan (18 samples)

Feb -Mar (18 samples)

Norfolk

Jan-Feb ( 8 samples )

Mar -Apr ( 8 samples)

0.52
0.39
0.39

-0.12
0

-0.26
■0.28
-0.33

-0.38
-O.38

0.26

0.11

0.06

-0.50
-0.38

Evaluation of Bacterial Densities

The data were analyzed by season for each of the four sampling
stations. Probability graphs were developed for each station by
plotting on logarithmic paper the MPN values against their occurrence
percentage -wise (Velz 1951)- For each season there is a distinct
curve on each of the graphs, with the exception of "shell oyster
washings" for Jun - Sep which could not be plotted as all results
were over 1100+ . These graphical summarizations allow immediate esti-
mations of the medians and of the percentages of "shell oysters",
"shell oyster washings", oysters "as shucked", and oysters "as packed"
which fall within any specified MPN range.

Assuming we are interested in what ranges 25$ of the "shell
oysters" fall, from season to season, we find in Figure 1 that in
Jun-Sep the range is 2300 MPN or less; Oct-Nov 600 MPN or less; Dec-
Jan 26 MPN or less; and Feb-Apr 31 MPN or less. Selecting the median
or 50$ to see into what ranges "shell oyster washings" (mud and
detritus) fall, we find from Figure 2 that in Oct-Nov the range is
9000 MPN or less; Dec-Jan 1700 MPN or less; and Feb-Apr 2100 MPN or
less. From Figure 3 it can be seen that in Jun-Sep 75$ of the "as
shucked" oysters fall into the range of 56,000 MPN or less; Oct-Nov
50,000 or less; Dec-Jan 1*1,000 MPN or less; and Feb-Apr 14,000 MPN
or less.

The "as packed" oyster is the product as it goes on the
market. We would like to know, by seasons, into what MPN ranges 90$
of the product will fall. Referring to Figure h* in Jun-Sep the
range is *i00,000 MPN or less, in Oct-Nov 100,000 MPN or less, in
Dec-Jan the range is down to 17,000 MPN or less, and in Feb-Apr it
rises to 30,000 MPN or less.

■33-

It can be seen from Figures 1-h, in the case of each sampling
station, that the curves group together naturally in pairs. On in-
vestigating the basic data it was found that during the latter part
of November air temperatures dropped abruptly to below 50°F. and sea-
water temperatures took a corresponding drop. Temperatures remained
in this category into April. The Jun-Sep and Oct-Nov curves with
tempex-atures above 50 F. form a natural pair, and the Dec -Jan and
Feb-Apr curves with temperatures below 50 F. from another natural
pair. This indicates a relation between air-water temperature and >
bacterial content of oysters.

A probability graph (Fig. 5) was made for Oct-Nov with
curves plotted for each sampling station. A similar graph (Fig. 6)
was made for Feb-Apr. it is apparent that at all four stations lower
bacterial content were obtained during Feb-Apr than in the warmer
months of Oct-Nov. Also the rise in bacterial content from "shell
oysters" and "shell oyster washings" to oysters "as shucked" and the
subsequent drop to oysters "as packed" can easily be estimated. It
can be seen that the "shell oysters" are relatively low in bacteria
as compared with "shell oyster washings " ; however, these two combined
contain less than oysters "as shucked", and oysters "as packed" con-
tain less than the product "as shucked".

The United States Public Health Service (1950) suggests a
limiting coliform MPN of 230 in' oyster shell stock in growing areas
or in shell stock or shucked oysters at point of shucking; MPN's of
higher values should be interpreted as indicative of unfavorable con-
ditions or practices surrounding the production and handling of the
product, however, in occasional samples, an MPN value of 2^-00 may be
tolerated. Kelly and Arcisz (195M utilized the additional MPN classi-
fiaction of 2^,000. The MPN of 16,000 has been included as an inter-
mediate. Ranges used in following tables are based on these MPN
values.

Table 2 was compiled from the "shell oysters" probability
graph and shows by season the percentage of shellstock that can be
expected to fall within the bacterial ranges. It can be seen that
during Dec-Apr about two-thirds of shell oysters can be expected to
be below 230 MPN and approximately 90$ below 2^00 MPN. It is also
apparent that, with no increase in pollution, oysters from the same
areas will increase in bacterial content during warm months. During
summer no shell oysters can be expected to be within the range of
230 MPN or less, and only about 25/j within 2^00 MPN or less.

.3)1

i.ooqooc-

500,000

100,000
50,000

10,000
5,000

1,000
500

100
50

10

"i r

-i 1 1 1 1 — i 1 1 r

t r

o
o

(Z
UJ
CL

CL

Cd

o

o

/

/

FIG I

WHITE STONE- NORFOLK
STUDIES

SHELL OYSTERS

— <- JUN-SEP

OCT-NOV

DEC-JAN

FEB-APR

_i i_

J 1 I I I I I I L

_1 1_

0.1 I 10 30 50 70 90 99 99.9

PROBABILITY (%) EQUAL TO OR LESS THAN

Fig. 1. Seasonal variation in coliform content of shell oysters,

•35-

,000,000

' lv-' ^ '-'1

500,000

00,000
50,000

_ «^

o
- o

cr

LU
Q_

10,000

z:

5,000

Q_

1,000

_ o

500

- o
o

100

50

i r

~i i i I r — i 1 1 1 1 r

1 0

/ /

FIG 2

WHITE STONE-NORFOLK
STUDIES

SHELL OYSTER WASHINGS

OCT- NOV

DEG-JAN

FEB-APR

-i i i i i i i i_

J L.

0.1 I 10 30 50 70 90 99 999

PROBABILITY (%) EQUAL TO OR LESS THAN

Fig. 2,
washings .

Seasonal variation in coliform content of shell oyster

•36-

i.ooopoc-

500,000-

00,000
50,000

_ ^

o
- o

LU
Q_

10,000

7T

5,000

a.

2

1,000

_ o

u_

500

_J
- o

100

-

50

-

n 1 1 1 1 — I 1 1 r

10

FI63

WHITE STONE- NORFOLK
STUDIES

AS SHUCKED

UUN-SEP

OCT- NOV

DEC- JAN

FEB-APR

j 1 — i — i i i i i i

0.1 I 10 30 50 70 90 99 999
PROBABILITY (%) EQUAL TO OR LESS THAN

Fig. j. Seasonal variation in coiiL'ux'm content of chucked oysters.

-37-

i,ooo,ooc-

500,000

100,000
50,000

10,000
5,000

1,000
500

100
50

10

o
o

cr

Id
Q_

~i 1 1 i — r

Q_

o

o
o

FIG 4

WHITE STONE-NORFOLK
STUDIES

AS PACKED

JUN- SEP

0CT-N0V

DEC-JAN

FEB-APR

j i i i i i i i_

0.1 I 10 30 50 70 90 99 999

PROBABILITY (%) EOUAL TO OR LESS THAN

Fig. k. Seasonal variation in coiiform content of packed oysters.

-38-

i.ooopoc-

500,000-

100,000
50,000

10,000
5,000

1,000
500

100
50

10

t — i — i — i — i 1 r

O
O

cr

Q_

cr
o
u_

_i
- o
o

FIG 5

WHITE STONE-NORFOLK
STUDIES

ALL SAMPLES 0CT-N0V

SHELL OYSTER WASHINGS

SHELL OYSTERS

AS SHUCKED

AS PACKED

j i i i i i i i u

Ql I 10 30 50 70 90 99 99.9

PROBABILITY (%) EQUAL TO OR LESS THAN

Fig. 5- Change in coliform content as oyster is processed
through plant; air-water temperature above 50 F.

■39-

I.OOO.OOC

500,000

100,000
50,000

10,000
5,000

1,000
500

100
50

10

"I 1 1 1 1 — r

o
o

QC
LJ
Q_

Q_

O

O

o

FIG 6

WHITE STONE- NORFOLK
STUDIES

ALL SAMPLES FEB-APR-

SHELL OYSTER WASHINGS

SHELL OYSTERS

AS SHUCKED

AS PACKED

_! I 1 I I I I-

0.1 I 10 30 50 70 90 99 999

PROBABILITY (%) EQUAL TO OR LESS THAN

Fig. 6. Change in coliforrn content as oyster is processed
through plant; air-water temperature below $0 F.

-40-

Table 2. Percentages of shellstock falling below certain MPN in
various seasons

MPN Jun-Sep Oct-Nov Dec-Jan Feb-Apr

230

2,400

16, 000

24,000

0

11

67

62

26

54

95

92

72

89

100

100

82

93

100

100

Table 3 was compiled from the "as packed" probability graph
and shows the percentage of packed oysters by season that can be
expected to fall within certain bacterial ranges. These figures in-
dicate again that packs during cold seasons have lower bacterial
content than those processed during warm seasons. None of the packs
met the standard 230 MPN during warm months and less than 10$ during
cold months.

Table 3- Percentages of packed oysters attaining certain MPN levels
by seasons

MPN Jun-Sep Oct-Nov Dec- Jan Feb-Apr

230

2,400

16,000

24,000

0

0

6

9

7

10

49

47

33

50

89

83

42

62

93

88

Summary

It has been known for sometime that shucking causes an in-
crease in the bacterial content of oysters. There is little difference
in this increase between oysters shucked under controlled commercial
conditions known to be clean and those shucked under regular commer-
cial practice. In the Virginia shellfish laboratories 121 lots of
oysters were examined from June 1955 through April 1956 to obtain
data on this increase. Each lot was sampled at 4 sampling stations,
i.e., "shell oysters", "shell oyster washings", oysters "as shucked",
and oysters "as packed". With few exceptions the oysters were caught
from beds in the lower Chesapeake Bay, in Mobjack Bay, and in the
Rappahannock and York Rivers near their mouths- -these beds are remote
from ship channels and on-shore pollution. The oysters were processed
in well-constructed and well-operated packing plants. The data were

-4l-

submitted to statistical analysis and it is concluded that shucking
contributed bacteria in significant numbers over and above the total
potential due to incorporation of detritus and mud from the outside
of the shell into the shucked oyster. During packing this bacterial
content decreased and although it did not return to the base line
("shell oysters" + "shell oyster washings"), the overall average rise
is not considered significant.

Probability graphs, prepared from the data, show the percent-
age of samples by season, for each sampling station, which fall within
any selected MPN (most probable number of coliforms per 100 milliliters
of sample) range. The United States Public Health Service suggests a
limiting coliform MPN of 230 in oyster shell stock or shucked oysters
at point of shucking; MPN's of that value or greater should be inter-
preted as indicative of unfavorable conditions or practices surround-
ing the production and handling of the product, however, in occasional
samples, an MPN value of 2^00 may be tolerated. In 195^ Kelly and
Arcisz, studying bacteriological control of oyster processing,
utilized the additional classification of 24,000 MPN. The MPN value
of 16,000 is included as an intermediate. Ranges used in following
examples are based on these MPN values.

From the "shell oysters" graph it can be seen that during
Dec -Apr about 2/3 of the shell oysters can be expected to be within
the range of 230 MPN or less and approximately 90$, within 2^00 MPN
or less. However, oysters from the same good areas, with no increase
in pollution to these areas during the warm months to justify an in-
crease in the bacterial content of the oysters, will show an increase.
We find that during the summer no shell oysters can be expected to
be within the range of 230 MPN or less and only about 25$ within 2^00
MPN or less.

From the probability graph for "as packed" oysters it can be
seen that during Dec-Apr about 10$ of the packed oysters can be
expected to fall within the range of 230 MPN or less: 50$, or the
median, within 2^00 MPN or less; 85$ within 16,000 MPN or less; and
90$ within the range of 2^,000 MPN or less. During the warmer months
of Jun-Nov, none of the packed oysters can be expected to be within
the range of 230 MPN or less and only about 8$ within 2*4-00 MPN or
less; 40$ within 16,000 MPN or less; and 50$ within 2^,000 MPN or less.

Literature Cited

Kelly, C. B., and William Arcisz. 195^« Bacteriological control of

oysters during processing and marketing. . Public Health Reports
69: 716-720.

-k2-

American Public Health Association. 19^7- Recommended procedure for
the bacteriological examination of shellfish and shellfish
waters. (Revision of recommended methods of procedure. Report
of the Standard Methods Committee for the examination of shell-
fish). Am. J. Pub. Health 37: 1121-1127.

Velz, C. J. 1951- Graphical approach to statistics - evaluation of
bacterial densities. Water and Sewage Works 98: 66-73*

U. S. Public Health Service. 1950* Manual of recommended practice
for sanitary control of the shellfish industry. P.H.S. Pub.
No. 33-

43-

UNUSED OYSTER SHELL IN SOUTH CAROLINA
SUITABLE FOR SEED OYSTER PRODUCTION

G . Robert Lunz

Bears Bluff Laboratories, Wadmalaw Island, S. C.

Abstract

Although South Carolina now has a surplus of
cultch from the steam canneries, this may dwindle as
an export seed oyster industry develops. A possible
substitute is to be found in old oyster shell thrown up
on the banks of many South Carolina creeks and rivers
by wave action and storms.

Experimental plantings indicate that this
washed shell is not very suitable as a spat collector
when broadcast on the normally soft oyster -producing
bottoms of South Carolina, but when screened to remove
small pieces it is useful as cultch in suspended tray,
bag or basket planting. Series of test plantings made
in different years at different times of the year show
that washed shell catches about half as many spat as
steamed shell from canneries. Since spatfall in South
Carolina waters is intense and long -continued, this
reduction in catch may be a valuable attribute.

Washed shell is available in large but as yet
unmeasured volume. One stretch of the North Edisto
River is estimated to have a half -million bushels of
washed shell on its banks. This is a "renewable"
resource in the sense that every period of bad
weather washes new shell up on the banks.

Fine and small shell from screening amounts
to 50 per cent of the volume of the material as it
is taken from river banks. In Low Country South
Carolina where rock is non-existent, fragments of
washed shell not suitable for oyster cultch may be
used as aggregate in concrete mix. This has been
done successfully at Bears Bluff Laboratories.

South Carolina waters are admirably suited to produce small
seed oysters. Naturally one of the principal requirements for seed
oyster production is suitable cultch. After the oyster industry in
South Carolina has satisfied its legal demands for shell planting
there is still approximately one-quarter million bushels of shell
remaining annually. In the past much of this shell has been sold for

-¥+-

purposes other than oyster cultivation. This surplus shell could be
used for the production of seed oysters. However, those companies
with surplus shell piles have sometimes shown an unwillingness to
release this shell at prices which would make it usable by the seed
oyster industry.

Various types of substitute cultch have been tried by Bears
Bluff Laboratories as well as by other laboratories in oyster produc-
ing areas. It is well known that oysters will set and survive on
many different materials, but none as yet has been found which can
satisfactorily compete with the shell of the steam oyster canneries.

A possible substitute is to be found in old shell thrown up
on the banks of many South Carolina creeks and rivers by wave action
and storms (Fig. l). As cultch this "washed" shell is about as
effective as reef shell of the Gulf Area.

South Carolina washed shell seems to lack resilience. It
feels chalky and it is easier to scratch with a knife than steamed
cultch. Microscopically, washed shell is pitted and eroded.

Steamed cultch seems thicker, is not as soft, and the inner
face is smoother and generally shiny. Microscopically the inner shell
surface does not show the pits and eroded areas seen in washed shell.

Reef shell, or mud shell - at least the sample obtained from
the Corpus Christi Bay area of Texas - is generally coated with a
marl or clay-mud mixed with small shell flakes. It is thicker than
washed shell and is more riddled with sponge. The Texas sample con-
tained a higher percentage of small broken shell than does the aver-
age sample of South Carolina washed shell.

The specific gravity of the three types of shell does not
differ greatly. South Carolina washed shell (5 samples) has a speci-
fic gravity of 2.335 steamed cultch (h samples) 2.i+0; and Texas reef
shell (3 samples) 2.37-

In the Gulf Area, Gunter (1938), St. Amant (1956), and Ingle
(1956), arrived at the conclusion that reef shell was suitable for
cultch, but found that generally it did not catch as many spat as
steamed cultch.

From time to time Bears Bluff Laboratories has attempted to
use washed shell as a substitute for steamed shell. Although washed
shell is useful for hardening soft bottoms, it settles too rapidly to
give good results as cultch. On bottom firm enough to hold up washed
shell it is too susceptible to wave action and movements by currents.
Generally, washed shell broadcast on oyster grounds is not worthy of
use .

45-

3 ■

...

.***'

Fig. 1. Washed shell is deposited along the edge of most
South Carolina coastal rivers.

-1*6-

Fig. 2. Washed shell (right) catches about half as many spat
as does steamed cultch (left).

-47-

MMMaMtttt

Fig. 3* Cultch can be suspended in wire troughs as well as
wire bags.

-48-

For seed oyster production in the manner described by Lunz
(1952) however, the results are more promising. In view of the in-
creased interest in seed oyster production for out of State markets,
and the possible shortage of steamed shell as the seed oyster industry
develops, it seems worthwhile to present some comparative date on the
spatfall on washed and steamed shell.

On June 1, 1955.* two half-bushel shell bags, one with washed

shell cultch and one with steamed shell cultch, were hung on cables

just above low water in We Creek at the laboratory. The steam cultch

was the usual cluster type with several lower valves attached together.

The washed shell was made up of selected deep-cupped shells, largely
individuals. On June 20, 1955 > spat on ten inner faces from each type

of cultch were counted. The spatfall ratio was 7 to 5 in favor of the
steamed shell cultch.

In August 1956, fifty half -bushel bags of shell were suspended
on cables in We Creek above low water. The bags of steamed and washed
shell were alternated. The washed shell had been screened through a
lxl inch wire to remove the fine shell. This removed approximately
50 per cent of the bulk of the washed shell as it was taken from the
banks. In November detailed studies showed that the intensity of
setting was 1.0 spat per square inch for the steamed shell and 0.51
spat per square inch for the washed shell.

Although it had been screened, the washed shell contained a
higher percentage of small and flat pieces of shell than the steamed
cultch. The bulk of the steamed shell was made up of cupped and
cluster shells. The washed shell was 65 to 7° per cent flat upper
valves of which 60 per cent had only one spat. Only 20 per cent of
the steamed shell had one spat per shell and 70 per cent had 3 or more
spat per shell.

At first inspection the spat on the steamed shell seemed
larger than those on the washed shell. However, a series of measure-
ments showed little difference. Measurement of 93 spat selected at
random showed that the mean size was 25 x 19 mm on the washed shell,
and of 138 spat' on steamed cultch the mean was 29 x 19 mm. The
largest spat found on steamed shell was 55 x 26 mm while on washed
cultch the maximum size was 46 x 25 mm. Thus the difference in size
of spat on the different types of cultch seems insignificant.

In early May 1957> before setting began, bags of washed shell
and steamed shell again were hung on cables in We Creek above low
water mark. The bags were so hung that every other bag was washed
shell. The washed shell had been screened as before. The small
screenings were discarded.

By May 21, both types of cultch had accumulated a fair catch
of spat. The steamed shell had k.h spat per square inch; the washed
shell 2.8 spat per square inch. As roughly translated (Lunz 1954),

49-

this is 20 spat per shell for steamed cultch and 12 spat per shell
for washed cultch. On June 6, 1957; shells from the two types of
cultch bags showed a catch and survival of 8.5 spat per square inch
for steamed shell and 9-5 for washed shell. By July 2, 1957 the
count was 10.2 for steamed cultch and 5-0 for washed cultch.

These experiments indicate that washed shell, even though
it is only about one-half as efficient as steamed shell, is satisfac-
tory for use in trays, baskets, or suspended bags for collecting seed
oysters (Fig. 2 and Fig. 3)- Because of the lower set and survival
rate on washed shell it is in some ways superior to steamed shell.
The use of washed shell for cultch would tend to eliminate overcrowd-
ing - a tendency of South Carolina seed criticized by Andrews and
McHugh (1956)' Thus, although washed shell is not the perfect sub-
stitute for steamed shell it can be used as cultch, particularly as
steamed shell becomes unavailable.

If the seed oyster industry does adopt the use of washed
shell as a substitute for steamed shell, how much washed shell is
available?

Along the edge of most of the larger coastal rivers in South
Carolina, particularly where the long reach of the river runs north-
west to southeast or northeast to southwest, fairly large seas are
built up at the time of moderate to fresh winds. Maximum tidal
current velocities in these coastal rivers and inlets exceed 2 knots.
When wind and tide oppose, the seas build up and shell from dead
oysters is piled up along the edge of the river. There are many
thousands of piles of shell along the banks of the creeks and rivers
of South Carolina.

Across from Bears Bluff Laboratories on the northern shore of
Wasmalaw River is a series of small but typical washed shell banks.
Moderate to fresh southwest winds driving against a 3-knot ebb tide
piled the shell up in the marsh. One of the series of shell banks
across from the Laboratories now is 125 feet long by 35 feet wide and
has a thickness or depth of approximately two feet. This bank con-
tains about 7>000 standard U. S. bushels.

Although no attempt has been made to calculate the volume of
washed shell available in all of South Carolina, the North Edisto
River system which begins at the ocean and extends up to Bears Bluff
Laboratories was surveyed to estimate the amount of available washed
shell. In the five statute miles from Bohicket Creek on the east
bank and South Creek on the west bank of the North Edisto River, on
up the river to Dawhoo entrance near Bears Bluff,- there are an esti-
mated 525)000 bushels of raw washed shell which could be taken from
this one of the many river systems in the State having washed shell
along their banks.

•50-

It was noted before, however, that approximately 50 per cent
of the washed shell as taken from the banks of the river would be
removed if the shell were screened through a 1 x 1 inch square mesh
wire screen. Thus, the North Edisto River would produce only slightly
more than 260,000 bushels of washed shell suitable for cultch.

No attempt has been made as yet to determine the cost of
removing this washed shell from the banks of the river. There is a
possibility that a sizeable part of the cost of removing the washed
shell from the banks could be paid from the sale of the screened shell.
This screened shell has a valuable use as aggregate. For over ten
years now, much of the concrete mixed and used at Bears Bluff has been
made from shell aggregate. The use of shell aggregate is age-old in
Low Country South Carolina, and prior to the availability of cement a
mixture of burned shell, unburned shell, and sand was used as a fore-
runner of present day concrete. A few ruins of structures made from
this material, called "tabby" in the l6th and early 17th Century, are
still in existence in coastal Carolina today. The manufacture of
tabby is a lost art, but screened shell with cement makes a perfectly
usable concrete.

It is of interest to note that washed shell is a "renewable"
resource. In 19^8 and 19^9 across from Bears Bluff, approximately
6,000 bushels of shell were removed from one bank now estimated to
contain 7,000 bushels of shell. Since 19^9 > between 500 and 600
bushels of shell have been removed annually, but the pile recovers
with each new fresh southwest wind and seems today to be as large as
it was in 19*4-8.

Literature Cited

Andrews, Jay D. and J. L. McHugh. 1956. The survival and growth of
South Carolina seed oysters in Virginia waters. Proc. Natl.
Shellfish. Assoc. ^7: 3-17-

Gunter, Gordon. 1938. A new oyster cultch material for the Texas
coast. Proc. Texas Acad. Sci . 21: Ik, (Abstract).

Ingle, R. M. 1956. Personal communication of Dec. 6, 1956.

Lunz, G. R. 1953. A preliminary report on some experiments in the
production and transplanting of South Carolina seed oysters
to certain waters of the Chesapeake area. Part 1: Production
of oyster seed. Proc. Gulf & Caribbean Fisheries Inst. Fifth
Ann. Session, 1952: 113-115-

195^. The general pattern of oyster setting in South Carolina.

Proc. Natl. Shellfish Assoc. *+5: ^7-51.

St. Amant, L. S. 1956. Some trends in the biological investigations

of various oyster problems in Louisiana. Conv. Addresses Natl.
Shellfish. Assoc, 48th Ann. Meeting, (Manuscript).

-51-

SURVIVAL OF SOME JUVENILE BIVALVES IN WATER OF LOW SALINITY

Paul E. C hartley
U. S. Fish and Wildlife Service, Milford, Conn.

Abstract

The minimum salinities at which juvenile
clams, Venus mercenaria, Ens is directus and Mya
arenaria, survived were 12. 5> 7-5 and 2.5 parts per
thousand, respectively. Juvenile oysters, Ostrea
edulis and Crassostrea virginica, survived in salini-
ties as low as 12.5 and 5«0 ppt, respectively, and
juvenile mussels, Brachidontes recurvus, survived at
2.5 ppt. E. directus and M. arenaria survived at
7.5 and 2.5 ppt only after first becoming acclimated
to intermediate salinities. They did not survive if
transferred directly to these salinities from 26. 0 -
27« 0 ppt. V. mercenaria ranging in size from 10.0
to 21.5 mm survived at 12.5 Ppt, but I5.O ppt was the
minimum salinity at which those ranging in size from
1.8 to 3-6 mm could survive. Growth was retarded at
salinities of 22.5 PPt and lower. The optimum salini-
ty for growth of recently set C. virginica was 15.0
to 22.5 ppt.

Introduction

Many commercially important bivalves occur in estuarine
environments where considerable fluctuations in salinity are common.
Some are found in areas where water may be almost fresh, while others
require a more saline environment. Intelligent management of these
fisheries, as well as proper evaluation of harbor improvements and
other engineering projects, requires a knowledge of the effects of
salinity on growth and survival of these animals. Such information
is also necessary for effective management of future commercial ponds
and hatcheries.

Some effects of low salinities and of changes in salinity on
adaptation and survival of juvenile bivalves are given in this report.
The term juvenile has been defined by Carriker (unpublished manuscript)
for Venus mercenaria as "very young clams in which the byssus gland
is no longer functional and the animal maintains its position in the
bottom by means of its foot alone". In this report a broader defini-
tion is necessary to include the several species of bivavles studied.
Juvenile bivalves, then, are small mollusks, generally less than one
year old, that have adult rather than larval characteristics and are
capable of either digging or attaching to substrates. The species

■52-

included in this report are the hard clam, Venus mercenaria, the soft
clam, Mya arenaria, the razor clam, Ens is directus, the American oyster,
Crassostrea virginica, the European flat oyster, Qstrea edulis, and the
hooked mussel, Brachidontes recurvus.

Methods

In addition to our laboratory sea water (salinity 26.0 to 28.0
parts per thousand, which will be referred to as full salinity), the
salinities used in these experiments were 22. 5^ 20.0, 17- 5> 15-0,
12.5, 10.0, 7-5, 5.0, 2.5 ppt and fresh water. The lower salinities
were obtained by diluting laboratory sea water with demineralized water
or with water from the Wepawaug River which empties into Long Island
Sound in Milford. The demineralized water was obtained from a Barn-
stead ED-2 demineralizer and stored in either polyethylene or pyrex
containers.

Experimental animals were kept in either enamel or plastic
containers of 3-, h- or 9-liter capacity and water was changed at
two-day intervals. Clams were provided with sand substrates to per-
mit burrowing. Food, generally a mixture of algae, flagellates and
diatoms, was added only when water was changed and before salinity
was adjusted. Temperatures ranged from 15-3° to 21.9°C but were uni-
form for all animals within an experiment. Feeding, ability to dig
in, growth, and survival were considered evidences of adaptation to
reduced salinities.

Results

Venus mercenaria

Our first experiment was designed to find the minimum salinity
at which hard clams can survive and also to determine how well they
recover after exposure to abnormally low salinities. Three sand-
filled plastic boxes, each containing 25 hard clams reared in the
laboratory and ranging in length from 10.0 to 21-5 mm, were trans-
ferred directly from full salinity to each of the experimental salini-
ties. When 10 per cent of the animals at any salinity had died, one
box was returned to full salinity. The second box was returned to
full salinity when 50 per cent of the remaining clams were dead. The
third box remained at the experimental salinity until all died or
until the survivors had become adapted to the salinity.

At full salinity and at 22.5 ppt, over 90- per cent of the
clams dug in and cleared the water of food during the first day. At
salinities of 20.0 and 17.5 ppt it required 7 days for 90 per cent of
the clams to become adjusted sufficiently to dig in and clear the
water of food. At 15-0 ppt none opened until the seventh day, but
by the 19th day over 90 per cent had dug in, were feeding and had
apparently adjusted to this salinity.

-53-

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■ 54-

There was little or no feeding at 10.0 ppt or lower, and in

these salinities all clams eventually died. At 10.0 ppt several clams

opened and one actually dug in on the 69th day, but at 5-0 ppt and lower
they neither opened nor dug in.

Although salinities of 10.0 ppt and lower eventually caused the
death of all clams, they withstood these salinities for several weeks
(Table l). Thus, at 10.0 ppt no deaths were observed until the 28th
day, and 32 days were required before 10 per cent died (Table 2).
After nine additional days of exposure to this salinity 50 per cent
were dead. Two individuals, however, survived until the 82nd and 90th
days of the experiment. At 5>0 ppt the first death occurred on the
18th day, and all were dead by the 5^th day. Even in fresh water
clams survived for 22 days before the first death, but all were dead
by the ^5th day.

Thirteen per cent of those clams returned to full salinity
from salinities of 0.0, 5«0 and 10.0 ppt after about 10 per cent had
died, eventually died. If they remained at these low salinities until
approximately fifty per cent were dead however, 51 per cent of the
survivors died eventually.

Table 2. Comparison of the length of exposure required to cause a
10 per cent and a 50 per cent mortality of juvenile V.
raercenaria in three different salinities and the percentages
of the surviving clams that died when returned to full
salinity.

Salinity

Number of days exposure
before a

Per cent of surviving clams
that died when returned to
full salinity after a

10 per cent
mortality

50 per cent
mortality

10 per cent
mortality

50 per cent

mortality

10.0 ppt

32

kl

Ik

71

5.0 "

29

37

k

50

Fresh water

2k

33

22

ko

At the conclusion of this experiment 25 clams that had sur-
vived at 15.0 ppt for 75 days were kept in water at 12.5 ppt for an
additional ^9 days without mortality. At this salinity, they dug in
normally and fed, but did not clear the water of food as rapidly as
in full salinity.

In a second experiment, 25 clams were transferred directly
from full salinity to 12.5 PPt and kept at this salinity for over

■55-

100 days. Only three died, but those that survived did not feed
normally.

Conditioning juvenile V. mercenaria at intermediate salinities
did not enable them to survive at lower minimal salinities. Most
survived at 12.5 ppt either after direct transfer from full salinity
or after conditioning in intermediate salinities. None adapted to a
salinity of 10.0 ppt regardless of whether they had been conditioned
at intermediate salinities or transferred directly from full salinity.

In another experiment it was found that smaller hard clams, *
ranging in length from 1.8 to 3*6 mm, reacted to the lowered salini-
ties more rapidly than did larger juveniles used in previous experi-
ments. At 15.0 ppt, for instance, 70 Per cent of the small ones dug
in by the seventh day, while 17 days were required for the larger
ones (Fig. 30- Moreover, at 10.0 ppt, the smaller clams all died
within 15 days although none of the larger ones died at this salinity
until the 28th day. In addition, all of the smaller group died at
12.5 ppt even though this salinity was not lethal to larger clams
(Table l).

Because of the possibility that hard clams from different
areas have different salinity requirements, a similar experiment was
conducted with specimens from Narragansett Bay. Like the large
Milford clams, those from Narragansett Bay dug in rapidly, adjusted
to higher salinities almost immediately and required much longer to
adjust to salinities of 17.5, 15-0, and 12.5 PPt. They survived at
12.5 ppt and all died at salinities of 10.0 ppt and lower.

Narragansett Bay clams did not survive in salinities of 10.0
ppt and lower as long as large clams from Milford. Thus, at 10.0 ppt
all from Narragansett Bay died within 3^ days, while only 5° Per cent
of those from Milford were dead after 4l days of exposure to this
salinity. This may have been due to the smaller size of the Narragan-
sett Bay animals and slight differences in the temperature of the
two experiments.

Although juvenile hard clams survived in salinities as low as
12.5 ppt, it seemed probable that growth would be retarded or prevented
at low salinities. Turner (1953) reported that adult hard clams did
not grow when the salinity was lowered to 19.0 - 21.0 ppt and that the
optimum salinity range for growth was 2^.0 to 28.0 ppt. Pratt and
Campbell (1956) reported that hard clams grew at salinities ranging
from 21.4 to 31.9 ppt in Narragansett Bay and that growth was apparently
not affected by salinity within this range.

To determine how reduced salinities affect growth of juvenile
hard clams, twenty-five of them were placed in each of five 9-liter
polyethylene containers of water ranging from 15.0 ppt to full salinity.
Water was changed every second day but no food was added during the
first three weeks to prevent growth of clams at higher salinities

-56-

• "EN9IS OIRECTUS

• • MYA ARENARIA

• • OSTREA E0UU3

o— o BRACHIOONTE3 RECURVU8

o o VENUS MERCENARIA

90
DAYS

Fig. l._J~Number of days needed by each of two size-groups of
"juvenile V. mercenaria to dig in at salinities of 15-0 and 27-0 ppt,

■57-

before those in lower salinities opened or dug in. After three weeks,
at least 70 per cent of the clams had dug in at each salinity and
food was then added whenever water was changed. At that time the
clams ranged from 4.9 to 9-9 nan and averaged from 7-3 to 7-6 mm in
length. They were measured at two-week intervals for 6 weeks. The
temperature during the experiment ranged from l6.5 to 22. 7°C-

The increase in average length was similar for each of the two-
week growing periods, which indicates that the animals had adapted to
the experimental salinities before feeding was started (Table 3)»
Otherwise those in low salinities would have grown more rapidly late
in the experiment, as they became adapted to the low salinities.
Growth was reduced progressively with decreases in salinities. At
15.0 ppt growth was negligible. Apparently, hard clams can survive
in salinities slightly lower than those in which they are capable of
growing.

