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1959 mOCEEDINGS

NATIONAL

SHELLFISHERIES

ASSOCIATION

Volume 50

TABLE OF COJJTENTS (^ I LIBRARY ] ^

SYMPOSIUM ON MORTALITIES OF SHELLFISH

Problems in analysis of mortalities DAVID E. DAVIS 1

Immunity in invertebrates, with special reference

to the oyster LESLIE A. STAUBER 7

Mortalities of oysters J. G. MACKIN 21

ESTIMATION OF MORTALITY RATES OF OYSTERS

A method of estimation of mortality rates in

oysters J . G . MACKIN Ul

PESTS, PREDATORS, AND DISEASES OF OYSTERS

Pacific oyster Crassostrea gigas mortalities with notes on
common oyster predators in Washington
waters CHARLES E. WOELKE 53

Hexamita sp . and an infectious disease in the
commercial oyster Ostrea

lurida J. E. STEIN, J. G. DENISON

and J. G. MACKIN 67

Effects of the flatworm Stylochus ellipticus
(Glrard) on oyster spat in two salt water
ponds in Massachusetts ANTHONY J. PROVENZAHO, JR. 83

Flatworm distribution and associated oyster

mortality in Chesapeake Bay JOHN R . WEBSTER and

ROYSTON Z. MEDFORD 89

PEST CONTROL

The effects of salt solutions of different strengths

on oyster enemies L. W. SHEARER and

C. L. MacKENZIE^ JR. 97

Chemical control of Polydora websteri and other

annelids inhabiting oyster~8hells . .CLYDE L. MacKENZIE, JR.

and L. W. SHEARER 105

INTRODUCTION OF NEW SPECIES

Trial introduction of European oysters (Ostrea edulis)

to Canadian east coast J. G. MEDCOF 113

GROWTH AMD QUALITY OF OYSTERS

Preliminary report on grovth and survival of the

Pacific oyster in Washington waters . ALBERT K. SPARKS and

KENNETH K, CHEW 125

Growth of the Pacific oyster Crassostrea gigas

in the waters of Washington State CHARLES E. WOELKE 133

Selection and evaluation of a method for

quantitative measurement of oyster condition
RONALD E. WESTLEY ik^

STUDIES ON CLAMS

Effect of hydraulic escalator harvester on

under-size soft-shell clams J. C. MEDCOF 1^1

Inheritance of shell markings and growth in the

hard clam, Venus mercenaria PAUL E . CHANLEY I63

PUBLIC HEALTH ASPECTS OF SHELLFISHERIES

Progress in the Cooperative State-Puhlic Health Service-
Industry Program for the Certification of Interstate
Shellfish Shippers EUGENE T. JENSEN I7I

A bacteriological study of the natural flora of
Pacific oysters (Crassostrea gigas) when
transplanted to various areas in

Washington R • R • COLWELL and

J. LISTON 181

ASSOCIATION AFFAIRS

Annual convention lo9

Officers and committees lo9

Information for contributors 190

Directory of members 191

PROBUa.B III AJIALYSIS OF MORTALITIES

David E. Davlsl

Laboratory of Comparative Behavior
Johns Hopkins School of Hygiene and Public Health

The interpretation of mortality data presents a variety of pit-
falls that may trap the investigator into making erroneous predictions
about the results of a change in mortality rates. The essence of man-
aging and harvesting a crop is to replace natural mortality by human
consumption. But unfortunately a m^re reduction of the mortality due
to one cause is no guarantee that this part of the crop will become
available for human use, because another cause may assume importance
and compensate for the previous losses. Thus compensatory mortality
may defeat efforts to reduce the number of deaths .

The determination of mortality rates is naturally the first step
in obtaining data for interpretation. First we shall define the various
kinds of mortality and briefly show the types of calculations. Then we
shall discuss the relation of these rates to populations and the compen-
satory effects. The definitions of mortality unfortunately are badly
confused. The following agree with those suggested by Ricker (1958),
and with demographic use (Spiegelman 1952). The basic necessity is to
be sure that the population under consideration is clearly defined.
Thus a crude rate is based on a population containing a miscellaneous
assortment of ages or sex while a specific rate refers to a particular
character. A rate could be specific with respect to age (i.e. not indi-
cate sex). Age and sex are the characteristics most commonly specified
but others (habitat, genetic strain, etc.) could be used. The term
"mortality rate" is often used to refer to two fimdamentally different
kinds of populations. In one definition (here called q, the probability
of dying) the number of deaths in a time interval is divided by the ini-
tial population or cohort. In the other (here called d, the death rate)
the number of deaths is divided by the average population and should be
restricted to stationary populations. As examples let lis consider a
group of 100 oysters on Jan. 1. Suppose that during a month 60 of these
die, then the probability of dying in this month is 60/1OO =0.6. Now
suppose that at the instant each oyster died it was replaced by another
so that the population instead of declining to Uo, always remained at
100. The death rate is 60/IOO =0.6 which is nimerically the same as
above. But note that probability of dying can never be greater than
1.0 (certainty) while death rate can be more than 1.0 due to replace-
ments. Thus it may happen that a population by instantaneous replace-
ments remains at 100 even though say, 200 oysters die, giving a death
rate of 2.0.

r Present addi-ess: Department of Zoology, Pennsylvania State Univer-
sity, University Park, Pennsylvania. Submitted July 1959-

The collection of data for determination of mortality rates re-
quires a knowledge of the number of deaths. Certainly the simplest
method is to determine the age of death of a sample of the population.
When suitably arranged as a frequency distribution, these records will
give a life table (Ricker 1958, Deevey I9U7) . Unfortunately this method
requires some assumptions about randomness of the sample and also a
method for determining the age of death.

Other methods require a knowledge of the population. One pro-
cedure depends upon recapture of marked individuals . Perhaps the most '
generally useful method is to estimate the number in a particular group
at two different times. If emigration is negligible, then the differ-
ence is due to deaths. All of these procedures are laborious and re-
quire fairly complicated calculations.

Assuming that by some method we have some information on num-
bers of deaths, the next step is to compute the values. Obviously the
probability of dying (q) is complementary to the probability of sur-
viving (p) so that q + p = 1. Now the relation of d, the death rate,
to q, the probability of dying, is 1-q = e"^, where e is the base of
natural logarithms. (This relation is not satisfactory for values of
q greater than about 0.7 because of discrepancies in the time intervals).
The advantage of this relationship is that one can change types of data
that are relatively easy to get (probability of dying), to the type
that is hard to obtain (death rate). An advantage of death rates is
that, being exponents, the death rates due to various causes can be
added. Thus 1-q = e-('ii+ d2+ d3), Nqw, if the death due to di or
d2 or d^ can be determined, then a prediction can be made of the value
of q vfhen this cause is removed. The difference between this predi-
ction and the observed death rate results from compensation. Before
indicating the nature of compensation it is desirable to explore some
other fundamentals .

The effect of disease and predation on a population spans a wide
spectrum from a level inadequate for the welfare of the species to ex-
tinction. Persons familiar with sport fishing know the many examples
of stunting and reproductive failure that occior in overpopulated ponds.
Here an increase in mortality rate would permit greater growth and re-
production and it is hoped that fishermen rather than disease will in-
crease the mortality. In other situations the effect of a disease or
predator is not measurable. By this we mean that whatever effect may
occur is so small that our methods of estimation cannot detect it. In
still other cases the disease or predator restricts the population since
it can be shown that without the disease the population will be higher
in number or mass. Cases of overfishing belong in this category (ex-
cept where the habitat has been altered). Finally the end of this wide
spectrum is reached in the examples of extermination of the population.
It is vital to recognize that this spectrum is continuous; only four
points on a continuous scale have been singled out for mention here.

The numerical relationship depends upon local circumstances .
At one time and place a disease may have one effect but at some other
time or place have another level of effect. Thus local conditions may

-2-

permit a disease to be harmless at one time and to be catastrophic at
another time. This variation in effect is bewildering to investigators
as well as practitioners.

Another fimdamentaJ. principle deals with the concept of density-
dependence. (Nicholson 195^). This notion considers the effect of a
mortality factor at various densities of the population. If the effect
increases percentagewise as the popxilation increases, the relation is
said to be density-dependent. If, on the other hand, the effect remains
the same percentagewise, the relation is said to be density- independent.
Unfortunately, in true biological fashion, animals refuse to be neatly
pigeon-holed into our categories but show a complex of intergading con-
ditions. Nevertheless the essence of the concept is important because
it shows that a density- independent factor by itself cannot restrict
the population. Thus if 95^ of the recruitment to a population is kill-
ed each year, the population will increase by about 5?^ each year for-
ever. In contrast if 100^ of the recruitment are killed then the
population will be limited. A factor whose effect can increase with
density is able to govern the population level. Disease by the very
nature of transmission is a classic example in theory of a density-
dependent factor. To determine in practice what quantitative relations
exist requires collection of certain data. To imderstand the relation
to density, the number that die m\ist be related to the number of new
individuals (births and immigration) . This recruitment is usually de-
fined on a realistic basis such as the number reaching a certain size
or age . It is at once apparent that the task of obtaining information
is great. One must know at several densities the deaths, the recruit-
ment and, of course, the population. Each of these statistics requires
a formidable effort. As might be expected few studies are sufficiently
complete to meet these exacting standards . Some programs have deter-
mined the percentage of the population killed by a disease or predator.
Others have calculated the amount consumed and others list the food
habits of the predator or the diseases found in the population. None
of these studies is sufficiently profound to indicate the effect on
the population .

To complicate the problem still further is the principle of
compensation. The essence is very simple. Every animal dies from some-
thing. If disease X is prevented then parasite Y may cause the deaths.
The important point is whether the time between these events is suffi-
cient to permit growth in size or number. Thus if a hawk catches today
a mouse that was destined to die of starvation tomorrow, then no measur-
able effect on population occurred. But if 6 months intervenes between
these potential events, a measurable change may occur. To manage a re-
source the causes of death must be determined and the compensatory as-
pects analyzed (figure l). Suppose that cause X and cause Y each pro-
duce a probability of dying of 0.6o. This means that the probability
of survival for each is O.kO which must be multiplied to give the joint
survival (0.l6). Hence the probability of dying from either X or Y is
0.8i|. Suppose further that X is eliminated but Y now alone causes a
probability of 0.84. Obviously no change has occurred due to the com-
pensation by Y. If, however, the total is now only O.7O then a change

-3-

has occiirred and more indlvlduala surArive. These rates may vary numeri-
cally with changes in density.

The procedure for analysis of a problem of mortalities is tedi-
ous and involved. First the causes capable of producing death must be

TIME

Figure 1, Diagrammatic representation of compensatory mortality.
The rate is constant (solid line) and hence ia a straight line on
arlthlog paper. At A a cause of mortality is removed, permitting some
Individuals to survive as indicated by the horizontal dotted line.
Then other causes operate until the point B where some cause has an
increased effect and compensates for the lack of mortality at A. If
the time intervals between A and B are very small then nothing is
gained by removal of cause at A, However, if interval is great, per-
mitting breeding or harvesting, then removal of cause A is worthwhile.

determined. Then they should be eliminated (one by one if feasible)
on an experimental basis in smaJJ. areas to determine the effect on
the death rate. If elimination of one or more causes does not alter
the probability of dying it is obvious that other causes are taking
the place and more caiises need to be eliminated. Extensive eradication
prograjns are not justified till this point has been established experi-
mentally. Failure to reduce the death rate may result from compensation
or from density-independence, since a factor that removes a constant
proportion has little chance to be flexible.

This article has not cited specific examples partly because
data on shellfish are scarce and partly to emphasize the principles.
The reference cited will provide additional information and examples
of these principles.

-If-

REFERENCES

Deevey, E. S. 19^7- Life tables for natural populations of animals.
Quart. Rev. Biol. 22(1^-) : 283-314.

Nicholson, A. J. 195^. An outline of the dynamics of animal popula-
tions. Australian Jour. Zool. 2(1)19-65.

Pickett, A. D., Putman, and E. J. LeRoiJX. I956. Progress in harmoni-
zing biological and chemical control of orchard pests in East-
ern Canada. Proc. 10 Intern. Congr. Ent. 3(359^) :l69-174.

Ricker, W. E. 1958. Hajidbook of computations for biological statis-
tics of fish populations. Fish. Res. Bd. Canada Biill. II9,
300 pp.

Spiegelman, F. C. 1952. Demography. Rinehart, New York, xi + 372
pages .

-5-

IMMUNITY IN INVERTEBRATES, WITH SPECIAL REFERENCE TO THE OYSTER

Leslie A. Stauber

Department of Zoology
Rutgers - The State University
New Brimswick, New Jersey

INTRODUCTION

There have "been several excellent reviews of the field of in-
vertebrate immunology and the author acknowledges his great indebted-
ness to men like Metchnikov (l905)^ Metalnikov (1920,1921), Chorine
(1931), Cameron (l93^). Huff (l9^), and Bisset (19^/') for the job they
have done in collating the large literature. Other reviews, like those
of Liebman (l946), George (19^*^1) and Wagge (l955) discuss the kinds and
functions of cells playing a role in invertebrate physiology, including
immunity. These and Steinhaus ' books on insect pathology (19^+9) and in-
sect microbiology (l9^6) have been helpful also. This article lays no
claim to being an exhaustive review but is intended as a statement of
principles with selected examples of what is considered to be of pos-
sible special interest to shellfish biologists.

In examining much of the literature in this field several points
are soon recognized: l) Most of the work was done 20 or more years ago;
2) most of it refers to observations on insects, especially insect lar-
vae; and 3) aside from reports of mortalities and descriptions of indiv-
idual parasitic organisms, almost none of it refers to molluscs. Michel-
son, in his recent review of predators and parasites of fresh water mol-
luscs, for example, cites only a few articles of possible interest in
our discussions here. Even the fine work of Cuenot on the phagocytic
organs of molluscs apparently stimulated none of the continuations and
extensions so easily and logically deduced from his findings. The oys-
ter, surprisingly because of its economic importance, has been one of
those so neglected. Effects of starfish, oyster drills, hurricanes and
freshets and the enormous problems of seed production and management
have probably led to the neglect of the development of oyster pathology.
Also some of the difficulties of working in the laboratory under rea-
sonably controlled conditions with oysters, portions of whose shells
must be removed to expose the heart for injection, are quite evident
and need not be stressed, though they probably have contributed to the
delay. Our interest in Immunology and in the oyster led us to enter
the field and some of the findings have already been reported ( Stauber
1950, Tripp 1958 a,b,c, Feng 1959a) .

Fortunately, Dr. J. G. Mackin also entered the field at about
this time and, with his associates, has made great contributions to the
field of oyster pathology for which we are deeply indebted. In spite
of these, however, our lack of specific knowledge concerning suscepti-
bility ajad resistance in the oyster will be disappointment to most of
you.

-7-

DEFINITIONS

In the earlier literature the so-called resistance of a host to
infection with another organism was considered as the reciprocal of its
susceptibility to that organism (Falk I928) . A highly susceptihle ani-
mal was thus one lacking in resistance. More recently Schneider (1951),
and especially Read (1958) have shown that this is much too simplified
an expression of the problem and that susceptibility and resistance "are
separate and distinct biological attributes" (Schneider).

If we begin the discussion with host insusceptibility, two kinds
are readily discernible. In the one case, the potential invader present
in the surroundings of the host cannot gain entry. Whether this be be-
cause of the protective shell of the host (like Limulus), the integrity
of its exposed epithelia or the normal secretions (like mucus) of its
food-gathering, digestive or other systems is of no moment. The fact is
that the "invader" cannot effect entrance. In the other case, even if
entry is effected, the organism cannot survive in the host since, as
Read defines insusceptibility, the "physiological state" of the host is
such that "the life needs of the parasite are not satisfied" in it.
This could mean, for example, for aa intercellular parasite, that a re-
quired free amino acid is not available, that a growth factor is present
in too low a concentration or that an inhibitor is present in too high
a concentration for the parasite to grow and develop. For an obligate
intracellular parasite, it might mean that the characteristic host cell
is not present in the species of host animal under study. If it is pre-
sumed that entry by the parasite is possible under natural conditions
or is obtained through experimental manipulations, then susceptibility
is "a physiological state of the host in which the parasite is supplied
with its life needs," and, therefore, does grow and develop (Read 1958) •

Read defines resistance as "those alterations of the physiolo-
gical state of the host which represent a response of the host to pre-
vious or present experience with the parasite or a chemically related
entity" (italics mine). The emphasis is on host response. Under such
definitions as given above, a host may be highly susceptible and show
no resistance, barely susceptible and show high resistance or even be
"a highly susceptible host which is also a highly resistant one" (Read).
Tr^anosoma rhodesiense, in the mouse, satisfactorily exemplifies the
first case since the parasites seem to accumulate in numbers exponenti-
ally until the death of the host, with no indication of any response to
the parasite by the mouse. On the other hand, the rat seems almost as
highly susceptible to T. lewisi as the mouse in the above example is to
T. rhodesiense, but the response (or resistance) of the rat brings a-
bout its eventual recovery with complete destruction of the accumulated
parasites (Taliaferro 1929 ) •

Resistance defined as host response implies that it takes time
to accomplish the end results, neunely, the manifestation of the resist-
ance. It has been customary to divide resistance into two categories,
innate and acquired. Acquired resistance is an individual host response
which develops after a primary exposure to the parasitic agent or its

-8-

products and after survival from this experience, and it may persist
for a very long time once developed. It is usually manifested as a
heightened response to the parasitic agent upon subsequent contact with
it, and in vertebrates like men, it is most often associated with the
presence of antibodies. Innate resistance relates to those responses
of the host appearing soon after its first contact with the invading
organism. It is usually a racial or species characteristic, though
it may be an individual one. It is obvious that, in the present state
of our ignorance, insusceptibility and innate resistance are probably
often used interchangeably since adequate evidence is not available
for decision; this would be the case when deciding whether a phagocy-
tized micro-orgajiism, is ingested before or after its death. In this
paper, we will retain the words "innate" and "acquired" as categories
for the remainder of this discussion and will attempt to illustrate
them by discussing the relative resistance of a series of rodents to
intracardial infection with a protozoan parasite, Leishmajiia donovani
(Grun 1958, Stauber I958) . It has been shown that the golden hamster
and the cotton rat show no innate resistance, parasites continuing to
increase in number in these hosts as long as the hosts live. By con-
trast, almost from the moment of inoc\ilation parasite numbers decrease
when introduced into the rat and rabbit. It might be argued that this
is mere insusceptibility in the rabbit and rat, but some increase in
parasites does seem to occur in one of the organs (spleen) of these
animals and normal-appearing parasites persist for appreciable lengths
of time. Until further evidence is available we are calling this in-
nate resistance. In the mouse, guinea pig and gerbil, the early rate
of increase of parasites is less than that seen in the cotton rat and
hamster. We have been calling this an effect of innate resistance,
although we do not know whether it means that some of the parasites
produced are being destroyed by the host from the beginning or that
the needs of the parasite are less ably met by these hosts. In the
latter instance, it woiHd be more appropriate perhaps to call this a
lesser degree of susceptibility. Guinea pig, mouse and gerbil are able
later to reduce the parasite numbers significantly enough essentially
to recover from this infection. This is acquired resistance and the
heightened resistance so produced has been demonstrated by following
the course of infection after a second injection of parasites.

INNATE RESISTANCE

Two points need introductory consideration in a discussion of
innate resistance in invertebrates; namely, the portal ajid mode of en-
try of the parasite and the size of the infecting dose. These factors
are applicable also to the phenomena of acquired resistance, but since
there is so little evidence for naturally acquired resistance in inverte-
brate hosts they are more likely to apply to innate resistance under
the conditions of infection of invertebrates in nature.

Any epithelial surface might be penetrated for entry into
deeper tissues including preliminajry entrance into natural cavities
such as mouth, anus, and genital and reproductive canals. While men-

-9-

tion has already been made of the possible role of secretions, such
as mucus or digestive juices, as impeding entrance, it is a fact that
examples exist of parasitic organisms using every conceivable portal
of entry into their hosts . Some might enter a host like the oyster
passively, swept along in the incurrent water to gain the digestive
tract or gill surface where ingestion by a phagocyte might carry them
into the deep tissues. Possibly Nematopsis is in this category. Or
they might enter the deep tissues only later from the gut, perhaps
like Hexamita, some bacteria and the commonly observed oyster ciliate.
Others might be able to penetrate by means of either their own motile
efforts or their digestive powers, like the miracidium of the flatworm
Bucephalus . Still others might require the assistance of a third
organism to break this initial line of defense. Bucephalus thus might
carry in bacteria, fungi or even viruses. The mantle erosions of the
boring sponge or the Dutch shell disease fungus and the gill erosions
produced by Pinnotheres are also breaches which might admit the entry
of microorganisms and the so-called piistule or "maladie du pied" of
oysters may arise in some such way. That infection of oysters could
occur in these ways is entirely logical from our knowledge of infecti-
ous processes in higher animals.