Table 3- Average growth increments of juvenile V. mercenaria kept
at various constant salinities.

Salinity

Average increment in millimeters from

in ppt

0-2 weeks

2-k weeks

k-6 weeks

Total average increment

15.0

.2

-.1

.0

.1

17.5

.2

.2

.k

.8

20.0

.k

•3

.k

1.1

22.5

• 5

.k

.k

1-3

Full salinity

• 7

• 7

.6

2.0

(26.0-28.0)

My a arenaria

When 25 soft clams, ranging in length from 5-0 - 3^.0 mm,
from each of five different areas were transferred directly from
their normal salinities to lower experimental salinities, it was
found that the minimum salinity for survival varied (Table l). Soft
clams from Chesapeake Bay, where salinity is low, survived direct
transfer to 2.5 ppt but soft clams from Milford Harbor and Narragansett
Bay, where salinity is higher, were killed by such a change. To
determine whether this variation was caused by conditioning or was
evidence of inherent differences, soft clams from Chesapeake Bay that
had been kept at full salinity for three weeks were then transferred
directly to a salinity of 2.5 ppt. Under these conditions all died

•58-

within three days as did those from Milford Harbor and Narragansett
Bay. On the other hand, when soft clams from Narragansett Bay and
Milford Harbor were adapted to a salinity of 12.5 ppt and then trans-
ferred to the lower salinities by steps of 2.5 ppt per week, they dug
in and fed at 2.5 ppt. Furthermore, after such gradual conditioning,
Milford clams which were transferred directly from 2.5 ppt to full
salinity all dug in and were feeding by the second day. After 8 days
at full salinity these same clams were again returned directly to 2.5
ppt. Two days later 9 out of 10 had dug in and were feeding. Another
group of 25 Milford soft clams that had been conditioned similarly to
survive at 2-5 ppt, also survived direct transfer to full salinity and
all but two dug in within kQ hours.

These experiments indicate that the minimum salinity at which
soft clams survive is determined by the salinity to which they have
become adjusted and not by inherent differences in clams from different
areas. Unlike V. mercenaria, the minimum salinity at which soft clams
survived was lowered by gradually conditioning the animals in inter-
mediate salinities. If conditioned properly, soft clams from any of
the .areas reported here survived at all salinities ranging from 2.5 to
28.0 ppt.

Ensis directus

Eighteen juvenile razor clams, ranging in length from 25-0
to ^7.5 mm, were transferred from full salinity to each of the experi-
mental salinities.

All clams transferred directly from full salinity to 15.0 ppt
or higher dug in rapidly and fed normally almost immediately (Table l).
Seven of the 18 placed in 12.5 PPt dug in, but all were dead by the
fifth day. At 10.0 ppt they were gaping widely one day after the
transfer and, except for feeble movements of the foot after stimula-
tion, were not able to move. Although three had partially dug in at
this salinity, all were dead by the third day. At 7-5 and 5-0 ppt
there was no evidence of digging in and all died within two days.
All kept at 2.5 ppt or in fresh water died in less than 2h hours.

Razor clams kept at 15.0 ppt and higher for 35 days were
later subjected to successively lower salinities at the rate of 2.5
ppt per week. They dug in and fed normally at salinities down to and
including 7.5 ppt. When transferred from 7.5 to 5-0 ppt all but two
dug in. Nevertheless, they began to die after 2k hours and within a
week all were dead.

Even though razor clams survived from 7-5 to 28.0 ppt, when the
salinity change was gradual, sudden changes were quickly lethal. Those
that had survived for 13 days at 7-5 Ppt all died within 48 hours after
they were returned directly to full salinity. Apparently, these clams
are incapable of adjusting to a rapid salinity change of this magnitude
in either direction.

-59-

Crassostrea virginica

The American oyster occurs naturally in areas of widely
varied salinities. Ingle and Dawson (1950) reported commercial pro-
duction of oysters in an area where the salinity ranged from fresh
water to 42.5 PP"t annually. Loosanoff (1932) found commercial sets
of oysters in an upper James River area where the salinity ranged
from 4.29 to 10.66 ppt and in 1950 he demonstrated that oysters from
Long Island Sound could stand rapid transfer from 3-0 to 27.0 ppt with
no apparent ill effects.

Our salinity work with juvenile oysters has been limited to
two growth experiments. In the first, groups of 50 recently set oys-
ters, averaging from 0.3 to 0. 5 mm in length, were placed directly in
experimental salinities from 2.5 ppt to full salinity. They were
measured after two weeks and again after four weeks.

All spat kept in 2.5 ppt at a temperature of 21.0 to 24.0°C.
died before the end of the first two-week period. Slightly over half
of the spat kept at 5-0 ppt died before the final measurements were
taken, but those that survived did show a slight but measurable amount
of growth. Oyster spat kept at 7-5 and 10.0 ppt showed substantial
but slow growth. Their average size at the end of the experiment was
only 1.9^ and 1.85 n™> respectively, while the spat kept at salinities
of 12.5 ppt and higher showed much faster growth and averaged from
3.69 to 4.91 mm at the end of the experiment.

In another experiment 29 to 37 recently set oyster spat
averaging from 1.0 to 1.4 mm in length were transferred gradually to
the experimental salinities over a period of seven days. They were
measured again after two and four weeks of exposure to salinities
ranging from 2.5 ppt to full salinity.

Once again oysters kept in salinities from 15-0 to 22.5 ppt
grew well with an average four -week increment of from 7*0 to 8.2 mm
in length. Those kept at 10.0, 12.5 ppt and full salinity grew less
with average increments of from 3-8 to 5-0 mm, while those at 5-0 and
7.5 PP"t had average growth increments of only 1.2 and 2.8 mm, respec-
tively. Spat kept at 2.5 ppt decreased in average length .3 mm during
the experiment. This may have been the result of selective mortality.
However, the shells of the spat may have been eroded at this salinity
since the shells of even the survivors were papery and soft. Eighty
to 100 per cent of the spat survived in all of the experimental
salinities except at 2.5 ppt where only 19 per cent survived and at
5 • 0 ppt where 66 per cent survived .

These results indicate that juvenile oysters differ somewhat
from larvae in their salinity requirements. Davis (personal communi-
cation) found that oyster larvae showed negligible growth at 7-5 ppt
and eventually died. Even at 10.0 ppt growth was slow and no larvae
reached the setting stage. However, both larvae and juveniles grew

-60-

normally at 12-5 ppt and higher. Juvenile oysters responded to
reduced salinities in a manner similar to that reported for adults
(Loosanoff 1952), that is, they failed to grow at salinities below
5.0 ppt, grew slowly at salinities below 12.0 ppt, and grew normally
from 12.0 to 27.0 ppt.

Ostrea edulis

The minimum salinity at which most 6-month-old European
oysters survived following direct transfer from full salinity was
15.0 ppt (Table l). One oyster, however, survived at 12.5 Ppt, fed
normally and appeared to thrive throughout the 35 days of the experi-
ment, although all other oysters at this salinity quickly perished.
After 35 days the oysters kept at 15.0 ppt were transferred to 12.5
ppt. They appeared to adjust to this salinity and only two died during
the 31 days of additional exposure.

Brachidontes recur vus

Twenty hooked mussels from Chesapeake Bay, ranging in length
from 17 to ^2 mm, were placed in each of 11 experimental salinities
from fresh water to full salinity. They were kept in 3-liter poly-
ethylene containers at temperatures ranging from I7.60 to 2^.0 C.

They were feeding normally in each salinity from 2.5 ppt to
full salinity four days after direct transfer and no appreciable
mortality occurred in these salinities during the 50 days of the
experiment (Table l). In fresh water, however, the first mussel
died after four days of exposure and all were dead within 30 days.

After 50 days, five animals from each salinity were opened
and the gonads examined. In general, gonad condition was poor, pre-
sumably because of the limited amount of food and water available
during the experiment. However, except at full salinity, at least
one mussel in each sample had recognizable gametes.

Discussion

Several factors must be taken into consideration before
efficient management of shellfish resources can be achieved with
respect to salinity. The minimum salinity at which bivalves can
survive is determined by several conditions that must be understood.
In addition, it is also important to determine how long unfavorable
salinities can be tolerated and what factors affect the length of
time a species can survive in unfavorable salinities. We shall first
discuss conditions that determine the minimum salinity at which
survival is possible and, finally, conditions that affect the length
of time bivalves can survive in unfavorably low salinities.

-61-

The minimum salinity at which survival is possible varies
with the size and/or stage of development of bivalves of the same
species. For example, in these experiments, large juvenile hard
clams (10. 0 - 21.5 mm) survived for over 100 day-, at 12.5 ppt but
smaller juveniles (1.8 - 3.6 mm) did not survive below 15. 0 ppt.
Moreover, growth of even the larger clams was retarded at lower sa-
linities and virtually stopped at 15.0 ppt. Larvae of the same
species have even higher salinity requirements. Davis (personal
communication) foiond that larvae grew slightly at 15.0 ppt but that
none reached setting stage at salinities below 17-5 ppt. Even at
this salinity the larvae were so sluggish and weak that they perished
during the setting and immediate post-setting stages. Turner (1955)
also found that although larvae grew at 20.0 ppt, one-day-old larvae,
which normally swim upward, were unable to do so between 15-0 and 20.0
ppt. Apparently, then, a population of hard clams cannot be sustained
below 20.0 or 17.5 ppt, although individuals might survive in salini-
ties as low as 12.5 ppt.

Geographically separated populations of bivalves may also have
inherently different salinity requirements. Races, physiologically
different in other respects, are known to exist and although little
supporting evidence was found in these experiments the same species
of bivalve from different areas may require different salinity ranges.

Another factor affecting the minimum salinity at which marine
bivalves survive is the source of fresh water. We found that hard
clams died at 20.0 ppt when the salinity was reduced by fresh water
very high in copper content, yet, they survived at 12.5 ppt when the
salinity was reduced by demineralized water.

The rate of salinity change is also important. Razor clams,
for example, were killed readily by rapid salinity changes of 15. 0 ppt,
while gradual changes over the same range were tolerated. Bivalves
capable of withdrawing all organs into a water-tight shell, such as
the hard clam, are not nearly as susceptible to rapid changes of
salinity.

In addition to a knowledge of the minimum salinity at which
a species will survive, grow, and reproduce, it is important to know
how long unfavorable salinities can be tolerated. Several conditions
affect the length of time bivalv s can exist in salinities too low
for indefinite survival.

Water temperature, for example, is of primary importance.
Loosanoff (19^8) found that oysters could survive in fresh water or
at a salinity of 3.0 ppt for 70 or 115 days, when the temperature
ranged from 8.0° - 12.0°C. At these same salinities all oysters died
within 15 days when the temperature ranged between 23.0° and 27.0°C.

Size is also important in determining how long unfavorable
salinities can be tolerated. As previously noted, small hard clams

■62-

CLAM SIZES

• •10 0-815 MM

• .1 8-3 6 MM

/

/

/

io le

DAYS

Fig. 2. Comparison of the mortality rates of five species of
mollusks in salinities which were 2.5 ppt lower than the minimum
salinity at which they survived.

-63-

(1.8 - 3.6 mm in length) all died within 15 days at 10.0 ppt hut none
of the larger ones (10.0 - 21.5 mm) died at this salinity until the
28th day of exposure.

Finally, some species are capable of enduring longer periods
of unfavorable salinity than others (Fig. 2). Hard clams survived
in unfavorable salinities for several weeks probably because they
could withdraw all organs into a water-tight shell and prevent exposure
of vital parts to unfavorably low salinities. However, other bivalves,
such as soft clams and razor clams, did not survive exposure to lethal,
salinities for more than a few hours or days' because their shells were
incapable of forming a water-tight cavity to protect the clams from
unfavorable salinities .

Acknowledgments

I would like to express my gratitude to Dr. V. L. Loosanoff
and Mr. H. C. Davis for their advice and cooperation in these experi-
ments, and for their assistance in preparing this report. I am also
indebted to Mr. C. A. Nomejko for preparing the graphs, and to our
associates in the Clam Investigations for supplying the experimental
animals from Maine, Narragansett Bay and Chesapeake Bay. I am also
grateful to the Office of River Basin Studies, U. S. Fish and Wildlife
Service, for the financial support that made these studies possible.

Literature Cited

Ingle, Robert M., and Charles E. Dawson. 1950. Variations in
salinity and its relation to the Florida oyster. Proc .
Natl. Shellfish. Assoc, 1950: 16-19.

Loosanoff, V. L. 1932. Observations on propagation of oysters in

James and Corrotoman Rivers and the seaside of Virginia. Va.
Comm. of Fish., Newport News, Va., pp. l-h6.

Loosanoff, V. L. I9U8. Survival, feeding and growth of oysters (0.
virginica) in low salinities. Anat. Rec . , 101: 1-55.

Loosanoff, V. L. 1950. On behavior of oysters transferred from low
to high salinities. Anat. Rec, 108: 91-

Loosanoff, V. L. 1952. Behavior of oysters in water of low salini-
ties. Proc. Natl. Shellfish. Assoc, 1952: 135-151-

Pratt, D. M., and D. A. Campbell. 1956. Environmental factors

affecting growth in Venus mercenarla. Limnol. & Oceanogr .
1: 2-17.

■6k-

Turner, Harry J. 1953. Growth of molluscs in tanks. Sixth Rept.
Invests. Shellfish. Mass. Dept. Nat. Res., pp. 35-39-

Turner, Harry J., and Carl J. George. 1955. Some aspects of the
behavior of the quahaug, Venus mercenaria, during the early
stages. Eighth Rept. Invests. Shellfish. Mass. Dept. Nat.
Res., pp. 5-lk.

•65-

CAUSES OF DEPLETION OF OYSTERS
IN ST. VINCENT BAR, APALACHICOLA BAY, FLORIDA !

R. Winston Menzel, Neil C. Hulings and Ralph R. Hathaway

Oceanographic Institute, Florida State University

Abstract

During the period from June 1955 through May
1957 a*1 ecological study was made of oyster reefs in
Apalachicola Bay, Florida. This report covers a phase
of the study on St. Vincent Bar. This reef is entire-
ly depleted although it has been very productive in
former years . Salinity over this reef has been high
and several enemies of the oyster have become estab-
lished here, especially the southern oyster drill,
Thais haemastoma, and the stone crab, Menippe
mercenaria. There was an abundant spatfall on the
old shells during both years of study, but no oyster
was found on the reef larger than 50 nun in length in
the first year and the maximum size was less than ^0
mm in the second year. An experiment was made dur-
ing the second year in which the oysters were pro-
tected from predation of both drills and stone
crabs, and not only was the mortality less for the
protected oysters, but a maximum size of between
70 and 80 mm in length was reached before the end
of the observations. Circumstantial evidence indi-
cated strongly that the predators, especially drills
and stone crabs, have caused depletion of the reef.

Introduction

An ecological study of oyster reefs in Apalachicola Bay, Florida,
was begun in June 1955- During the first year's study four oyster reefs
were sampled at approximately monthly intervals for oysters and
associated organisms, especially predators. The method of sampling
involved removal by hand of all shell from a measured area, usually
one to three square meters. Aqua-lungs were used in deep water.
Samples were brought back to the Oceanographic Institute, Florida
State University, Tallahassee, for examination. All large animals

1 Contribution No. 85, Oceanographic Institute, Florida State Univer-
sity. This study was supported by Contract No. 1^-19-008-230, U. S.
Fish and Wildlife Service. The writers wish to thank the Florida
State Board of Conservation for boat transportation and personnel.
Our thanks are due also to Dr. P. A. Butler, U. S. Fish and Wildlife
Service, for many helpful suggestions.

-66-

were either counted and recorded as number per square meter, or were
estimated as to abundance. The second year was devoted to a more
intensive study of one of the reefs, St. Vincent Bar, while the other
reefs were sampled seasonally. This is a report of part of the study;
a more detailed report will appear later.

This report deals only with St. Vincent Bar, which was sampled
from September 1955 through May 1957 • Only a small portion of this
reef was systematically sampled, but numerous observations indicate
that it was entirely depleted of large oysters. There were many old
shells and fragments of shells, but large oysters were never seen.
Swift (1898) shows dense growth of oysters in this area in his survey
of 1895-I896. Danglade (1917) stated that St. Vincent Bar was show-
ing signs of depletion and was closed by the State to recover. During
the investigation of Pearse and Wharton (1938) there was an indication
that St. Vincent Bar was still productive. Ingle and Dawson (1953)
list the production of this bar as "none". Mr. Joseph Martina, Jr.,
agent of the Florida State Board of Conservation, has stated (private
communication) that the last commercial oysters were taken from this
reef more than five years ago, and that it has been depleted since
then with only sporadic tonging by oystermen. Many of the oysters
taken in recent years probably came from isolated lumps in adjacent
areas, and not from the bar itself.

Results

St. Vincent Bar was first sampled 21 September 1955. At this
time over 500 live oysters were found per square meter. Nineteen per
cent of the oysters were of the species Ostrea equestris Say, but
nearly ^00 were of the commercial species, Crassostrea virginica
(Gmelin), and the density was considered fairly heavy. No oyster was
over 50 mm in length, and it is fairly certain that all had attached
during the summer of 1955' When the next examination was made, less
than one month later (ik October 1955), only 165 live oysters of both
species were found per square meter, and the largest oyster was less
than 20 mm in length. By the following 9 May 1956 examination, 72
oysters of both species were recorded per square meter and all were
under kO mm in length. The percentages of Ostrea equestris decreased
from 32f0 of the total number of oysters found in the October 1955
examination to a low of 6$ in the May 1956 examination.

It is evident that the oyster mortality was high. The most
serious aspect of the mortality was that the oysters died before they
attained market size. Sometimes mortality was so rapid among large
oysters that the size of the largest oysters decreased with each
succeeding month.

It appears certain that an increase in salinity, caused by
rainfall below normal over the past four or five years, has occurred
on many of the outlying reefs in Apalachicola Bay. This has allowed

-67-

the invasion, often in large numbers, of many animals that cannot
tolerate salinity as low as can the Virginia oyster. The small non-
commercial species, Ostrea equestris is one example. The southern
oyster drill or conch, Thais haemastoma Conrad, is less tolerant of
low salinities than the commercial oyster, and the same is true for
the stone crab, Menippe raercenaria Say. Both the drill and the stone
crab are oyster enemies of importance in the Gulf area (Butler, 195^-j
Chapman, 1955; Menzel and Hopkins, 1955- ) Drills occurred in numbers
as high as six per square meter and averaged 2.75 per square meter for
all of the 20 examinations during the period of the study. No young >
drills were seen, although egg capsules were deposited during the
spawning season of 1956. The size of the adult drills ranged from 52
to 8^ mm. The abundance of stone crabs, 50 to 100 mm wide, was esti-
mated to be about one per square meter; no satisfactory quantitative
sampling method was developed for stone crabs in the subtidal area.
It is not possible to determine the mortality of oysters caused by
T. haemastoma on a reef because of the method of attack, however,
some drill "marks" were seen. Many broken shells were seen and these
are evidence of damage by stone crabs . Because of the abundance of
these two predators, it is assumed that they were responsible for much
of the mortality.

Other enemies occur in this area also, but the effect of
salinity on their distribution apparently is not as critical as with
the two predators mentioned above. There were many snails of the
genus Qdostomia (0. impressa Say), but no correlation was possible
with oyster mortality, e. g. they were as numerous in other areas
where oyster mortality was much lower. Also present in large numbers
on four occasions was the "oyster wafer", Stylochus frontalis Verrill.
At the December 1955> and the January, February and April 1956 exami-
nations they numbered up to 50 per square meter. From one to four
worms were found per square meter in four other examinations of the
natural bottom. The mortality rate did not reflect their presence or
absence.

On 9 May 1956, wire baskets of one-half inch mesh hardware
cloth were made. These baskets measured 26 by 51 cm, and were 9 cm
high. Each basket held 12 liters of shell, which was tonged from St.
Vincents Bar. All large organisms, especially drills and stone crabs,
were removed before the material was placed in the baskets. It has
been noted above that sampling showed 72 live oysters per square meter
and therefore some live oysters were included in the baskets. Enough
baskets were made so that two could be examined at approximately
monthly intervals from the first examination on 27 June 1956 to the
termination date on 11 May 1957- Eleven examinations were made during
the period. Meter square samples of the bottom were taken concurrently
with each examination of the baskets.

The volume of shell in the two baskets was approximately equal
to the amount usually taken from one square meter of bottom, but the
numbers of living organisms were not comparable. The baskets were

-68-

raised 9 era from the bottom and hence provided more surface for the
attachment of sessile organisms, especially oysters, and also seemingly
provided a better habitat for many of the small sedentary organisms.

On the first examination in June 1956, there were 605 live
oysters in the two baskets, ranging up to 40 mm in length. The
majority, however, were below 20 mm and 51$ were Ostrea equestris.
The number of oysters in the two baskets was over 1500 by October and
November 195 6, but had decreased to 79*+ by the last sample taken 11
May 1957. The number of oysters per square meter of bottom ranged
from a high of 363 in November to a low of 117 when the last sample
was taken in May.

Previous to the May 1957 sampling, the salinity ranged from
20 to over 30 ppt. Occasionally the salinity was lowered to about
10 ppt for a short period of time as a result of freshets. These
lowered salinities had no observable effects on the fauna of the reef.
During the late spring of 1957* rainfall increased considerably lower-
ing the salinity to below 10 ppt. On the May 1957 examination the
lowered salinity (7.5 ppt) had considerable effect on some of the fauna.
No living Ostrea equestris were found. Two Thais haemastoma were found
but they were inactive, buried in the substrate.

There were always considerable oysters per unit area, but none
of the oysters on the bottom exceeded kO mm in length during the second
year's study. Ingle and Dawson (1952) have shown that growth of the
commercial oyster is rapid and continuous throughout the year in
Apalachicola Bay. Yet on St. Vincent Bar the maximum size of the
oysters on fome sampling dates was less than the maximum of the month
before. On the other hand, some of the oysters in the baskets during
the latter part of the investigation reached a length of between 70
and 80 mm. These oysters were deep-cupped and fairly thick-shelled.

Although conditions were not the same in the baskets as on
the bottom, all known enemies of oysters, except large stone crabs
and drills, were present. In fact some animals were more abundant
in the baskets than on the bottom. The "oyster wafer" was found in
the baskets in seven of eleven examinations, and only four times on
the bottom during the same period. In no instances were many worms
found, the maximum at any examination being five in the two baskets
and four per square meter of bottom. Small stone crabs in the baskets
rarely measured as much as 30 mm in width of carapace, but even crabs
this small are capable of killing oysters.

Dermocystidiura marinum Mackin, Owen and Collier was known to
be present in the area (Dawson 1955)- Dr. J. G. Mackin, Texas A and
M Research Foundation kindly examined slides of eight survivors from
a mortality experiment conducted on the bar. These were large oysters
that had been transplanted from another reef in the Bay and were
placed in wire baskets to protect them from large predators. These
oysters were taken in October 1956 and had a mortality of over 5°$>

•69-

during the previous month. Examination showed two oysters to be
negative, and six others to have light to moderate infections. It
is thought that the fungus caused the heavy mortality of the large
oysters in this particular experiment. Mortality on the bottom,
however, was not characteristic of that caused by the fungus (Mackin
1951> Ray 1953)' The oysters were small and the mortality proceeded
rapidly during the winter months. The best evidence against the
mortality being caused by D. marinum is that the oysters lived better
and were of a larger maximum size in the baskets than on the bottom.

The results of the basket experiments from which drills and
stone crabs were eliminated, confirmed the assumption that predation
by these animals was the major factor in the oyster mortality and
the lack of any large oysters.

Summary

St. Vincent Bar, Apalachicola Bay, is a depleted oyster reef
that was formerly very productive. For the past four or five years
rainfall has been below normal in the area, with the result that
salinity has increased and has allowed the establishment of several
important oyster predators on the reef. Beginning in the spring of
1957* rainfall increased. In May 1957 the salinity over the reef was
low (7-5 PPt ) and there was a scarcity of predators. There was abun-
dant spatfall during the two years of the study, but mortality was
very high, although the numbers of oysters remaining were probably
adequate to replete the reef. Even though the numbers of oysters per
unit area were always considerable, no oyster was ever found over 50
mm in length. Shells from the bottom were placed in wire baskets in
June 1956, and two baskets were examined monthly; -the last examina-
tion was in May 1957- Drills and large stone crabs were effectively
eliminated from the baskets and mortality of the oysters was con-
siderable less than on the unprotected bottom. More important, some
of the oysters in the protected baskets reached a length of 70 to 80
mm before the end of the period. This is strong circumstantial evi-
dence that predation, especially by drills and stone crabs, was the
primary cause of mortality and the lack of large oysters. Thus with
increased rainfall and decreased salinity, and with elimination of
predators, there is hope that St. Vincent Bar will recover.

Literature Cited

Butler, P. A. 195^. The southern oyster drill. Proc. Natl. Shell-
fish. Assoc. 45: 67-75.

Chapman, C. R. 1955. Feeding habits of the Southern oyster drill,

Thais haemastoma. Proc. Natl. Shellfish. Assoc. k6: I69-I76.

-70-

Danglade, E. 1917- Conditions and extent of the natural oyster beds
and barren bottoms in the vicinity of Apalachicola, Florida.
Rept. U. S. Cororo. Fish., 1916: 1-68.

Dawson, C. E. 1955- Observations on the incidence of Dermocystidium
marinum infection in oysters of Apalachicola Bay, Florida.
Texas Jour. Sci. 7(l): ^7-56.

Ingle, R. M., and C. E. Dawson. 1952. Growth of the American oyster,
Crassostrea virginica (Graelin) in Florida waters. Bull.
Marine Sci. Gulf and Caribbean 2(2): 393-404.

1953- A survey of Apalachicola Bay, Florida. Florida State

Board of Conservation. Tech. Ser. 10.

Mackin, J. G. 1951- Incidence of infection of oysters by

Dermocystidium in Barataria Bay area of Louisiana. Proc.
Natl. Shellfish. Assoc. k2: 22-35-

Menzel, R. W. and S. H. Hopkins. 1955- Crabs as predators of oysters
in Louisiana. Proc. Natl. Shellfish. Assoc. 46: 177-184.

Pearse, A. S., and G. W. Wharton. 1938. The oyster "leech", Stylochus
inimicus Palombi, associated with oysters on the coasts of
Florida. Ecol. Monogr. 8: 605-655-

Ray, S. M. 1953. Studies on the occurrence of Dermocystidium marinum
in young oysters. Proc. Natl. Shellfish. Assoc, kk: 80-88.

Swift, F. 1898. Report on the survey of the oyster regions of St.

Vincent Sound, Apalachicola Bay, and St. George Sound, Florida.
Rept. U. S. Fish. Comm. I896, 22: 187-221.

-71-

THE SEASONAL SIGNIFICANCE OF TOTAL SOLIDS OF OYSTERS
IN COMMERCIAL EXPLOITATION

James B. Engle

Fishery Research Biologist

Clara & Chesapeake Oyster Investigations

U. S. Fish and Wildlife Service

Annapolis, Maryland

Abstract

Per cent dry weight and total solids of oyster
meats were determined from 1,000 samples taken monthly
or raore frequently for 10 years. Seasonal fluctuations
are shown by monthly averages of 11 stations for all
years. Lowest solids occurred regularly in August and
highest solids in November and December. The average
monthly low was 13-l4$ solids and the average monthly
high was 19-20$. The lowest solids content was 6.1$
in a sample taken during a period of abnormally low
salinity in August 19^-6. The highest solids content
was 26.5$ in February 19^7* Solids, seasonally were
low in summer and early fall, high in late fall and
early winter, relatively low in late winter and high
in late spring. This indicates that for optimum
quality of oysters, the best seasons for harvesting
are late fall and late spring.

The quality of oyster meats is a factor of foremost considera-
tion to consumer, producer and scientific personnel of State and
Federal conservation departments, pure food agencies and health
departments. From the commercial aspect, quality of oysters is in
part yield and in part appearance. Each of these is related to a
so-called fatness. "Fatness" in this instance is a misnomer because
most of the accumulated material is glycogen, a carbohydrate, and
not a fat .

The accumulation and utilization of glycogen are seasonal
phenomena of great importance in determination and evaluation of
quality in oyster meats. Fluctuations in quality and condition of
oysters may be attributed to gonad development, discharge of gametes,
type and availability of food, and effects of extreme changes in
environment. Adverse environmental influences such as (l) rapid sa-
linity depression caused by hurricane storms, freshets following
wet seasons and melting ice and snow in the water shed (Engle 19^6,
Beaven 19^6), and (2) accumulation of H2S and accompanying depletion
of oxygen (ito and Iraai 1955) inhibited growth and fattening of
oysters. Industrial pollutants, such as pulp mill effluents, may

-72-

change the local environment sufficiently to interfere with normal

fattening of oysters (Galtsoff et al 19^7 )• These factors are linked

and overlap and a summation of their effects on the condition of
oysters can be measured.

Indices of condition of oysters such as per cent glycogen,
per cent dry weight or total solids, and "condition factor" are
acceptable means of measuring quality in oyster meats. Chipman (19^7)
Engle (1950) and Engle and Chapman (1951) described in detail the
methods of determining condition.

The dry weight or per cent solids is the simplest of these
methods of determining condition or quality of oyster meats. It is
used here for that reason and because pure food agencies use it as
an index of adulteration with water. It may be defined briefly as
the dry weight or solids per unit quantity of wet meat determined by
the following procedure:

1. Shuck oysters without cutting the body except at adductor
muscle.

2. Drain meats on a stainless steel grid for 12 minutes after
shucking.

3. Weigh drained meats and homogenize in Waring blender.

h. Weigh two aliquots of blended meats and dry for 72 hours
in an oven at 90° •

5. Per cent dry weight is the dry weight of the aliquot
divided by the wet weight of the aliquot x 100. Total
solids are calculated by multiplying the wet weight of
the whole sample of meats by the per cent dry weight.
Per cent dry weight and per cent solids are synonymous
in this discussion.

Condition or quality of oysters is far from stable. Individual
oysters vary within a bar or bed and oysters from one bar or community
may differ from those in another. These differences in quality are
seasonal also, but within a geographical area seasonal changes are
often similar. If oysters from widely separated geographical areas
are compared, differences in seasonal patterns of fattening are
observed. For instance, oysters in Long Island Sound appear to
"fatten" earlier in the fall than do those in Chesapeake Bay. Varia-
tions in seasonal temperatures of waters affect the condition cycle.
Continued high temperatures in the fall of the year usually delay
fattening of oysters. Hopkins et al (1953) indicated that Louisiana
oysters reach peak fatness in late winter and early spring. The pres-
ent studies are confined to seasonal changes in condition or quality
of oysters in Chesapeake Bay and the influence on commercial use.

-73-

Fig. 1. Per cent total solids of oysters, salinity and
temperature in Chesapeake Bay on monthly average basis, 19^9
to 1955.

M M 4 S N
f 1 J » 0 0

Fig. 2. Per cent total solids of oysters, salinity and
temperature in Eastern Bay on monthly average basis 19^9 to
1955.

-Ik-

Condition of oysters, expressed as per cent dry weight or
solids, has been observed in oysters from several locations in upper
Chesapeake Bay during the past ten years. Emphasis has been focused
on seasonal changes with respect to cyclic occurrence and to annual
amplitude. More than 1,000 determinations of per cent solids were
made from 19^5 to 1955- Samples were taken from 11 stations in upper
Chesapeake Bay and its tributaries.

The solids content of oysters ranged from a low of 6.1$ at
Tea Table in upper Chesapeake Bay in August 19^6 to 26.5$ at Thomas
Bar in the Patuxent River in February 19^7- For oysters in Chesapeake
Bay proper, the average annual low, based on seven years of continuous
observations from 19^9 to 1955 inclusive, was 1^$ and the average
annual high for the same period was 20$. Eastern Bay, a major oyster-
producing tributary of upper Chesapeake Bay, which was under observa-
tion for the same period, produced an average annual low of 13$ and a
high of 19$. Excessively wet seasons were included during 19^+5 to
19^7 which accounted for the extremely low figure of 6.1$ solids.
Per cent solids is the complement of water content, hence, an oyster
with a solids content of 6.1$ is 93-9$ water and an oyster with a
solids content of 26-5$ is 73-5$ water.

Monthly averages of per cent solids taken over the seven
years indicate a seasonal periodicity as follows: (l) The lowest
level of solids occurred regularly in August at all stations. Almost
as regularly the highest level of solids occurred in December in
Chesapeake Bay and in November and December in Eastern Bay and other
tributaries. High solids occurred regularly in May and June follow-
ing a short period of decreasing solids in February, March and April
(Figs. 1 & 2).

The principal cause of seasonal fluctuation in solids is
changes in glycogen. In June glycogen is converted to gonadal pro-
ducts (Engle 1950). By August, glycogen and gametes are most
expended and oysters are in their poorest state. The annual cycle
of condition can be seen in Fig. 3 in which the points for each month
represent the average for seven years.

Commercial utilization of oyster meats requires that the pro-
duct contain as much meat and as little water as possible. A pure
food measure of adulteration is the per cent solids and a tentative
level of solid content proposed as the dividing line between good and
poor oysters is 10.4 per cent. To have oysters go below that percent-
age in nature took a long exposure to low salinities such as prevailed
in upper Chesapeake Bay in 19^5-46. Salinities at that time, from
March I9J+5 to August 19^6, did not exceed 10 parts per thousand except
for several weeks in September 19^5 while for at least half this
period the salinity was less than 5 parts per thousand and oysters
were poor. During these unusual conditions from September 19^+5 to
August 1946 solids were under 10 per cent. Long exposure to reduced
salinity achieved this but it can be accomplished by over exposure to
fresh water during shucking house cleaning (Pottinger e_t al 19^-1 )•

-75-

Q

o

CO

21

20
19

18
17
16
15
14
13

CHESAPEAKE BAY
TOLLYS BAR

M

o

M

20

EASTERN BAY

MILLHILL BAR
BODKIN BAR

1?
18

17

>* /TV

16
15

14
1 3
1?