Mode of entry is also important because it might lead to ef-
fective host resistance or none at all. Entry directly into the blood
streajn, as in our injections of foreign substances and microorganisms
into the oyster heart, may lead directly to phagocytosis and destruction
or directly into those very cells in which an obligate intracellular
parasite must eventually find itself in order to grow and develop.
The same organism entering a tentacle of the mantle wo\ild find a con-
siderably different set of clrcijmstances facing it.

The numbers of the infecting organisms gaining entry is also
of prime importance . An excellent example may be studied in the work
of Hewitt, Richardson and Seager (l9^2) with an avian malaria parasite,
Plasmodium lophurae , in the duckling. Per cent mortality, time to
death, numbers of i)arasites at the height of the infection, and even
whether parasites reach a density detectable by the counting proce-
dures used, are all directly correlated with the size of the infecting
dose of parasites injected. Even xmder more natural conditions of
infection, McConnell and Cutkomp (l95^) have shown recently that the
median lethal spore concentration of Bacillus thuringiensls necessary
for 100^ kill of the first instar larva of the corn borer, Pyrauata
nubilalis, is approximately 50^000 spores per ml. The plants were
dipped in the spore suspensions and the larvae exposed to the plants
for 36 hours to produce this result. The important point is that
significant reductions of mortality occiir when less concentrated spore
suspensions are xosed. The analysis has not been carried farther, but
presumably it takes these conditions either for one spore to reach
the place of penetration into the host tissues or, what is more likely,
to permit entrance of sufficient numbers of infecting organisms to
render ineffective the innate resistance of the host.

-10-

Innate resistance is usually disciissed under the categories of
cellular and humoral mechanisms of response, depending on whether host
cells or body fluids are chiefly involved. My studies and those of my
students have largely concerned the cellular aspects, more specifically
phagocytosis and some of its consequences . We have injected into the
oyster's heart and adductor muscle such materials as India ink, starch
grains, erythrocytes (red blood cells of fish, mammals and birds, es-
pecially cells of the duckling infected with a malaria parasite, Plas-
modium lophurae , with its relatively indigestible malaria pigmentj]!
bacteria (as both vegetative cells and spores), soluble starch, bovine
hemoglobin, human serum albumin and diptheria toxoid (Stauber 1950j
Tripp 1958 a,b,c; Feng 1959 a,b). Most of the living organisms used
are non-pathogenic for the oyster (except possibly Adelson's A-3 bac-
terium isolated from gaping oysters). We may briefly summarize the
results as follows: l) The injected "particles" are quickly phagocyt-
ized or pinocytized (unless arterial blockage occurs as when very large
numbers of particles are injected) by cells which we call leucocytes or
phagocytes and which ordinarily are found to be abimdant, especially
in the smaller blood vessels and sinuses. 2) The "particle "-laden
phagocytes soon become distributed widely throughout the oyster. 3a)
If the "particle" is digestible by the oyster leucocyte (vegetative
bacteria, erythrocytes, malaria parasites, hemoglobin), digestion will
proceed toward completion. 3b) If the "particle" in indigestible
(India ink, bacterial spores and malaria pigment), leucocytic migration
will carry it eventually across an epithelial surface to be voided in
feces or in the mucus masses ejected from the oyster. The time requir-
ed for the removal of approximately 90^ of the material from the blood
varies with the material injected and the method of determination, be-
ing longest for indigestible material, like bacterial spores and India
ink, where migration seems to be the only mechanism for removal, and
shortest for soluble organic substances like hemoglobin.

Another manifestation of host celliilar response in inverte-
brates is leucocytosis, the mobilization of phagocytic cells in the
blood stream. In the work of Feng and Canzonier (1959) even the intra-
cardial injection of sterile sea water may cause an increase in abund-
ance of cells in the heart blood within 2k hours. The proportions of
the various types of blood cells may also change after the injection
of foreign material (ink, bacteria) or even after hemorrhage. This
has been well described by Cameron (193^) for the larvae of the wax
moth caterpillar. The shift is toward greater proportions of cells
called lymphocytes by Cameron and others, and is similar to some of
Feng's (1959^)) observations for the oyster. It is believed by Cameron
and others that the lymphocytes differentiate into phagocytic cells
like the ordinary leucocytes. That phagocytosis often leads to de-
struction of the organism engulfed may have been best demonstrated by
Shirodkair et al. (1958) with whole Limulus blood in roller tissue
culture when as many as 2k million bacteria, of a species isolated
from oysters, were destroyed in six hours at room temperature.

-11-

Encapsulation is another cellular aspect of innate resistance
and usually appears as a response either to a large mass of foreign
material or to a special type of indigestible organism or material.
The former is well described by Salt (l955^ 1956) as host response to
the introduction of the whole egg of a parasitic insect into the body
cavity of another species of insect larva. In innately resistant hosts
the phagocytes or hemocytes come together in large numbers around the
foreign body, sometimes even differentiating into a fibrous -like con-
nective tissue capsule around it. Dr. Mackin (l95l) has described and
has permitted \is to examine abscesses in oysters infected with Dermo-
cystidium, the outer margin of which conforms to this type of response .
In the oysters injected by Tripp with avian red cells there is evidence
for encapsulation of large masses of the cells and also for the fact
that lymphocytoid as well as leucocytic cells are involved in the en-
capsulation. Newton (1952) reports cellular infiltration and fibrotic
reaction in certain snails to invading flatworm larvae. In the case
of the special type of indigestible organism, acid-fast bacilli are most
often involved, though nodules may even be formed around India ink
(Metalnikov and Chorine 1930, Cameron 193^). A similar situation has
recently been described for freshwater gastropods by Mlchelson (1958)
where very large masses of acid-fast bacilli are surrounded by a kind
of fibrous cell capsule. Huff's review indicates that this type of
capsule has been foijnd around encysted gregarines and nematodes in
crickets, a flatworm in a beetle and a sporozoan in a mealybug. That
encapsulation, or phagocytosis, does not necessarily result in the
death and destruction of the inciting agent should be emphasized (for
recent studies in rabbits see Rogers 1958) • This is especially true
of the acid-fast bacilli, which several workers have shown may remain
viable (Cameron) and may even increase in numbers within the nodules
(Michelson, I958) .

In considering humoral aspects of Innate resistance, the as-
sumption must be made that cellular fragmentation or secretion (Wagge
1955) or some biochemical alteration confers bacteriostatic, lytic or
other properties on the body fluid. If these properties are present
even before the introduction of the microorganism then they more pro-
perly should be called insusceptibility factors. Some insect bloods
do have striking bacteria-dissolving properties and many have agglu-
tinating activity on microorganisms which may facilitate phagocytosis
and encapsulation. In insects, melanin deposition, which may enshroud
large objects or be deposited within small nodules containing India
ink or bacteria, is also a typical innate host response. As for detox-
ification of toxic products of organisms much more work is required to
resolve the difference between Feng's finding of the almost immediate
removal of diphtheria toxoid and bovine hemoglobin from oyster plasma
and the reports of Metchnikov (1905) and Bengston (192I+), e.g., that
tetanus or botullnimi toxin may remain for several weeks in the blood
of injected Insects without losing toxicity even through metamorphosis
to the adult winged insect.

-12-

The oyster has been little investigated for hijmoral factors
though Tripp (l958) and Feng (l959) have found some indications of
both agglutinating and properdin-like properties in oyster blood.

One aspect of innate resistance (or insusceptibility) deserves
emphasis, namely, the many reports of racial and even individual re-
sistance among invertebrates to specific infectious agents. Pasteur
in 1870 became the savior of the silkworm industry by observing that
all the larvae were not infected by the microsporidian of pebrine and
that selection of the uninfected larvae led to the establishment of
resistant stocks. Huff's (l935) demonstration, by selection of culi-
cine mosquitoes, that susceptibility to the avian malaria parasite
Plasmodium cathemcrium behaved as a simple Mendelian recessive, is
another striking example of hereditary influence on susceptibility or
resistance. In the molluscan field, although little has been done ex-
perimentally, the resistance of the local survivors of the several
epizootics of "Malpeque disease" in Canadian waters is evidence of
similar individual differences among oysters and of the effects of
rigorous selection on the susceptibility of the population (Logie I958,
Needier and Logie 19^7). The observations of Andrews and Hewatt (1957)
on oysters from different areas exposed to Dermocystidium marinum
should also be mentioned here. Newton's (1952,1953) work on the in-
heritance of susceptibility of a freshwater gastropod, Australorbis
glabratus, to the flatworm Schistosoma mans on i is an excellent be-
ginning in the molluscajn field. Exposure of the Puerto Rican strain
to the parasite yielded Infections in 95^ of the snails whereas in
similar exposure of Brazilian snails none became infected. In the
resistant snails the invading parasites were destroyed in 2k to k&
hours, with marked cellxilar infiltration and a fibrotic walling off.
Matings between the two strains were accomplished and three generations
of progeny studied. The findings show that resistance is a heritable
character, but that the picture is complicated, several genetic factors
being involved.

It is probable that many factors influence innate resistance.
The most obvious for the infected oyster are temperature, salinity,
nutritional state and the flora and fauna of its environment . Little
has been done along this line experimentally, though the studies of
Andrews ajid Hewatt (l957) with Dermocystidium illustrate what can be
done. We have conducted but one experiment so far, with the A-3 bac-
terium, to determine the role of temperature in the oyster's response
to the intracardial injection of this organism. We can report at this
time only that environmental temperature does influence the rate of
initial clearing and subsequent fate of the organisms in the oyster.
This phenomenon is well-known to those interested in the biological
control of insects where percentage kill by infection with a micro-
organism may be directly correlated with environmental temperatures
(Heimpel 195^).

The influence of nutrition (metabolites and their inhibitors)

-13-

I believe is best illustrated by the experiments of Terzian, Stabler
and Ward (l952) on mosquitoes infected with avian malarial parasites.
Before and after the infecting blood meal the mosquitoes were fed on
sugar alone or on sugar containing vitamins or inhibitors. It has
been shown not only that specific compounds may have significant effects
on resistajice, but that the maximum effects depend upon a specific op-
timal rajige of concentrations above which the specific effects of the
compounds are depressed or eliminated. These findings suggest a fun-
damental relationship between such substances and innate resistance >
(or susceptibility).

As for the influence of other living microorganisms found in
and about a given animal, it first should be noted that infectious
agents can be isolated regularly from the bloods or tissues of many
otherwise normal animals. This is especially true of arthropods
(Steinhaus 19^6, Cameron 193^) and indeed is a necessity if those ar-
thropods are to transmit infections like malaria parasites, bacteria,
rickettsiae and viruses to vertebrates and even to their own offspring
(through the egg) . Some of these organisms isolated from tissues may
be true pathogens of the host under study, others may be mere secondary
invaders unable to invade by their own capabilities, but whose inherent
pathogenicity may seal the fate of the host if they do get in. For ex-
ample, Cameron states, with respect to virulent strains of certain
bacteria in the alimentary tract of larval moths, that they "tend to
invade the body cavity if for any reason the defensive mechanism is
disturbed."

Drs. Cort, Hussey and Ameel have given me the privilege to re-
port their unusual and as yet unpublished findings with certain fresh-
water gastropods. These snails, if infected with flatworm larvae, may
then be infected with a microsporidian protozoan which invades and
damages not the snail but the flatworm larvae within the snail . It
seems probable that the microsporidian hyperparasites are regularly
invading these snails, but can be established only when the snails con-
tain a suitable tissue for their growth, namely, the trematode larvae.
An analogous situation, also in snails with trematode larvae, was re-
ported much earlier (Cort, Olivier and Brackett 19^l) • The point I
wish to emphasize from these examples is that the epithelial surfaces
of animals are being penetrated regularly, and perhaps most often, by
adventitious non-pathogenic organisms present in relative abundance at
points where entry can most frequently occur. It is believed by some
that this explains in part the so-called normal agglutinins in man and
other animals to so many essentially non-pathogenic organisms (Wagner
1959^ Springer, Horton and Forbes 1959).

ACQUIRED RESISTANCE

In this section naturally acquired resistance will be discussed
chiefly; there will be only casual reference to that artifically in-
duced by Injections of dead organisms . If thus restricted and if

-Ik-

defined as a heightened responGc of a host as the result of previous
contact with the infecting agent, then the following statement from
Stelnhaus is pertinent: "It is somewhat surprising that so few ob-
servations have been made on naturally acquired resistance in insects.
We know practically nothing, e.g., concerning the residual immunity in
insects that have survived an epizootic wave" (italics mine). Huff
(1930), with avian malaria parasites in mosquitoes, clearly indicated
no change in susceptibility with reinfection, but there is a large
literature showing that injections, especially of old cultures of
bacteria, into the body cavity of caterpillars (Metalnikov and others)
have immunized the caterpillars in as short a time as 2i<- hours to
doses of virulent organisms lethal to unvaccinated controls . Huff
(19^) writes that "insects have poor powers of overcoming parasitic
protozoa, fungi, and insects once these organisms have invaded their
tissues." Concerning the oyster we know even less. The apparent de-
crease in the numbers of Dermocystidium in oysters during the winter
(Mackin 1953^ Ray I958, Andrews and Hewatt 1957) or of Nematopsis
when infected oysters are transplanted to clean water (Feng 19 58) fur-
nish us with instances which could be investigated for heightened re-
sistance to subsequent infection.

That so few exajnples of acquired resistance are known among
invertebrates may even be quite logical. Because of their relatively
short generation times, their usual small size and often enormous re-
productive capacities, subsequent epizootics would be much more likely
to be circumvented by the appearance of resistant stocks through na-
tirral selection, as in "Malpeque disease." Even with very high mor-
tality rates a residual stock of animals under favorable conditions
later might repopulate an area. Indeed, this seems to be our chief
hope in the present catastrophic mortalities of oysters in New Jersey
and has been given consideration in the discussions of mortalities
caused by Dermocystidium (Andrews and Hewatt I957).

If this reasoning is adequate to explain the lack of evidence
for the occiirrence of acquired resistance in most of the invertebrates,
perhaps those invertebrates with a long life span, like Limulus , should
be investigated more fully as likely hosts capable of demonstrating
acquired resistance.

Since acquired resistance must yet be demonstrated for the
oyster it seems unnecessary to discuss this topic further in this
paper .

CONCLUSIONS

It is fair to state that although invertebrate immunology has
been under study for a long time, little precise knowledge is avail-
able for the oyster and its relatives . Since some of the mechanisms
of resistance reported here for invertebrates may not even occur in
molluscs and more specifically in oysters (like melanin deposition).

-15-

I am concerned that I should not mislead you. The only satisfactory
conclusion to draw is that much painstaking effort is needed to fur-
nish the information we so greatly need at this time .

ACKNCWLEDGMEM'S

I am deeply indebted to Dr. Thurlow C. Nelson and Dr. Harold
H. Haskin for inspiration, encouragement, counsel and criticism over
the years spanning my interest in this work. Gratitude also goes to ,
the former and present students of all three of us; they have bene-
fited us in many ways by their work and enthusiasm. Those needing
special mention here are Drs . M. R. Tripp and K. 0. Phifer, Messrs.
L. M. Adelson, W. J. Canzonier, A. F. Eble and S. Y. Feng.

The research reported in this paper was supported in part by
a research grant (E-781) from the National Institution of Allergy and
Infectious Diseases, Public Health Service, and in part by funds from
the Oyster Research Laboratory, Agricultural Experiment Station, Rut-
gers - The State University.

LITERATURE CITED

Andrews, J. D. and W. G. Hewatt, 1957- Oyster mortality studies in
Virginia. II. The fungus disease caused by Dermocystidium
marinum in oysters of Chesapeake Bay. Ecol. Monogr. 27:1-26.

Bengston, I. A. 192if. Studies on organisms concerned as causative
factors in botulism. Hyg. Lab. Bull. 132:1-96.

Bissett, K. A. 19^7- Bacterial infections and immunity in lower ver-
tebrates and invertebrates. J. Hyg. 45:128-135-

Cameron, G. R. 193^. Inflajiimation in the caterpillars of Lepidoptera.
J. Pathol. Bacteriol. 38:^^1-466.

Chorine, V. 1931. L'immunite chez les insectes. Bull. biol. France
Belg. 65:291-393.

Cort, W. W., L. Oliver, and 3. Brackett. 19^1. The relation of physid
and planorbid snails to the life cycle of the strigeid trema-
tode, Cotylurus flabelliformis (Faust, I917) . J. Parasitol.
27:437-448.

K. L. Hussey, and D. J. Araeel. 1959a- Studies on a micro-

sporidian hyperparasite of strigeoid trematodes . I. Prevalence
and effect on the parasitized larval trematodes. (in Press).

K. L. Hussey, and D. J. Ameel. 1959b. Studies on a mi-

crosporidian hyperparasite of strigeoid trematodes. II. Ex-
perimental transmission. (in Press).

-16-

Cuenot, L. 191^. Les organes phagocytaires des Mollusques. Arch.
Zool. Exp. Gen. 5^+: 267- 30 5.

Falk, I. S. 1928. A theory of microhic virulence. In "Newer Know-
ledge of Bacteriology and Immunology," edited by E. 0. Jordan
and I. S. Falk, Univ. Chicago Press, Chicago, pp. 565-575 •

Feng, S. Y. 1958* Observations on distribution and elimination of

spores of Nematopsis ostrearum in oysters. Froc . Natl. Shell-
fish. Assoc. 48:162-173.

1959a'. Defense mechanism of the oyster. Bull. New Jer-
sey Acad. Sci. kil^J.

1959^ • Unpublished manuscript report.

and W. Canzonier. 1959- Unpublished manuscript report.

George, W. C. 194l. Comparative hematology and the functions of the
leucocytes. Quart. Rev. Biol. 16:426-439,

1952. The digestion and absorption of fat in lamelli-

branchs. Biol. Bxill. 102:118-217.

Grun, J. 1958. A study of experimental leishmaniasis in the mouse,
Mongolian gerbil, hamster, white rat, cotton rat and chin-
chilla. Ph.D. Thesis, Rutgers Univ. 82 pp.

Heimpel, A. M. 195^- A strain of Bacillus cereus Fr. and Fr. patho-
genic for the Beech sawfly, Pristophora exichsonii. Can. Ent.
86:73-77.

Hewitt, R. D., A. P. Richardson and L. D. Seager. 1942. Observations
on untreated infections with Plasmodixjm lophurae in twelve
hundred young white Pekin ducks. Am. J. Hyg. 36:362-373.

Huff, C. G. 1930. Individual immunity and susceptibility of Culex

pipiens to various species of bird malaria as studied by means
of double infectious feedings. Am. J. Hyg. 12:424-44l.

1935- Nat\iral immunity and susceptibility of culicine
mosquitoes to avlaji malaria. Am. J. Trop. Med. 15: ^27-^3^.

1940. Immunity in Invertebrates. Physiol. Rev. 20:68-88.

Liebman, E. 1946. On trephocytes and trephocytosis; a study on the

role of leucocytes in nutrition and growth. Growth 10:291-329.

Logie, R. R. 1958. Epidemic disease in Canadian Atlantic oysters

(Crassostrea virginica) . Ph.D. Thesis, Rutgers Univ. l43 pp.

-17-

Mackin, J. G. 1951- Histopathology of infections of Crassostrea vir-
ginica (Gmelin) by Dermocystidium inarinum, Mackln, Owen and
Collier. Bull. Mar. Sci. Gulf Carib. 1:72-87.

1953. Incidence of infection of oysters by Dermocystidlimi

in the Baratarla Bay area of Louisiana, Natl. Shellfish. Assoc.
Convention Adc'jesses, 1951> PP- 22-35 •

McConnell, E. and L. K. Cutkomp. 195^. Studies with Bacillus thurin-
giensls in relation to the E\iropean corn borer. J. Econ. Ent.
i^7:107if-1082.

Metalnlkov, S. 1920. Immunite naturelle et acquise chez chenilles de
Galleria mellonella. Ann. Inst. Pasteur 24:888-.

1921. Immunite naturelle et acquise chez les chenilles
de Galleria mellonella. Ann. Inst. Pasteur 35:363-.

and V. Chorine. 1930. Etude sur 1' immunite naturelle

et acquise de Pyrausta nubilalis. Ann. Inst. Pasteur kh-.ZJ'^-
278.

Metchnikov, E. I905. Immunity in infective diseases. Translated from
the French by F. G. Binnle. Cambridge Univ. Press. 591 pp.

Michelson, E. H. 1957. Studies on the biological control of schisto-
somebearing snails. Predators and parasites of fresh-water
Mollusca: A review of the literature. Parasitology h'J:kl3-
426.