1

1 1 1 1

\ ^' ' /

• i i i i i i i

Fig. 3- Composite curves showing monthly averages of
per cent total solids on basis of seven years, 19^9 "to 1955>
to indicate general seasonal changes.

-76-

In September oyster meats are thin on most bars in upper
Chesapeake Bay because little glycogen has accumulated since spawning
(Fig. 3). At this time yield of shucked meats is low and plumpness
and esthetic appeal, which in large measure are reflections of glycogen
content, are lacking. The curve of seasonal condition indicates that
oyster meats are prime in November. Yield in pints of meats per
bushel of shell oysters may increase from 4 in August to 8 in November.

It would seem logical to postpone harvesting of oysters until
October to obtain higher yields of a superior product. Oysters for
September and summer use could be quick frozen in May when they are
usually in prime condition. This is a departure from precedent and
in upper Chesapeake Bay (Maryland) would require changes in laws to
extend the harvesting season beyond April 15 • There is no regulation
that prohibits sale of frozen oysters in summer months.

With oyster production declining in many parts of the Atlantic
seaboard, the necessity of getting the most out of nature and the oyster
is imperative. One way of doing this is by applying the knowledge
gained by studies such as these to the harvesting and processing of
this food. The data disclosed by these observations show seasonal or
cyclic variation in the quality of oyster meats. The higher level of
solids and glycogen in oyster meats occurs in late fall and late spring
and the lowest in late summer. Harvesting and processing oysters at
these periods of peak quality would benefit the consumer by offering
the food at its best and the producer by offering the food at its
maximum yield.

Literature Cited

Beaven, G. F. 1946. Effect of Susquehanna stream flow on Chesapeake
Bay salinities and history of past oyster mortalities on
upper Bay bars. Natl. Shellf isheries Assoc. Conv. Add.
1946: 38-41.

Chipman, W. A. 194-7. Seasonal changes in the fattening of oysters.
Natl. Shellf isheries Assoc. Conv. Add. 1947: 28-32.

Engle, J. B. 1946. Commercial aspects of the upper Chesapeake Bay

oyster bars in the light of recent oyster mortalities. Natl.
Shellf isheries Assoc. Conv. Add. 1946: 4-2-46.

Engle, J. B. 1950. The condition of oysters as measured by the
carbohydrate cycle, the condition factor and the per cent
dry weight. Natl. Shellf isheries Assoc. Conv. Add. I95O:
20-25.

Engle, J. B., and C. R. Chapman. 1951- Oyster condition affected by
attached mussels. Natl. Shellf isheries Assoc. Conv. Add.
1951 : 70-78.

■77-

Galtsoff, P. S., W. A. Chipman, Jr., J. B. Engle and H. N. Calderwood,
19^7- Ecological and physiological studies of the effect of
sulfate pulp mill wastes on oysters in the York River, Vir-
ginia. Fish. Bull. 43, U. S. Fish and Wildlife Service 51:
58-186.

Hopkins, S. H. , J. G. Mackin and R. W. Menzel, 1953- The annual
cycle of reproduction, growth and fattening in Louisiana
oysters. Natl. Shellf isheries Assoc. Conv. Add. 1953:
39-50.

Ito, S. and T. Imai, 1955- Ecology of oyster bed I. On the decline
of productivity due to repeated cultures. Tohoku Jour.
Agric. Res. 5(4); 251-268.

Pottinger, S. R., J. F. Puncochar and W. H. Baldwin, 19^1. Results
of some preliminary experiments on the effects of washing
and blowing on the mineral content of oysters. Fishery
Market News, U. S. Fish and Wildlife Service, Wash., D. C.
June (19^1): 3-6.

-78-

AW AUTOMATIC WATER SAMPLER FOR MARINE SHORE STATIONS

Ronald E. Westley

State of Washington Department of Fisheries
Shellfish Laboratory, Pt . Whitney, Quilcene, Washington

Abstract

An automatic machine was designed which takes
hourly samples of natural waters for a week. The
machine was housed on shore with a pump located near
high tide level and an intake hose buoyed at proper
depths. The apparatus is suitable for measurement of
salinities, some pollutants, and possibly bacteriolog-
ical or plankton samples subject to the particular
requirements of storage, evaporation and contamina-
tion of samples.

In many investigations of inshore marine environments it is
necessary to measure changes in waters that occur during short spans
of time. It has been determined that significant changes in chemical
and physical properties of sea water may occur even in the space of
an hour and that these changes may have a decided influence on the
biological populations present in an area. Thus, in an investigation
it is necessary to ascertain if such changes occur and to determine
their magnitude to evaluate observed biological phenomena.

Some of the properties of sea water can be determined directly
and automatically with recording instruments and these present no
major problems. The properties that can be measured only by chemical
determinations from water samples are quite a different matter. To
station men in an area to collect the necessary samples is expensive
and wasteful. For example, to collect hourly samples for a week would
require h man-weeks of work. Usually it has not been possible to do
this and continuous data have not been available.

During the past few years the Washington Department of
Fisheries has been investigating unexplained mortalities of the
Olympia oyster (Ostrea lurida) in the bays of south Puget Sound,
Washington. In the course of these investigations a pulp mill
located in the area became one of the suspected causes of mortali-
ties. It was then deemed advisable to collect continuous data on
pulp mill effluent and salinity values. To gather this information
the University of Washington Department of Oceanography, working on
contract for the Department of Fisheries, designed and built an auto-
matic water -sampling machine which collects a sample of sea water
each hour for a week. The machine was designed by Dr. R. G. Paquette
and Mr. E. L. Scott, and constructed by Mr-. Scott. The University of
Washington is applying for commercial patent rights on the machine.

-79-

Fig. 1. Automatic machine for taking hourly samples of natural
waters for a week.

-80-

The basic design of the machine is as follows. The 180
citrate bottles are held in exact position in rows by metal separators
in a large, flat tray. The tray also serves as a large sink to
carry off excess water. Mounted on the top of the tray, on a track,
is a miniature, walking-gantry crane which holds the fill pipe, which
in turn is connected by flexible tubing to the sea-water pump. The
operation of the machine is governed by an electrically-timed motor
operating a series of cams and micro-switches which in turn initiate
and terminate the various functions of the machine.

Operating sequence is as follows: The walking crane motor
starts, moving the crane and fill pipe to a point halfway between
two bottles. The sea-water pump starts and flushes all the water in
the system from the previous sample. The crane then moves forward
again, stopping when the fill pipe is directly over the bottle to be
filled, and after the bottle is filled the pump shuts off. From start
to finish this operation requires about ten minutes. The complete
cycle is repeated once an hour, filling one new bottle each hour.
After walking crane moves forward to the end of one row of bottles a
wedge on the traveler comes in contact with a fixed roller which
causes the fill pipe to slide over to the next row of bottles. In
this same motion a switch is activated, the direction of movement of
the walking crane is reversed, and it moves backward over the next
row of bottles.

Exact physical location of such a machine is governed largely
by the information needed, tidal currents of the area, and special
considerations of the specific problem. In the particular situation
being investigated in south Puget Sound, the machine was located on
Hammersley Inlet, a narrow channel connecting the pulp mill with the
oystering areas. Complete mixing of water occurs in this restricted
channel, and enables the machine to obtain a representative sample of
everything leaving the mill estuary.

Installation of the present model must conform to a few basic
requirements. The machine must be located near a source of electric
power, and because of the number of samples collected, it should be
readily accessible. The machine itself is housed in an 8 x 12 ft.
building set on the shore. The sea-water pump is located in a water-
proof casing at extreme high tide level on the beach. Care must be
taken that vertical distance from the pump to extreme low water does
not exceed the ability of the pump to lift water. From the pump a
chemically-inert plastic line runs to an anchor placed in about 10
feet of water at low tide. From the anchor the hose is attached to a
buoy floating on the surface which suspends the intake one foot below
the surface of the water. A readily-accessible check valve is
located at the buoy.

Operation of the machine proves to be fairly simple and trouble-
free. The greatest source of trouble is the check valve; it is impera-
tive that the check valve be reliable, resistant to the action of sea

-81-

water and capable of complete closing even under low water pressure.
The only remaining source of trouble with the check valve arises
through jamming by small particles of foreign material entering the
line through the intake screens. To avoid freezing, lines are buried
in the ground and the building housing the machine is insulated and
provided with a small, electric heater.

A large number of sample bottles is required to keep the
machine in constant operation. In practice, about four sets of
bottles are needed to insure against shortages caused by delays in
sample analysis and bottle washing. The large number of samples
collected, and the frequency with which each bottle is reused,
requires that bottles be numbered in sequence. Extreme care in
recording data is necessary to avoid confusion.

Analysis of samples is a major item. In this investigation
a method of screening samples was worked out. Initially, all samples
were run to determine expected variation; later, since our interest
was primarily in the quantities of sulfite waste liquor leaving the
mill, we were able to consider mainly ebb-tide samples. After deter-
mining the time period involved in concentration changes, we decided
to run every other sample on the ebb tide, plus one sample on the
start of the flood. If the values derived from these samples fall
within the expected range no additional samples for the time period
are analyzed. However, if variance is encountered, the rest of the
samples are analyzed until the extent of the variation is determined.

With a little ingenuity, the basic principles of this machine
could be applied to many different types of investigations requiring
frequent observations. In situations where samples are desired with
greater or less frequency, it would be a simple matter to change the
electric -timing device which regulates the sampling period. If
samples are desired only at certain stages of the tide, a pressure
switch activated by increasing tidal height could be installed. In
areas where tidal range exceeds the ability of the pump to lift water,
the pump could be located on a float at water level. Some considera-
tions necessary for adaptation of the machine to other investigations
are:

1. Length of time samples can be stored.

2. Prevention of evaporation.

3. Prevention of contamination of the sample by components
of the machine or extraneous sources.

In addition to various types of samples that can be collected
for chemical analyses, it is believed that modifications of the basic
setup would permit collection of bacteriological or plankton samples.

In summary, it has been found possible with this machine to
obtain data with a minimum of effort and expense. Servicing and
repairs to the machine required about one man-day per week (an average
during six months operation). Overall cost of the machine, building
and installation was about $U,500.

-82-

BURIAL AS A METHOD FOR CONTROL OF
THE COMMON OYSTER DRILL, UROSALPINX CINEREA,
OF LONG ISLAND SOUND

V. L. Loosanoff and C. A- Nomejko

U. S. Fish and Wildlife Service, Milford, Conn.

Abstract

A method is suggested by means of which the
oyster drill, U. cinerea, an important enemy of oys-
ters, may be controlled. The method consists of
burying drills under several centimeters of bottom
material. This can be accomplished by using modi-
fied agricultural plows, oyster and clam dredges,
etc., to turn over layers of bottom deposit. Labora-
tory experiments demonstrated that when buried at a
depth of 3 cm kO per cent of the drills could not
reach the surface and eventually died; at k cm 75
per cent died; and at a depth of 6 cm 92 per cent
did not reach the surface. Additional experiments
showed that all drills buried under a 6 cm layer
of bottom material at 25°C. died in five days; at
20°C, in seven days; at 15°C . , in 12 days, and at
10°C. 19 days were needed to kill all the drills.
Approximately 52 days were necessary to cause com-
plete mortality of the group which was buried in
the mud when the water temperature ranged from 0°
to 5°C

Biologists and oyster growers have thought for some time
that the oyster drill, Urosalpinx cinerea, is not able to emerge when
buried beneath several inches of substratum (Carriker 1955)- Since
reliable field observations and studies under controlled conditions
were not available to support this conception, we decided to deter-
mine the mortality of drills when buried at different depths beneath
a mixture of mud and sand.

Preliminary experiments were undertaken during the summer of
1956 and have already been described (Loosanoff 1956). In general, it
was found that when drills were buried beneath 2 cm or less of bottom
material there was virtually no mortality because all of them emerged
within a short time. When buried at a depth of 3 cm, however, approx-
imately 40 per cent could not reach the surface and eventually died;
at h cm, 75 per cent died; at a depth of 6 cm, 92 per cent did not
reach the surface.

-83-

It was found that the rate of emergence from soft mud was
higher than from harder substratum . Drills experienced no difficulty
in moving along mud surfaces; both large (25 mm) and small (8 mm)
drills moved through mud, even though submerged to one -half their
height. The experiments also indicated that small drills emerge in
approximately one -half the time needed by large individuals.

Since preliminary results appeared promising, studies were
continued at Milford Laboratory during the winter of 1956-57. Two
principal aspects interested us. First, we wanted to determine the
best time of year to kill drills by covering them with bottom deposit.'
We thought that one such period is late fall or early winter when
temperatures are low enough to render drills inactive. Under such
conditions, drills would not be able to emerge from even a thin layer
of bottom deposit and, regardless of the lowered metabolic activities
would eventually suffocate and die. Nevertheless, it was also possi-
ble that burying of drills could be done most efficiently when water
temperatures were high, because under such conditions the mud-covered
drills would suffocate more quickly. To find the most favorable time
for burial, a series of experiments was conducted to determine the
depth from which oyster drills cannot emerge from a mixture of mud and
sand at different temperatures.

These experiments were conducted during the winter months
when the temperature of the water in our laboratory can be rigidly
controlled. Drills were placed in a mixture of eight parts of sand
and one part of heavy mud, which closely resembles the soil composi-
tion of the Long Island Sound oyster beds. The drills selected for
this experiment were attached to the walls and bottom of the aquarium,
which indicated that they were healthy. Before being used in these
experiments, the drills were conditioned for one week at the tempera-
ture at which they were to be buried.

At the end of the conditioning period the drills were placed
in special boxes filled with a mixture of mud and sand. These boxes
were placed in larger ones through which water of the desired tempera-
tures ran continuously. The temperatures were 5°> 10°, 15°j 20° and
25°C. To be certain that the mixture in the boxes was of proper
temperature, the water was permitted to run over them for about three
days before the drills were introduced.

Twenty drills were used for each temperature. They were of
approximately the same size, measuring from 20 to 22 mm in length.
They were pressed into the substratum by means of lucite forceps, to
which a plastic ruler was attached for measuring the proper depth.
The depths were measured from the top of the drill shell (the drill
was held horizontally with the operculum down) to the top of the mud-
sand layer. The drills were considered to have emerged when their
siphons were exposed above the bottom. A period of three days was
allowed because earlier experiments showed that if the drills did
not emerge within this period, they would never emerge and would
eventually die. The experiment was repeated, and the results were in
close agreement.

-84-

100

eo

Ul 60

o

Ui
£L

40

20

TEMPERATURE °C
10 15 20

a ss

25

I

-ssa.

246 246 246 246

CENTIMETERS

2 4 6

Fig. 1. Percentage of U. cinerea of Long Island Sound, which
were able to emerge within three days when buried at different tem-
peratures at depths of 2, k and 6 cm in a mixture of mud and sand.
Average of two experiments.

■85-

0-5

10 15 20

TEMPERATURE °C.

Fig. 2. Number of days needed at different temperatures to
cause complete mortality of U. rinerea of Long Island Sound, buried
at a depth of 6 cm in a mixture of mud and sand . Average of two
experiments.

-86-

All drills buried beneath 2 cm of bottom material at tempera-
tures of 25°, 20° and 15°C emerged (Fig. l). At 10°C, however, only
85 per cent of the drills in the first experiment and 80 per cent of
those in the second experiment reached the surface. At 5°C there was
no emergence in either experiment.

Drills buried at a depth of h cm showed a considerably lower
percentage of emergence than those at 2 cm. At 25°C only 10 per cent
of the drills in the first experiment and 5 per cent in the second
experiment were able to escape. At 20°C 5 per cent emerged in each
experiment. Five per cent of the drills in the first experiment and
none in the second experiment emerged when kept at 15°C. In the 10°
and 5°C groups none of the drills emerged.

Of all the drills buried beneath 6 cm of bottom material and
kept at different temperatures the only individual that emerged
belonged to the group kept at a temperature of 25°C (Fig. l).

The second series of experiments was conducted to determine
the time needed to suffocate drills buried under a thick layer of
bottom deposit. Groups of 100 drills were buried at a depth of 6 cm
in large boxes measuring 32j?" long, l6f" wide and h^" deep. A mixture
of mud and sand, as described above, was used. These boxes were placed
in even larger boxes measuring 36" long, 20" wide and 8" deep through
which a continuous flow of water of the desired temperature was allowed
to run. The same temperatures as in the previous series of experiments,
namely, 25°, 20°, 15° and 10°C, were used. The temperature of the
flowing water in the fifth box was not maintained strictly at 5°C, as
in the previous series of experiments, but fluctuated between 0 and
5°c.

Samples of drills, consisting of ten individuals, were taken
out and examined; periodically; the time between the examinations was
dependent upon the temperature at which the drills were kept. Complete
mortality was determined in this manner: if ten drills constituting
the sample were found dead, ten more were removed and examined a day or
two later. If the latter were all dead, then the time when the first
ten were found dead was taken as the time of complete mortality. After
the second record of complete mortality was obtained the remainder of
the population was removed and examined as an additional check.

The results of these experiments were as follows: at 25 C it
took five days for all drills to die (Fig. 2). At 20°C all drills
buried under 6 cm of bottom material died in seven days. Complete
mortality of all the drills kept at 15°C was recorded in 12 days,
and in the 10°C group 19 days were required. In the final group,
kept in water the temperature of which varied between 0 and 5°C,
approximately 52 days were needed to kill all the drills.

In these, as well as in the previous experiments already
described (Loosanoff 1956), it was noted that if the drills did not

-87-

emerge, they were usually found at the termination of the experiment
at the same depth as originally placed. This indicates that there
was little or no movement after the drills had been placed in position.
It also appeared that those U. cinerea, which started to ascend to the
surface, were always successful in reaching their goal. In other words,
they never stopped at intermediate depths.

The results of our experiments suggest a simple method which
may be practical for controlling the common oyster drill in many areas.
The method consists of burying drills under several centimeters of
bottom material. Usually a layer 5 to 6 cm deep kills most drills.
This depth of burial might be accomplished by using modified agricul-
tural plows, oyster and clam dredges, etc., to turn over layers of
bottom deposit. We have in mind several types that may be inexpensive
and efficient. It may be necessary to remove from the bottom accumu-
lations of shells heavy enough to hinder operations of the plow or
other device used.

If the mechanical aspects can be properly developed, the method
should help oyster growers in many sections of this country and abroad,
where the bottom is soft enough for plowing and where drills or closely
related species are abundant.

Construction and use of plows should be considerably cheaper
than other devices such as suction dredges. It is also probable
that their efficiency will be greater than that of other methods. For
example, Carriker's 1957 observations indicate that as many as 4°- per
cent of newly hatched drills may attach to plant fragments and be
transported by tidal currents to new areas. Carriker thinks that this
type of passive emigration accounts for greater dispersion of the
drills than does phoresis. It may also explain sudden appearances of
heavy concentrations of drills in certain areas where parent drills
were virtually absent. Obviously, barriers built to protect oyster
beds from drill invasions may be inadequate under such conditions, and
either plows or suction dredges or both may be needed to combat drills.

In conclusion we want to emphasize that our studies have been
conducted only with the common drill of Long Island Sound. Therefore,
results obtained with drills in other geographical regions may be
different .

We wish to express our thanks to E. F. Snoek, formerly of
this laboratory, who participated in the early stages of this work.

References

Carriker, M. R. 1955- Critical review of biology and control of

oyster drills, Urosalpinx and Eupleura. U. S. Fish and Wild-
Li Te Serv., Spec. Sci . Rept. Fisheries. No. LkQ, 150 pp.

-88-

Carriker, M. R. 1957- Preliminary study of behavior of newly hatched
oyster drills, Urosalpinx cinerea (Say). Jour. Elisha Mitchell
Sci. Soc. 73(2): 328-351-

Loosanoff, V. L. 1956. Preliminary experiments on development of a
new mechanical method for control of oyster drills. Bull.
Milford Biological Lab., U. S. Fish and Wildlife Service.
20(13): 1-2.

-89-

THE VERTICAL DISTRIBUTION OF OYSTER LARVAE IN DELAWARE BAY

Donald E. Kunkle

Rutgers University* Oyster Research Laboratory, Bivalve, N. J.

Abstract

During the summers of 195^> 1955> 1956, and 1957 an extensive
larvae sampling program was conducted by this laboratory. The objec-
tive of the program was to gather as much information as possible about
the distribution and movements, both vertical and horizontal, of oys-
ter larvae in Delaware Bay. Since a thorough knowledge of vertical
movements of larvae is essential to eventual understanding of hori-
zontal movements, only the former aspect will be discussed here.

This report is based upon an analysis of larvae counts made
during the first three years. Surface and near bottom samples were
counted from 636 stations distributed throughout the oyster-growing
area of Delaware Bay. Samples were grouped according to tide current
phases at times of collection, i.e., low slack, early flood, maximum
flood, late flood, high slack, early ebb, maximum ebb, and late ebb.

Five stages of larval development -- straight hinge, early
umbo, late umbo, mature, and eyed -- were recognized by size and
physical characters; each was treated individually in the analysis.

Totals of each larval stage at each tide current phase were
obtained. By plotting the percentages of these totals at surface and
bottom, a vertical distribution pattern for each stage was derived.
Mature and eyed larvae exhibited an almost identical pattern whereas
the three earlier stages showed no consistent pattern. The first
three stages were then combined and termed "early stages", while the
mature and eyed were combined as "late stages". The combined early
stages showed almost uniform vertical distribution throughout the
tide current cycle which is in agreement with the findings of most
investigators.

The late stage larvae showed a marked tendency to congregate
on or near the bottom during both slack water periods and throughout
the ebb tide as well. The early flood and maximum flood periods
revealed a nearly uniform vertical distribution, while the late flood
again showed a trend toward the bottom. These results are closely
in accord with M. R. Carriker's (1951) findings in Barnegat Bay, and
further strengthen his conclusion that late stage larvae move toward
the headwaters of an estuary by alternately riding the flood tide and
remaining directly on the bottom throughout the ebb. The latter theory
is further advanced by the fact that 1^3 flood tide stations averaged
20.8 late stage larvae (surface plus bottom) per station whereas 122
ebb stations averaged 10. 5 per station. Since we could not sample
directly on the bottom, this 50% reduction in larvae on the ebb is
our best evidence that many larvae were directly on the bottom.

-90-

It appears obvious that oyster larvae could not perform any
significant movements by their own means in any but weak current
velocities, and that aiJ horizontal and most verticaJ movements are
the result of tidal currents and turbulence. The laboratory observa-
tion by T. C. Nelson and E. B. Perkins (1931), that eyed larvae are
stimulated to active swimming by the introduction of higher saline
water and, conversely, to inactivity by lower saline water, could
explain the reaction of late stage larvae following each slack water
period. However, the rate and minimal salinity change required for
such stimulation is still unknown.

This study is being continued in an attempt to further estab-
lish these observations as well as to evaluate the factors of wind-
driven currents, localized current variations, salinity sensitivity,
and other influences on the distribution of oyster larvae.

Literature Cited

Carriker, M. R. 1951- Ecological observations on the distribution
of oyster larvae in New Jersey estuaries. Ecol. Monogr.
21: 19-38.

Nelson, T. C, and E. B. Perkins. 1931. Annual Report of the Depart-
ment of Biology for the year ending June 30, 1930- N. J. Agr.
Exp. Sta. Bull. 522: I'-kj.

-91-

STUDIES ON THE COMPARATIVE UTILIZATION OF OXYGEN
BY LIVING AND DEAD OYSTERS

A. K. Sparks, J. L. Boswell and J. G. Mackin

Texas A & M Research Foundation

Marine Laboratory

Grand Isle, Louisiana

Abstract

Studies were conducted using sealed mason
jars and blackened paraffin-sealed battery jars.
In each experiment one jar contained a live oyster,
one a dead or dying oyster of the same weight and
one jar contained only sea water. "Dead" oysters
were killed by removing one valve and severing the
heart. Disintegration of dead oysters did not
require appreciably greater amounts of oxygen than
that used by living oysters in respiration. Oys-
ters survived for several days in water containing
less than 1.0 ppm dissolved oxygen.

Introduction

Within the scope of responsibilities of the authors is the
investigation of claims of damages by oystermen of Louisiana tide-
water against various oil companies operating in the same area. In
recent years there have been a number of such claims which allege
that mass mortalities of oysters result from oxygen depletion caused
by some operation of the industrial concern involved. These claims
are all alike in that the mortalities are alleged to occur in two
steps. First, there is a mechanical or chemical destruction of a
few oysters on a bed. Second, these few, as they disintegrate, use
oxygen from the surrounding water which results in suffocation of
adjacent oysters. These, in turn, spread the area of oxygenless
water until finally all oysters on the bed are dead. An example of
this type of allegation was that of Mr. August Pitre (Petition,
August R. Pitre vs. The Texas Company, May 3, 195^, number 32013,
24th Judicial District Court, Parish of Jefferson, State of Louisiana)
which stated that "upon information and belief petitioner further
avers that there was a sufficient burial (under a few drilling mud
sacks) of the live oysters so as to initiate a successful oyster
mortality, in that as the live oysters died it thereby caused an

Oceanography and Meteorology Series Contribution No. 112 from the

Department of Oceanography and Meteorology, The A & M College
of Texas.

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added increase in oxygen demand, which resulted in a progressive
mortality to the live oysters from one end of the lease to the other".

The authors know of no data which substantiate such claims,
but there were no data which directly contradict the theory of pro-
gressive deoxygenation of a section of a bay beginning with a small
nidus of dead oysters. In view of the persistency of the complaints
and the prospect that it would some day be necessary to provide the
court with something more substantial than opinion, it was decided to
test the theory experimentally by determining whether or not the oxygen
required by a dead oyster in disintegration exceeds materially that
required by a live oyster for respiration.

Materials and Methods

Six experiments were conducted using mason jars or battery
jars as aquaria. In each experiment groups of aquaria were set up
as follows: a live oyster was placed in an aquarium containing sea
water of known salinity, temperature, and oxygen content. In an
adjacent aquarium an oyster, matching in weight, which had had one
valve removed and the heart severed, was placed in sea water of the
same content. A third aquarium contained sea water only. All three
aquaria in each group were sealed, oxygen consumption was allowed to
continue for a predetermined period of time after which a sample of
water was siphoned from each aquarium and titrated by a modified
Winkler technique for oxygen content. Each aquarium was sampled only
once for oxygen content hence each figure in the tables represents
the measureiaent of oxygen consumption of one live or dead oyster for
a given period of time.

Experiments and Results
Experiment I. This experiment was set up as follows:

1. Fifteen sealed mason jars each containing 800 ml of sea
water of 21.8 parts per thousand salinity taken from the running
salt water system at the Texas A & M Research Foundation Marine
Laboratory at Grand Isle, Louisiana were used as aquaria.

2. Water temperature was 24°C when the experiments began.

3. Five jars contained live oysters. Five jars contained
oysters killed by removing one valve and destroying the heart. These
oysters were designated as experimental "dead oysters", although ciliary
activity continued for a considerable time. The five remaining jars
contained only sea water and were designated as controls.

-93-

4.72

2.81

2.02

1-33

0.00

3-87

3-35

2.26

1.29

0.00

4.68

4.00

5.40

5-77

5-89

Table 1. Studies of utilization of oxygen by live and
dead oysters. Experiment 1.

O2 content in ppm after lapse of
Time Set 1 Set 2 Set 3 Set 4 Set 5
is hr. lgr hr. 2g hr. 5 hr . 22? hr

Live oysters

Dead oysters

Controls

Discussion: The data from this experiment is contained in
Table 1. Reduction of oxygen in the live- and dead-oyster jars in
this experiment was approximately the same, and oxygen was completely
exhausted in both "live" and "dead" jars within 22 hours. In Set num-
ber 5 the dead oysters, which had been killed 22^ hours previously,
had a strong odor. The oxygen in the control rose during the experi-
ment, apparently as the result of photosynthetic activity of
phytoplankton .

Experiment 2. This experiment was like Experiment 1 with the
following exceptions:

1. The dead oysters were killed and held out of water for
18 hours before the experiment was begun.

2. Water temperature was 26.0°C and salinity 21.3 ppt.

3. Different periods were used.

Discussion: The data from this experiment are presented in
Table 2. The dead oysters utilized oxygen at a slightly faster rate
than the live oysters; the dead oyster in Set 11 exhausted the oxygen
in its aquarium while 0.24 ppm remained in the live oyster jar. All
dead oysters had a strong odor at the end of the experiment and were
too "soupy" to retain their shapes.

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Table 2. Studies of utilization of oxygen by live and
dead oysters. Experiment 2.

Og content in ppm after lapse of
Time Set 6 Set 7 Set 8 Set 9 Set 10 Set 11

1 hr . 2 hr . 3 hr • 4 hr . 5 hr . 6 hi'.

Live oysters

2.58

1.63

1.69

0.89

1.25

0.24

Dead oysters

2.38

1.41

1.4l

0.97

1.13

0.00

Controls

4.04

4.06

4.o4

4.68

4.35

5. 24

Experiment 3- This experiment was like Experiment 1 except
that:

1. Longer time intervals were used.

2. Water temperature was 23.0°C and salinity was 24.46 ppt.
Discussion: There was no essential difference in the utilization of
oxygen in this series of experiments (Table 3)» Oxygen was exhausted
in jars containing dead and live oysters between 8 and 24 hours after
the experiment started.

Table 3- Studies of utilization of oxygen by live and
dead oysters. Experiment 3-

O2 content in ppm after lapse of
Time Set 12 Set 13 Set 14 Set 15 Set 16 Set 17
2 hr. 4 hr. 6 hr. 8 hr. 24 hr. 26 hr.

Live oysters 2.18

Dead oysters 2.98

Controls 3.87

Experiment 4. The following changes were made in Experiment

1. Battery jars painted black were substituted for mason
jars. This was to prevent photosynthesis which apparently had
occurred in the earlier studies (Fig. l).

2. 3100 ml of sea water were used in each jar.

2.82

1.37

1.37

0.00

0.00

2.34

2.38

0.60

0.00

0.00

3.95

4.03

4.51

3.31

3.71

-95-

3- For siphoning samples, a pair of glass tubes were placed
in each jar, and melted paraffin was poured on the surface of the
water around the tubes, sealing the jar.

h. The oysters in these experiments were smaller, averaging
57 gms.

5. Water temperature was 22.0 C, salinity was 29«8 ppt, and
oxygen in all aquaria was 3-71 ppm.

6. Water samples were siphoned for . determination of oxygen
content after 6, 7; 8, 9, 10, 11 hours.

Discussion: In this series, with more water and smaller oys-
ters, oxygen was not exhausted in any aquaria containing live or dead
oysters (Table h). Oxygen was reduced at about the same rate in both
sets of aquaria. As considerable quantities of oxygen remained in
all jars after 11 hours, it was decided to repeat the experiments but
to wait longer before sampling.

Table k. Studies of utilization of oxygen by live and
dead oysters. Experiment k.

O2 content in ppm after lapse of
Time Zero Set 18 Set 19 Set 20 Set 21 Set 22 Set 23
hr. 6hr. 7 hr. 8 hr. 9 hr. 10 hr. 11 hr.

Live oysters 3.71 2.02 1.6l 2.10 2.14 1,17 1-93

Dead oysters 3-71 2.98 2.90 2.94 2.42 2.10 1.77

Controls 3-71 3-51 3-51 3-83 3-80 3.39 3.55

Experiment 5- This experiment was like Experiment k, with
the following exceptions:

1. The time intervals for sampling oxygen consumption were
from 15^ to 20^ hours (see Table 5).

2. The oysters used in this series averaged 6l gms.

3. At the beginning of the studies the water temperature was
23 •5°C, the salinity was 3O.65 ppt, and the oxygen measured 5. 00 ppm.

Discussion: After 15^ hours the dead oysters had reduced the
oxygen to a point below 1.0 ppm while the oxygen in live oyster aquaria
remained above 2.0 ppm (Table 5)- By seventeen hours the live oysters
reduced the oxygen to less than 1 ppm. In set 28 and set 29> the
oxygen was above 2.0 ppm after 19^ and 20^- hours. It is uncertain that

-96-

Fig. 1. Battery jars painted black and sealed with paraffin.

-97-

Table 5- Studies of utilization of oxygen by live and
dead oysters. Experiment 5-

Oq content in ppm after lapse of
Time Zero Set 24 Set 25 Set 26 Set 27 Set 28 Set 29
hr. 15? hr. 16^ hr . 17^ hi-. 1&? hr. 19^ hr . 20^ hr.

Live oysters 5. 00 2.02 2.26 0.64 0.60 2.18 2.24

Dead oysters 5. 00 O.32 1.17 O.36 0.85 0.00 O.76

Controls 5. 00 4.60 4.44 4. 96 4.28 4.35 4.60

the latter is the case, since rises occurred at approximately the same
time in the dead oyster aquaria and in the control aquaria. Because
the living oysters had failed to utilize all the oxygen within the time
limits of these experiments and because the oxygen appeared to be
rising in the dead oyster and control aquaria, it was decided to con-
tinue the experiments, but to sample at longer intervals. All dead
oysters in this series had a strong odor and were beginning to lose
their shape and become "soupy".

Experiment 5- These studies duplicated the previous series
with the following exceptions:

1. The oysters used ranged from 12 to 30 grams in weight,
averaging 21.6 grams.

2. Samples were taken 24, 48, 72, 144, 168 and 192 hours
after the experiments were started.

3. At the beginning of the studies the water temperature was
17.0°C, the salinity was 25. 0 ppt, and the oxygen measured 7-32 ppm.

Discussion: During the experimental period the dead oysters
reduced the oxygen more quickly than did the live oysters (Table 6).
Within 72 hours, however, the live oysters had reduced the oxygen to
less than 1 ppm. It is of interest that there was a decided loss of
oxygen in the control vessel, due, it is presumed, to respiration
and bacterial decomposition of the plankton. The live oysters in
this series were all alive at the end of each period; the oyster in
Set 35 presumably survived at least 120 hours in water containing
less than 1.0 ppm of oxygen. The dead oysters had a strong odor and
were almost completely disintegrated at the end of the series.

-98-

FIG. 2

STUDIES OF UTILIZATION OF OXYGEN BY
LIVE AND DEAD OYSTERS
EXPERIMENTAL GROUPS l,2,AND3

6

J**/*t2Htt<u*£S\.*l * » « ■ » *

TIME IN HOURS

Fig. 2. Utilization of oxygen by live and dead oysters,

-99-

Table 6. Studies of utilization of oxygen by live and
dead oysters. Experiment 6.

O2 content in ppm after lapse of
Time Zero Set 30 Set 31 Set 32 Set 33 Set 3I+ Set 35
hr. 2k hr. k-8 hr. 72 hr. ikk hr. 168 hr . 192 hr,

Live oysters 7.32 4.15 4.76 0.6l 0.6l 0.12 0.24

Dead oysters 7-32 4.19 0.1+1 0.12 0.00 0.00 0.00

Controls 7.32 7. 24 7.20 6.26 3.1*1 2.6k 0.81

Summary

The principal difference between the living and dead oysters
in these experiments is that use of oxygen by living oysters is affected
by opening and closure of the shell, while dead oysters utilize oxygen
at a rate determined by the type and load of bacteria present at the
beginning of the studies. Because of reactions to the stimuli of
handling and moving when the experiments were set up, oysters remained
closed for considerable lengths of time and apparently failed to remove
any oxygen from the water. Mitchell (1914), working on oxygen require-
ments of shellfish, found that "a light tap on the table or water bath,
a heavy step in the room, the slamming of the door in a neighboring
part of the building was surely registered by some movement of the
shell."

A graph of the first three series of experiments (Fig. 2)
shows that, in general, the oxygen utilization of the live oysters
and the dead oysters follows the same trend. The control jars in
each of these series showed that a factor other than utilization by
oysters was influencing the oxygen content. It was assumed that this
factor was photosynthetic activity of phytoplankton. For this reason
the remaining experiments were carried out in battery jars painted
black and sealed with paraffin.

Experiments k and 5 are plotted in Figure 3- Experiment k
indicates a more rapid utilization of oxygen by the living oyster for
the first ten to eleven hours. The increase in oxygen in all battery
jars at the eighth to ninth hour may be due to light penetrating the
paraffin seal. The increase in oxygen occurred at 1:00 p.m., a time
of maximum light in the laboratory.

Oxygen in the battery jars was not exhausted by live or dead
oysters within the eleven-hour duration of Experiment k. Subsequently,
another series of experiments was set up in which the first samples
were taken after 15^ hours. The results of these experiments, plotted
on Figure 3> appear to fit rather well on the end of the previous

-100-

STUDIES OF UTILIZATION OF OXVCEN f\Qf 3

BY LIVE AND DEAD OYSTERS
EXPERIMENTAL GROUPS 4 AND 5

I 2 3 4 5 6 7 6 9 10 II 12 13 14 15 16 17 18 19 20 21
TIME IN HOURS

FIG. 4

STUDES OF UTILIZATION OF OXVGEN

&f LIVE AND DEAD OYSTERS

EXPERIMENTAL GROUP 6

0 20 40 60

TIME IN HOURS

oo

120 140 BO 160 20

Fig. 3- & h.
groups h to 6.

Utilization of oxygen by live and dead oysters,

-101-

curves. The peaks in these curves appear at 10:30 a.m. and 1:30 p.m.,
again periods of brightness in the laboratory. These experiments in-
dicate that dead oysters continued to utilize oxygen in decaying, but
live oysters ceased to use oxygen before it was completely exhausted.

To determine if this were true, a sixth experiment was con-
ducted, with the first samples taken after kQ hours. The results of
these experiments are shown in Figure k. Presumably, the oyster in
the sample taken at kQ hours had remained closed for a considerable
time and had not used oxygen at the same rate as the live oyster in
the 24-hour sample. The curves indicate that the live oyster reduced
the oxygen to less than 0.5 ppm, then no longer used oxygen. From
ikk hours to 192 hours apparently no oxygen was used by the living
oysters. It can be seen from the graph that the live oysters reduced
the oxygen in their containers to considerably less than 1.0 ppm
within three days, then survived five additional days at the low oxygen
level. Oxygen was exhausted in the jars containing dead oysters some-
time between 72 and ikk hours, probably shortly after 72 hours.

It is presumed that the oxygen depletion in the control of
Experiment 6 is due to bacterial decomposition of plankton.

Conclusions

It has been demonstrated that dead oysters in disintegration
do not use appreciably greater amounts of oxygen than living oysters
of the same size use in respiration. It has also been shown that
oysters are able to survive for several days in water containing
less than 1.0 ppm dissolved oxygen.

Literature Cited

Mitchell, Phillip H. 1912. The oxygen requirements of shellfish.
Bull. U. S. But-. Fish. 32: 209-222.

■ I Oi • -

NOTES ON THE CALIFORNIA ABAJ..ONF FISHERY

Keith W. Cox

Marine Resources Operations
California Department of Fish and Game

Abalone are widely distributed throughout the world in tropi-
cal and temperate waters, and are much sought after for their shells
and as food. There are fisheries for these mollusks in California,
Japan, Mexico, South Africa, and on a smaller scale, in the Channel
Islands off England.

History of the California Fishery

Until the Chinese came to California during the gold rush
days, local Indians were the only ones to utilize abalone to any
extent; these people had been using the meats for food and the shells
for ornaments, fish hooks, and money, since pre-Columbian times.
Descendants of the Spaniards and immigrants from the United States
did not use abalone, presumably because of the pentiful supply of
other foods. Chinese, however, were happy to find this mollusk which
they knew in their homeland as a great delicacy. They at first
gathered abalone only for their own consumption but in 1864 they began
to dry the meat for shipment to the Orient. Collections were made
only from rocks that could be reached at low tide, and soon this
rather limited area was over-exploited. Various seaboard counties
soon began imposing legal restrictions (See Laws and Regulations).

At first San Diego was the center of operations and when
nearby rocky shores became unprofitable, camps were established in
Lower California and on several of the Channel Islands off southern
Calinf ornia.

About 1900, after gathering of abalone on shore was no longer
profitable, the Japanese came into the industry, introduced diving
suits, and the fishery moved into deep water. Japanese divers worked
out of Monterey from Pacific Grove south to San Simeon. Nearly all
operations were carried on between Pt . Lobos in Monterey County and
Picos Creek in San Luis Obispo County, the abalone being landed at
Monterey. In 1929 the area from Picos Creek to Pt. Buchon was opened
and a number of Caucasian divers started operating out of Morro Bay.
The center of the industry gradually shifted to this more southern
port and to this more southern fishing area, which extends roughly
from Cayucos (Morro Bay) to a few miles above San Simeon, a distance
of approximately 30 miles.

This area has been in continuous production since 1929 and is
the source of most red abalone, (Haliotis rufescens ) landed in

-103-

• EUREKA

ABALONE

E23 SPORTFISHING

COMMERCIAL FISHING
a RED ABALONE
'.PINK ABALONE

SAN FRANCISCO

"MONTEREY

L!SAN SIMEON

i'morro BAY

^ SANTA BARBARA
\ o

''••V;

•SAN DIEGO

Fig. 1. Location of abalone fisheries along California coast.

-10U-

California today. Red abalone produced in this area are of the highest
quality and bring a higher price than the pinks (H. corrugata), which
come from southern California.

During the first years of World War II, few abalone were
landed. The Japanese were forbidden to dive and other divers were
aiding the war effort by working in southern California gathering
the marine alga, Gelidiura, from which agar -agar is manufactured.
Prior to the war 90$ of our agar had been imported from Japan, and
it was necessary to develop a local supply of this product which is
used extensively in bacteriological work.

In 19U3, the north coastal area from Mendocino County to
Pigeon Point, San Mateo County, was opened to commercial diving. It
is not generally known but most of the north coast was open to
commercial diving from I9O9 to 1937 and again from 19^3 through I9I+5
(See Laws and Regulations). Among difficulties encountered by the
few commercial divers attempting to operate along the North Coast,
not the least was the attitude of some local residents. In the 1920' s
a group of citizens loaded an old brass cannon that was on the bluff
over-looking Mendocino Bay and fired at the abalone boats that were
at anchor. The boats left and did not return. Again, during World
War II, abalone boats attempting to work along the Marin County coast
were fired on. This opposition, combined with the characteristic rough
weather and rugged bottom of the northern California coast, has tended to
restrict the numbers of divers attempting to work in this area. As a
result, only a very small amount of abalone was taken from the area dur-
ing the periods when commerical abalone fishing was permitted in Northern
California.

Due to wartime demand for additional protein foods, the main-
land from Santa Barbara south to the Mexican Border and the Channel
Islands were opened to commercial diving in 19^3- In 1955 the main-
land from Santa Barbara to San Diego was closed again and at the
present time diving in this area is carried on almost exclusively at
Santa Cruz, Santa Rosa, San Miguel, and Santa Barbara Island. Most
abalone are pinks, a few reds are brought in, but greens (H. fulgens),
which at one time were an important constituent of the catch, are no
longer wanted by markets and consequently are seldom taken by divers.

Among the fisheries of California, the commercial abalone
ranks 12th to 15th in importance both as to total poundage landed
and to monetary value. It is a luxury item, however, and as such
invites attention and efforts to increase production. Although it
is a unique fishery in the United States, abalone are found through-
out the world and are taken for commercial purposes in several
countries, but only in California are abalone taken by both commer-
cial and sport fishermen.

The commercial fishery has had a varied and stormy history
and even today all is not serene along the Pacific's shores. The
areas open to commercial fishing are changed from time to time and

-105-

today abalone are taken from only two general areas. The red abalone,
which at the present time is providing the major portion of the catch
(2.35 million pounds in 1956), is taken almost entirely in Central
California. The pink abalone, which produces the balance of the
catch (1.88 million pounds in 1956), is taken in southern California
exclusively from the Channel Islands. No commercial abalone fishing
is permitted north of San Francisco. Although there has been at
various times considerable pressure by commercial interests to open
this region, recent findings of the Department of Fish and Game's
abalone investigation indicate that the area would not support an
abalone industry and that commercial abalone fishing is impractical.

Abalone are taken by divers who operate under a revocable
permit issued by the Department of Fish and Game. Equipment must meet
specifications and size limits must be observed. Present rules require
that the diver must have a surface air pump operated from the boat,
100 feet of air hose, two baskets, and a measuring device. Divers
must operate only in certain areas and must keep 150 feet offshore in
water not less than 20 feet deep. Present minimum size limits for
abalone are as follows: for commercial operations -- red 8", green
7-l/U", black 5", all other species 6"; for sport fishing -- red 7",
green 6-l/V, pink 6", and black 5".

The commercial size limits, originally arrived at by a fortunate
combination of economics and compromise, have proved adequate to pro-
tect the supply of abalone. If the market had been based on canned
or dried abalone, it is debatable whether there would be a fishery in
California today. The present fresh frozen market is built upon large
abalone; labor costs automatically prohibit the utilization of abalone
6" in diameter.

There are no bag limits other than those imposed by the pro-
cessors. At the present time there are approximately 60 boats oper-
ating in the industry. Each boat carries a diver, a line-tender and
a boat operator. There is a $40 boat fee and each member of the
crew and the diver pay a $10 commercial license fee. There are 15
processing plants which employ from 5 to 30 workers who prepare the
abalone for market. Almost the entire catch is packaged quick frozen
in 5 lb. containers; the rest is sold fresh in local markets and
restaurants. At one time some of the product was canned and some was
was dried but this never amounted to much and is now prohibited by
law.

Divers are paid according to the amount of saleable meat
that can be recovered from the abalone. Red abalone is the most
desirable since it grows the largest and the meat is of better quality.
Consequently it brings a higher price. At present market prices,
red abalone bring from $7 to $9 per dozen to the diver. Pink abalone,
on the other hand, are generally smaller and the meat is not of such
high quality. As a result present prices are between $4 and $6 per
dozen. In the fresh market abalone steaks sell from $1.95 per lb. for

■106-

large red abalone to 75^ per lb. for the better grade pink abalone.
Divers can make from $5*000 to $30,000 per year depending on weather
and ability in that order.

Abalone may not be shipped from California in either the fresh
or frozen state. It is felt that present stocks are sufficient for
local market demands and that any large additional increase in produc-
tion would tend to deplete severely the available supply. It is
reported, however, that California abalone may be found on menus of
restaurants in several large eastern cities.

Transplanting

Transplanting of abalone in California had not been attempted
prior to the Department's first experiment in 1956. At that time
over 800 red abalone were successfully transported over a distance of
several hundred miles to a new area where there were none. These
abalone were carried in bait tanks (approximately V x 51 x 6') aboard
the research vessel YELL0WFIN. Because of abnormal spacing of the
holes in the shells, injuries to soft parts and a general weakened
condition, only 660 of the original 800 were tagged and replaced by
divers on the bottom. A later check revealed that only 19 of the
660 had failed to survive transplanting. The remainder have
apparently adapted themselves to the new environment.

Since this first large scale transplant, other experiments
have been conducted by the Department in an attempt to improve the
methods and techniques. It has been determined that abalone will
survive transport but the length of time they can remain out of water
depends primarily on the species. Survival and general condition
are improved if during transport abalone are not confined to a bait
box with circulating sea water, but placed in nets or sacks, kept out
of the direct sunlight, and a constant flow of water maintained over
them. If kept submerged in freely circulating sea water red abalone
may be stored in live boxes in which they will live without food for
at least 70 days. The pink abalone of southern California require
more careful handling than the reds; they cannot remain out of water
for such long periods of time and must be kept constantly covered by
streams of sea water and out of the sun while in transit.

Further transplanting experiments are planned and it is hoped
that it will be possible (l) to establish abalone in favorable areas in
which they do not occur, (2) to establish abalone where they are in such
limited concentration that the addition of new stocks might help build
up the population, and (3) to use abalone as biological indicators,
since they appear to be sensitive to certain chemical changes in the
water. This fact has possibilities as an aid in pollution studies.

It should be kept in mind that transplanting is far from an
established procedure. From the little we have done, however, we

■107-

know that, if certain precautions are observed, it is possible to
transplant abalone from one area to another . An adequate supply of
food, i.e., the seaweeds, Nereocystus or Macrocystus, and an area
more or less exposed to the open sea are required.

The Japanese have done a limited amount of experimental trans-
planting. The abalones transplanted from northern Japan transformed
themselves into what was formerly thought to be a distinct species
confined to southern Japan.

Movement and Migx'ation

Abalone inhabit rocky shores in certain areas along the coast.
They are found from high tide mark out to depths of over 300 feet,
with the maximum concentration between 25 and ^0 feet.

Tagging experiments have shown that there is no migration and
very little movement among abalone. Those tagged and released in
shallow water (5 to 8 feet deep and 10 feet offshore) were later
collected by shore fishermen within a few feet of original sites.
Those released in deeper water 20 to 25 feet offshore never appeared
on shore; when checked by divers a year later these abalone were
still in the same general area. Abalone tagged and released in still
deeper water h-0 to 50 feet deep and up to l/k mile offshore are still
in the area of release after two years. This would indicate that
although individual abalone may move around considerably they never
move any great distance. In the tidal zone they have been observed
to move over 100 yards parallel to the shore, but none has ever been
observed to move from shallow to deep or deep to shallow water.

Life History

There has been no attempt to cultivate abalone artificially
in California. All the abalone taken have been the result of natural
propagation. Until recently too little was known of the early life
history to attempt culture experiments. It is now known that spawning
takes place in spring and summer j it was formerly thought that spawn-
ing took place during winter. There is a free-floating stage which
probably lasts from eight to ten days. The small abalone then sinks
and becomes attached to the bottom and starts to grow into an adult.

Although we now have a better knowledge of the early life
history, other considerations enter into attempts to cultivate abalone.
One of the principal difficulties is in collecting spat. The fishing
grounds face the open ocean and weather conditions being what they
are along the Pacific Coast, it is a matter of conjecture whether
collecting equipment could be set and maintained during the spawning
season. Spat collecting nets, prepared according to methods suggested
by Cedric Lindsay, Supervisor Shellf isheries Investigation, Washington

■108-

Department of Fisheries, were exposed but abalone had not matured at
the expected time. At this time in previous years in this area the
majority of abalone were in spawning condition.

It is planned to continue to attempt to collect abalone spat;
experiments will have to be conducted to determine the best methods.

Growth rate is not accurately known but it is strongly suspected
that young abalone grow to about one inch in diameter the first year,
and when they reach k to 5 inches in diameter they are probably 3 to 4
years old. From then on the growth rate varies. In some areas growth
is fairly uniform with the majority reaching a size of 8 inches or
more. Most of the growth takes place during winter months with some
abalone adding as much as 1-1/2 inches in shell diameter during this
period. Some abalone will reach a certain size and grow no more;
others apparently keep on growing. Abalone up to 11 inches in diameter
are known. In some locations most abalone are under 7-l/2 inches,
while in other areas, often times close by, specimens of the same
species are almost all 8 to 9 inches in diameter. The causes of this
difference in growth are not known. Previously it was thought that
growth was constant and that the size of an abalone was directly
proportional to its age. It is only recently that the Department's
investigation has disclosed that growth rate is not constant but
varies from area to area for the same species, also from species to
species in the same location. In areas where abalone are being
harvested by divers growth rate is more rapid than in those areas
where they are left undisturbed. Whether harvesting surplus animals
is the deciding factor, or whether growing conditions are superior
in these areas, are moot questions.

-109-

THE MARYLAND SOFT SHELL CLAM INDUSTRY:
ITS POTENTIALS AND PROBLEMS

J. H. Manning and H. T. Pfitzenmeyer

Maryland Department of Research and Education, Solomons, Maryland

Abstract

Four of Maryland's tidewater counties are now
producing about half the total U. S. catch of soft
shell clams. Maryland's production in 1957 is esti-
mated at about 250,000 bushels, worth $1,000,000 to
the dredgers. Some of the problems confronting the
industry are briefly discussed.

A preliminary report on the Maryland soft shell clam fishery
was presented at the Baltimore meeting of the National Shellf isheries
Association in 1955- At that time 60 hydraulic clam dredges were
licensed in the State, and annual production of soft shell clams was
estimated at 110,000 - 120,000 bushels, worth about $500,000 to the
dredgers. Figure 1 shows the growth of the fishery since the beginning
of dredging on a commercial scale--from seven dredges in 1952 to 132
in July 1957- The accelerated rates of expansion between 195^ and
1955 and between 1956 and 1957 are attributable, at least in part, to
the enactment of legislation recognizing and regulating the industry.

Value of the fishery to primary producers during the past
three years is indicated in Figure 2. These estimates, which are
considerably in excess of estimates based solely on collection of
the production tax, are believed to be conservative. The soft shell
clam resource should be worth about $1,000,000 in gross income to
dredgers during the current year.

Operation of the clam dredge fishery is limited by law to
waters of four counties, and within three of these counties it is
further limited by local restrictions. Effective operation of the
gear is limited to depths not exceeding about ten feet by a legal
restriction on the length of the conveyor system (19 feet, axle to
axle). As shown in Figure 3> less than 25 per cent of the "barren
bottom" within the ecological range of the soft shell clam and the
operational range of the hydraulic clam dredge is open to dredging.
If the clam resource were distributed proportionally throughout
Maryland tidewater and a ready market existed for increased produc-
tion, full territorial expansion of the fishery should result in a
four-fold increase in dockside value --to about $4,000,000 annually.
Whether the full potential of the Maryland clam fishery will be
reached is a question only time can answer. The clam fishery must
co-exist with other commercial fisheries of long-established value

-110-

K

a

u

Ik
O

o

z

1952-19^7. 1- Gr°Wth °f thS S°ft She11 Ciam fishe^ ln Maryland,

Fig. 2. Estimated value of Maryland's soft shell clam catch
to primary producers.

-Ill-

within an estuary rapidly growing in importance as a residential
and recreational center. Many of the problems confronting the clam
industry involve economic and social rather than biological relation-
ships.

In general, Maryland production of clams is controlled by
demand. The dockside price has remained steady at about $4 a bushel
for more than two years. Any development that depressed the price
would be most unfortunate for the fishery, since costs of operating
a clam dredge are high. Figure h indicates some reason for optimism ,
concerning capacity of the market to absorb increased production.
The New England catch has declined from more than 600,000 bushels
in 1950 to less than 250,000 bushels in 1956. The Maryland catch
has taken up some of the slack in recent years, but Atlantic Coast
production is still far short of the 1950 level. Currently, Mary-
land's catch accounts for about one half of the total.

Maryland is not getting the ultimate value per unit catch
from the clam resource. A very high percentage of the catch is
shipped out of the State, and the price differential on the New Eng-
land market favors New England clams by about 75 psr cent. This is
not justified by the difference in shucking yield, which is not more
than about 25 per cent, often less. Maryland clams, taken from
brackish waters, may be inferior to Maine clams as steamers, but for
other purposes they need no apology. It seems likely that at least
part of the price differential can be erased by quality control.
Shell stock which is exported must be in first-class condition when
shipped, and substantial, sanitary containers are needed to replace
the fruit baskets now used. A most encouraging recent development
in the industry is the trend to local processing. Before 1956
practically the entire catch was shipped as shell stock. Twelve
shucking houses are now operating in Maryland, with resultant gains
to tidewater economy and simplification of transportation and sani-
tation problems.

Creation of local demand is a virtually unexplored approach
to market development. A few dealers have taken steps in this direc-
tion, but there has been no concerted effort by the industry. Fried
clams, when placed on the menus of local restaurants, have received
enthusiastic acceptance.

Perhaps the most serious internal problem of Maryland's clam
industry is the occurrence, from early autumnto early spring, of red
coloration in clams. Pink yeast has been isolated from a few ship-
ments of Maryland clams, but there is no clear evidence that con-
tamination originated in Maryland or that pink yeast is the sole or
primary cause of the pigmentation. At least one Federal and two State
agencies are preparing for a concerted attack on this problem in the
coming months.

-112-

Fig. 3. Proportion of actual and potential clam-producing
bottoms in Maryland.

Fig. h. Production of clams in New England (1950-56) and
Maryland (1955-56).

■113-

We do not feel that any of the problems of Maryland's clam
industry, internal or external, are unsolvable. The industry is now
recognized as a major factor in Maryland's tidewater economy, and as
such deserves full support and attention from State and Federal
agencies concerned with the harvesting, processing, and marketing of
seafood resources. If the industry will make known its needs to
these agencies and displays a willingness to cooperate, it will find
them more than willing to help. With adequate organization, represen-
tation, and management, the Maryland clam industry can look forward
to a promising future.

-114-

FILTERING EFFICIENCY OF HARD CLAMS IN MIXED SUSPENSIONS
OF RADIOACTIVE PHYTOPLANKTON

Rebecca Joyce Smith

Fishery Radiobiological Laboratory

U.S. Fish and Wildlife Service

Beaufort, North Carolina

Abstract

Simultaneous removal of two species of
phytoplankton by Mercenaria (= Venus ) mercenaria L.
was measured by labeling one species with T?S^ and
the other with Ca^5. Since both species are removed
from the same volume of water pumped, a faster removal
of one indicates a greater efficiency of the clam in
filtering that species. A species of Carteria was
always one of two species in suspension, since it
alone of 15 species, tested had sufficient uptake
and retention of Ca for use in these experiments.
In general, algal cells k/*- or larger in diameter
were filtered with uniform efficiency. Cells 2 to
3y* usually were not filtered as efficiently as larger
cells in the same suspension, even though clams
secreted much mucus, probably in an effort to retain
these smaller cells. A dinoflagellate, Gymnodinium
sp., caused most clams to close soon after filtering
activities began. When clams remained open, they
efficiently filtered Gymnodinium from suspension,
but ejected chains of compact cells, which were tyk^o
Gymnodinium, through their incurrent siphons. The
population density, varying from 2 to 20 million
cells per liter in most suspensions, did not affect
the efficiency with which the species were removed.

Although extensive studies of the feeding behavior of filter-
feeding lamellibranchs have been made, there remain some basic problems
relative to filtering efficiency and factors affecting it. Variations
in filtering rates of the hard clam, Mercenaria (= Venus ) mercenaria
L., result from a change in efficiency of particle removal or in
volume of water pumped. In recent investigations Rice and Smith (in
press) found that filtering rates of hard clams varied with different
species of phytoplankton. Since these filtering rates were based on
radiological measurements of P^ incorporated into the cells of only
one species, it was not possible to determine whether these variations
involved filtering efficiency of gills or volume of water pumped. A
comparison of the simultaneous removal of two species from the same
suspension would eliminate changes in rate of water propulsion as a

-115-

factor. With both species in the same water, a faster removal of one
would indicate greater efficiency in filtering that species. Removal
of two species of algae from the same suspension can readily be fol-
lowed when each species is labeled with a different radioisotope.
The purpose of this investigation was to study the filtering efficiency
of hard clams for different species of phytoplankton.

The radiosotopes employed for labeling algae were P^ and Ca^-3.
Their radioactive characteristics differ sufficiently to allow sepa-
rate determination of each isotope when both are present in a mixture.
Calcium, like phosphorus, is essential in metabolism of algae. Only
a small per cent of the calcium which occurs naturally in sea water is
necessary for algal growth. Since the uptake of Ca ■* is the same as
that for non-active calcium, the dilution which occurs when this
radioisotope is added to natural sea water prevents much Ca 5 from
being taken up by the algae. To increase uptake of Ca , artificial
sea water was prepared without the addition of calcium (Rice 195&).
Fifteen species of algae were tested for rate of cell division and
uptake of Ca -^ when grown in this artificial sea water containing
Ca 5. In all experiments Carteria sp. was labeled with Ca -3, since
it was the only species that had sufficient uptake and retention for
use in these experiments. In suspension with Carteria, other species
were labeled with P-^ following the methods of Rice (1953) for obtain-
ing highly radioactive cells.

The phytoplankton used may be grouped according to their length
as follows:

Algae

Classification

Size (m)

Chromulina pleiades

Chrysophyceae

2

Nannochloris atomus

Chlorophyceae

3

Chlamydomonas sp.

Chlorophyceae

8

Gymnodinium sp.

Dinophyceae

9x12

Platymonas sp.

Chlorophyceae

9x14

Carteria sp.

Chlorophyceae

10x14

Amphydinium klebsi

Dinophyceae

12x17

Nitzschia sp.

Bac i liar iophyceae

4x17

Nitzschia closterium

Bacillariophyceae

5x38

The twelve clams used in this study were collected in the
vicinity of the Fishery Radiobiological Laboratory, Beaufort, North

-116-

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O CARTERIA SP.

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HOURS

Fig. 1. Similarity of filtering efficiency of clams for two
species of planktonic algae present in the same suspension in two
tests in which the rate of filtration was different.

-118-

Carolina and were kept in running sea water when not being used in
experiments. They ranged from 80 to 88 mm in length and from 66 to
77 mm in height. Each of three clams was placed in ten liters of
filtered sea water to which had been added measured numbers of Carteria
cells containing Ca^5 and cells of a second species of algae labeled
with PJ . The suspensions were adequately stirred to prevent any
settling of cells. In control jars containing no clams, the numbers
of cells in suspension at the beginning and the end of experiments
were the same. When clams were placed in suspensions of some algal
species, considerable numbers of cells quickly adhered to shell sur-
faces. Further tests showed that coating the shells with thin layers
of paraffin prevented significant losses of cells from suspension.

Samples were taken of the suspension before adding a clam and
at 30-minute intervals for 3 hours after it had opened. After filtra-
tion of cells from samples with millipore filters, measurements of
their radioactivity due to Ca ? and P^^ were made. Rates of removal
of both algal species in suspension were calculated from these measure-
ments.

It is believed that the decrease in cell numbers in suspension
represents filtration by the clam, although it is not definitely known
whether filtered cells which may be rejected are sufficiently free of
mucus to go back into suspension. There is reason to believe, however,
that rejected cells are bound in mucus and do not break up and re-enter
the suspension.

With the exception of tests using Nannochloris atomus, Chromulina
pleiades and Gymnodinium sp., the decrease in cell numbers of any species
during any half hour of a 3-hour observation period never varied over
10 per cent, and usually not over 5 per cent, from that of Carteria. In
only 6 out of 80 experiments was the rate of removal of one species dif-
ferent from that of the other (Table l). Even in these instances the
difference in filtering efficiency was only significant during the
first hour of filtering activity.

Whether cells of these algal species were completely filtered
from the water pumped was not determined. When algae of different
sizes such as Nitzschia and Carteria were filtered with the same
efficiency, it is believed that all cells were removed from water
passing through the gills. It is improbable that algae of such unlike
size and shape could escape the gills at the same rate. If the species
in suspension are similar in size, a uniform percentage of escapement
is possible.

The rate of filtration apparently did not affect the efficiency
with which clams removed either species. Figure 1 shows results of
two representative experiments. During the observational time the
clam in Test 1 removed cells of Nitzschia sp. and Carteria sp. as
efficiently as the clam in Test 2, yet filtered them at a much slower
rate, due to less water being pumped through its gills. Figure k

-119-

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Fig. 2. Differences in filtering efficiency of clams for two
species of planktonic algae in the same suspension.

-120-

shows that all three clams removed both algal species with the same
relative efficiency during the 3 hour period, yet filtered the cells
at a faster rate during the first 1 l/2 hours. When both species
were removed at a faster rate during one time interval than another,
this indicated a change in volume of water pumped, since the filtering
efficiency for each species had not changed.

In 20 of 3^ experiments when Chromulina pleiades or Nannochloris
atomus were in suspensions with Carteria, the clams removed Carteria
at a faster rate than either of the other species (Table l). Figure 2
shows the results of one of these experiments in which many small cells
escaped the gills. A more abundant formation of mucus was observed in
tests containing N. atomus and C. pleiades, but this was ineffectual
in preventing considerable escapement of cells.

In Ik of 3^ experiments the percentage decrease in cell num-
bers of N. atomus and C. pleiades during each hour did not differ
more than 10 per cent from that of Carteria cells. Rice and Smith
(in press) have shown that filtering rates of hard clams in certain
unialgal suspensions were altered considerably when other algae were
added. A more efficient retention of N. atomus was observed when
Nitzschia closterium was present; this may have resulted from a
clogging of gills by the large cells of Nitzschia. In other experi-
ments yet unpublished, the percentage escapement of N. atomus through
the gills of oysters averaged 88 per cent; this was reduced to 51 per
cent by addition of Carteria to the suspension. It seems possible,
therefore, that the presence of relatively large Carteria cells in-
creased the efficiency of retention of small cells of N. atomus and
C. pleiades by the hard clam.

In 9 of 13 experiments Gymnodinium and Carteria were removed
with similar efficiency (Curve B in Figure 3). In the other h tests
the presence of Gymnodinium resulted in variations in rates of removal
of Carteria. Curve A shows these fluctuations in the filtering rate
of Carteria, but a steady rate of removal of Gymnodinium.

Apparently the use of different cell densities of each species
did not change the efficiency of removal. The total number of cells
in each suspension .was kept low, never exceeding 20,000,000 cells per
liter, except in 6 tests containing 73,000,000 Nitzschia closterium
cells per liter and 26,000,000 Carteria cells per liter. Previous
experiments (Rice and Smith, in press) have shown that dense suspen-
sions stimulate secretion of mucus with a resultant increase in pro-
duction of pseudofeces. A comparison of the results of tests using
suspensions of different population densities (Figure k) shows that
cells of Nitzschia closterium and Carteria sp. are removed with
similar efficiency at the various time intervals.

The presence of Gymnodinium sp. seemed to have a definite
effect on the feeding behavior of clams. In many tests it was
impossible to obtain data since the clams closed soon after filtering

-121-

100

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O CARTERIA SP.

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suspensions were removed with similar efficiency (Curve B) but in
the remaining four experiments the presence of Gymnodinium affected
the rate of filtering of Carteria (Curve A).

-122-

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-123-

activities began. When they did remain open, chains of compact cells
were ejected through the incurrent siphons. Some of these rejected
cells were collected, washed several times in sea water and dissolved
in acid so that thin samples could be prepared to minimize self
absorption of beta particles of Ca 5, These samples were measured
for their p32 and Ca45 content. Ninety-four per cent of the cells in
these chains were Gymnodium, which demonstrated that the clams were
selectively rejecting this dinoflagellate after filtering it from the
water. This ejection of chains of cells was frequently observed only
in tests using Gymnodinium.

Identification of the species of Gymnodinium used in this
investigation needs to be made, since different results have been
obtained with other species of this dinoflagellate. Ballatine and
Morton (1956) found that Gymnodinium veneficum caused the small clam,
Lasaea rubra, to cease filtering and to close its shell. It is
known that Gymnodinium brevis produces a substance that is highly
toxic to marine animals. Gymnodinium splendens, however, has been
reported to be an excellent food organism for oysters.

The results of these studies demonstrate that the hard clam,
Mercenaria mercenaria, does not filter algal cells of different sizes
with the same efficiency. Cells of 2 to 3/^ are not removed as
efficiently as those of larger size in the same suspension, even
though these larger cells aid in retention by gills. In general,
cells which are 4/i-or larger in1 diameter are filtered with uniform
efficiency.