1958. Observations on an acid-fast pathogen of fresh-

water snails. Unpubl.

Needier, A. W. H. and R. R. Logie. 1947. Serious mortalities in

Prince Edward Island oysters caused by a contagious disease.
Trans. Roy. Soc. Can. kl, Ser. Ill; sec. 5, pp. 73-89-

Newton, W. L. 1952. The comparative tissue reaction of two strains
of Australorbls glabratus to infection with Schistosoma man-
soni. J. Paras itol. 38:362-366.

1953. The inheritance of susceptibility to infection

with Schistosoma mansoni in Australorbls glabratus . Exp.
Paras itol. 2:242-257.

Ray, S. Personal Communication.

Read, C. P. 1958. Status of behavioral and physiological "resistance,
In symposium on "Resistance and Immunity in Parasitic Infec-
tions." Rice Inst. Pamphl. 45:36-54.

-18-

Rogers, D. E. 1958- The cellular management of bacterial parasites.
In "The Pasteur Fermentation Centennial I857-I957." pp. 6I-73.
Chas. Pfizer & Company, Inc., New York. 207 PP«

Salt, G. 1955- Experimental studies in insect parasitism, VIII.
Host reactions following artificial parasitization. Proc.
Roy. Soc. London, Ser. B. l4i+: 38O-398.

1956. Experimental studies in insect parasitism. IX.

The reactions of a stick insect to an alien parasite. Proc.
Roy. Soc. London, Ser. B. 1^+6:93-108.

Schneider, T. A. •1951- Nutrition and resistance-siJisceptibility to in-
fection. Am. J. Trop. Med. 31:17^-182.

» C
Shirodkaft, M. V., F. B. Bang, and A. Ijarwick. I958. Antibacterial

action of Limulus blood in an in vitro system. Biol. Bull.

115:3^1.

Springer, G. F., R. E. Horton, and M. Forbes. 1959. Origin of anti-
hurflan blood group B agglutinins in germfree chicks. Ann. New
York Acad. Sci. 78:272-275.

Stauber, L. A. 1950. The fate of India ink injected intracardially
into the oyster, Ostrea virginlca Gmelin. Biol. Bull. 98:
227-241.

1958' Host resistance to the Khartoum strain of Leish-

mania donovani . In symposium on "Resistance and Immionity in
Parasitic Infections." Rice Inst. Paraphl. ^5:80-96.

Steinhaus, E. A. 19^6. Insect microbiology. Comstock, Ithaca, New
York. 763 pp.

19^9- Principles of insect pathology. McGraw-Hill, New

York. 757 pp.

Taliaferro, W. H. 1929- The immunology of parasitic infections.
The Century, New York, ij-l^l pp.

Terzian, L. A., N. Stabler, and P. A. Ward. 1952. The effect of an-
tibiotics and metabolites on the Immunity of mosquitoes to
malarial infection. J. Infect. Dis . 90:ll6-130.

Tripp, M. R. 1958a. Disposal by the oyster of intracardially injected
red blood cells of vertebrates. Proc. Natl. Shellfish. Assoc.
1+8:11+3-1^7.

19581" • Studies on the defense mechanism of the oyster.

J. Parasitol. kk {k, Sec. 2): 35-36.

-19-

1958c. Studies on the defense mechanism of the oyster.

Crassostrea virginica. Ph.D. Thesis, Rutgers Univ. 131 PP.

Wagge, L. E. 1955. Amoebocytes. Intern. Rev. Cytol. if:3l-78»

Wagner, M. 1959. Serologic aspects of genafree life. Ann. New
York Acad. Sci. 78: 261-272.

-20-

MORTALITIES OF OYSTERS

J. G. Mackin^

Department of Oceanography and Meteorology

Agricult\Jiral and Mechanical College of Texas

College Station, Texas

INTRODUCTION

Mortality may mean many things. It can be either the basic
annual mortality, below which losses never drop, or the peak waves of
mortality which periodically sweep the oyster beds and which account
for losses of high percentages of the total population. Or it may mean
an increasing annual loss resiilting in complete extinction of oysters
over considerable part of the original range. It is hoped to discuss
all of these types, and as far as possible to analyze the factors which
produce the mortalities. Additionally it is aimed to point out recog-
nition characteristics of each of the general types to be discussed.
There is ample illustrative material. As pointed out by Gross and
Smyth (1946) decimation of oyster populations is one of the most im-
portant biological phenomena of the first half of the twentieth century,
and is worldwide in scope.

Studies of mortality are essentially studies of one phase of
population ecology. On a broad basis there are only two phases of
population studies . One is the exajnination of all of those forces
which tend to build up and maintain a population, the other focuses
attention on all agencies which tend to reduce populations (produce
mortalities) or to prevent them from exploding. Ideally these two
classes of forces should balance each other to maintain a static popu-
lation. Actually any population, at any one time, is either crescent
or it is declining, the two processes alternating ajid progressing by
a series of waves. These waves may average out, over a long period of
time, in an increase, or in a decline. For oysters, decline has been
the rule.

In the following sections, the most important types of mortal-
ities classified as to origin are discussed. Emphasis is placed on
those types which are generally classed as of "unknown origin." The
literature is full of these enigmas, and it is this type which has
produced the most spectacular declines in population of oysters, some-
times extending over many years, and destroying entire industries over
certain areas . Mortalities deriving from predation are subordinated
in this paper, not because they are unimportant, but because symposiums
of rather exhaustive nature have been organized and completed in the

1 Contribution from the Department of Oceanography and Meteorology,
Agricultural and Mechanical College of Texas, Oceanography and Mete-
orology Series .

-21-

past, which dealt with the principal predators. Oyster biologists
quite generally have dedicated a very high percentage of their efforts
to study of problems of predation, and the author believes that there
is less reason for extensive treatment of this phase of oyster popu-
lation studies at this time, important as it is.

TYPE I. MORTALITIES CAUSED BY EXTREMES OF THE
NATURAL PKYSICAL ENVIRONMENT

Examples are extremes of cold and heat, low salinity caused
by floods, and hurricane damage. Cases of mortalities of this type
are numerous and nearly everyone can recall several. I chose the
mortality in the Mississippi Sound caused by flood of the Mississippi
in 19^5 as a prime example (Gunter 1950^ Owen 1950). Flood crests of
the Mississippi rose so high that the Army Engineers opened the Bonne
Carre Spillway locks in early 19^5- Floodwater from the Mississippi
was shunted through Lake Fonchartrain and thence into Lake Borgne and
Mississippi Sound. Water was fresh or near-fresh over the oyster beds
of Mississippi Soiond from Grand Island (not the same as Grand Isle)
eastward for about 10 miles. The great oyster-producing areas of the
Louisiana marsh were similarly affected in the northernmost part. The
kill was 100 per cent through much of the area but graded off to zero
in the more easterly and southerly parts of the Sovond and marshes .
Many other cases could be cited: The Santee floods of South Carolina,
(Lunz 1936) upper Chesapeake area affected by floods of the Susquehaxma
(Beaven 19^6), and many others. Details of such losses are too well
known to need further description.

In some areas, mortalities by flood and low salinity are not
so drastic. Marginal areas which lie on the landward side (or river-
ward side) of all estuaries are annually threatened with loss to fresh-
water kill. In some years loss is 100 per cent, in others zero, but
in most years a certain percentage of the oysters are lost, but the
number fluctuates between the maximum and the minimum. Oysters planted
in marginal low-salinity areas are spared most sources of mortality
other than low salinity.

Characteristics of losses to extremes of physical environments
are as follows:

(1) The duration of the mortalities is apt to be sharply limited.

(2) Recovery is rapid and complete and the oysters rebound to
a level of population higher than that prior to the flood (because the
floods also destroy predators and foci of disease, and reduce fouling).

(3) The effects of extremes of physical environment extend to
many marine forms other than oysters, and the oyster community as a
whole is affected to greater or lesser extent.

-22-

(k) The mortalities are density independent.

(5) The mortalities are quite apt to be seasonal, since ex-
tremes of temperature, floods, etc., are usually seasonal.

There may be difficulty in some instances in distinguishing
between mortalities caused directly by heat and cold, or other natural
environmental extremes, and those caused by disease. However, the
application of other yardsticks should eliminate confusion. Disease
is selective and spotty in occurrence, and is density dependent, while
the opposite is true of environmentally induced mortality due to phy-
sical extremes.

TYPE II. MORTALITIES DUE TO DISEASE

Of the greatest interest to present studies are those mortal-
ities known to have been caused by disease or in which disease is sus-
pected to be the cause. These cases are fairly numerous and the
mortality waves attributed to disease may affect wide areas over the
host range. Disease is known to have caused high rates of mortality
of oysters in Europe, Australia, Japan, and North America for well
over a half century. Generally the study of diseases of oysters in
the United States has lagged far behind other studies. In spite of
that, much has been accomplished in the past ten years. This has
been the resiilt of development of method, and a better understanding
of the nature of the diseases of oysters. In earlier years, with few
exceptions, workers assumed that etiologic agents of disease in oysters
must be bacteria, and that these bacterial parasites could be isolated
by the time-honored methods used in hvmian diseases . Crude methods
were used in testing for pathogenicity, with little or no regard for
epidemiological factors . The crudity of the methods was primarily
responsible for failures to establish etiological relationships. Often,
even when disease was suspected, no efforts at isolation of a pathogen
were made. The science of the study of diseases of marine inverte-
brates is lagging at least a half century behind disease studies in
insects and crop pleints .

It seems certain at the present time that all oyster-producing
bays are endemic areas for one or more diseases . It would be exceed-
ingly strange if this were not true. It has been the general opinion
until recent years that oysters were somehow unique in that diseases
are rare. A great preponderance of evidence now indicates that not
only are bivalve molluscs frequent hosts for pathogens, but that they
are regularly parasitized by a lonique group of low fungi, which are so
far off the beaten path of scientific inquiry that knowledge of taxo-
nomy, relationship, life cycles, physiology, and epidemiology is only
beginning to be accumulated. An entire new field of research is being
opened in oyster biology. This is not intended to suggest that only
this low group of fungi is important. Diseases involving bacteria
and protozoa are known, ajid the oyster is afflicted with a wide variety

-23-

of diseases. It is predicted that viral diseases will be found in
the near future.

It is proposed to examine here several of the mortality waves
attributed to disease and to analyze the factors peculiar to these
waves of high mortality. It is believed that a study of representative
types may be of value.

A. The Mortalities of 1919-192^ on the coast of Europe

Beginning in 1919^ a series of mortalities decimated oysters
(Ostrea edulls) in various parts of Europe. Orton (192U) investigated
this mortality wave and wrote two voluminous reports . Mortalities
occurred in Italy, Atlantic coast of France and Holland, Ireland, and
south England. The time of deaths is believed by Orton to have been
mainly suimner and spring, but a careful reading of accounts shows that
the data given reflect periods when the mortalities were noted and not
when they actually occurred. There are frequent mentions of mortali-
ties continuing into the winter. This part of the accounts is con-
fused, as are the reports of the numbers of oysters dying. It is my
opinion that "unusual" mortalities were recorded which were far from
being out of the ordinary, and were observed mainly because everyone
was, at the time, mortality conscious. Be that as it may, it seems
certain that the mortalities continued through several years; they
began in either 1919 or 1920, and in some areas had not subsided until
the mid-1920s. Most of the deaths appear to have been in warm weather,
but some may have been in the fall, winter, or spring. The mortalities
in Italy probably had no real relation to those of the Atlantic coast
of Europe. Spottiness of locale of mortalities was marked.

It was at first believed that munitions diimped into the sea
after World War I were responsible for the mortalities. Oil from
siinken tankers was also considered as a possible cause, as was arsenic.
These hypotheses were eliminated by experimentation, and the pollution
hypothesis generally discarded. There were also those hypotheses which
involved weather extremes as directly responsible for the mortalities,
and lastly, disease was considered. Study of weather data led Orton
to conclude that extremes of weather were not responsible for the mor-
talities and he was not able to find parasites in the oysters. As
stated, crude experiments with bacteria were negative, but it is cer-
tain that there was nothing in the results which actually ruled out a
bacterial parasite. Incidentally, some of Orton 's figures show what
appear to the author to be intracellular stages of Hexamita in the
tissues. However, if Orton was correct, and the mortalities were con-
centrated in svmmier months, Hexamita probably was not a cause of the
mortalities. He was handicapped in that his investigations were al-
ways several months behind the mortalities . It seems probable that
he made most of his studies of disease on some other than the one
operative when oysters were dying at the greatest rate .

-2U-

Although Orton stated that he failed to find a cause of these
mortalities, it is clear that he believed they were due to disease.
He listed a number of histopathologies characteristic of "hockley"
(sick) oysters and in his book on oyster culture (1937) referred to
the mortalities as an "epidemic". Dollfus (1922) listed these mor-
talities as caused by a disease of unknown origin. Korringa (19^7)
referred to the mortalities in France and England in I92O-I92I as
"mysterious and catastrophic", and later (l952) stated that what is
known indicates that they were caused by a disease. The mortalities
are so treated here, with the reservation that it is probable that
there was more than one disease, and that in the efforts aimed at
completeness of data, deaths from causes other than disease were intro-
duced to compoimd the very evident confusion. For example, Korringa,
although he accepted the disease hypothesis so far as mortalities of
French oysters were concerned, believed that the Dutch mortalities
occurring at the same time were due to low salinity. Gaarder and
Alvsaker (l9^l) fiorther befogged the issue by suggesting that all of
these oysters starved to death.

Groping through the interminable non-pertinent details, the
author believes that the following things are probably true of the
European oyster mortalities of 1919-1925:

(1) A great mortality of oysters occurred on the Atlantic
coast of Europe and the south coast of England, and the mortalities
peaked in at least two years, I92O-I92I.

(2) The mortalities were concentrated in summer. The two
summers involved were warmer than usual, but not as warm as some
years in which no "abnormal" mortalities occurred.

(3) Affected oysters showed strongly marked histopathologies,
the most important of which was probably myolysis. However, oysters
tended to die fat. Other histopathologies included severe cellular
reactions, reduction of liver pigmentation, reduction of Leidig cell
tissue, excess development of mucoid glands in epithelia, abscesses,
and degeneration of the gonads .

{h) Mortalities were spotty in distribution, affected areas
and apparently unaffected areas sometimes lying close together.

(5) The mortalities followed a long period of ' neglect of
oyster beds during World War I.

(6) The mortalities failed to affect organisms other than oys-
ters , Even the Portuguese oyster (Crassostrea angtilata) was not af-
fected.

(7) The mortalities were superposed on a background of regu-
larly occurring "normal" mortality variously estimated at 10 to 25
per cent. Certain studies cited by Orton tend to indicate that the

-25-

background mortalities were grossly underestimated, as they nearly
always are in every great mortality.

(8) There are data which indicate that there were earlier
mortalities which at least approached the severity of the I92O-I92I
wave. Circumstances indicate very similar conditions for some of
these. Others were definitely winter mortalities which probably
should be kept separate from those of high temperature periods.

(9) Oyster stocks apparently never recovered completely from
the I92O-I92I mortalities, suggesting a higher level of endemic dis-
ease following the epidemic.

B. The Australian "winter disease"

Roughley (1926) described a disease of oysters (Crassostrea
commerclalls) occurring in the George's River of New South Wales,
Australia. Oysters in this estuary had for 8 or 9 years previous to
192^4- (the year of Roughley 's study) died in varying numbers. From
Roughley 's accoimt the following facts relating to the mortalities
may be stated:

(1) The mortalities occurred in late winter and spring, and
were accentuated by unusually cold weather.

(2) Although mortality was associated with cold weather Roiighley
showed by several studies that cold was not a direct cause of death,
and indeed those oysters exposed to lowest temperatures were not af-
fected to the extent that others were.

(3) The mortalities were associated with cessation of feeding
activity which occurs in C. commercialis at 10° C.

(k) The sick oysters showed various histopathological conditions
when sectioned and stained. Diapedesis was marked, the digestive gland
was pale, and some oysters had severe ulcerations and abscesses, espe-
cially in the gonadal region. Myolysis was marked, and in some oysters
the gills disintegrated.

(5) Most mortalities were in the lower half of the intertidal
zone although not confined to that level.

(6) Winter disease was not confined to the George's River, but
appeared intermittently elsewhere in southeast New South Wales.

(7) Plankton studies showed that a normal fauna and flora was
present while oysters were dying nearby in large numbers. No mention
was made of mortalities of animals other than the one species of
oyster. Oatrea angasl inhabits the same region but is not considered
to be a commercial oyster.

-26-

(8) Oysters died "fat" in most cases.

(9) Mortalities were "spotty." They might cover only a small
section of a bed, or occiar on one side of an estuary and be absent on
the other, or involve only some sections of one part of an estuary.
Mortality rates also varied greatly in different beds.

(10) Different years differed radically in the extent of the
mortalities. Death rates might be high in one winter, and quite low
in others.

Roughley failed to find the cause of these mortalities, but
believed that they were caused by disease of bacterial origin. It has
been my privilege to study Orton's slides of diseased oysters as well
as slides made from oysters dying of winter disease in Australia in
recent years. Some of these latter (and perhaps all) were infected
with Hexamita. The histopathologies were identical to those described
for oysters (0. edulis ) from Holland with "pit disease" (Mackin, Kor-
ringa, and Hopkins I952). Oysters in holding basins in Holland are
subjected to low temperatiires (5° C). The histopathologies in these
oysters also correspond with those observed in Ostrea lurlda from
Puget Sound in Washington.

Winter disease of Australian oysters was chosen as an example
of an intermittently recurring mortality producer which apparently is
world-wide in distribution. While Hexamita is associated with the
disease, there is some evidence that there may also be another simul-
taneously operative disease, or there are phases of Hexamita attack
which have not as yet been definitely associated with the parasite.
There may be several species of Hexamita involved.

A careful reading of much of the European literature dealing
with the decline of the Ostrea edulis industry indicates that devastat-
ing losses of oysters associated with cold winters have played a con-
siderable part in elimination of the species from certain areas as,
for example, parts of Scotland. Epidemiological data suggest that
cold was a factor only when coupled with other agencies. A very simi-
lar picture is found in Washington (State), where 0. lurida has been
eliminated from certain areas for no known cause, except overfishing.
Overfishing may explain radical reduction on natiural beds only, but
cannot explain failiire to recover when fishing ceases over a period
of years .

C. Fungus disease caused by Dermocystidium marinum.

This is by far the best documented of the diseases. Because
the details are relatively well known, only a very brief analysis is
here presented. The major factors are as follows:

(1) The caiisative agent is a low fungus, Dermocystidivmi marinum.

-27-

closely related to the Synchytriaceae . General outlines of the life
cycle are known with some details of the biochemistry of the parasite.

(2) The disease caused by this fungus produces annual summer

epidemics in its host (Crassostrea virginica), the severity of which

increase southward in its range, sometimes reaching high peaks, e.g.,

death rates of 90 per cent in a single summer in Louisiana. Various

environmental factors modify these death rates in a quite regular

manner .

>

(3) The known range is apparently from New Jersey to Texas,
with some areas of low concentration or even complete absence scat-
tered along the coast within the range.

(k) Scouring of bays by fresh water, and low salinity generally
may control the disease in certain years. Conversely, a build-up of
high salinity and high temperature over several years results in de-
cimation of oyster populations.

(5) Dense planting of large numbers of susceptible seed oysters
produces maximiom conditions for development of the fungus . Under- con-
ditions of high temperature, high salinity, and dense pop\ilations of
susceptible oysters, the maximum losses occur.

(6) Immature oysters under field conditions are resistant to
infection, and susceptibility increases with age. In Louisiana, mar-
ket oysters two years old and older are most often victims of attack.
Resistance of young oysters probably is due to lesser feeding volumes
(Andrews and Hewatt 1957) and possibly a tendency of yoimg oysters

to reject infective cells. Epidemics sweeping beds of older oysters
and leaving freshly planted young oysters barely touched are often
observed in Louisiana.

(7) There is evidence that resistant strains of Crassostrea
vlrginica exist.

(8) There is also some evidence of acquired immunity.

D. Characteristics of mortalities caused by disease.

A study of the conditions surrounding mortalities of oysters
produced by disease permit the formulation of some general character-
istics of this tj'pe of mortality. There are as follows:

(1) Mortalities due to disease are almost always specific,
that is, one species of oyster, or possibly two, may be affected, but
the oyster community as a whole is unaffected. It is, of course, not
impossible that a disease-producing organism might attack a variety
of related hosts, but the commiinity as a whole will be unaffected.

-28-

(2) Mortalities due to disease are almost always seasonal.
Peaks of mortality are more or less sharply defined in limited periods
of the year, while low-level losses may spread into other seasons .

(3) Peaks of mortalities may be ciomulative over several years,
sometimes building up to a peak year and then declining. These cyclic
effects may sometimes be correlated with climatic cycles.