Thanks is given to Dr. W. A. Chipman and Dr. T. R. Rice of
this laboratory. Dr. Chipman offered helpful suggestions and assisted
in evaluating and reporting results. Dr. Rice advised in investiga-
tional procedures and made available unialgal cultures of phytoplankton.

Literature Cited

Ballantine, Dorothy and J. E. Morton. 195&- Filtering, feeding, and

digestion in the lamellibranch Lasaea rubra. Jour. Mar. Biol.
Assoc. U.K., 35: 241-272.

Rice, Theodore R. 1953- Phosphorus exchange in marine phytoplankton.
Fishery Bull. U. S. Fish and Wildlife Serv., ^k (80): 77-89-

Rice, T. R. 1956. The accumulation and exchange of strontium by
marine planktonic algae. Limnol. & Oceanogr. 1: 123-138.

Rice, T. R. and Rebecca J. Smith. Rates of filtration by Venus

mercenaria (L. ) determined with radioactive phytoplankton.
Fishery Bull. U. S. Fish and Wildlife Serv., 58 (129 ): (in
press).

-124-

flTSTOPHYSIOLOGY OF THE OYSTER KIDNEY1

Milton Fingerman and Laurence D. Fairbanks

Department of Zoology, Newcomb College, Tulane University,
New Orleans 18, Louisiana

Abstract

Histophysiology of the oyster kidney, Milton
Fingerman and Laurence D. Fairbanks, Newcomb College,
Tulane University, New Orleans 18, Louisiana

The morphology and physiology of the kidney
(organ of Bojanus) in the oyster Crassostrea virginica
are described. The morphology was determined by in-
jection of Evans Blue and by histological section.
Two bladders are present, one on each side of the ani-
mal with a secretory portion in between and posteriorly.
A funnel-shaped duct runs from the floor of the peri-
cardium to each bladder and opens close to the urinary
pore. The latter is immediately posterior to the
genital pore . Both openings are covered by a common
flap. Injection of dye revealed that the tubules of
the organ of Bojanus are bathed in blood from the
adductor muscle which is supplied directly by the
posterior artery from the ventricle. Blood then
collects and flows into a large contractile vessel
adjacent to the visceral ganglion. This vessel
proceeds directly to the portion of the gills pos-
terior to the intestinal loop and bifurcates antero-
posterior ly as the afferent interbranchial vessel.
In extremely dilute sea water the blood was hyper-
tonic to the environment. The situation was reversed
when oysters were placed in concentrated sea water.
Some osmoregulation must have taken place. Only in
the most dilute medium does the possibility exist
that the organ of Bojanus serves in an osmoregulatory
capacity. The wall of the ventricle appears to be
the primary organ of filtration. In the lowest
salinity tested the pericardial fluid was hypotonic
to the blood whereas at higher concentrations the

"^This investigation was conducted under a contract between Tulane
University and the United States Fish and Wildlife Service. Funds
were made available under provisions of P. L. 466, 83rd Congress,
approved July 1, 195^, commonly called the Saltonstall-Kennedy Act,

-125-

Fig. 1. An oyster whose pericardium was injected with Evans
Blue to reveal the location of the bladder of the organ of Bojanus.
A, adductor muscle; P, pericardium; B., bladder. The line indicates
where the section shown in Figure 2 was taken.

-126-

pericardial fluid was more concentrated, which indi
cates salt elimination to keep the oyster's tissue
fluids more dilute than the surrounding waters.

Introduction

Several aspects of the morphology of oysters have been
studied by a number of previous investigators (Leenhardt, 1926; Hopkins,
193^; Nelson, 1938; Shaw and Battle, 1957). However, very little is
known of the urinary system of bivalve mollusks except for the physio-
logical investigations of Picken (1937) and Florkin and Duchateau
(19^8) on the freshwater genus Anodonta and the morphological study
of Awati and Rai (1931 ) on the Bombay oyster, Crassostrea cucullata.

Fingerman and Fairbanks (1956a, b) demonstrated that the
commercial oyster, Crassostrea virginica, has some ability to osmore-
gulate. After 12 hours the salinity of body fluids had not changed
to the same extent as the environment. Fingerman, Fairbanks, and
Plauche (1957) determined chloride concentrations of fluids taken
from several portions of the body. These investigators found that
the urine of oysters kept in diluted sea water was hypotonic to
blood. They postulated, therefore, that the organ of Bojanus functions
in osmoregulation by conserving salt when oysters are in a dilute
environment. The investigation described below was undertaken to
obtain more information on the morphology and physiology of the organ
of Bojanus in Louisiana specimens of Crassostrea virginica.

Morphology

Differences of opinion concerning the orientation of non-
cephalized animals are found in the literature. We shall use the
descriptive terms defined below in reference to the oyster.

Anterior, oral or in the direction of the hinge.

Posterior, anal.

Ventral, toward the gills.

Dorsal, opposite the gills.

Right, toward the side of the flat valve and promyal chamber.

Left, toward the side of the cupped valve.

The location of the organ of Bojanus and related excretory
structures is best demonstrated in the living animal by injection
of a dye such as Evan Blue into the pericardial cavity (Figure l).
Four to five ml of dye solution are usually required when a large

■127-

animal is used. The dye enters each bladder of the organ of Bojanus
from the pericardial cavity. A bladder and separate duct from the
pericardium are present on each side of the animal. Urinary orifices
open into the right and left epibranchial chambers. The bladders lie
ventral and adjacent to the pericardial cavity. As the bladder fills,
dye is forced out through the urinary orifice located in the mid-
ventral edge of each bladder. The genital orifice is immediately
anterior to the urinary orifice (Figure 2). Both openings are covered
by a common flap. A large nerve, visible without magnification extends
anteriorly from the visceral ganglion along the mid-lateral wall of each
bladder. The lateral wall of each bladder is capable of contracting
to such a degree that the greater part of the fluid is forced out of
the bladder. This has been observed in several oysters, apparently
in response to distension of the bladder caused by fluid flowing in
from the pericardial cavity. If an incision is made in the lateral
wall of the bladder dorsal to the nerve and perfusion into the peri-
cardial cavity is continued, dye can be seen flowing from the peri-
cardial cavity through a rather large duct that empties into the
bladder near the external urinary orifice. The proximal openings of
the ducts leading from the pericardial cavity are in the ventral wall
of the pericardium at the antero-lateral base of each of the two
auricles. The ducts run along the medial wall of each bladder. They
are flared at their origins in the pericardial wall and taper distally.
The diameter of the outlet in the wall of the bladder is about one-
third the size of the opening in the pericardial wall. The ducts are
slightly curved with their distal ends directed posteriorly and are
lined with cilia that beat distally from the pericardial cavity.

Histological sections reveal that both bladders of the organ
of Bojanus are bordered medially and posteriorly with blind tubules
that diminish in size with distance from the bladders (Figure 2).
The line in Figure 1 shows where the section in Figure 2 was taken.
The majority of the tubules lies ventrally adjacent to the adductor
muscle. The most anterior tubules are at the level of the anterior
end of the bladder, next to the antero -ventral portion of the peri-
cardium, and lateral to the gonadal ducts. In this region the tubules
are relatively large, rounded and few in number. Considerable space
surrounding the tubules indicates a liberal supply of blood. The
tubules are thick-walled structures composed of tightly-packed well-
differentiated columnar cells having bulbous or vacuolated ends
exposed to the lumen.

When both adductor muscle insertions are left intact and ties
are placed around the anterior artery and the auricles, injection of
dye into the ventricle reveals that the posterior artery sends blood
directly to the adductor muscle. This blood next bathes the tubules,
collects in the region of the visceral ganglion and posterior
extremities of the tubules, and then flows out into a large contractile
vessel adjacent to this ganglion. The vessel next proceeds directly
to the portion of the gills posterior to the intestinal loop and
bifurcates antero-posteriorly as the afferent interbranchial vessel.

-128-

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Fig. 2. Histological section through the organ of Bojanus.
B, bladder; T, tubules of organ of Bojanus; U, urinary pore; G,
genital pore; D, digestive tract; M, mantle; C, ctenidia; E,
epibranchial chamber; H, heart; P, pericardial cavity; L, lower
end of duct from pericardial cavity to bladder.

-129-

The excretory system of Crassostrea virginica differs in two
major respects from that of Crassostrea cucullata as described by
Awati and Rai (1931)- In the latter species the urinary and repro-
ductive openings are not separate as in Crassostrea virginica but
unite to form a single reno-gonidial aperture on each side of the
body. Secondly, Crassostrea cucullata has an interne phridial canal,
connecting the two bladders, that does not appear to be present in
Crassostrea virginica.

Physiology

In an attempt to understand the physiology of the organ of
Bojanus, determinations of the chloride concentration in fluids taken
from several portions of the body were performed according to the
method described by Schales and Schales (19^-1) on oysters maintained
at three different salinities. Body fluids were sampled from 30
oysters kept at each salinity for periods up to 14 days. These samples
were taken from the bladder of the organ of Bojanus, the ventricle,
the mantle, the pericardial cavity, and the blood vessel running from
the secretory portion of the organ of Bojanus to the gills and mantle.
The chloride concentration of the water in which the oysters were
maintained was also determined. The results are presented in Figure
3 as differences in chloride concentration between the blood from the
ventricle and the other body fluids. The concentration of the sea
water and the difference in concentration between the sea water and
blood in each of the three instances is also shown. Data obtained
with oysters in the most concentrated sea water are at the top of
the figure and data from oysters in the most dilute sea water at the
bottom. At the right of the figure are probability coefficients,
determined by t test, based on the difference between the heart blood
and each fluid. At low salinities (l6l.7 milliequivalents of chloride
per liter) all body fluids were hypertonic to the environment. How-
ever, only the mantle fluid was hypertonic to blood from the ventricle.
At the intermediate concentration (227-0 meq/l), mantle and pericardial
fluids were slightly more concentrated than the environment whereas
the remaining fluids were less concentrated. At the highest concentra-
tion (515-6 meq/l) all fluids were hypotonic to the environment; the
fluid from the organ of Bojanus was least concentrated followed by
blood from the ventricle.

Evidently some sort of osmoregulation occurred to keep the
oysters hypertonic in diluted sea water and hypotonic in concentrated
sea water. Only in the most dilute medium does the possibility exist
that the organ of Bojanus serves in an osmoregulatory capacity,
tending to eliminate water from the blood to keep the latter hyper-
tonic to the environment. Prosser, Green, and Chow (1955) studied
the role of the antennary glands of the shore crab, Pachygrapsus
crassipes, in osmoregulation and arrived at the same conclusion con-
cerning these organs as that found for the organ of Bojanus. The
antennary organs may function in hyper-osmotic regulation but not at
all in hypo-osmotic regulation.

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-131-

In the oyster, as in Anodonta, the wall of the ventricle
appears to serve as the major filtration structure. In the lowest
salinity tested, pericardial filtrate was hypotonic to the blood in
the ventricle showing that salt was being conserved to keep the
oyster hypertonic to its environment. At the two higher salinities,
the pericardial filtrate was hypertonic to ventricular blood showing
that salt was being eliminated in order to keep the oyster hypotonic
to its environment. The opening of the duct from the pericardium to
the bladder of the organ of Bojanus lies close to the urinary pore.
Presumably, contraction of the pericardial wall could result in elimi-
nation of the contents of the pericardial cavity with little mixing
of this fluid and the urine in the bladder.

Literature Cited

Awati, R. R. and H. S. Rai . 1931- The India Zoological Memoirs.

III. The Bombay Oyster, Ostrea cucullata. pp. 107, figs. 51>
Rs. 2-8.

Fingerman, M. and L. D. Fairbanks. 1956a. Osmotic behavior and

bleeding of the oyster Crassostrea virginica. Tulane Stud.
Zool. 3: 149-168.

Fingerman, M. and L. D. Fairbanks. 1956b. Investigations of the

body fluid and "brown-spotting" of the oyster. Proc. Natl.
Shellfish. Assoc. ^7: 146-147.

Fingerman, M., L. D. Fairbanks, and W. C. Plauche. 1957» Body fluid
of the oyster Crassostrea virginica. Publ. Instit. Mar. Sci.
Vol. 4, pages 284-292.

Florkin, M. and G. Duchateau. 1948. Sur 1 'osmoregulation de
l'anodonte (Anodonta cygnea L. ) Physiol. Comp. Oecol.
1: 29-45.

Hopkins, A. E. 1934. Accessory hearts in the oyster, Ostrea gigas.
Biol. Bull. 67: 346-355-

Leenhardt, H. 1926. Quelques etudes sur Gryhea angulata (Huitre

du Portugal. Annales de L'institut Oceanographique, Monaco,
Nouvelle Serie, 3: 1-90.

Nelson, T. C. 1938. The feeding mechanism of the oyster. I. On the
pallium and the branchial chambers of Ostrea virginica, 0.
edulis, and 0. angulata with comparisons of other species of
the genus. Jour. Morph. 63: l-6l.

Picken, L. E. R. 1937- The mechanism of urine formation in inverte-
brates. II. The excretory mechanism in certain Mollusca. Jour.
Exp. Biol. 14: 20-34.

-132-

Prosser, C. L., J. Green, and T. J. Chow. 1955- Ionic and osmotic
concentrations in blood and urine of Pachygrapsus crassipes
acclimated to different salinities. Biol. Bull. 109: 99-IO7.

Schales, 0. and S. S. Schales. 19^1. A simple and accurate method

for the determination of chloride in biological fluids. Jour,
Biol. Chem. 1^0: 879-884.

Shaw, B. L. and H. I. Battle. 1957- The gross and microscopic
anatomy of the digestive tract of the oyster Crassostrea
virginica (Gmelin). Canad. Jour. Zool. 35: 325-3^8.

-133-

SOME FACTORS IN THE USE OF NANNOPLANKTON CULTURES
AS FOOD FOR LARVAL AND JUVENILE BIVALVES

Robert R. Guillard

The Oyster Institute of North America

at the

Marine Biological Laboratory

U. S. Fish and Wildlife Service, Milford, Conn.

Abstract

To guide in estimating food quantity required,
densities of larvae and phytoplankton in our cultures
are compared with natural densities of grazers and
food cells. Orders of magnitude are similar, which
supports use of our present feeding schedule, 0.01
ml of wet packed algal cells per day per liter of
larval culture containing 3 -15 larvae per ml.

Selection of food species is critical. Thirty-
seven isolates of 26 genera in 6 algal classes were
tested as food for larval or juvenile oysters
(Crassostrea virginica) or clams (Venus mercenaria).
Small naked flagellates are necessary for oyster
larvae, best for clam larvae, and adequate for
juveniles of both species, though the latter grow
best on cryptomonads or small diatoms. The dino-
flagellates Amphidinium carter! and Gymnodinium sp.
are useless as food. Species such as Chlorella "A"
or Stichococcus sp. are moderately toxic, particu-
larly to oyster larvae ; the chrysophyte Prymnesium
parvum is extremely toxic to all stages of both
bivalves.

Mass unialgal cultures of good foods such as
Isochrysis galbana and Monochrysis lutheri (chryso-
phytes) have become toxic. This correlates with
bacterial contaminants and high growing temperatures
of 23 - 26°C. The toxin appears to be heat-labile
and adsorbable by paper or membrane filters. Certain
bacterial cultures isolated from toxic algae have
similar properties. Algal food cultures should be
started from pure stocks and grown at the lowest
temperatures the algae tolerate.

A major problem involved in raising bivalves, whether for
experimental purposes or in a commercial hatchery, is to supply
adequate food. Because no synthetic diet has been devised, the only
available foods are cultures of various nannoplankton organisms.

-13^-

This discussion is offered in the hope that it will be of
help to those responsible for selecting and growing food organisms.
It is based largely upon work carried out at the Milford Laboratory
during 1956-57- Feeding experiments with larvae and juveniles of
the oyster (Crassostrea virginica) and hard clam (Venus mercenaria)
were carried out in cooperation with Harry C. Davis and Paul Chanley
of the Milford Laboratory. Davis and Guillard (in press) have
presented results on the use of ten genera of micro-organisms by
larvae. Ecological data are drawn in part from studies of the micro-
biota of two brackish ponds on Martha's Vineyard. Bioassays of toxins
produced by micro-organisms were made in cooperation with Mr. Davis.

The comparatively simple question of food quantity is con-
sidered first. Food organisms must not only be present in adequate
total quantity, but also in suitable concentration. Loosanoff, Davis,
and Chanley (195^) have shown that larvae must not be too crowded,
but whether there is an optimum concentration, due perhaps to some
social phenomenon, is still unknown. We are concerned first with
whether or not conditions in our larval cultures differ markedly from
natural conditions in respect to the relative abundance of phytoplank-
ton and grazing organisms.

In culture methods commonly used at Milford, also by Walne
(1956), the larvae are maintained at concentrations of 3 to 15 per
ml, while phytoplankton concentrations vary from 10,000 to 100,000
cells per ml, depending upon sizes of the cells.

Data on zooplankton concentrations in nature are frequently
deficient because small organisms are not quantitatively retained by
collecting devices. Deevey (19^8) recorded from 10 to 500 zooplankters
per liter, exclusive of ciliates, in samples collected with a #10 net
in West Tisbury Great Pond. She noted (1956) a maximum of about 200
animals per liter, again exclusive of ciliates, in Long Island Sound
in late summer. The writer's observations of preserved whole samples
from W. Tisbury and Oyster Ponds indicate that small ciliates (probably
of the suborder Oligotrichia) often outnumber all other zooplankters.
Lackey et al (1952) reported that ciliate concentrations in the Woods
Hole region were generally of the order of a few per ml, but that
exceptional concentrations of the order of 100 per ml occurred.

It is seen that total animal population densities in nature
approach our culture densities at times. The volume of animals per
unit volume of water, however, is estimated to be somewhat higher in
the cultures. A culture having ten 200/* oyster larvae per ml has
about 20ccof larvae per cubic meter. Deevey (1956) found the maxi-
mum plankton displacement volume (collected with a #10 net) to be 6
cc per cubic meter in Long Island Sound. Note that bivalve larvae
are often but a small proportion of the animal population. Rees
(1954) cites maximum larval concentrations of the order of 10 per
liter. However, a recent report by the State of Washington Depart-
ment of Fisheries (1957) describes concentrations of 2 larvae per ml

-135-

in the region of Willapa Harbor. Perhaps high concentrations in con-
fined waters are not unusual.

To compare the concentrations of food organisms in culture
with those in nature one must refer to studies made by methods allow-
ing small and fragile organisms to be counted. Lackey et al (1952),
by examining the entire contents of small water samples concentrated
by sedimentation or centrifugation, determined that the concentrations
of micro-organisms over oyster grounds (except for places like Great
South Bay, Long Island) varied from about 300 to 300,000 per ml.
Undesirable blooms of Stichococcus and Nannochloris in Great South Bay '
exceeded 2 million cells per ml at times. Hulbert (1956) made a care-
ful study of the living phytoplankton of Great Pond, Falmouth, Mass.
Summer maxima were of the order of 100,000 cells per ml; more general
levels were about 10,000 cells per ml. Conover (1956), using formalin
preserved samples, found that summer maxima in Long Island Sound were
of the order of 10,000 cells per ml. Our samples taken in the summer
of 1956 from West Tisbury Great Pond and Oyster Pond, Martha's Vine-
yard , were preserved with acid I2-KI, allowed to settle, and examined
with a water immersion lens. Holmes (1956) discusses the accuracy of
this method. Total counts varied from 4,000 to 40,000 cells per ml
in these samples.

The range of summer maxima found in nearby waters (excluding
tidal pools and local blooms) is similar to the range of food con-
centrations in the larval cultures. It is suggested that the feeding
schedule described by Davis and Guillard (in press) be used as a guide
in estimating the quantity of algal cells needed to feed given numbers
of larvae. This schedule provides 0.01 ml of wet -packed algal cells
per day per liter of larval culture (ca. 10 larvae per ml). The wet-
packed cell volume of an aliquot of the algal culture is found by
centrifuging it at 1000 g. for 25 minutes in Hopkins or Goetz tubes.
The number of cells per feeding will vary, depending upon the average
cell size of each food species.

There are fewer data to serve as a guide in estimating juvenile
food requirements. Some data from experiments designed to compare
growth of oyster spat on different foods are of interest. For example,
50 spat averaging 0.7 mm in maximum dimension were kept 30 days in 3
liters of water (changed every other day) and fed an unidentified
cryptoroonad (Rhodomonas ? ) at three times the rate used in larval
cultures. The average increase in size was 4.5 mm. Unfed controls
grew 0.6 mm in the same time. Martin (1928) and Prytherch (1924)
observed faster growth of spat under favorable circumstances in
nature, suggesting that more food may be required, perhaps supplied
continuously, rather than batchwise. These data may serve as a
starting point for estimating food requirements for Juveniles of this
size.

It is more important and difficult to supply the right kind of
food than the right amount. Growth of the animals is the only criterion

-136-

by which values of different food organisms can be measured at present.
Larvae fed equal volumes of different foods (according to the schedule
mentioned above) are compared with unfed controls and with others
given known good foods. Methods are given by Davis (1953 )> and Davis
and Guillard (in press). Assay is most indicative of true value of
food organisms themselves if cultures are bacteria -free and harvested
just before the end of the logarithmic phase of growth. This yields
maximum cell density and minimum accumulation of debris and soluble
metabolites. The assay should be repeated using unialgal but not
bacteria-free cultures if the organism to be used routinely as a food.
Impure cultures may become toxic, especially if growth conditions are
sub-optimal.

From such experiments one cannot make an unqualified evalua-
tion of the worth of a given species of micro-organism in nature,
partly because growing conditions there are not known, and also be-
cause the interactions with other micro-organisms and with physical
factors of the environment are more numerous. For example, Shilo and
Aschner (1953) found that the toxicity due to Prymnesium parvum was
decreased by bacteria, by adsorption of the toxin on materials such
as mud, and by oxidizing agents.

During the past 20 months we have assayed the food value of
37 isolates of 26 different genera of micro-organisms. These were
classified as follows: 18 green algae, 7 diatoms, 6 chrysophytes,
3 cryptomonads, 2 dinof lagellates, and one unicellular blue-green
alga. In some experiments only larvae were fed, in others only
juveniles, and in some, both larvae and juveniles were fed from the
same algal cultures. In some experiments the cultures were bacteria-
free, in others only unialgal. Over 100 different assays have been
made using hard clam or oyster larvae, and roughly half that number
with juveniles.

Of the organisms we have tested, only certain small naked
flagellates (no larger than 12^ have benefited oyster larvae in their
earliest stages (k8 hrs. after fertilization). The chrysophytes
Isochrysis galbana and Monochrysis lutheri were best. As the larvae
grew, after about the sixth day, they became capable of using certain
other forms, such as Platymonas sp., Phaeodactylum tricornutum and
Chlorella #580. Clam larvae have been able to use more different
types of foods than oysters of the same age. Juveniles of both species
utilized a still wider range of organisms. Chrysophytes and diatoms
have been notably successful. Those naked flagellates which were best
for larvae were relatively good also for juveniles, but those foods
best for juveniles (cryptomonads, Skeletonema costatum, Actinocyclus
sp. ) were useless to larvae. Factors besides nutritive composition
of the foods are obviously involved in their acceptability to larvae
of different sizes and species. Davis and Guillard (in press) discuss
the influence of presence and thickness of algal cell walls (external
to the plasma membrane) and of production by the algae of toxic sub-
stances. It is significant that certain organisms, in particular the

-137-

dinoflagellates Amphidinium carter 1 and Gymnodinium sp., were useless
to larvae and juveniles alike although they did not kill larvae. The
chrysophyte Pryronesium parvum, on the other hand, was extremely toxic
to both larval and juvenile clams and oysters. This organism, which
unfortunately is widely distributed, produces a toxin of high molecular
weight. Exposure to normal feeding concentrations of Rrymnesium pre-
vented the development of oyster eggs and caused heavy mortality in
cultures of larval clams and oysters. In a 17-day feeding experiment
with 3 ram oyster spat and 3>8 mm juvenile clams (average sizes) 100$
of the oysters and 75$ of the clams died. During this period the con- ,
trol oysters fed a mixture of good foods grew 28$ in length, while the
clams grew M±$ in length. Unfed controls grew less than 5$>

The fact that a micro-organism may be toxic when fed to larvae
or juveniles does not necessarily imply that it can prevent the develop-
ment of eggs to the straight hinge stage. Chlorella isolate 'A' is an
example. It is of interest that two different Stichococcus-like
organisms, one from Great South Bay and one from the Martha's Vineyard
ponds, were also relatively toxic. Gibor (1956) considered the
Stichococcus sp. which he fed to Artemia not toxic but only indigestible.

Again, it is instructive to compare ideas derived from labor-
atory experiments with those derived from observations in nature.
Lackey et al (1952), from analysis of the plankton of such places as
St. Mary's River and Peconic Bay, supported the idea that small
flagellates are good larval food and that diatoms and dinoflagellates
are better food for adult oysters. The idea generally agrees with
out findings, although we have not yet found any dinoflagellates which
are useful as food to larvae or juveniles. The samples of phytoplankton
taken from the Martha's Vineyard ponds, had, as far as we know now,
a favorable composition for both larvae and juveniles. The fraction of
cells over 12 microns in greatest dimension was less than 20$ in all
samples except one in which about 50$ were large pennate diatoms.
This came from a shallow inlet of low salinity. Very samll chrysophyte-
colored cells were usually most numerous. Some of these were obviously
flagellated; others were minute diatoms. The frustules of these small
diatoms can be demonstrated only with difficulty by the methods normally
employed. At some times cryptoraonads were dominant; often small green
flagellates were numerous, some of them possibly zoospores. Hulbert ' s
findings (1956) in Falmouth Great Pond were generally similar, although
at times the plankton was dominated by diatoms brought in from Vineyard
Sound .

To summarize, our experience suggests that for best results
the algologist should provide a mixture of cultures of two or three
small naked flagellates - especially chrysophytes - for larvae, and
should supplement this with diatoms and cryptomonads for juveniles.
Note that this is for best results -- larval clams grow fairly well
on such forms as Phaeodactylum or Chlorella #580, and very well on
Chlorococcum isolate 'C'.

■138-

Still another problem besets the use of cultures as foods.
Presumably assay of a healthy bacteria-free culture measures the
effects of such relatively constant properties as cell size, wall
thickness, and average chemical composition. However, one cannot
always wait to get a culture bacteria-free; moreover, large cultures
for routine feeding can seldom be kept pure, and may not always be
in the log phase of growth. At times this may make little difference,
but there is clear evidence that impure cultures may be toxic under
some circumstances.

Toxicity of good food organisms was first recognized in carboy
cultures of Isochrysis galbana and Monochrysis lutheri grown at room
temperature, exceeding 23°C at times. These cultures had been used
successfully through the fall and early winter; erratic growth of the
algae and toxicity to larvae became apparent in late winter.

Assays were made with various preparations from a toxic
Isochrysis culture and with bacteria derived from this culture.
Newly fertilized oyster eggs give clear results in k8 hours or less.
Highly toxic preparations' cause the eggs to disintegrate. Dilutions
of such preparations usually allow eggs to develop into ciliated
stages or deformed shelled larvae. On occasion larvae develop
apparently normally to straight hinge stage, then die.

Certain properties of toxic culture medium resemble those of
toxins derived from pure cultures of other organisms. The following
brief summary will make this clear. The toxic principle from the
Isochrysis cultures is destroyed by boiling and is not carried over
in the distillate. It is not destroyed by heating to 60°C. An
aliquot of toxic culture is made non-toxic by Millipore filtration,
which removes the algae and bacteria and adsorbs certain molecules.
Filtration through Whatman #5 filter paper, which removes few algae or
bacteria, also removes most of the toxicity. Rapid centrifugation,
which removes most of the algae and some of the bacteria, leaves the
toxicity. Exposure to low pressure which occurs during filtration
does not remove toxin. The material therefore has many of the
properties of the toxin found in pure cultures of Prymnesium parvum,
as described by Shilo (Shelubsky) and Aschner (1953T It differs
from the Gymnodinium brevis toxin described by Ray and Wilson (1957)
in that it is adsorbed on both Millipore and paper filters, while
Gymnodinium toxin is adsorbed only on paper. The Gymnodinium
venef icum toxin studied by Abbott and Ballantine (1957) has a molecular
weight greater than 1000 and is dialyzable and adsorbable. The toxin
described by Shelubsky (1951) from the blue-green alga Microcystis is
a compound of small molecular weight with quite different properties.
For the sake of completeness we mention that the toxicity of Chlorella,
Chlamydomonas, Stichococcus, and certain other algae may well be due
to the fatty acids they liberate. This is well reviewed by Proctor
(1957).

-139-

Pure and mixed cultures of bacteria were obtained by streaking
toxic algal cultures and by inoculating aliquots into various enrich-
ments of sea water. Preliminary studies on toxicity, adsorption, and
heat lability showed that the preparations made from bacterial and
from toxic algal cultures had similar properties, but definitive
experiments have yet to be made.

Efforts to induce toxicity in pure algal cultures by the
addition of bacteria or aliquots of toxic cultures were not uniformly
successful, but succeeded more often when the algal cultures were
grown at 23 - 26°C, than when they were grown in the 20° room.
Cultures grown at 20° sometimes became toxic when transferred to
23 - 26°C, and once altered, remained toxic when returned to 20°.
Isochrysis is less tolerant of high temperatures than Monochrysis,
and becomes toxic more often. We have never observed toxicity in
pure cultures of either Isochrysis or Monochrysis. Impure cultures
may become toxic when grown in artificial sea water (medium ASP 2 of
Provasoli et al 1957 )> which eliminates the possibility that toxicity
is due to some interaction between the micro-organisms and organic
substances in sea water.

The hypothesis is offered, therefore, that toxicity is due to
bacteria that get into cultures in the course of handling. Toxin pro-
duction, presumably by bacteria rather than by algae, takes place
when algae provide the necessary substrate. This is influenced by
the condition of the algal culture. We noticed, for example, that a
dense culture in a carboy sealed to an aerating system became toxic
in a day when the supply of air and C02 was accidentally shut off.
The fact that larvae too young to feed can be killed or injured
suggests that some toxin is liberated into the medium.

In our experience, erratic results in feeding experiments
have tended to coincide with the use of impure cultures, especially
those grown at room temperature. Possibly some of the irregularities
mentioned by Walne (1956) were caused by bacteria. Lest bacteria be
blamed for all ills, let us record that they can also detoxify
cultures. The report that bacteria destroy Prymnesium toxin led us
to incubate an aliquot of toxic Isochrysis culture with a mixed
culture of marine bacteria. This treatment removed the toxicity.
In another trial, toxicity was only reduced.

It is recommended that whenever possible large algal cultures
be started from bacteria-free stocks and grown at the lowest tempera-
tures that provide rapid growth. Simpler methods of control may be
devised when the nature of the toxicity is determined and factors
responsible for its production are better understood.

-li+0-

References

Abbott, B. C, and D. Ballantine. 1957. The toxin from Gymnodinium
venef icum Ballantine. Jour. Marine Biol. Assoc. U.K. 36:
169-189-

Conover, S. A. M. 1956. Phytoplankton. in "Oceanography of Long
Island Sound", by Gordon A. Riley et al. Bull. Bingham
Oceanogr. Coll. 15: 62-112.

Davis, H. C. 1953- On food and feeding of larvae of the American
oyster, C. virginica. Biol. Bull. 10U: 33^-350.

Davis, Harry C, and Guillard, R. L. Relative value of ten genera of
micro-organisms as foods for clam and oyster larvae. U. S.
Fish and Wildlife Serv. Fish. Bull. (in press)

Deevey, G. B. 19^8. The zooplankton of Tisbury Great Pond. Bull.
Bingham Oceanogr. Coll. 12(l): 1-44.

Deevey, G. S. 1956. V. Zooplankton in "Oceanography of Long Island
Sound," by Gordon A. Riley et al. Bull. Bingham Oceanogr.
Coll. 15: 113-155.

Gibor, Aaron, 1956. Some ecological relationships between phyto-
and zooplankton. Biol. Bull. Ill: 230-234.

Holmes, Robert W. 1956. The annual cycle of phytoplankton in the

Labrador Sea, 1950-51. Bull. Bingham Oceanogr. Coll. 16: 1-74.

Hulbert, E. M. 1956. The phytoplankton of Great Pond, Massachusetts.
Biol. Bull. 110: 157-I68.

Lackey, J. B., G. Vanderborgh, Jrv and J. B. Glancy. 1952. General
character of plankton organisms in water overlying shellfish-
producing ground. Natl. Shellfish. Assoc. Conv. Add. 1952:
152-156.

Loosanoff, V. L., H. C. Davis, and P. E. Chanley, 1954. Food require-
ments of some bivalve larvae. Proc. Natl. Shellfish. Assoc.
66-82 .

Martin, G. W. 1928. Experimental feeding of oysters. Ecology 9(1 ):
1+9-55.

Procter, V. W. 1957- Studies of algal antibiosis using Hematococcus
and Chlamydomonas. Limnol. and Oceanogr. 2: 125-139-

Provasoli, L., J. J. A. McLaughlin, and M. R. Droop. 1957- The

development of artificial media for marine algae. Arch, fur
Mikrobiol. 25: 392-428.

•i4i-

Prytherch, H. F. 1924. Experiments in the artificial propagation of
oysters. Bur. Fish. Doc. #96l: 1-14.

Ray, S. M., and W. B. Wilson. 1957. The effects of unialgal and
bacteria-free cultures of Gymnodinium brevis on fish and
notes on related studies with bacteria. U. S. Fish and
Wildlife Serv. Spec. Sci . Report - Fisheries No. 211.

Rees, C. B. 195*+ • Continuous plankton records: The distribution of
lamellibranch larvae in the North Sea, 1950-51* Bull. Marine
Ecol. 4(27): 21-46.

Shelubsky, M. 1951 • Observations on the properties of a toxin pro-
duced by Microcystis. Proc. Internatl. Assoc. Liranol. 11:
363-366.

Shilo (Shelubsky), M., and M. Aschner. 1953- Factors governing the

toxicity of cultures containing the phytof lagellate Prymnesium
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Walne, P. R. 1956. Experimental rearing of the larvae of Ostrea
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Washington, Dept. Fish. 1957. Willapa Harbor Pacific Oyster Bulletin.
Ser. XVI No. 3, July 15, 1957 .

-lJ+2-

DISPOSAL BY THE OYSTER OF INTRACARDIALLY INJECTED
RED BLOOD CELLS OF VERTEBRATES1

M. R. Tripp

Department of Zoology
Rutgers University

Abstract

Various vertebrate erythrocytes were injected
intracardially into the oyster, Crassostrea virginica,
and their fates were determined histologically"! In
many vessels and sinuses the particles are phagocytized
within a few hours. Some phagocytes then migrate
through tissue and surface epithelium to be voided.
Those erythrocytes not removed in this fashion are
digested intracellular ly. It is suggested that the
ability of the oyster to defend itself against foreign
particles is dependent upon both the migratory and the
digestive capabilities of its leukocytes.

Introduction

The present study is one of a series of steps to be taken
in elucidating the defense mechanisms of the oyster, Crassostrea
virginica Gmelin. The first step was taken by Stauber (1950) when
he injected india ink intracardially and traced its fate. The series
of experiments to be reported here is concerned with the fate of a
raetabolizable particle -the vertebrate red blood cell-placed in the
circulatory system of the oyster. This work confirms the findings of
Stauber (1950) and extends the concept of the phagocyte as an active
participant in the defense of the oyster body.

Methods

The experiments were designed to reveal phagocytic, dispersal
and digestive activities at several times following intracardial
injection of red blood cells. Rabbit, weakfish, and normal duck
erythrocytes, as well as cells from a duck infected with the avian
malaria parasite, Plasmodium lophurae, were used as inocula. The
red cells were washed thoroughly and resuspended in sea water. The
heart was carefully exposed by filing an opening through the left
valve of the shell directly over the pericardium. Then, by means of

"T^his investigation was supported by a research grant (E-78l)from the
National Institute of Allergy and Infectious Diseases of the National
Institutes of Health.

■143-

a tuberculin syringe and a 30 gauge needle, 0.2 ml of the inoculum was
introduced into the ventricle. The oysters were replaced in tanks of
sea water and one was sacrificed at each of the following times:
10 minutes, 30 minutes, 6 hours, 1 day and 2 days, and then every
other day up to about three weeks. Each oyster sampled was relaxed
in cold magnesium sulfate solution overnight, then fixed in Bouin's
solution, washed, dehydrated in a series of alcohols, cleared in xylol,
and mounted in paraffin. Twelve transverse sections were obtained
from each animal. Two sections were cut from each of the following
levels: palps, stomach, visceral mass posterior to the stomach, heart,
adductor muscle and the most posterior portion of the gills. The
sections were stained with hematoxylin and eosin.

In each animal certain vessels, sinuses and tissues were
examined under high dry and oil immersion objectives. The presence
or absence of phagocytosed red blood cells in epithelium was ascer-
tained by oil immersion examination.

The following blood channels were sampled: heart, anterior
and posterior aortae, subepithelial spaces of the gut at all levels,
large arteries of the visceral mass, sinuses between the digestive
diverticula, dorsal and ventral circumpallial arteries, proximal mantle
vessels, medial and lateral gill axis sinuses, vertical gill vessels,
palp vessels and the blood spaces of the adductor muscle and kidney.

The movement of phagocytes containing red cells through
epithelium was determined by examination of the following sites:
walls of esophagus, stomach and rectum, walls of about twenty
digestive diverticula, inner and outer aspects of the palps, outer
aspect of the mantle in several areas, nephridial tubules, gonaducts,
external lining of the heart, and internal lining of the pericardium.

In addition to the sections, smears of heart blood were made
before the oyster was relaxed. These preparations were fixed with
methyl alcohol and stained with Giemsa stain. Also, in vitro experi-
ments with mixtures of oyster heart blood and chicken red cells have
confirmed the phagocytic abilities of oyster leukocytes.

Results

Following intracardial injection of red blood cells, five
phases of oyster response are discernible: l) large masses of red
cells virtually occlude many arteries; 2) these particles are
phagocytized; 3) phagocytes migrate into tissue; k) phagocytes
migrate through epithelium resulting in elimination of the particles
from the body; and 5) those red blood cells retained in the body
within phagocytes for twelve days are digested.

Phagocytosis of red blood cells begins immediately after
injection. Within ten minutes large numbers of red cells are engulfed

-3M-

by phagocytes. This is confirmed by sections and blood smears. From
one to eight red cells per phagocyte are usually seen and not uncommon-
ly up to fifteen or twenty are found within a single cell. Six hours
after injection approximately 95% of the red blood cells in oyster
blood vessels are within phagocytes.

Masses of phagocytized red cells in arteries are removed in
two ways: l) blood pressure forces the particles through smaller
arteries into sinuses, and 2) phagocytes containing red blood cells
begin to migrate from the vessels and sinuses to tissue. As the
emboli are resolved, blood flows more freely.

As early as 48 hours post-injection, the migration of red
cell-laden phagocytes across epithelial layers can be detected. This
movement to the exterior reaches a peak at about eight days. The
surfaces traversed are: gut wall at all levels, gonaducts, mantle,
digestive diverticula, pericardium and inner and outer aspects of the
palps. After emerging into lumina, red cell-laden phagocytes are
easily voided.

In all experiments, evidence of intracellular red blood cell
destruction was obtained. Experiments using avian red blood cells
containing malaria parasites were particularly clear in this respect.
The pigment associated with malaria is the product of hemoglobin
destruction wherein the parasite splits off globin, leaving the acid
hematin residue behind. This acid hematin is extremely resistant to
destruction. In the oyster, when a parasitized red blood cell is
destroyed, the readily recognizable pigment remains as a marker.
Twelve days after injection, malaria pigment in oyster phagocytes
was distributed in the tissues while intact red cells were difficult
to find. Destruction by osmotic effects can be ruled out since an
occasional free red cell could be found which was still intact. The
amount of free pigment due to rupture of cells during inoculation
was negligible and virtually undectectable in early samples. These
observations, coupled with the evidence for intracellular proteases
and lipases (Yonge 1926), make inescapable the conclusion that
vertebrate red blood cells injected intravascularly are digested by
oyster phagocytes. Further evidence for this process is supplied by
the pioneer work of Yonge (1926), who has clearly shown that when
shark corpuscles are fed to Qstrea edulis they are phagocytized and
digested to a mass of fat globules in 12 hours.

Discussion

As Stauber (1950) has noted, it is to be expected that in
the oyster, a poikilothermic animal, the degree of activity of
phagocytes would be affected by temperature changes. In his experi-
ments, at temperatures from 12° to 21° C, ink-laden phagocytes were
seen migrating across epithelial layers after eight days. With
temperatures at 21 C. movement of phagocytized red cells across

-145-

epithelium was first recorded at two days. This early movement may
have occurred in the india ink work also, but it was not discovered
because no samples were taken between 22 hours and eight days. More
recent data show the period between one and eight days post-injection
to be a very significatnt one in the oyster's attempt to rid itself
of foreign particles.

The response of an oyster phagocyte to particulate matter is
to engulf the particle and then migrate. If the particle is not
metabolizable, it may be eliminated by phagocytic migration. When
the particle is metabolizable, such as a red blood cell, this migratory
response may be supplemented by intracellular digestion. In the
experiment discussed here, some red cells were quickly removed by
phagocytic transport before they could be digested. After twelve
days, most of the red cells in phagocytes had been digested. Degrada-
tion of red cells seems to be the more important means of elimination.
Although migration may be able to remove a large number of particles
over a long period of time, digestion of these particles would greatly
enhance the efficiency of the defense mechanism. In this manner per-
haps some potentially pathogenic microorganisms are destroyed by
digestion while such intracellular parasites as Nematopsis and
Dermocystidium, which appear to be resistant to the intracellular
enzymes of oyster leukocytes, may be eliminated by migration of host
cells to the exterior.

No matter what the defense mechanism, it depends on the oyster
maintaining a physiological optimum. Any added stress might reduce
the efficiency of the phagocytes, allow parasites to accumulate, and
lead to the death of the oyster.

Summary

When vertebrate red blood cells are injected intracardially
in the oyster:

1) Phagocytosis begins immediately and is over 90% complete
within six hours.

2) Phagocytes carry these particles from blood channels
through tissues and epithelia to various lumina from which some are
voided.

3) Those erythrocytes not removed in twelve days are digested
within phagocytes.

h) Digestion seems to be more important than migration, as a
means of eliminating vertebrate red blood cells from the oyster.

-146-

References Cited

Stauber, L. A. 1950. The fate of India ink injected intracardially
into the oyster, Qstrea -yirginica Gmelin. Biol. Bull. 98(3):
227-2U1.

Yonge, C. M. 1926. Structure and physiology of the organs of feeding
and digestion in Qstrea edulis. Jour. Marine Biol. Assoc.
14: 295-386.

-1^7-

SOME OBSERVATIONS ON BLOOD
CIRCULATION IN THE OYSTER, CRASSOSTREA VTRGINICA

Albert F. Eble2

Department of Zoology, Rutgers,
The State University, New Brunswick, N. J.

Abstract

It was found that the American oyster has
three veins that return blood to the heart, the
branchial, the pallio-branchial and the pallio-
cloacal.

Each accessory heart was found to possess
a branch which follows closely the adductor muscle
dorsad. Each branch empties its blood into the
shell side of the mantle.

A study of the gill circulation reveals an
afferent and an efferent system. The medial gill
axis sinus is the major afferent branchial vein and
the pallio-branchial vein is the major efferent
vessel. In the demibranchs, afferent and efferent
systems are connected by spaces in the interf ilamentar
junctions via the vessels associated with the princi-
pal filaments.

The present study deals with the venous return and gill
circulation of the American oyster, Crassostrea virginica (Gmelin).
Other investigators (Kellogg 1890, Leenhardt 1926, Hopkins, A. E.
193*0 have shed much light on the arterial system of the oyster but
details of venous circulation and its relation to the gills have never
been adequately explained.

There are three pairs of major vessels which return blood to
the heart:

l) the branchial vein, which drains blood from the anterior
portion of the lateral gill axis sinus and the lateral
portions of the visceral mass,

"T?his investigation was supported by a research grant (E-T81) from

the National Institute of Allergy and Infectious Diseases, Public

Health Service.

2
Present Address: Department of Life Sciences, Trenton Junior

College, Trenton, New Jersey.

-148-

2) the pallio-cloacal vein, which drains blood from the
cloacal chamber and the dorsal portions of the mantle
and visceral mass and,

3) the pallio-branchial vein, which drains blood from the
ventral regions of the mantle and the entire posterior
portion of the gills.

The branchial vein is a dorsal extension of the lateral gill
axis sinus. It ascends in a dorso-posterior direction to join the
atrium. During its course toward the atrium it lies embedded in the
epibranchial septum, which divides the anterior epibranchial chamber
on each side into two separate channels. The pallio-cloacal vein
originates in the mantle of the cloacal chamber and runs close to the
adductor muscle as it proceeds dorsally over the muscle then bends
sharply ventrad to join the atrium. The pallio-branchial vein origi-
nates in the posterior mantle just where the mantle joins with the
most lateral gill lamella. It proceeds anteriorly and, at the level
of the end of the oral process, turns dorso-anteriad and runs up to
the atrium.

Blood which drains from the sinuses of the organ of Bojanus
(kidney) empties into the accessory hearts first described by Hopkins
(193*0- The accessory hearts beat in a proximo-distal direction and
propel blood into the circumpallial artery and, via numerous short
connectives, to the shell side of the mantle all along its course.
In addition, there is a branch of the accessory heart which, instead
of crossing the cloacal chamber, follows the adductor muscle dorso-
posteriad for approximately half its length and empties, via numerous
terminal sprouts, into the shell side of the mantle. Blood after
filtering through the organ of Bojanus has probably lost what little
pressure it had hence the accessory heart takes on the important
function of increasing the pressure in this critical region. The
accessory heart sends blood out the circumpallial artery into the
pallial curtain and, through numerous small branches, to the shell
side of the mantle. This blood, then, filters through the numerous
tissue spaces of the mantle where it collects either in the pallio-
branchial vein, which receives blood from the ventral side of the
mantle, or the pallio-cloacal vein, which receives blood from the
general area of the cloacal chamber.

One of the major veins that drains the visceral mass as well
as the medial palps runs along the medial axis that separates the
two gills, and is known as the medial gill axis sinus. All the blood
that enters and circulates through the gill filaments comes from this
major vein in which the blood flows in a posterior direction. This
vessel then, marks the beginning of the afferent branchial system
(Figure 1-top water tubes). As it proceeds, it gives off branches
to every other principal filament in the adjacent gill lamellae.
It also gives off in every inter lamellar junction a vessel which runs
across to the outermost lamella of each side and empties into branch

-149-

Gill Gucolatk

rrAlUO-
Ve,„

Fig. 1. Dorsal view of the demibranchs and water tubes on the
left side in the region of the adductor muscle. The afferent bran-
chial system is indicated by the vessels associated with the middle
water tubes and the combined circulation is indicated in the lower
water tubes.

-I5O-

vessels serving every other principal filament in these lamellae.
From here the blood can go across the sub-inter lamellar junctions,
which connect opposing principal filaments of the demibranch at
different levels across the water tube, to the corresponding princi-
pal filament of the adjacent lamellae (see upper two water tubes,
Figure l). Finally, at all levels, blood moves in the spaces of the
interf ilamentar junctions to the vessels of the interdigitating
adjacent principal filaments which constitute the beginnings of the
efferent system. This efferent branchial system can be broken down
into three separate parts which all eventually empty into the pallio-
branchial vein (see middle water tubes, Figure l).

1) The blood vessels of the principal filaments of the
medial lamellae of the inner demibranchs: these efferent vessels
collect into a common vessel at the top of the water tube then pro-
ceed to the anterior end of the water tube, then turn laterally across
the interlamellar junction toward the pallio -branchial vein.

2) The vessels of the principal filaments of the lateral
lamellae of the outer demibranchs empty separately into the pallio-
branchial vein.

3) The efferent vessels in the principal filaments on
either side of the lateral gill axis: these vessels drain into a
common vessel known as the lateral gill axis sinus; previously this
vessel was thought to be continuous the whole length of the gill,

but there it only proceeds to the anterior end of the water tube which
it drains, turns sharply laterad, and thence proceeds across the inter-
lamellar junction to join the pallio-branchial vein. As it turns
laterad to join the pallio-branchial vein it receives the vessel
that drains the medial lamellae of the inner demibranchs (part "1" of
efferent branchial system) and the two empty into the pallio-branchial
vein as a common vessel.

Literature Cited

Hopkins, A. E. 1934. Accessory hearts in the oyster, Ostrea gigas.
Biol. Bull. 67: 346-355, 2 figs.

Hopkins, A. E. 193 6. Pulsation of blood vessels in oysters, Ostrea
lurida and 0. gigas. Biol. Bull. 70: 413-1+25, 4 figs.

Kellogg, J. I89O. A contribtuion to our knowledge of the morphology
of lame llibranchi ate mollusks. Bull. U. S. Bur. Fish.
10: 389-436, 97 figs.

Leenhardt, H. 1926. 1926. Quelques estudes sur Gryphea angulata
(Huitre du Portugal). Inst. Oceanogr. 3: 1-90, 36 figs.

-151-

AN ABNORMAL VIRGINIA OYSTER WITH A BIFURCATED MUSCLE

Gordon Gunter
Gulf Coast Research Laboratory, Ocean Springs, Mississippi

In October, 1955 Mr. Jack Ross brought in an oyster which he
had taken in Davis Bay, on which this Laboratory is situated. He had
shucked this oyster and noted that it was abnormal.

The muscle was split into two parts where it attached to the
left valve. The more forward and dorsal part was oblong in shape,
being 2 cm long and about 1.3 cm wide, with the long axis parallel to
the axis of the body. The lower part of this muscle was somewhat
irregular in shape and it was approximately 1.4 by 1.2 cm. The
posterior margin of the dorsal part of the muscle extended almost 2
centimeters posterior to the margin of the ventral smaller portion.
Those two parts were separated by epithelial tissues of the body wall
about 1.2 cm across. The left valve itself showed three dark areas.
Two of them corresponded to the muscle attachment but one was ventral
and posterior. Apparently there was no muscle attachment along this
smaller area. The muscle scars on the shell were approximately 2.5
cm apart and, apparently, the measurements of the muscles and their
separation, made on the alcoholic specimen, were somewhat modified by
shrinkage. The left valve showed an iregularly ridged, white area
splotched with irregular purple markings, apparently a former muscle
attachment, between the real attachments. The shell at this point
was approximately 2.5 cm thick. The remainder of the shell was less
than a cm thick. The writer did not cut the shell or break it open.

The left valve of the oyster was 1^.0 cm long. The muscle
attachment to the right valve was somewhat hourglass-shaped with the
lower lobe being much the larger and spread posteriorly. The "waist"
of this muscle was slightly less than 1 cm accross. The smaller lobe
was a cm across and the other one was slightly over 2 cm across.
This hourglass figure extended with the long way at right angles to
the anterior-posterior axis of the oyster and with the small lobe
towards the promyal chamber. It is probable that the abnormal pull
of the double muscle of the left valve caused this abnormal shape of
the attachment on the right valve.

The three attachment portions of this muscle all showed the
closing and locking muscle, although the areas were somewhat irregu-
lar. The attachment area of the upper valve showed in general the
hourglass shape described for the muscle of that side.

The shell itself was not particularly heavily infested with
associates and boring organisms. There were a few small barnacles
and signs of some worm holes. There were no signs of organisms with-
in the shell which would have ■ caused this abnormality.

-152-

The oyster was in excellent condition and was a creamy white
in color. It had a considerable amount of glycogen. Apparently the
muscle abnormality interfered very little with its life processes.
The cause of this aberration is unknown.

I have presented this specimen to Dr. Paul S. Galtsoff as an
addition to his collection of abnormal oysters.

■153-

OBSERVATIONS ON MUSCLE ATTACHMENTS, CILIARY MOTION, AND THE PALLIAL

ORGAN OF OYSTERS

Paul S. Galtsoff

Shellfish Laboratory, Bureau of Commercial Fisheries
U. S. Fish and Wildlife Service, Woods Hole, Mass.

A detailed study of the anatomy and functions of the oyster
which I have conducted during past years has revealed several facts
which heretofore have remained unnoticed. The present discussion
deals with the adductor muscle, the skeleton and muscular apparatus
of the gills, the ciliary tracts of the mantle and gills, and the
structure of the so-called pallial organ.

Attachment of Adductor Muscle

The adductor muscle of all pelecypods is a very conspicuous
and powerful organ. Experiments which I described at the 1952 meet-
ing of the Association show that the muscle of a fully grown Crassostrea
virginica withstands a pulling force up to 22 pounds (10 kilograms).
Under this force the muscle fibers usually break in the middle and the
remaining parts still adhere to the valves. Only rarely is the muscle
separated at the place of attachment. On the other hand, by applying
heat to the outside of the shell over the area of the muscle scar the
connection between the muscle and the valve may be severed or at least
greatly weakened.

The muscle scar is always smooth and glossy. Examination of
sections made across the decalcified shell with the muscle attached
to it show that the fibers end in a layer of epithelial cells next to
the inner surface of each valve (Figure l). The boundaries of these
cells are not visible and the entire layer has the appearance of a
syncytium. The nuclei are very large. Fibrillar protoplasmic strands
extend across this layer and end at the inner surface of the shell.
In the areas adjacent to the adductor muscle the mantle is covered
with a typical epithelium which gradually changes into a specialized
layer as the mantle approaches the adductor muscle. Bruck (1913) who
described this epithelium in Anodonta and Cyclas called it "adhesive"
epithelium and considered that it provides a mechanism for attachment
of muscle fibers to the shell. He did not notice, however, an organic
film, no more than 2 microns in thickness, by which the muscle fibers
are cemented to the shell of Crassostrea virginica. This film is
always present in the oyster but is usually destroyed when muscles
are cut from the shell. The cement by which muscles are attached to
the shell probably consists of collagen, a protein generally found
in connective tissue. To varify this assumption various connective
tissue stains were used. The staining reactions were not too reliable,
however, and better results were obtained by observing effects of
proteolytic enzymes.

-154-

Fig. 1. Attachment of adductor muscle to shell. A thin layer
of cement, in one place detached from the muscle, lies over a row
of epithelial cells through which pass muscular fibrillae. Drawing
from decalcified preparation of oyster shell with muscle attached.
Hematoxylin and eosin--X 65O.

-155-

It is known that under proper conditions collagen may be
digested by an enzyme called collagenase. A series of experiments
conducted last month at Woods Hole indicates that this protein plays
a major role in the attachment of muscles in oysters. The experiments
consisted of injecting small amounts of phosphate buffer solution con-
taining 1 mg of collagenase per ml into the adductor muscles through
small holes drilled in the valves. In another set of experiments the
muscles of small oysters with the shells attached to them were immersed
in the solution of collagenase and were kept at a temperature of
2i|-25°C. for 24-48 hours. Solutions of trypsin and of phosphate buffer,
without enzyme were used for control experiments. In all cases muscles
treated with collagenase became detached within 36 hours, but controls
remained attached to shells. There is no doubt that the oyster uses
collagen as a cement for the attachment of its adductor muscle.

Gill Apparatus and Ciliary Motion

The muscle fibers of the gills present a different picture.
They end in root -like processes which are deeply embedded in chitinous
material. There is no sign of any cement by which they may be glued
to the skeletal bars. The framework of an oyster gill is an inverted
V-shaped structure with two sets of muscles. One set is located
between the two converging arms of the frame; the contraction of these
muscles brings the arms together and closes the gills. The other set
of muscles located on the outside of the frame surrounds the sharp
point where the arms are joined together. The contraction of these
muscles pulls the arms apart and opens the gill. In this way access
of water to the gill lamellae is facilitated. The opening and closing
of the gill lamellae is one of the essential mechanisms by which the
oyster regulates the rate of pumping of water through the gills.

Control of size of ostia is another mechanism which the
oyster uses to regulate passage of water through the gills. The
importance of ostia in feeding and respiration was correctly emphasized
by T. C. Nelson (1940). Observing live oysters with the gills exposed
on one side, I found that muscular contraction of the gills closes the
ostia and that this method of regulating the dimensions of the ostia
is much more important than filling and emptying of blood vessels of
the gill filaments. The latter view has been maintained by Elsey (1935)
but was not confirmed by my observations.

The lateral cilia which surround the ostia force the water
into the water tubes and at the same time sort the particles suspended
in water by throwing the large ones back to the surface of the fila-
ments and by permitting the smaller ones to be drawn inside the gills.
It is generally considered that the removal of particles from water
is the function of the latero -frontal cilia. My observations show
that this removal is incomplete and that the lateral cilia constitute
another barrier which prevents the larger particles from passing
through the ostia. In order to study ciliary currents the gills were

-156-

..»,... :«i ,^V"^ ■.■.'r-T'v-'.V-'-'^-.'y-'

3Vwg

Fig. 2. Small portion of exposed surface of gill with expanded
ostia. Horizontal arrows indicate direction of roetachronic waves of
the two rows of lateral cilia. Small particles of carbon (right)
are sucked in while large particles (left) are thrown off by the
recovery strokes of the cilia. Drawn from life--X 250.

-157-

exposed by removing parts of the shells, and phosphorescent powder
(observed in ultraviolet light) or a suspension of colloidal carbon
was added to the water. The behavior of the lateral cilia was
recorded by the artist. It was clearly seen that while minute
particles were sucked into the water tubes through the ostia, the
larger ones were thrown back by the recovery strokes of the lateral
cilia. In several instances the larger lumps of powder were tossed
back and forth for a long time until they finally were discarded
(Figure 2).

The control of ciliary motion is a problem of great physiologi-
cal interest. Since the ciliary motion continues after the severance
of the tissue from nerve ganglia, it is generally considered that
ciliary activity is not under control of the nervous system although
the possibility of such control was suggested by Lucas (1929> 1935)
and others. I found that in the oyster with exposed gills, but other-
wise intact, the motion of the frontal cilia and of the cilia of the
terminal grooves of the gills ceases with the spontaneous contraction
of the adductor muscle and is restored when the muscle relaxes. There
is undoubtedly some sort of connection between muscular contraction
and ciliary motion, but whether this control is exercised through the
nervous system or by stimulation of the protoplasm of ciliated cells
cannot be answered without a detailed physiological study. Electric
shock applied directly to the ciliated epithelium produced no effect
on ciliary rhythm.

The Pallial Organ

The pallial or abdominal organ of Crassostrea virginica is an
inconpicuous little protuberance on the side of the adductor muscle
in the right suprabranchial chamber. The surface of the organ is
covered by long cells with still cilia (Figure 3)- Numerous sensory
cells with long processes are found compressed between the epithelial
cells. Typical connective tissue underlies the epithelium. The
large nerve with numerous branches ending at the periphery of the
organ suggests that the pallial organ is of great importance to the
oyster. The location of the organ inside the superbranchial chamber
makes study of its physiology difficult (Figure h) . The organ probably
serves for detection of mechanical disturbances in the water--a view
which was advanced by several investigators who described this struc-
ture in various lamellibranch.es, (Dahmen 1923* Awati and Rao 1931 )•
It does not seem reasonable that the organ is concerned with chemical
sense. The location of the organ in the suprabranchial chamber speaks
against this possibility. Furthermore, it is a well established fact
(Hopkins 1932) that the edge of the mantle, with tentacles which come
in contact with water before it enters the mantle cavity, is very
sensitive to irritating or poisonous substances and gives the oyster
a warning signal to close the shell. The long stiff cilia for the
pallial organ seem to be more suitable for detecting mechanical dis-
turbances than for responding to chemical stimulation.

■158-

Fig. 3- Cross section of pallial organ showing long epithelial
cells, narrow sensory cells on right and left sides of protuberance,
and very large nerve trunk in connective tissues under the organ--
X 100.

■159-

r

a./, ok

ac.h

Fig. k. Location of pallial organ (pal or) on wall of adductor
muscle (ad m) in suprabranchial chamber which was cut and the oppos-
ing sides of mantle pulled apart. Accessory heart (ac h), mantle (m),
and rectum (r). Drawn from life.

-160-

The observed details are of minor nature but they provide
better understanding of the operation of the feeding and respiratory
organs. Such an understanding is essential for the solution of
practical problems of self-purification, feeding, and fattening of
shellfish. The aim of these investigations is to learn more about
the life of the mollusk in order to find ways of producing better
oysters.

References

Awati, P. R., and H. S. Rao. 1931* Ostrea cucullata (the Bombay
oyster). Ind. Zool. Mus. Lucknow 3: 1-107.

Bruck, A. 191^' Die Muskulatur von Anodonta cellensis Schrot. Ein

Beitrag zur Anatomie and Histologic der Muskelfasern. Zeitschr.
wiss. Zool. 110: 481-619-

Dahmen, P. 1923- Anatomie von Ostrea chilensis Philippi. Jena.
Zeitschr. f. Naturwiss. 52: 573-626.

Elsey, C. R. 1935- On the structure and function of the mantle and
gill of Ostrea gigas and 0. lurida. Trans. Roy. Soc. Canada,
Sec. 5: 131-158.

Hopkins, A. E. 1932. Chemical stimulation by salts in the oyster,
Ostrea virginica. Jour. Exp. Zool. 6l: 13-28.

Lucas, A. K. 1929- The distribution of the branchial nerve in

Mytilus edulis and its relation to the problem of nervous
control of ciliary activity. Anat. Rec. hk: 230.

Lucas, A. M. 1931* An investigation of the nervous system as a

possible factor in the regulation of ciliary activity of the
lamellibranch gill. Jour. Morph. and Physiol. 51: 1^7-19^ •

Nelson, T. C. 19^0. On the nature and action of diantlin a new
hormone -like substance carried by the spermatozoa of the
oyster. Jour. Exp. Zool. 85: 299-338.

■161-

OBSERVATIONS ON DISTRIBUTION AND ELIMINATION OF SPORES
OF NEMATOPSIS OSTREARUM IN OYSTERS*

Sung Yen Feng**

Virginia Fisheries Laboratory
Gloucester Point, Virginia

Abstract

A non-random distribution of cysts of
Nematopsis along the mantle margin of oysters was
discovered. Sampling sites must be carefully chosen
for comparative studies. When oysters with high
and low initial infections of Nematopsis were trans-
planted into areas of low and high infestation respec-
tively, the transplants attained the characteristic
level of infections of native oysters in that area.
Presumably a dynamic equilibrium of elimination and
reinfection of the parasite was reached. Spores dis-
charged from living oysters may infect crabs.
S. Y. Feng.

INTRODUCTION

Nematopsis ostrearum, a sporozoan parasite of oysters, was
first described by Prytherch (1938). Sprague (1950) emended the
description. The known definitive hosts for N. ostrearum are three
species of xanthid crabs, Panopeus herbstii, Eurypanopeus depressus, and
Eurytium limosum, according to Prytherch (19^0) and Sprague (195CJ.
The most commonly recognized stages in crabs are mobile sporadins
or gregarines and spherical gametocysts which produce gymnospores.
Only vegetative spores or sporozites are found in oysters. These
spores occur most abundantly in mantle tissues, but also in adductor
muscle, heart, gills, labial palps, and perhaps other organs
(Prytherch 19^0, Sprague and Orr 1955). In this paper the peculiar
distribution of cysts in the mantle margin and evidence of discharge
of spores by oysters are investigated.

♦Contributions from the Virginia Fisheries Laboratory, No. 80. Part
of a thesis submitted in partial fulfillment for the degree of Master
of Arts, College of William and Mary, September, 1957-

**Author's current address is Department of Zoology, Rutgers University,
New Brunswick, N. J.

-162-

FUSION OF MANTLES
EDGE OF MANTLE
ED6E OF FLAT VALVE

T *~i

MILLIMETER RULE

1

EXCISED MANTLE

Fig. 1. To determine distribution of cysts, the edge of the
mantle was excised, measured, and subdivisions designated by letters.
In three oysters, cysts were counted in a continuous band on the
entire excised strip (Fig. 2) but in later groups carefully spaced
samples were taken. In subsequent discussions and figures, data are
arranged in sequence A to C (ventral mantle) and C to E (dorsal
mantle). The shaded area beneath the adductor muscle was selected
as a sampling site for other studies.

-163-

RESULTS

Distribution of Nematopsis ostrearum Cysts in Mantles of Oysters

Sprague discovered that in mantles of oysters, N. ostrearum
cysts occurred predominantly in a band two mm wide, near and parallel
to the margin. He also reported uniform distribution of cysts within
the band, but this conclusion presumably was based upon examination
of short sections of the mantle margin. In June and August, 1956,
the author counted cysts in the band along the entire mantle margins '
of three oysters. A strip of tissue five mm wide was excised from
the margin of the mantle, and lengths of ventral (A to C) and dorsal
(C to E) mantles were measured as shown in Figure 1. The counts,
from 305 to 393 microscopic fields of one mrn^ each, were grouped by
zones, each representing 10 per cent of the mantle margin (Figure 2).
Great variations in counts were noted but typically there were many
cysts opposite the adductor muscle and few cysts at the anterior and
posterior ends of the mantles.

The burden of counting the entire mantle margin in heavily-
infected oysters led to refinements of technique and the choice of
oysters from an area of relatively light infections for further study
of the pattern of distribution of cysts. On October k, 1956, 20
oysters were collected from Hoghouse Bar in the Rappahannock River
and opened by removing their cupped valves. In each oyster, incisions
were made in the mantle of the flat valve at B and D to mark the
position of the adductor muscle (Figure l); the strip of tissue was
excised, and the subdivisions were measured. Instead of counting
cysts in the whole margin, the strips were divided into 10 equal
sections. An 8 by 5 mm sample of tissue was taken from the center
of each section and treated with 10 per cent KOH solution for one
minute. The partially-digested tissue was placed between two slides
and compressed until it attained a length of 10 to 12 mm. The pre-
paration was examined at 100 X magnification under a microscope
equipped with an ocular Whipple cell. The intensity of infection in
each section was determined from the mean number of cysts per mm
calculated from the count in 10 fields. Each point in Figure 3
represents the mean value for 20 oysters.

Counts of 20 oysters confirmed the pattern observed in the
first three oysters (Figure 3)- The lowest numbers of cysts were
observed at the anterior (oral) ends of the dorsal and ventral
mantles. The number increased rapidly from the anterior ends of both
mantles towards the adductor muscle. The greatest concentration of
cysts occurred immediately above the adductor muscle in the dorsal
mantle and slightly posterior to it in the ventral mantle. A decrease
in the number of cysts was found posteriorly in the region of the
pallio -branchial fusion.

This general pattern is usually shown by individual oysters
including those with low counts but seems to be accentuated in those

-164-

70

60

50

40

MARGIN OF VENTRAL MANTLe

FUSION OF MANTLES

— - — MAROIN OF DORSAL MANTLE-

10 -

10 20 30 40 50 60 70 80 90 100

LENGTH OF THE MANTLE MARGIN IN PER CENT

Fig. 2. Variation in individual oysters in distribution of
Nematopsis ostrearum along margin of flat -valve mantle. Each line
represents counts made along the entire mantle margin of a single
oyster. Areas enclosed by the two sets of broken lines mark position
of adductor muscle.