{k) The rates of mortality in epidemics nearly always are den-
sity dependent. That is especially true if there is only one host
(the oyster) and no free-living stages. In cases where alternate hosts
are involved, abundance of the parasite may be controlled by the alter-
nate host, or by conditions obtaining during free-living stages . Shell
disease of Ostrea edulis is such a case, where the abundance of the
fungus causing the disease is controlled by the abundance of dead shell
of molluscs other than oysters. But most disease-producing parasites
of oysters seem to have only one host and free-living stages exist as
spores which may not reproduce, but simply bridge an intervening ecolo-
gically unfavorable period for the parasite.

(5) Recovery from the effects of disease is slow. Introduction
of new host susceptibles as seed in an area, may largely counteract the
natural agencies working toward reduction of peaks of mortality. Den-
sity reduction and elimination of imports will speed up recovery from
disease-produced mortalities .

(6) Mortalities from disease are "spotty", i.e., they affect
different beds in one locality in a seemingly haphazard manner, esp-
ecially in the beginning of a cyclic wave of mortalities. Unequal
effect on different plantings in the same locality will continue be-
cause of difference in the local rates of elimination of susceptibles
and because of varying densities of plantings, variations in suscepti-
bility due to different origins of seed, and perhaps other factors .

(7) Survivors of an epidemic generally are found to be in good
physiological condition. They survive either because of chance escape
or because of individual resistance. In either case growth and re-
productive capacity will be unimpaired. However, those oysters attack-
ed by disease but not becoming fatalities may be found to be variously
affected.

(8) Sections of oysters dying of disease will show character-
istic histopathologies which will contrast markedly with the normal
tissues of siirvivors in general. Some of the survivors, infected but
not so heavily as to cause death, will inevitably show developmental
stages in the histopathological conditions, and often the parasite it-
self will be found, also in developmental stages of infection. But
most survivors will be largely free of characteristic histopathologies.

The author believes that, of all causes of mortality, disease

-29-

ranks first. Disease not only produces spectacvilar major declines in
production, but also accoimts for much "background" mortality. Studies
of disease in invertebrates have lagged behind those of commercially
valuable vertebrates, with the exception of those diseases of insects,
which form a segment of research well worth review. Best general works
on Insect diseases are the volumes by Steinhaus (19^6, 19^9)*

E. Some basic principles of epidemiology

The development of epidemics (technically epizootics) is de-
pendent on three things. These are (l) variance in virvilence and in-
fectivity of the pathogen, (2) variance in the susceptibility of a
population, and (3) the effectiveness of the methods of transportation
of the parasite. All three of these are influenced by variation in
the physical and chemical environment, and all vary with time. Thus
epidemics develop, or fail to develop, develop partially, develop fully,
or terminate because of an almost unlimited interaction of variables,
which progressively change from the beginning to the end of an epidemic.
Without going into those factors having to do with virulence, infect-
ivity, and immunity, the population composition factors effective in
epidemic development are discussed briefly.

^y popiilation of animals is made up of several well defined
categories of individuals so far as disease is concerned. These are
as follows :

(1) Susceptible individuals, i.e., those which can be infected
by a pathogen, and which will develop typical disease following infec-
tion.

(2) Immime non-carriers, those individuals which, if they can
be infected, do not develop disease, and which rid themselves com-
pletely of the pathogen.

(3) Immune carriers, which do become infected and harbor the
pathogens but do not react with the typical disease syndrome.

(k) Infected individuals, which will later develop disease
(latent infections) .

(5) Cases with typical disease.

(6) Atypical cases. (For a full discussion of these six cate-
gories, see Topley and Wilson 1936).

If a case of oyster mortality is considered in which the dis-
ease is a new import, and has not been endemic in the area in question,
there are only two types of individuals in the host population: the
susceptibles and natural Imraunes. The degree of development of an
epidemic under such conditions would depend on the relative numbers

-30-

of these two categories, with the likelihood that innnunes will be
scarce. Given proper density of the host population, epidemics under
these conditions are apt to he very severe.

However, after a disease has become endemic, all six categories
will appear and as the epidemic progresses, their relative numbers
change. Susceptibles become fewer, and with development of induced
immunes, the percentage of the population capable of becoming infected
decreases. With significantly high death rate, the population of sus-
ceptibles becomes more and more thinly scattered with proportionately
greater difficulty in transmission of infective elements. The epidemic
is thus self -limiting.

Introduction of a new host population into the area, either by
natural accretion of spat, or by planting, will again tip the scales
in favor of the disease. As a result, in the face of an endemic dis-
ease, an oyster population is itself self limiting. When density of
the population of susceptibles reaches a point where transmission be-
comes easily accomplished, a new epidemic is triggered.

The matter of host susceptibility varies very greatly with
changes in the external environment. For example, in recent studies
on Dermocystidium marinum in Louisiana it was fo\md that deaths per
thousand cases increased from 23 in April to 207 in August, a result
of increased temperature; this was an overall increase of more than
900 per cent (Mackin and Sparks 1959) •

The effect of the introduction of non- Immunes into an endemic
area also was shown in the study cited above. One thousand oysters
from non-endemic territory for Dermocystidium marinum were placed be-
side 1000 oysters in the endemic area. Culture tests of samples showed
that 690 out of the thousand endemic . oysters were in one or the other
of the groups of carriers, ranging from a few typical cases to numerous
lightly infected oysters. The non-endemic oysters, in the following
summer epidemic, all became infected with fungus disease and developed
the highest rate of mortality to disease ever observed, kjQ deaths per
1000 population in one month's time at the peak period, while the en-
demic oysters attained only a rate of I85 deaths per 1000 population
in the peak month. Just what part acquired immunity played in this is
not certain. Other studies have showed that a reimiant population left
after a severe epidemic may develop as high death rate due to disease
in the following summer as the original population attained in the ini-
tial subjection to disease.

TYPE III. MORTALITIES DUE TO STARVATION

This category is discussed more because the literature con-
tains supposed cases, than because of a personal belief that mortalities
due to starvation occur on a large scale. There are no cases supported

-31-

by conclusive data. On the basis of certain studies and observations
made in past years the author believes that it is almost impossible to
starve an oyster to death in the natural habitat.

Hoek (1902) studied mortalities of oysters in Holland which
occurred in the latter years of the past century. The mortalities
were accompaaiied by failure of oysters to fatten and grow properly.
Hoek concluded that the oysters had been starved to death, and that
for any given area there would be a maximum number of oysters which
could successfully be grown without starvation. For the limited area
of Dutch ground under study, the number was stated to be 100 million.
Korringa (19^7) agreed with Hoek that certain mortalities of Dutch
oysters were due to starvation.

The bases for the decision that Dutch oysters starved to death
when the populations became too great were (l) the large population
Itself, and (2) the fact that the oysters failed to fatten properly.
Other than these two reasons there seems to be no basis for this theory.
But mortality following over-population can be caused by disease.
Thinness, and even death of oysters can be a result of over-concentra-
tion of food (Loosanoff and Engle 19kk) , or a result of disease. The
possibility of initiating a significajit mortality by means of plankton
blooms due to overfertilization, as shown for the Great South Bay area,
is more impressive than is the starvation theory. Phytoplankton has
been shown to have toxic properties in some cases, and may control a
habitat by means of metabolites . In any event, if oysters are starved
to death, the mortalities must necessarily involve other plankton feed-
ers and cannot be so selective that the oyster is the only organism
affected. No data showing a similar effect of the hypothetical lack
of food causing starvation of Dutch oysters on other general plankton
feeders was presented by Korringa or Hoek, and the theory must be con-
sidered to be unproved.

In the early part of the century, there were two years (1905
and 1906) in which oysters in Louisiana were very poor, and in fact
largely unmarketable. This condition was attributed to lack of food
(Oyster Commission of Louisiana I906) . H. F. Moore of the U. S.
Bureau of Fisheries investigated this matter, but failed to find a
deficiency of food plankters in the water (Moore and Pope I9IO). The
periodic failure of oysters to fatten properly is characteristic in
many parts of the world and is not a result of so simple a factor as
lack of food. It is believed that lack of metabolites, or over-con-
centration of metabolites is a better hypothesis, but this too remains
to be demonstrated.

TYPE IV. MORTALITIES RESULTING FROM SPATIAL COMPETITION

Korringa (l9^7) described a case of oyster mortality due to
competition with the slipper limpet, Crepidula fornicata, in Dutch

-32-

waters. This Crepldula was an import from the United States, and
shortly after importation developed tremendous populations on old
cockle shells. They were so numerous that space normally utilized
by spat for setting was preempted, resulting in setting failure.
Korringa ranked the crisis in the industry produced by the sD.ippers
along with that caused by shell disease. Apparently both crises were
met by cleaning all beds in the affected area down to the bare mud.
Use of Crepidula on a commercial basis during World War II completed
the counter measures against the limpet.

Space competition between yoiong oysters and various foulants
is common everywhere. Barnacles are perhaps the most important of
these competitors, but in some areas encrusting Bryozoa, serpulids,
or others may become important. All this usually may be classed as
background mortality to be expected in average years . Such competition
may sometimes be helpful when set of young oysters is so plentifiil as
to be embarrassing, as it is in some parts of the Gulf Coast. New
Englanders will find that difficult to understand, but it is a very
real handicap in the South.

TYPE V. MORTALITIES DUE TO PREDATORS

Along with mortalities due to disease, predator-produced losses
take front rank in importance. Oysters have an unusual number of the
most effective kinds of predators . At least several of these are ef-
fective in mortality in any area where oysters are grown. Most of
them are well known, but a few have only recently been described.
There are five major groups of animals which prey an oysters:

(1) Fishes. Most important of the fishes are the drum, sheeps-
head, and skates or rays . Locally any of these may produce major dam-
age, especially to young oysters on newly planted beds. Fishes as
oyster predators seem to be more common in subtropic areas than they
are in temperate zones . Predation due to fishes is apt to be very
local. In Louisiana, oystermen, lontil very recently, were in the habit
of fencing oyster beds against this type of predation. It is believed
that much of the damage attributed to fishes actually may be due to
other causes, especially crabs.