■165-

FUSION OF MANTLES

~MMQM OF OORSAL MANTLE"

1

10 20 30 40 50 60 70 80 90 100

LENGTH OF THE MANTLE MARGIN IN PER CENT

Fig. 3- Mean distribution of Nematopsis ostrearum in margin of
flat-valve mantle of oysters, October 1956. The mean count of 20
oysters is plotted. The area enclosed by the two sets of broken
lines marks position of the adductor muscle.

-[(,(..-

with high counts. In any one oyster the highest concentration of
spores may occur in either the dorsal or ventral mantles.

Elimination or Disappearance of N. ostrearum Cysts
from Mantle Margins of Oysters

Controversial opinions concerning the nature of Nematopsis
infections in oysters have been found in the literature. Erytherch
(19^0), Lanadau and Galtsoff (1951), and Sprague and Orr (1955) note
that spores were more abundant in older oysters and, therefore,
believe that Nematopsis was cumulative in oysters. The occurrence
of spores in walled cysts probably encouraged this viewpoint. Owen
et al. (1951) were of the opinion that oysters eliminate spores be-
cause some old oysters comparatively free of spores were encountered.
Prytherch ( 19*4-0 ) estimated that the sporozoites suffered a better than
50 per cent mortality from phagocytosis before they reached the final
stage in oysters. Stauber (1950) noted, "It is possible that in
addition to the mortality mentioned above, there is an important
sporozite loss due not to intracellular digestion by phagocytes but
to emigration and elimination of the sporozoite-laden phagocytes"
(p. 239). In the present studies, field experiments were carried
out to determine whether oysters did retain spores once they were
encysted in the tissues.

Experiment I

On May 2, 1956, three stations, representing the full range
of salinities in the oyster -growing areas of the James River-Hampton
Roads system, were selected for holding oysters in trays. The lower
station was Darling's Watchhouse (J*4)* in the relatively high-salinity
(20 parts per thousand) waters of Hampton Roads; the intermediate
station was Miles' Watchhouse (<J12) with salinities of 16 ppt; and
the upper station at Deep Water Shoal (J2*+) had mean salinities of
about 7 ppt. Two groups of oysters were collected for the experiment
-- one from jU where infections of Nematopsis were moderately heavy
(21 cysts per mm2) and the other from J 2k where infections averaged
0.1 cysts per mm2. At each of the three stations, oysters from the
two sources were placed in separate compartments of a tray which was
set on the bottom. On September h, 1956, a sample of 25 oysters was
examined from each lot at the three stations.

After a period of four months, oysters with high initial in-
fections showed slight increases in number of cysts at Jk and J12,
and a significant decrease at J2M Figure k). Oysters with low initial
infections had a marked increase in number of cysts at the lower
stations, but no increase at J2*4.

*Jk indicates distance in nautical miles above the mouth of the
James River.

-167-

4"i

_ 3"

O
O

o

4-1

D W. M. W D.W.S.
OYSTERS WITH HIGH
INITIAL INFECTIONS

D. W. M. W. D.W.S.
OYSTERS WITH LOW
INITIAL INFECTIONS

Fig. h. Changes in level of Nematopsis infections when oysters
were moved to areas characterized by lower and higher intensities of
parasites. At each station (D.W., M.W., and D.W.S. representing
Darling's Watchhouse, Miles Watchhouse, and Deep Water Shoal Light-
house respectively), oysters with low and high initial infections
(represented by horizontal lines ) were placed in opposite ends of a
tray on the bottom. The levels of infections four months later
(September h) are denoted by vertical bars. N is the mean number of
cysts per mm .

■168-

At each station it appears that a characteristic level of
infection was reached in a relatively short time. If cysts do
accumulate in tissues, the numbers in oysters at jk and J12 should
have increased as much in heavily-infected individuals as in lightly-
infected ones. At J2^, oysters with heavy initial infections showed
a decline in number of cysts from 21 to 6 cysts per ram , and this
can be explained only by removal of cysts from the mantle. This
decrease occurred during the warm summer season when spores are
probably most abundant. Little is known of seasonal variations in
infections and there is a possibility that spores may be redistributed
in the oyster. However, this experiment strongly suggests that oysters
can destroy spores or remove them from their tissues. Unfortunately
the trays were lost after September and subsequent changes could not
be followed.

Experiment II

The hypothesis that oysters eliminate or discharge cysts of
Nematopsis is further supported by observations made on oysters in
suspended trays. On June 1, 1955 > oysters collected from Hoghouse
Bar in the Rappahannock River, an area of relatively low level of
infection, were placed in Trays 56, 57 and 59 suspended at Gloucester
Point in the York River. These oysters had been fully acclimated
during an 8- to 12-month period at Gloucester Point where both
salinities and level of Nematopsis infections in oysters on natural
grounds are high. On January 6, and June 5, I956, respectively,
Trays 56 and 59 were moved to the Fleet Pier near J24 in the James
River. In this low-salinity area the level of Nematopsis infections
in oysters is low. Tray 57 was retained at Gloucester Point and Tray
67 containing native Deep Water Shoal (J24) oysters was suspended at
the Fleet Pier in the James; these two trays served as control groups
for high- and low-incidence areas. On September 23 and October 27,
1956, samples of 25 oysters from each tray were examined and the
results are given in Table 1.

The mean number of cysts in oysters decreased significantly
in all trays with the exception of Tray 57 which was retained in a
high-intensity area (Table 1 *2 values). Since all trays were
suspended off the bottom, most mud crabs were kept out and this may
explain the relatively low intensity of Nematopsis infections at
Gloucester Point. It must be remembered also that the experimental
groups were moved to the low-salinity area several months before these
examinations were made. The experiment was designed for purposes
other than Nematopsis studies.

Level of Nematopsis Infections and Distribution of Mud Crabs

Surveys of James, York and Rappahannock Rivers revealed that
species of crabs known to be hosts for Nematopsis were scarce in low-
salinity (under 15 ppt ) waters. Low incidence of Nematopsis at J24

-169-

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can probably be attributed to paucity of mud crabs and low death rate
among oysters. The high-level of infection at jk may have been
accentuated by abundance of mud crabs around the watchhouse which had
unusually favorable habitat niches. Furthermore, the death rate of
oysters--from causes other than Nematopsis--is high at jk.

DISCUSSION

The non-random distribution of cysts in the oyster mantle
reveals that standard counting techniques and sampling methods are
essential; it also suggests that samples must be taken from various
parts of the mantle margin in order to estimate accurately the total
number of cysts in a particular oyster. For most purposes, however,
it should not be necessary to make counts from numerous parts of the
mantle, for by sampling carefully in a chosen site, comparisons between
oysters and locations can be made. The shaded area of the left ventral
mantle (Figure l) where infections approach a maximum, is suggested as
a sampling site.

The question arises: What are the causes of this peculiar
distribution? Hopkins (193^> 193&) first discovered the accessory
heart in oysters, observed much surging back and forth of blood in
the circumpallial arteries, and advanced the theory that the
circumpallial arteries received both arterial and venous blood.
Stauber (unpublished data, 1957) has clearly demonstrated the pattern
of blood circulation in the mantle margin by injection of green
vegetable dye into the ventricle, which supplies the anterior circum-
pallial network, and red dye into the accessory heart which feeds the
posterior circumpallial arteries at the fusion of left and right
mantles; the two colors were found to meet in dorsal and ventral
mantles in areas where the highest counts of cysts were observed.
This suggests that distribution of N. ostrearum cysts in the mantle
is probably associated with the pattern of circulation. It is possi-
ble that cysts tend to accumulate where blood flow is slowest. There
are indications, however, that localization of cysts can not be
explained, solely on the basis of mechanical transport; the special
affinity of cysts for gill tissue in N. prytherchi and for mantle
tissue in W. ostrearum suggests specific physiological requirements.

Seasonal patterns of infection, about which much more informa-
tion is needed, are now of great importance. For example, if oysters
are continually discharging spores which reinfect crabs, there is no
need for an oyster to die to complete the cycle, and the incidence
and intensities of infections may not be as closely related to the
pattern of mortalities of oysters as previously suspected. Further-
more, the whole cycle may be speeded up considerably. It is already
evident that Nematopsis infections are more common and the number of
spores produced by crabs is much greater than had been assumed under
the theory of accumulation of spores in the oysters. Stauber (1950,
p. 239) conjectured that, "unless the hypertrophy of the sporozoite-

■171-

infected phagocyte restricts amoeboid activity, it is possible that
elimination of developed spores in this fashion may constitute the
more normal route of infection for crab hosts". The finding of sub-
stantial Nematopsis infections in James River, where deaths of oysters
are few, tends to support this supposition. Viability of discharged
spores could be easily tested by placing starved mud crabs (free of
gregarines) in closed aquaria with live oysters heavily-infected with
Nematopsis. If crabs become infected, it would prove that some spores
are discharged; if not, the problem of disposition of spores would
remain unsettled.

The exact mechanisms by which oysters remove spores is still
a mystery; however, Stauber (1950) and Tripp (1957) have demonstrated
the rates and methods by which india ink and metabolizable vertebrate
red blood cells are digested or eliminated by phagocytes. Presumably
Nematopsis is eliminated by similar activities. Probably temperature
plays an important role in regulating the rate.

Many phases of the ecology of this parasite are still unsolved,
though it now appears that cysts, which have been reported to be non-
toxic, are even less harmful to oysters than was previously supposed--
as indicated by the ability to discharge them and thereby prevent
massive accumulations over long periods of time.

SUMMARY AID CONCLUSIONS

Distribution of Nematopsis ostrearum along mantle margins of
oysters was non -random. Lowest numbers of cysts were observed at the
anterior ends of dorsal and ventral mantles. Numbers increased
posteriorly toward the adductor muscle. The greatest concentration
of cysts occurred immediately above the adductor muscle in the dorsal
mantle and slightly posterior to it in the ventral mantle. A decrease
in number of cysts was found posteriorly in the region of pallio-
branchial fusion. Techniques for estimating number of cysts in the
mantle margin of oysters and the possible significance of this unusual
distribution of cysts are discussed. A standard sampling site on the
mantle is suggested for estimating levels of infection.

Evidence for the hypothesis that oysters eliminate or discharge
encysted spores of Nematopsis ostrearum has been presented. It is
possible that spores discharged from living oysters may infect crabs.
Seasonal patterns of infection, mechanisms of discharge of spores, and
toxicity of this parasite to oysters have not yet been adequately
studied.

-I72-

Literature Cited

Feng, Sung Yen. 1957- Ecological and epidemiological studies of
Nematopsis ostrearum, a sporozoan parasite of the oyster
Crassostrea virginica, in lower Chesapeake Bay and its tribu-
taries. Unpublished Thesis for M.A., College of William and
Mary, Williamsburg, Va.

Hopkins, A. E. 193^- Accessory heart in the oyster, Ostrea gigas.
Biol. Bull. 67: 3^6-335.

. 1936. Pulsating blood vessels in the oyster. Sci.

83: 581.

Landau, H. and Paul S. Galtsoff . 1951- Distribution of Nematopsis
infection on the oyster grounds of the Chesapeake Bay and in
other waters of the Atlantic and Gulf States. Texas Jour.
Sci. 1(3): 115-130.

Owen, H. M., L. L. Walters, and L. A. Bregan. 1951- Etiological

studies on oyster mortality I. Nematopsis ostrearum Prytherch,
19^0 (Sporozoa: Porosporidae). Jour. Mar. Res. 10: 82-90.

Prytherch, H. F. 1938.' Life cycle of a sporozoan parasite of the
oyster. Sci. 88: 451-^52.

. 19^0. The life cycle and morphology of Nematopsis

ostrearum sp. nov., a gregarine parasite of the mud crabs
and oysters. Jour. Morph. 66: 39-65.

Snedecor, George W. 1956. Statistical Methods. Fifth ed. The Iowa
State College Press. Ames, Iowa. 53^ PP-

Sprague, V. 1950* Studies on Nematopsis prytherchi Sprague and N.

ostrearum Prytherch, emended. Texas A & M Research Foundation.
59 PP-> [Mimeo • ) •

Sprague, V. and P. E. Orr, Jr. 1955- Nematopsis ostrearum and N.
prytherchi (Eugregarinina: Porosporidae) with reference to
the host -parasite relations. Jour. Parasit. kl: 89-104.

Stauber, Leslie A. 1950. The fate of india ink injected intracardially
into the oyster, Ostrea virginica Gmelin. Biol. Bull. 98:
227-241.

. 1957* Unpublished data on the circulatory system of

the oyster. Dept. of Zoology, Rutgers Univ.

Tripp, M. R. 1957- Disposal by oysters of intracardially injected

red blood cells of vertebrates. Proc. Natl. Shellfish. Assoc.
48: 1^3 -147.

-173-

INCIDENCE AND LIFE HISTORY OF PARORCHIS ACANTHUS, A DIGENETIC
TREMATODE, IN THE SOUTHERN OYSTER DRILL, THAIS HAEMASTOMA

Nelson R. Cooley

Gulf Oyster Investigations, Bureau of Commercial Fisheries
U. S. Fish and Wildlife Service, Gulf Breeze, Florida

Abstract

This paper is the initial report of a program
to develop biological control of the Southern Oyster
Drill, Thais haema stoma, cause of considerable economic
loss in some areas on the Gulf of Mexico.

Information on the life cycle, known hosts,
reported localities, synonymy, and morphological
descriptions of developmental stages of the digenetic
treraatode Parorchis acanthus has been compiled from
various sources.

The pathology of Parorchis infection in T.
haemastoma is briefly described and figured.

Preliminary data indicate that incidence of
natural infection of P. acanthus in T. haemastoma is
about 2% in Northwest Florida, 3«M& in Mississippi
and less than 1$ in Texas. All natural infections
were massive, parasite larvae constituting 70 to 90$
of the liver.

Parorchis infections experimentally induced in
herring gulls (Larus argentatus), in ring -billed gulls
(L. delawarensis ), and in oyster drills are reported.
Sexually mature Parorchis adults were first recovered
from the cloacas of four out of four gulls 30 days
after feeding each gull approximately 200 encysted
metacercariae suspended in sea water. In oyster drills
continuously exposed to droppings of Parorchis-infected
gulls, the first infection was found on the 21st day of
exposure and on the 33d day of exposure, four of 33
examined were infected. The elapsed time and the
recovery of only first generation rediae indicate
that these were true experimental infections.

Possible use of P. acanthus as a biological
control agent for T. haemastoma is discussed.

-174-

Introduction

Although very few data have been published, the Southern
Oyster Drill, Thais haemastoma, is probably responsible for extensive
economic loss to the Gulf oyster fishery. In an attempt to develop a
method of biological control, the natural enemies of this snail are
being investigated. Scattered observations on the parasites of Thais
have been reported in the literature. Few of the papers containing
these observations were concerned directly with the parasites.
Schechter (19^3) reported the emergence of Parorchis acanthus
cercariae from occasional Thais from Barataria Bay, Louisiana. Butler
(1953) noted that at least two trematodes parasitizing Thais in
Louisiana and in the Pensacola, Florida, area may cause considerable
damage to the gonad. During a study of the reproductive cycle of
Thais, Butler (1956) found some drills heavily infected with larvae of
parasitic worms which damaged the gonad so badly as to render it unfit
for cytological study. The latter two observations suggested the
possibility of biological control of Thais populations. Accordingly,
in the summer of 1956, a survey of the parasites of this drill was
begun in hope of finding a parasite which could be used for this
purpose.

In September, 1956, heavy infections of a larval digenetic
trematode, later identified as Parorchis acanthus Nicoll, 1907> were
found in the livers of drills from Gulfport, Mississippi, and a study
of this worm was begun. Since then, the parasite has been found in
a number of drills from Biloxi, Mississippi, from Pensacola, Florida,
and from Galveston, Texas. This paper is the first report of these
investigations .

Life Cycle of Parorchis Acanthus

The life cycle, based on the descriptions of Stunkard and
Cable (1932) and Rees (19^0), is shown in Figure 1. The sexually
mature worm, living in the cloaca and bursa Fabricii of the herring
gull, releases eggs, each of which contains a completely developed
miracidium. The miracidium contains a single well-developed redia.
The egg hatches immediately after release into the cloaca of the
gull, and the miracidium is discharged in the feces of the gull into
the water. If the free-swimming miracidium does not enter a snail
host, it dies in less than a day. Upon finding a snail, the miracidium
apparently enters the aperture of the shell, passes up between the
shell and body of the animal, penetrates the tunica propria of the
liver at the spire and disintegrates, releasing redia (Redia i). The
germ balls of Redia I give rise to a second generation of rediae
(Rediae II ) which penetrate deeper into the liver, ultimately lodging
in the connective tissue between the hepatic tubules. In massive
infections, Rediae II also invade the gonad and, according to Stunkard
and Shaw (1931)> may cause parasitic castration in Urosalpinx cinerea.
The germ balls of Rediae II develop into cercariae only, no additional

■175-

GULL

ENCYSTED
METACERCARIA

(CLOACA a BURSA FABRICII)

EGGS CONTAINING

MIRACIOIUM 8

ITS INCLUDED

REDIA

SEA WATER

FREE-SWIMMING
MIRACIDIUM

Fig. 1. Life cycle of Parorchis acanthus (based upon Stunkard
and Cable 1932, Rees 19AO).

-176-

generations of rediae being produced. The mature cercaria, released
through the birth pore of Redia II, leaves the snail, swims about
briefly in the water, then settles down on any available submerged
object and rapidly secretes a double-walled cyst membrane about it-
self while detaching its tail. The larva is now an encysted meta-
cercaria. The metacercaria, when attached to food, is ingested by
the gull, passes through the digestive tract and excysts in the lower
intestine. The juvenile worm attaches itself, by means of a powerful
ventral sucker, to the wall of the cloaca, bursa Fabricii and, less
commonly, the lowermost part of the intestine and develops into a
sexually mature adult .

A list of hosts of P. acanthus, sites of infection in drills,
localities from which infections were reported and authorities are
given in Table 1.

Morphology of Parorchis Acanthus

The morphology of the different developmental stages had been
described by a number of authors (Table 2) under the names of
Zeugorchis acanthus, Parorchis acanthus, P. a Vitus, Cercaria sensifera,
and C. pur pur ae . As far as I can determine from the literature, all
are generally regarded to be synonyms of P. acanthus. I have seen all
developmental stages, and although there are minor differences, my
material is in general agreement with the published descriptions. A
complete description of my material will be published elsewhere at a
later date .

Effect of Larval Stages of Parorchis Upon Thais

Invasion of the snail host by any sizeable number of miracidia
results in production of a very large number of second generation
rediae (Figure l). In all natural infections examined by dissection,
rediae were easily seen with the naked eye through the transparent
tunica propria covering the liver. In cross sections of unfixed
livers they appeared as enormous numbers of tiny white wormlike objects
interspersed among irregularly distributed small masses of liver tissue,
All natural infections were rated massive, since 70 to 90$ of the liver
had been replaced by rediae. Any slight tear in the tunica propria
permitted the escape of a flood of rediae and cercariae into the
surrounding medium.

The gross appearance of cross sections of livers of infected
and uninfected snails may be compared in Figure 2. The liver of the
uninfected drill is variable in color, usually being light gray or
beige, but may be very dark, almost black. Its consistency is like
that of soft cheese. The surface is covered by a fairly tough tunica
propria. The swollen liver of the infected drill is less variable in
color, usually being lighter gray or light yellow. Its consistency
is softer and its tunica propria more easily torn.

-177-

Table 1. Known hosts of Parorchis acanthus

SITE OF

HOST

INFECTION

LOCALITY

AUTHORITY

ADULT STAGE

Gulls

Herring

Cloaca, bursa

Scotland

Nicoll 1906

Fabricii

Mass

Linton 1914, Cable 1937

Wales

Rees 1939

Florida

Unpublished

Ring -billed

Cloaca

Florida

Unpublished

Western

Calif

Penner 1957> pers. com.

Common

Rectum

Scotland

Nicoll 1907

Terns

Common

Cloaca

Mass

Stunkard & Cable 1932

Roseate

Cloaca

Mass

Stunkard & Cable 1932

Shore birds

Willett

Florida

Penner 1957? pers. com.

Dowitcher

Florida

Penner 1957> pers. com.

Water turkey

Florida

Penner 1957> pers. com.

LARVAL STAGE

Snails

Urosalpinx cinerea

Liver, gonad

Mass

Stunkard & Shaw I93I, 19.

Thais lapillus-'-

Liver, gonad

Wales

Rees 1939

Thais lapillus

Liver

Scotland

Lebour 191^, Lebour &
Elmhirst 1922

Wales

Rees 1937

Thais lapillus

Liver, gonad

Mass

Stunkard & Cable 1932

Thais haemastoma

Liver, gonad

Miss

Florida

Texas

Unpublished

Louisiana

Schechter 19^3

Littorina planaxis

Calif

Penner 1957> pers. comra.

Melogena sp.

Liver

Florida

Penner 1957> pers. comm.

Batillaria sp.

Florida

Penner 1957* pers. comm.

Cerithium sp.

Florida

Penner 1957* pers. comm.

XSee Clench (19^7: 86-87) for synonymy.

-178-

Table 2. Developmental stages, synonymy, and sources of morphological
descriptions of Parorchis acanthus.

Developmental stage

Name given

Author i

ty

Adult

Zeugorchis acanthus

Nicoll 1906

Parorchis acanthus

Nicoll 1907

Parorchis avitus

Linton 191U,

1928

P.

avitus (immature)

Stunkard & Cable 1932

Egg

Z.

acanthus

Nicoll 1906

P.

acanthus

Nicoll 1907

P.

avitus

Linton 1914

Miracidium

Z.

acanthus

Nicoll 1906

P.

acanthus

Nicoll 1907,

Rees 19*4-0

P.

avitus

Linton 1914

Redia

Cercaria purpurae

Lebour 19l4,

Rees 1937

C.

sensifera

Stunkard & Shaw 1931

P.

acanthus

Rees 19^0

Cercaria

C.

purpurae

Lebour 1907,
Rees 1937

1914

C.

sensifera

Stunkard & S

haw 1931

P.

acanthus

Lebour 19 lk,
19^0

Rees 1937 &

Metacercaria

C.

purpurae

Rees 1937

c.

sensifera

Stunkard & Shaw 1931

p.

avitus

Stunkard & Cable 1932

p.

acanthus

Lebour & Elmhirst 1922

-179-

Fig. 2. A. Liver of uninfected drill, transverse section,
X 7«5» Note homogeneous appearance of normal tissue. B. Liver
of drill with massive Parorchis infection, transverse section X 7«5-
Whitish objects occupying most of liver are parasite larvae, gray
patches are remaining liver tissue.

-180-

A comparison of stained cross sections of the livers of
infected and uninfected drills (Figure 3) reveals the extensive
displacement of the liver tissue produced by developing rediae. The
histopathology of liver infections is characterized by:

(1) extensive displacement and reduction of liver tissue to
10 to 30% of the surface area of the cross section;

(2) compression of liver tubules resulting in obliteration
of most if not all of their lumina;

(3) presence of basophilic inclusion granules of uncertain
significance in the cytoplasm of the large triangular
cells of the hepatic tubules, revealed by hematoxylin-
azure B-eosin staining;

(h) presence of certain cells, thought to be phagocytic blood
cells, in groups next to some of the parasite larvae; and

(5) apparent absence of direct destruction of liver tubules
by invading parasites.

Invasion of the gonad was observed in a few specimens. The
gonad of the uninfected drill is cream-colored or dark beige to brown,
its size and thickness varying according to seasonal changes in the
reproductive cycle; that of infected animals has been thin or patchy
and dark yellow or orange in color. Whether the differences observed
are an effect of infection or constitute normal seasonal changes is
uncertain at this time.

Some Results of Experimentally-Induced
Infections with Parorchis Acanthus

Gulls :

One herring gull (Larus argentatus ) and three ring-billed
gulls (L. delawarensis), known by repeated examinations over a one-
month period to be Parorchis -free, were used in this experiment.
Large numbers of cercariae obtained by dissection of naturally in-
fected drills were permitted to encyst on the bottom of a Petri dish
of sea water. The encysted raetacercariae were scraped free with a
scalpel, suspended in sea water, and several hundred pipetted into
the esophagus of each gull before feeding the daily meal of chopped
fish (usually mullet). This treatment was repeated a week later in
order to insure infection of all birds. The fish, obtained locally,
were quick-frozen, cut up into small pieces, and stored at 0° F for
several weeks to prevent possible infection of the gulls with fish
parasites. The fish were thawed just before feeding time. At
intervals of seven to ten days, the cloaca of each bird was aspirated
carefully with a medicine dropper pipette and the aspirate examined
for worms.

-181-

Fig. 3. Effect of Parorchis infection upon Thais liver.
Bouin, Weigert's iron acid hematoxylin and eosin, transverse
section, T u, ca. X 50. A. Uninfected liver. B. Infected
liver. Note extensive displacement and compression of liver
tissue by parasites, occluded lumina of liver tubules.

-182-

No worms were found until the 30th day after first feeding
encysted raetacercariae, when sexually mature worms were recovered from,
each of the birds. For three months after being fed encysted
raetacercariae, none of the birds exhibited any symptoms of illness.
However, three months and eight days after being fed cysts, the herring
gull died suddenly. Three days later, one of the ring -billed gulls
died, apparently in the same manner. Post-mortem examination failed
to reveal the cause of death in either case, but mature Parorchis
adults were recovered from the cloaca of each bird. The other two
infected ring-billed gulls have shown no symptoms of illness during
the slightly more than four-months period of their infections, although
they are now (July) somewhat less greedy at feeding time than they
were when captured last January.

Drills:

This experiment was designed to determine whether (l) Parorchis
infections could be induced in large numbers of Thais in outdoor tanks
and (2) whether there is any relationship between the size of the drill
and its susceptibility to infection.

The experiment was performed in two 10 by 20 foot concrete
tanks. Running sea water, at a constant depth of about h inches
(roughly 2 cubic meters of water), was supplied to each tank at the
rate of approximately 2 gallons per minute, permitting a complete
change of water in about h\ hours.

Into each tank a lot of 653 snails was introduced from the
rocky shore of our laboratory island in Santa Rosa Sound. Each lot
consisted of 408 small drills (15 to 35 mm long) and 2^5 large drills
(^5 to 60 ram long). These size ranges were chosen arbitrarily to
facilitate recognition of each size class. During the 33 days of the
experiment, no food was supplied to the drills.

Three ring-billed gulls and one herring gull, known to be
infected with sexually mature Parorchis adults and discharging
miracidia in their feces, were hung in hardware cloth cages from the
walls of the experimental tank; a single ring-billed gull, known to
be free of Parorchis infection, was placed similarly in the control
tank. The droppings of the gulls fell freely and directly into the
water in each tank. The birds were fed daily either frozen small
pinfish (Lagodon rhomboides) or frozen chopped mullet (Mugil cephalus).

Repeated sampling of the drill population about our laboratory
island over a period of several months has never revealed infected
snails, suggesting that incidence of Parorchis infection in the popu-
lation is very low, if not absent. The incidence in nearby Pensacola
Bay, on the other hand, is slightly above 2$. Thus, it was decided
that a sample of 5% of the total number of drills in each group
(roughly 2.5 times the natural incidence in the bay) should be adequate

-183-

to reveal any induced infections. Accordingly, 33 drills (about
equal numbers of large and small individuals) were removed from each
group initially and on the 13th, 21st, and 33**d days of exposure to
gull droppings. All snails without visible infections when the liver
was examined under a dissecting microscope were fixed in Bouin's
fixative (Allen's modification) and stained sections were examined
with a compound microscope to detect light infections.

No infections were observed in the samples prior to the 21st
day, when one small infected snail was found. On the 33r(3 day, 2 large
and 2 small drills (about 12$ of the sample) proved to be infected.
All infections were light and stained sections contained only first
generation rediae, indicating that these were bona fide experimental
infections. Although the time required for the development of each
larval stage is unknown, these observations indicate that an extended
period is required.

It is difficult to explain satisfactorily the low incidence
of infection in this experiment in which it appeared certain the
experimental snails would be exposed to a high concentration of
miracidia. One might speculate that these results were due to (a)
resistant snails (see Hyman 1951> PP- 307-308), or death of most of
the miracidia before they could penetrate snails (See Linton 1914,
Rees 19^0, pp. 379-381), or a combination of these causes. However,
this experiment must be repeated under various conditions before a
precise explanation can be given.

On the basis of this study we may conclude that (l) Parorchis
infections were experimentally induced in Thais; (2) For some un-
explained reason the rate of infection was low; and (3) There seemed
to be no important difference in the susceptibility of large and small
drills to infection with Parorchis.

Incidence of Natural Infections of Parorchis Acanthus in Thais

Although the survey of the natural incidence of infection in
drills from oyster-producing areas of the Gulf Coast region begun in
the fall of 1956 has not been completed, some preliminary data from
2050 drills can be reported at this time (Table 3)- These drills
were collected in Galveston Bay, Texas, from Mississippi Sound, at
Biloxi and Gulf port, Mississippi, and in Pensacola and Apalachicola
Bays, Florida. Only massive infections detected by dissection are
reported.

During repeated sampling of the drill population of the
Pensacola Bay area, some samples of a hundred or more drills contained
no infected snails, while in other samples of comparable size from
different locations up to 7$ were infected. Samples from one point
on the north shore of the bay, where large numbers of herring and
ring-billed gulls roosted during the fall and winter, almost invariably

-184-

Table 3. Incidence of Parorchis acanthus infections in Thais from
three oyster -producing areas.

Area

Snails

Number
examined

Infected

Number

Per cent

Northwest Florida

Mississippi

Texas

1123
^52

23

16

3

2.05

3-37
0.66

Totals

2050

k2

2.05

-185-

contained infected drills, while another area on the south shore
where few gulls were noted, sampled on four occasions from November
1956, to February 1957* yielded only one infected snail out of 196
examined.

Of several hundred drills collected from the rocky shore of
our laboratory island in Santa Rosa Sound, none was infected. Only
a comparatively few gulls frequented the laboratory area during the
past fall and winter.

a

The large gull populations which build up during the fall
and winter, although important, are not the only source of Parorchis
infection in the Pensacola area, for willets, dowitchers, and water
turkeys, reported by Penner (1957) to harbor the adult worm elsewhere
in Florida, also occur here to some extent, and could constitute an
additional source of infection.

Possible Use of P. Acanthus in
Biological Control of the Oyster Drill

The serious drill predation existing in certain areas of
the Gulf oyster fishery requires development and institution of
effective drill-control measures.

Although the natural incidence of Parorchis infections in
Thais is low, it is evident that it causes considerable damage to
the individual drill. This experimental work demonstrates that Thais
can be experimentally infected with P. acanthus. The percentage
experimentally infected was low, but still higher than natural inci-
dence. Further study to determine how the rate of infection may be
increased to the extent necessary to utilize this parasite as a
biological control agent is warranted by this initial success.

Acknowledgments

The personnel of the United States Fish and Wildlife Service
Shellfishery Laboratory, Gulf Breeze, Florida, furnished varied
assistance: field collections of drills were made; Figure 1 was
drawn by Charles R. Chapman and photographed by Jack I. Lowe; and
Dr. P. A. Butler and Charles R. Chapman contributed certain unpub-
lished data. Other field collections of drills were made by Dr.
Abraham Fleminger, United States Fish and Wildlife Service, Gulf
Fishery Investigations, Galveston, Texas, and William Demoran, Gulf
Coast Research Laboratory, Ocean Springs, Mississippi. Dr. Lawrence
R. Penner, Zoology and Entomology Department, University of Connecti-
cut, kindly permitted the use of unpublished data. The aid of these
individuals is gratefully acknowledged.

-186-

Literature Cited

Butler, P. A. 1953- The Southern oyster drill. Proc. Natl. Shell-
fish. Assoc, hh: 67-75.

Butler, P. A. 1956. Unpublished data on the reproductive cycle of
Thais haemastoroa.

Cable, R. M. 1937- The resistance of the herring gull, Larus

argentatus, to experimental infections of the trematode,
Parorchis acanthus. Jour. Parasitol. 23(6): 559-

Clench, W. J. 19^7- The genera Purpura and Thais in the Western
Atlantic. Johnsonia 2(23): 61-91.

Hyman, L. H. 1951. The Invertebrates... Vol. II. 1st ed.; 2d
imp. vii + 55° PP- N. Y., McGraw-Hill Book Co., Inc.

Lebour, M. V. 1907- Larval trematodes of the Northumberland

Coast. Trans. Nat. Hist. Soc. Newcastle, N. S., 1: ^37-
45J+; 500-501.

Lebour, M. V. 191^- Some larval trematodes from Millport.
Parasitology 7(1 ): 1-11.

Lebour, M. V., and R. Eimhirst. 1922. A contribution towards the
life -hi story of Parorchis acanthus Nicoll, a trematode in
the herring gull. Jour. Marine Biol. Assoc. United Kingdom,
N. S., 12: 829-832.

Linton, E. 191^- Notes on a viviparous distome. Proc. Natl. Mus.
^6: 551-555.

Linton, E. 1928. Notes on trematode parasites of birds. Proc. U. S.
Natl. Mus. 73(1): 1-36, pis. 1-11.

Nicoll, W. 1906. Some new and little known trematodes. Annals and
Mag. Nat. Hist., 7th Ser., 17(102): 517-527-

Nicoll, W. 1907. Parorchis acanthus, the type of a new genus of

trematodes. Quart. Jour. Micros. Sci. 151: 3^5-355* PI- 21.

Penner, L. R. 1957. Personal communication: letters dated 8 April
and 11 May 1957-

Rees, G. 1937- The anatomy and encystment of Cercaria pur pur ae

Lebour, 1911. Proc. Zool. Soc. London 107, Ser. B: fe'5-73*
Pis. I -II.

•187-

Rees, G. 1939- Studies on the germ cell cycle of the digenetic
trematode Parorchis acanthus Nicoll. Part I. Anatomy of
the genitalia and gametogenesis in the adult. Parasitology
31(4): 417-^33.

Rees, G. 19^0. Studies on the germ cell cycle of the digenetic
trematode Parorchis acanthus Nicoll. Part II. Structure
of the miracidium and germinal development in the larval
stages. Parasitology 32: 372-391.

Shechter, V. 19^3 • Two flatworms from the oyster -drilling snail,
Thais floridana haysae Clench. J. Parasitol. 29(5): 362.

Stunkard, H. W., and R. M. Cable. 1932. The life history of
Parorchis avitus (Linton), a trematode from the cloaca
of the gull": Biol. Bull. 62(3): 328-338.

Stunkard, H. W., and C R. Shaw. 1931. The effect of dilution

of sea water on the activity and longevity of certain marine
cercariae, with descriptions of two new species. Biol. Bull,
6l(2): 2U2-271.

-188-

THE CROWN CONCH MELONGENA CORONA GMELIN;
ITS HABITS, SEX RATIOS, AND POSSIBLE
RELATIONS TO THE OYSTER. 1

Ralph R. Hathaway

Oceanographic Institute
Florida State University

Abstract

Studies on the crown conch in northwest Florida
from St. Marks to Panama City indicate large populations
living on soft, protected intertidal bottoms, often
associated with oysters, and usually displaying sex
ratios in favor of females. Animals in natural habitats,
tagged and observed for up to 193 days, confined their
movements to small areas. Tagged animals removed up to
150 m from their habitat quickly moved back into it.
Attempts to observe captive crown conchs feeding on
oysters were unsuccessful, although this was often seen
among free animals on oyster reefs. Sample populations
from oyster reefs were made up of large, predominantly
female crown conchs. Adjacent soft bottoms and high
intertidal areas of protected beaches yielded smaller
animals and greater numbers of males. The ability of
an intertidal oyster to hold its valves closed upon
the proboscis of a crown conch for hours during low
tide is cited an an argument against the idea that
Melongena kill their prey by secreting a toxin from
the proboscis. It is concluded that crown conchs
are not serious predators of oysters.

Introduction

In recent years biologists and fishermen alike have become
more aware of the importance of fundamental biological information
in solving problems faced by commercial fisheries. This is a well
recognized fact in the oyster industry, as is shown by its interest
in the National Shellf isheries Association. Here, and in many other
otner places, the biological and physical milieu of the oyster
Crassostrea virginica Gmelin is explored in great detail. Animals
associated with oysters account for a large part of this effort, and

"'"Contribution No. 86 Oceanographic Institute, Florida State Univer-
sity. This work was supported by contract 14-19-008-2307, U. S.
Fish and Wildlife Service. The data are taken from a thesis in
partial fulfillment of the requirements for a Master of Science
degree, Florida State University, under the direction of Dr. R. W.
Menzel.

-189-

of these, other mollusks are a prominent element. Gastropods known
to prey on Crassostrea are subjects of active research, while other
snails, whose relation to oysters is uncertain, remain to be investi-
gated. The crown conch, Melongena corona Gmelin, is one of this latter
group .

Previous studies on the crown conch are reported b;> Clench and
Turner (1956). This report indicates there is no planktonic larval
form, that the snails feed on a variety of mollusks and detrital mate-
rial, and that there is a remarkable extension of the proboscis in
the act of feeding. These authors define two subspecies, M. corona
corona and M. corona johnstonei, blending in a "cline" in the region
of Apalachee Bay, Wakulla County, Florida.

The present study was undertaken on the crown conch because
of its abundance and frequent association with oysters on the west
coast of Florida. Sex ratios, habitats, migrations, and possible
relations to the commercial oyster were examined.

Methods and Areas Studied

Methods consisted of field observations of undisturbed animals,
sex and length measurements, and techniques devised to study feeding
and migration. These animals are sexed easily and accurately upon
extraction from their shells after immersion in boiling water for five
minutes. It has been necessary to define four sex classes: definitive
male, definitive female, presumptive female, and male-with-small-penis.
A large penis or capsule gland characterizes the definitive male and
female classes, respectively. Absence of any kind of penis or capsule
gland defines presumptive females, and male-with-small-penis is defined
as it is named. These sex classes cannot be differentiated by any
live-sexing technique. Hargis (1957) emphasizes the advisability of
finding either a penis or a capsule gland in order to determined posi-
tively sex in Urosalpinx cinerea. Similar emphasis must be given to
sexing in Melongena. Length was measured to the nearest mm from the
apex of the spine to the end of the siphonal canal. Crown conchs
were kept in laboratory aquaria and in wire cages in natural habitats
with the intention of getting numerical information on oyster predation.
Migration was checked by marking or tagging several dozen animals in
the field and attempting to follow their movements by repreated trips
to the habitats. The subspecific differences noted by Clench and
Turner (1956) were not studied and both groups are treated together in
this paper.

Areas covered by this study consist of the Northwest coast of
Florida from Apalachee Bay to St. Andrew Bay. Specific areas were:
(l) St. Marks estuary; (2) Alligator Harbor; (3) Indiana Lagoon in
Apalachicola Bay; (h) St. Joseph Sound; and (5) St. Andrew Bay near
Panama City, Florida. Apalachee Bay has a poorly defined shoreline
consisting of great salt marshes and expanses of grass flats of the

-190-

species Syringodiuro f ilifonne, Diplanthera wrighti, Halophila
engelmanni, and Thallassia testudinum. The bay is shallow, and
influx of fresh water from numerous rivers, especially the St. Marks
and the Ochlockonee, give much of it an estuarine character. The
sounds to the west, including Alligator Harbor and the complex called
Apalachicola Bay, are shallow and muddy, and protected by offshore
barrier beaches. The oyster industry of Apalachicola Bay is centered
where fresh water discharge from the Apalachicola River maintains
lower salinities favored by Crassostrea virginica. West of Cape San
Bias, where the harbors of Port St. Joe, Panama City, and Pensacola
open to deep oceanic waters a few miles offshore, influence of fresh
water is less obvious. Oyster beds occur only in the upper reaches
of these bays.

Results

Results may be considered under three headings: habitats,
populations, and relations to oysters. Habitats along the varied
coastline from Apalachee Bay to Panama City abound with Melongena.
They occur on intertidal bottoms that are protected and soft, and in
a high salinity range. Very young animals occur in high intertidal
zones, especially in salt marshes. Older animals are found on inter-
tidal oyster reefs and nearby subtidal flats, as well as in areas
where there are no oysters. Nevertheless, crown conchs are always
found in or near an intertidal zone. Those on oyster reefs will bury
in winter, or if the reef is too hard for this, will move to adjacent
soft bottoms-

Populations have been analyzed in regard to sex ratios, length
distribution, and migration.

Sex ratios vary significantly from unity. Those found in
this study are tabulated below. Samples from the reef in Indian
Lagoon indicate more females than males in a population that lacks
small animals. Snails from Indian Lagoon bottom are mainly small
male animals. Snails from St. Marks were collected both on and off
oyster reefs. These samples range in size from very small to large,
and display variable sex ratios. Crown conchs from St. Andrew Bay
and St. Joseph Bay were not associated with oysters. These samples
were of great size range and variable sex ratios.

Migration studies indicate fast and direct movements by
Melongena towards its original position after it has been removed
up to 150 meters from its habitat. Undisturbed crown conchs remain
within small areas of their preferred habitat for many months. These
results are based on observations of 329 animals in four locations for
up to 193 days. Evidence for large scale migratory activity was not
seen, however, absence of small individuals suggests that populations
on oyster reefs are immigrants. Crown conchs associated with oysters
appear to be older, predominantly female animals that have moved in
from other areas.

-191-

Table 1. Sex ratio of Melongena corona.

Place and time of

No. of females

Size of

Average Length

Sampling

per 100 snails

Sample

mm

Indian Lagoon Reef

11 Sept. 1956

69

79

117

17 Nov. 1956

62

133

117

30 Jan. 1957

60

137

114

Indian Lagoon Bottom

30 Jan. 1957

28

46

77

St . Marks

9 Sept. 1956

64

146

71

23 Sept. 1956

50

64

54

17 Nov. I956

64

36

57

16 Jan. I957

552

29

79

14 Feb. 1957

43

67

75

Ik Feb. 1957

582

29

55

30 Mar. I957

482

144

74

St. Andrew Bay

13 June 1957

39

96

84

18 June 1957

57

90

81

St. Joseph Bay

19 June 1957

62

144

65

Include definitive female and presumptive female sex classes.

o
Not significantly different from 50 by Student's t test.

-li>.'-

Relations to oysters are obscured by conflicting observations.
It is common to find a Melongena feeding on an oyster -whose valves
are firmly closed upon the snail's proboscis. Feeding of crown conchs
on oysters has been studied in basket experiments and in aquaria. In
none of these experiments was a crown conch seen to successfully attack
and consume an oyster, although an unsuccessful attack was observed.
This was seen among six Melongena kept in aquaria for 2 months. It
occurred in the manner described by Gunter and Menzel (1957)> the snail
making a prolonged investigation of the oyster, and finally ejecting
its proboscis in an attempt, presumably, to insert it between the
oyster's valves. This mode of attack appears to be clumsy when direct-
ed towards oysters since the opening between the valves is relatively
small in this species.

Crown conchs confined in wire baskets did not increase the
mortality of oysters kept in the same baskets. Six Melongena were
observed under these conditions for up to 38 days. Snails in captivity
were seen to continue such activities as mating and laying, but it is
difficult to know if this behavior is representative of that which
occurs in natural habitats. Feeding activity may be especially sensi-
tive to unnatural conditions. A snail with the carnivorous habits of
M. corona would be expected to be attracted to an oyster reef and
there to make numerous attacks on oysters. If only a small percentage
of attacks are successful, the limited number of observations on cap-
tive conchs might not include any successful attacks, while one might
be observed occasionally among the much larger number of snails on a
reef. Any reduction in feeding activity due to captivity would make
the observation of successful attacks by captive animals even more
unlikely. Also, there is the consideration that high summer mortality
among Florida oysters may induce crown conchs to feed on dead and
weakened animals, producing an exaggerated picture of Melongena preda-
tion on healthy oysters.

On an intertidal oyster reef exposed at low tide the sight of
an oyster tightly closed upon the proboscis of a crown conch raises
questions regarding the manner in which these snails kill their prey.
If a toxin were being released from the proboscis the oyster would
not be expected to remain closed long after the proboscis was in-
serted. It would appear more likely that Melongena kills oysters by
mechanical action of the proboscis, since it is possible to find
oysters that have held tightly to the proboscis for many hours, or
at least since the receding tide exposed them. Upon exposure to air
the conch attacking an oyster reduces its body activity, including
that of the proboscis. Predator and prey then maintain a status quo
until the tide comes in and covers them, at which time action of the
proboscis begins again.

-193-

Conclusions

M. corona are abundant along the miles of shoreline in Worth-
west Florida where soft, protected, and intertidal bottoms provide
ideal habitats for this animal. They usually remain within a small
area throughout the year, and probably throughout their lives. Small
individuals are not found on oyster reefs, indicating that Melongena
populations associated with oysters are immigrants. Sex ratios vary
from unity, most often in favor of females. This is most definite
among crown conchs on oyster reefs. In addition, Melongena on oyster
reefs are larger than those found elsewhere. Predation by crown
conchs on oysters occurs under natural conditions, but experiments
indicate Melongena is not a serious predator on Crassostrea.

Literature Cited

Clench, W. J., and Ruth D. Turner. 1956. The family Melongenidae
in the Western Atlantic. Johnsonia 3. (35): 161-188.

Gunter, G., and R. W. Menzel. 1957» The crown conch, Melongena
corona, as a predator upon the Virginia oyster. Nautilus
7: 84-87-

Hargis, W. J., Jr. 1957- A rapid live-sexing technique for Urosal-
pinx cinerea and Eupleura caudata, with notes on previous
methods. Limnol. and Oceanogr. 2: 4l-42.

-194-

ASSOCIATION AFFAIRS

ANNUAL CONVENTION

The 1957 Convention of the National Shellf isheries Association
and the Oyster Growers and Dealers Association of North America was
held July 21 to 25 at the BeLmont Plaza Hotel, New York City. Through
the courtesy of Frank M. Flower and Sons of Bayville, Long Island,
oystermen and scientists were able to see a suction dredge and other
equipment in operation. A new feature of this convention was the
reports of laboratory directors on research in progress and plans
for the future. This half -day session was sponsored by the Research
Committee which was composed of industry and scientific members.
Presentation of papers and attendance by oyster planters and biologists
from the West Coast were encouraging signs of progress.

OFFICERS OF THE NATIONAL SHELLFISHERIES ASSOCIATION FOR 1^57-1958

President- -Melbourne R. Carriker, Department of Zoology, University
of North Carolina, Chapel Hill.

Vice-President--L. Eugene Cronin, Maryland Department of Research and
Education, Solomons, Maryland.

Secretary-Treasurer — Philip A. Butler, U. S. Fish and Wildlife Service
Shellf ishery Laboratory, Gulf Breeze, Florida.

Executive Committee--M. R. Carriker, Chairman, L. E. Cronin, P. A.
Butler, G. F. Beaven, H. C. Davis, and G. R. Lunz .

APPOINTED COMMITTEES FOR 1957-1958

Joint Program Comraittee--L. E. Cronin, Chairman, D. H. Wallace, J. B.
Glude, J. R. Nelson, G. R. Lunz, P. A. Butler, D. B. Quayle,
and J. D- Andrews.

Editorial Committee-- J. D. Andrews, Editor (two years); T. C. Nelson,
Associate Editor (one year); S. H. Hopkins, Associate Editor
(three years).

NSA Brochure Committee--G. F. Beaven, Chairman, A. F. Chestnut, and
J. B. Engle.

Joint 1958 Convention Committee-- J. N. McConnell and T. C. Nelson.

-195-

A BRIEF HISTORY OF THE NATIONAL SHELLFISHERIES ASSOCIATION

During the early years of the present century, a need was
felt for a national organization wherein those state and federal
officials charged with conserving and administering the extensive
shellf isheries of our nation might meet, exchange ideas, and gain
information from scientists who were studying biological and sanitary
problems related to the industry. The first organizational meeting
was held in New York City on January 15> 1909 and on May 5 of that
year a constitution and by-laws for permanent organization of a
National Association of Shell Fish Commissioners was presented.

Successive meetings attracted increased attention from state
officials, scientists and industry representatives. As scientists in
fishery biology, sanitation, and nutrition became active in the
Association, a revised constitution and by-laws was adopted on August
19> 1930> and a more descriptive name, The National Shellf isheries
Association, was chosen.

The first joint meeting with the Oyster Growers and Dealers
Association was held in 1929- In 1937 the Oyster Institute of North
America became a part of the Annual Oyster Convention. Annual joint
meetings of the three organizations, held in major cities along the
Atlantic Coast, have been centers of attraction for all people con-
cerned with shellfish.

The National Shellf isheries Association is a unique organiza-
tion in that fishery administrators, business men, and fishery
biologists meet to discuss their problems with mutual benefits.
Reports of the latest research on shellfish are brought to the
attention of administrators and members of the industry. During
recent years, these reports and addresses have been reproduced and
distributed annually to members as the Proceedings.

-196-

MEMBERSHIP

Candidates for membership in the Association should submit
completed application forms to the Secretary-Treasurer for review by
the Membership Committee.

NATIONAL SHELLFISHERIES ASSOCIATION
Application for Membership

Date_
Name

Mailing Address

Official Title or Position
Special Interests

Mail completed blanks to Secretary-Treasurer, Dr. Philip A. Butler,
U. S. Shellfishery Laboratory, Gulf Breeze, Florida.

Dues are $2.00 per year and include the Annual Proceedings. Make
check payable to the National Shellf isheries Association.

-197-

EDITORS' NOTES

Volume kQ has been reproduced by the Xerox-offset process by
the Duplicating Department of the University of North Carolina at
Chapel Hill. Part of the cost of duplication was borne by the Oyster
Institute of North America, whose help is greatly appreciated.

The Secretary-Treasurer reports that 419 copies of Volume 47
were distributed which included 208 to industry members, 135 to
Association members, 62 to libraries in the United States, and Ik to
foreign libraries. Copies are sent to libraries without charge.
Author abstracts are collected and sent by the Editor to Biological
Abstracts.

At best, editorial work is burdensome to the Editorial Commit-
tee and often irritating to the author. Much time and effort can be
saved for both parties if authors will examine carefully for style
the most recent volume of the PROCEEDINGS and use this as a model.
Please note that all figures and tables must fit on an 8 x 10^ inch
page with ample margins. Style, spacing, and general format should
follow closely the examples in Volume kQ.

Considerable confusion has arisen about submitting papers
written in a style best fitted for oral presentation. For publication
in the PROCEEDINGS, papers written in the best scientific style are
desired. The content does not need to be limited to material presented
at the Convention- Other papers will be considered for publication.

INFORMATION FOR CONTRIBUTORS

Original papers given at the Annual Association Convention,
and other papers on shellfish biology or related subjects submitted
by members of the Association will be considered for publication.
Manuscripts will be judged by the Editorial Committee or by other
competent reviewers on the basis of originality, contents, clarity
of presentation and interpretations. Each paper should be carefully
prepared in the style followed in previous PROCEEDINGS before sub-
mission to the Editorial Committee. Papers published or to be pub-
lished in other journals are not acceptable.

Manuscripts should be typewritten and double -spaced : original
sheets are required but carbon copies, if available, will facilitate
reviews. Tables, numered in arabic, should be on separate pages with
the title at the top. Scientific names should be underlined.

Illustrations should be reduced to a size which fits on
8 x lOjjj inch pages with ample margins. Glossy photographs are
preferred to originals. Illustrations smaller than a page should be
carefully oriented and loosely attached to plain white paper with

-198-

rubber cement. Legends should be typed on separate sheets and numbered
in arabic.

Use the following style for literature citations: "Smith,
Rebecca Joyce. 1958. Filtering efficiency of hard clams in mixed
suspensions of radioactive phytoplankton. Proc. Natl. Shellfish.
Assoc. kQ: 115-124. Note in Volume h8 punctuation for literature
citations in text. In abbreviations for names of serial publications,
follow Biological Abstracts (see 29(5): v-xxxv, 1955)- Abbreviations
for units of weight and measure in the Handbook of Chemistry and
Physics, 36th Edition, pages 3108-313^ will be followed. Punctuation
will be strictly limited, consistent with clarity, for abbreviations
of common measurements, literature references in the text, and certain
other usages. Note usage in recent PROCEEDINGS.

Each paper should be accompanied by an abstract which is con-
cise yet understandable without reference to the original article.
It is now our policy to publish the abstract at the head of the
paper and to dispense with a summary. A copy of the abstract for sub-
mission to Biological Abstracts will be requested at a later date.

Reprints and covers are available at cost to authors. Master
sheets will be retained for one year after publication. When proof
sheets are returned to authors, information about ordering reprints
will be given. The present agency from which authors may obtain
reprints is the Duplicating Department, Bingham Y, University of North
Carolina, Chapel Hill, N. C, Mr. J. Nelson Callahan, Head.

For inclusion in the PROCEEDINGS of the current year, all
manuscripts should reach the Editor prior to October 1 following the
summer Convention. Send manuscripts and address all correspondence
to the Editor, Jay D. Andrews, Virginia Fisheries Laboratory,
Gloucester Point, Virginia.

■199-

DIRECTORY OF MEMBERS OF THE NATIONAL SHELLFISHERIES ASSOCIATION

(To July, 1958)

Aldrich, F. A., Assistant Curator of Limnology, Academy of Natural
Sciences, 19th & The Parkway, Philadelphia 3> Pa.

Allen, J. Francis, Department of Zoology, University of Maryland,
College Park, Md.

Andrews, Jay D., Oyster Biologist, Virginia Fisheries Laboratory,
Gloucester Point, Va.

Arcisz, William, Bacteriologist, Shellfish Sanitation Laboratory,
U. S. Public Health Service, P. 0. Box 68, Gulf Breeze,
Florida.

Atlantic Biological Station, Fisheries Research Board of Canada, St.
Andrews, N. B., Canada.

Ayers, Robert J., Oregon Fish Commission, 1236 W. Marine Drive,
Astoria, Oregon.

Ball, Eric T., 212 Summit Street, New Haven 13, Conn.

Baptist, John P., U. S. Fish & Wildlife Service, Fishery Radiobiological
Laboratory, Beaufort, N. C.

Beaven, G. Francis, Oyster Biologist, Maryland Department of Research
& Education, Solomons, Md.

Berry, W. R., Bureau of Shellfish Sanitation, 5709 Sellger Drive,
Norfolk 2, Va.

Blake, John W., Department of Zoology, University of North Carolina,
Chapel Hill, N. C.

Blount, F. Nelson, Blount Seafood Corporation, 383-393 Water Street,
Warren, R. I.

Brittain, G. J., Jr., Shellfish Consultant, 56^3 Flamingo, Houston 21,
Tex.

Butler, Philip A., Chief, Gulf Oyster Investigations, U. S. Fish &
Wildlife Service, Gulf Breeze, Fla.

Carlson, Frank T., Scientist, Oyster Institute of North America,
U. S. Fishery Laboratory, Beaufort, N. C.

■200-

Carriker, Melbourne R., pepartraent of Zoology, University of North
Carolina, Chapel Hill, N. C.

Carver, Thomas C, Jr., Fishery Research Biologist, U. S. Fish &
Wildlife Service, Gloucester Point, Va.

Chanley, Paul E., Fishery Research Biologist, U. S. Fish & Wildlife
Service, Biological Laboratory, Milford, Conn.

Chapman, Charles R., Fishery Research Biologist, O.R.B.S., Slidell,
La.

Chestnut, A. F., Director, University of North Carolina, Institute of
Fisheries Research, Morehead City, N. C.

Chipman, Walter, Director, Fishery Radiobiological Laboratory, U. S.
Fish & Wildlife Service, Beaufort, N. C.

Collier, Albert, 1009 Church Street, Galveston, Texas.

Cooley, Nelson R., Biologist, U. S. Fish & Wildlife Service,
Shellfishery Laboratory, Gulf Breeze, Florida.

Cronin, L. E., Director, Maryland Department of Research & Education,
Solomons, Md.

Currier, Wendell, Ass't. to Vice President, Research & Development,
Campbell Soup Co., Camden, N. J.

Darling, J. S. & Son, P. 0. Box 412, Hampton, Va.

Dassow, John A., Technological Laboratory, U. S. Fish & Wildlife Ser-
vice, 2725 Montlake Blvd., Seattle 2, Wash.

Davis, Harry C, Fishery Research Biologist, Biological Laboratory,
U. S. Fish & Wildlife Service, Milford, Conn.

Dawson, C. E., Biologist, Bears Bluff Laboratory, Wadmalaw Island,
S. C.

Deiler, Frederick G., Biologist, Freeport Sulphur Co., Port Sulphur,
La.

Dow, Robert L., Director, Marine Reserach, Department of Sea and Shore
Fisheries, Vickery-Hill Building, Augusta, Me.

Dumont, William H., U. S. Fish & Wildlife Service, Department of
Interior, Washington 25, D. C.

Dunnington, Elgin W., Oyster Biologist, Department of Research &
Education, Solomons, Md.

-201-

Edwards, Malcolm B., Pacific Coast Oyster Growers Association, South
Bend, Wash.

Engle, James B., U. S. Fish and Wildlife Service, Clam and Chesapeake
Oyster Investigations, 800 Dreams Landing, Annapolis, Md.

Esveldt, George D., Manager, E. H. Bendiksen Company, 820 Broadway,
South Bend, Washington.

Pahy, William, University of North Carolina, Institute of Fisheries
Research, Morehead City, N. C

Fingerman, Milton, Newcomb College, Tulane University, New Orleans, La.

Florida State Board of Conservation, W. V. Knott Building, Tallahassee,
Fla.

Flower, Frank M. & Sons, Growers of Pine Island Oysters, Bayville,
Long Island, N. Y.

Fox, Leo, Associate Sanitary Biologist, Department of Public Health,
511 A State House, Boston 33 > Mass.

Galtsoff, Paul S., Director, Shellfish Laboratory, U. S. Fish & Wild-
life Service, Woods Hole, Mass.

Ganaros, Anthony E., Fishery Research Biologist, Biological Laboratory,
U. S. Fish & Wildlife Service, Milford, Conn.

Gibbs, Harold N., A-71 Sowams Road, Barrington, R. I.

Girard, John G., State Department of Health, Smith Town, Seattle,
Wash.

Glancy, Joseph B. , Shellfish, Inc., Box 212, West Sayville, Long
Island, N. Y.

Glude, John B., Chief, Clam and Chesapeake Oyster Investigations,

U. S. Fish & Wildlife Service, P. 0. Box 151, Annapolis, Md.

Grice, George D., Fish and Wildlife Service, Box 113^> Juneau, Alaska.

Gunter, Gordon, Director, Gulf Coast Research Laboratory, Ocean
Springs, Miss.

Gustafson, Al, Professor Biology, Bowdoin College, Brunswick, Me.

Hanks, James E., Woods Hole Oceanographic Inst., P. 0. Box 682,
Woods Hole, Mass.

-202-

Hargis, William J., Jr., Associate Biologist, Virginia Fisheries
Laboratory, Gloucester Point, Va.

Harrison, George T., President The Tilghman Packing Company, Tilghman,
Md.

Haskin, Harold H. , Director, New Jersey Oyster Research Laboratory,

Department of Zoology, Rutgers University, New Brunswick, N. J.

Haven, Dexter, Virginia Fisheries Laboratory," Gloucester Point, Va.

Hedrick Brothers Oyster Company, 730 Auster City Street, New Orleans,
La.

Hewatt, Willis G., Biology-Geology Department, Texas Christian Univer-
sity, Forth Worth, Tex.

Heydecker, Wayne D., Atlantic States Marine Fisheries Commission,
22 West First Street, Mount Vernon, N. Y.

Hofstetter, Robert P., Rt . #1, Box 132, La Porte, Tex.

Hopkins, Sewell H., Biology Research Laboratory, Texas A & M Research
Foundation, College Station, Tex.

Ruber, L. Albertson, Hydrographic Engineer, 297 E. Commerce Street,
Bridgeton, N. J.

Hulings, Neil C, Oceanographic Institute, Florida State University,
Tallahassee, Fla.

Jensen, Eugene T., U. S. Public Plealth Service, Shellfish Branch,
Washington 25, D. C.

Kahan, Archie M., Executive Director, Texas A & M Research Foundation,
College Station, Tex.

Kelly, C. P., Shellfish Sanitation Laboratory, U. S. Public Health
Service, Gulf Breeze, Fla.

Kelley, R. L., R. L. Kelley Farm, Atlantic, Virginia.

Kogenezawa, A., Biologist, Tohoku University, 15 Yakushido, Minami
Koizumi, Sendai City, Japan.

Lednum, J. M., Town Engineer, Town of Islip, N. Y.

Lester & Toner, Inc., c/o Royal Toner, 208 Front Street, New York 38,
N. Y.

-203-

Lindsay, Cedric, Director, Shellfish Laboratory, Fisheries Department,
Washington State, Quilcene, Wash.

Littleford, Robert A., Seafood Processing Laboratory, Crisfield, Md.

Loesch, Harold, Marine Biologist, Department of Zoology, Texas A & M,
College Station, Texas.

Logie, R. R., Department of Zoology, Rutgers University, New Brunswick,
N. J.

Loosanoff, Victor L., Director U. S. Fish and Wildlife Biological
Laboratory, Milford, Conn.

Lunz, G. Robert, Director, Bears Bluff Laboratories, Wadmalaw Island,
S. C.

Mackin, J. G., Director, Marine Laboratory, Texas A & M Research
Foundation, Galveston, Tex.

Macomber, Ronald, 11 Prescott Avenue, Montclair, N. J.

McConnell, James L., P. 0. Box 103^, Bay Towing & Dredging Company,
Mobile, Ala.

McConnell, James N., Director, Division of Oysters and Water Bottoms,
Department of Wildlife and Fisheries, 126 Civil Courts Build-
ing, New Orleans 16, La.

McIIugh, J. L., Director, Virginia Fisheries Laboratory, Gloucester
Point, Va.

Manning, Joseph H., Clam Biologist, Chesapeake Biological Laboratory,
Solomons, Md.

Mansueti, Romeo, Fishery Biologist, Maryland Dept. of Research &
Education, Solomons, Md.

Marshall, Nelson, Dean of Liberal Arts, Alfred University, Alfred,
N. Y.

Menzel, R. Winston, Oceanographic Institute, Florida State University,
Tallahassee, Fla.

Messer, Richard, 1205 Dinwiddie Avenue, University Heights, Richmond
26, Va.

Miles, J. H. & Co., Inc., Norfolk 1, Virginia.

National Fisherman, Goffstown, New Hampshire.

-204-

Nelson, J. Richards, The F. Mansfield & Sons Co., 610 Quinnipiac Ave.,
New Haven, Conn.

Nelson, Thurlow C, 8 North Main St., Cape May Court House, N. J.

New Jersey Department of Conservation, Trenton, N. J.

Pellissier, Carroll E., Editor of Fishing Gazette, 46l Eighth Ave.,
New York 1, N. Y.

Perlmutter, Alfred, Research Foundation, State University of New
York, Israel Project, 12 Harakevet St., Tel Aviv, Israel.

Poraeroy, Lawrence, Marine Biology Laboratory, Department of Biology,
University of Georgia, Sapelo Island, Ga.

Porter, Hugh J., University of North Carolina, Institute of Fisheries
Research, Morehead City, N. C.

Price, T. J., Fishery Radiobiological Laboratory, U. S. Fish & Wild-
life Service, Beaufort, N. C.

Pritchard, Donald W., The Johns Hopkins University, 121 Maryland Hall,
Baltimore 18, Md.

Provenzano, A. J., Jr., 11 Morton Avenue, Saugus, Massachusetts.

Quayle, Daniel B., Rt . #2, Box 573, Poulsbo, Washington.

Ray, Sammy M., 3127 Avenue R, Galveston, Tex.

Rice, Theodore R., Fishery Radiobiological Laboratory, U. S. Fish &
Wildlife Service, Beaufort, N. C.

Rhode Island Department of Agriculture & Conservation, John L. Rego,
Director, Veterans Memorial Building, 83 Park Street,
Providence 2, Rhode Island.

Ropes, John W., U. S. Fish & Wildlife Service, 29 Linden Street,
Salem, Mass .

Russell, Henry D., Springdale Avenue, Dover, Mass.

Rust, John D., 5031* Bradley Boulevard, No. 22, Chevy Chase 15, Md.

Sangree, John B. , Glassboro State Teachers College, Glassboro, N. J.

Sayce, Clyde S., Fishery Biologist, Box 205, Ocean Park, Wash.

Sellmer, George, Department of Biology, Upsala College, East Orange,
N. J.

-205-

Shearer, L. W., Biologist, 8 Elm Street, Milford, Connecticut.

Shuster, Carl N., Director, Marine Laboratory, Department of Biological
Sciences, University of Delaware, Newark, Del.

Sieling, Fred W., Department of Research & Education, Box 186, Snow
Hill, Md.

Silliman, Ralph P., Chief, Section of Anadromous Fish, Interior

Department, U. S. Fish & Wildlife Service, Washington 25, D. C.

Smith, F. G. Walton, Director, The University of Miami Marine Labora-
tory, Coral Gables k6, Fla.

Sollers, Allen A., 1305 Park Avenue, Baltimore 17, Md.

Sparks, Albert K., Chief Biologist, Assistant Director, Box 203,
Thibodaux, La.

Sprauge, Victor, Rochester, New York

St. Amant, Lyle S., Wildlife and Fisheries Commission, 126 Civil
Courts Building, New Orleans 16, La.

Staples, George M. Ill, Manager, Osprey Fishery, Box 66, Crisfield,
Maryland.

Stern, Joseph A., School of Fisheries, University of Washington,
Seattle, Wash.

Trezise, William R., Fishery Aide, 318 - 12th Street, Raymond, Wash.

Truitt, Reginald V., Great Neck Farm, Stevensville, Md.

Udell, Harold, N. Y. Conservation Department, Bureau of Marine
Fisheries, Freeport, Long Island, N. Y.

Valaer, Edward P., Shellfish Sanitarian, D. C. Department Public
Health, Washington 1, D. C.

Virginia Commission of Fisheries, Newport News, Va.

Wallace, Dana E., Department of Sea & Shore Fisheries, Vickery-Hill
Building, Augusta, Me.

Wallace, David H., Director, Oyster Institute of North America, 6 Mayo
Avenue, Bay Ridge, Annapolis, Md.

Webster, John R., U. S. Fish & Wildlife Service, P. 0. Box 151,
Annapolis, Md.

-206-

Weiss, Charles M., Department of Sanitary Engineering, School of
Public Health, University of North Carolina, Chapel Hill,
N. C.

Welch, Walter R., U. S. Fish & Wildlife Clara Investigations, R.F.D.,
Boothbay Harbor, Me.

Westley, Ronald E., Fishery Biologist, Washington State Department
of Fisheries, Shellfish Laboratory, Quilcene, Wash.

Whaley, Horace H. , The Johns Hopkins University, 121 Maryland Hall,
Baltimore 18, Md.

Wilde, Frank W., Box 5, Shady Side, Md.

Wilson, Alfred J., Jr., U. S. Fish & Wildlife Service, Shellfishery
Laboratory, Gulf Breeze, Florida.

Woelke, Charles E., Fishery Biologist, Box 323> Quilcene, Wash.

Wolraan, Abel, The Johns Hopkins University, Whitehead Hall,
Baltimore 18, Md.

Wurtz, Charles B., 32^7 Disston Street, Philadelphia, Pa.

-207-

MB1- WHO! I.IHKAKY

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