(2) Crabs. Any of the larger crabs may destroy oysters, de-
pending on the size of the oysters, size of the crabs, environment,
etc. However, the large Cancers and their relatives are most effective
in destruction of oysters. On the Gulf Coast the most active is Menippe
mercenaria, a crab with very heavy and strong claws, which caji crack
large, heavy shelled oysters (Menzel and Hopkins 195^). These authors
found densities of crabs of 3500 to an acre of oyster reef in parts of
Louisiana. They showed that these crabs could kill spat at the rate

of 10 per day, and all sizes of oyster were killed in experimental
tests at the overall rate of 219 oysters per crab per year. Extended,

-33-

these data could be taken to show that it is possible for an average
population of stone crabs (Menippe) to destroy 7^6, 500 oysters per acre
per year. If only l/3 of these oysters were market-size this would
mean around 800 Louisiana sacks, or about the maxira\jm capacity of oys-
ter bedding grounds to hold oysters . Experimental figures may not be
projected directly to field conditions and the normal natural kill is
unquestionably only a fraction of the experimentally demonstrated pos-
sibilities; nevertheless, losses to the large crabs are unquestionably
very much heavier than generally recognized. The extent of damage by '
crabs is very difficiilt to measure because of fragmentation of the
shell by the predator.

(3) Predaceous snails . Mortalities of oysters due to preda-
ceous snails are probably better known, and certainly have had wider
publicity and have been subjected to more research than mortalities of
any other kind. On the Atlantic Coast, for many years, intensive re-
searches have been directed at problems of the drill (Urosalpinx cinerea) -
Carriker (1955) tias recently summed up these researches and there have
been recent seminars directed at the predaceous snail problem alone.
Because of these thorough reviews, it is not thought necessary to
attempt to add anything here . However, a high percentage of the back-
groimd mortality of oysters on the Gulf and Atlantic coasts, and in
the Pacific northwest, is caizsed by various species of predaceous
snails, which are present on certain oyster grounds in all parts of
the country.

(k) Echinoderms . Starfishes, where they are present, consti-
tute the caiise, both of continuing background mortality in years of
normal abundance, and of catastrophic mortalities in those years when
the starfish cyclically produce enormous populations. Burkenroad (19^6)
predicted a peak of abundance of starfish in Long Island Sound for 1957^
a remarkably accurate forecast. Burkenroad studied I85 cases of sub and
super-normal abundance, which he found to alternate at about "J-year
intervals . The most interesting point in the study of the intermittent
"plagues" of starfish is that the peaks of abundance are in no way de-
pendent on abundance of oysters . Just what cyclic changes are operative
is not known with any degree of certainty. Indeed, a study of the pre-
dators of oysters indicates that their abundance may not be based on
oyster abundance, since most oyster predators have alternate prey and
may prefer some food source other than the oyster.

(5) The predaceous f latworms . The predaceous flatworms maJce
up the last major group of oyster predators. Some genus of polyclad
is present in most oyster producing areas. Extensive mortalities have
been attributed to these worms in various places. In the United States,
the outstanding examples have been Florida and the Puget Sound area of
Washington. In the latter case, the flatworms actually drill a hole
through the shell (Woelke 1957) • This author estimated the population
of flatworms (Pseudostylochus ostreophagus ) in one area at 600,000 per
acre, and indicated a loss of 88 per cent of spat in a one-year period
(1953-5^). The worms were fo\ind on nearly all oyster beds in South Puget
Sound.

-3h-

Generally speaking, the characteristics of mortality waves due
to predators are of the same nat\ire as those outlined for diseases .
However, the less pronounced dependence of predators on any one "host"
species or taxonomic group of prey species, makes their population
variation less density-dependent, and it is often completely independent
of numbers of oysters. Herein, the classic picture of predator-prey
population interdependence breaks down. Otherwise mortalities due to
predators are generally simple to detect, because of the large size of
the predator itself, and the more or less obvious attack of the pre-
dator. The factors of (a) seasonal development, (b) non-physiological
effect on escapees, (c) non- involvement of the community as a whole,
parallel the same characteristics as given for diseases .

TYPE VI. MORTALITIES DUE TO TOXINS

The literature is full of studies dealing with the effect of
suspected toxic substances on oysters. Most of them stand in the cate-
gory of the so-called pollutional toxins, and nearly every recent mor-
tality that had no readily ascertainable cause has been claimed to be
of pollutional origin, irrespective of whether the characteristics fit
or not. But the nimiber of proved cases of pollutional damage to oysters
is surprisingly small, and all such are local in nature, and the facts
obvious to all concerned.

The best examples of destruction of oysters by toxins are
those caused by red tide organisms in the Pacific area. There are
several reports of such cases from Australia, and they appear to be
common in Japan. There have been claims that oyster mortalities due
to red tide have occiorred in the northeast Pacific (i.e., Willapa Har-
bor), and on the west coast of Florida. Gonya-ulax poisoning appears
to affect humans more than it does the oyster, and mussel poisoning is
well known.

A. Characteristics of mortalities of toxic origin.

(1) The mortalities are non-specific. Considerable numbers
of animal species other than the oyster affected are also destroyed.
These are not necessarily related molluscs. Industrial wastes are
generally toxic rather than specifically toxic and would be expected
to destroy a great part of a fauna, irrespective of taxonomic relations
of the species, if oysters are affected. At the same time that most

of the fauna is being destroyed, a few species may be stimulated to
develop larger populations . One would then expect profound changes
in the community of organisms associated with oysters . These changes
woxild not take the form of reductions in numbers of individuals but
would appear as wholesale complete eliminations of dominant and sub-
dominant species, genera or families.

(2) When a mortality of oysters is caused by toxic substances,
and that mortality destroys any considerable part of the oyster

-35-

population, a continuation in time of subjection to the toxin will
destroy the entire popiJiLation . It is not possible, for example, to
reach the LD50 for oysters which is then followed by a revival of the
oyster population and cessation of mortalities if the pollution is
continuing. Continuation in time of such lethal concentration must
result in destruction of the popiilation, since the LD50 is essentially
that level of toxicity which is lethal threshold to the population as
a whole. Additionally, when a level of toxicity is reached which will
destroy a significant part of an oyster population, an increase in
concentration of the pollutant will, in the same time, destroy the
whole popiilation.

(3) A level of toxicity which can directly destroy a part of
a pop^ilation will so affect survivors that the individuals of the
population will be physiologically altered. The two most basic of
physiological yardsticks, growth and reproduction, may be used to test
for sub-lethal levels of pollution. It is not possible for a mortality
due to pollution to be followed or accompanied by normal gonadal de-
velopment, spawning, ajid setting in the face of continued pollution.
Neither can survivors of pollutional mortality continue to grow and
fatten as long as pollution continues. Before lethal levels of pol-
lution are reached these basic functions will be destroyed and both
growth and reproduction will cease.

(k) Mortalities due to pollution are non-seasonal. While it
is obvious that resistance to toxic effect may vary with the metabolic
level of the oysters, which in turn varies with seasonal temperature
changes, such variation only modifies in some small degree the amount
of the lethal dosage. In the face of such drastic effect as death
from toxin, the small threshold modification produced by seasonal
temperature or other change is hardly measurable. Certainly a concen-
tration of toxic substance effective. to the point of producing death
of oysters in one season cannot be so ineffective in a succeeding sea-
son that deaths and physiological depression do not occur in any degree.
Physiological damage is a yardstick often overlooked in measiorement of
effect of pollution.

(5) The effects of pollutional damage are greatest at the
source of the toxin and the effects on oysters diminish with distance
away from the soxoxce. This criterion would seem to be self-evident.
The decrease in effect is due to two factors, the most important being
dilution. The other is biological and chemical modification of the
pollutant which tends to reduce toxicity.

(6) Deaths from toxic effects are not density-dependent.
Concentration of the oysters or population densities have no effect
on death rates .

(7) In a given area of approximately equal pollution, all
oyster beds will be fo\Jnd to be affected. It is not believed to be

-36-

possible for beds with high mortality from toxic substances to alter-
nate or be interspersed with bedti in good condition and with little
or no mortality. Extremes of variation with respect to mortality rate
within a limited area are not consistent with nat\aral effects of pol-
lution.

Pollution may not involve a toxin, but instead may modify the
habitat in some indirect manner. The usual non-toxic effect is to ex-
ert a strong oxygen demand. Deaths are due to asphyxiation rather
than to toxic effects. All of the characteristics mentioned above
apply equally well irrespective of whether or not the action is direct
or indirect.

VII. MORTALITIES DUE TO METABOLIC COMPETITION

It is not known for certain that mortalities due to metabolites
actually exist, but, because of the developing interest in this field,
the matter is explored briefly. For a recent discussion of researches
on metabolites in the sea, see Lucas (1955)- I't tias become increasing-
ly apparent that dominant organisms produce substances, which, excreted
or secreted into the sea, exercise control over other organisms . More
to the point, large and rapidly growing organisms may suppress growth
of smaller, less rapidly growing organisms of the same or closely re-
lated species (Rose 1959). Thus, when a large fast-growing species of
oyster is introduced into an area originally populated by a small, slow-
growing species it is apparent that there may develop a competition ion-
related to competition for either food or space. Modern oyster pro-
duction requires populations of oysters of such density that artificial
dominance is set up and sometimes maintained. In some cases attempts
have been made to maintain two such oyster dominants in the same area.
There are three such cases, in which species of Crassostrea appear to
be in direct competition with species of Ostrea. The first of these
is the Crassostrea angulata - Ostrea edulis combination on the French
coast. C. angulata was introduced to the French coast originally in
the middle of the 19th century in the basin of Arcachon. In I868
another introduction was made in the Glronde estuary. Natural reefs
developed from these introductions, and the conditions seemed to be
well suited to the imported species. It successively became numeri-
cally dominant over estuaries farther and farther north on the French
coast. Oystermen found it easier axid more profitable to cultivate
than was the native 0. edulis. Lambert (l9^6) described the reduction
in numbers and importance of 0. edulis throiigh the years. He believed
that C. angiilata will take over the entire French coast and replace
the native.

In Australia a similar situation exists. Crassostrea commer-
cialls originally coexisted with the flat oyster, Ostrea angasi, which
is very similar to 0. edulis . 0. angasi disappeared from the north
part of its range and is now restricted to the colder waters of the

-37-

south of Australia where C. commercialls does not grow.

In the South Puget So\md area of Washington^ the introduced
Japanese oyster, Crassostrea gigas, is now preempting the area for-
merly occupied exclusively by 0. l\rrida, the excessively small native
species. The C. gigas population has increased rapidly in late years,
with a corresponding decrease in 0. lurida. Oystermen have increasing
difficulty raising the latter, while at the same time the C. gigas
industry has grown enormously.

In all of these cases, there is reason to believe that economics
may play a part in the substitution of a fast-growing, large oyster for
a slow-growing, smaller oyster. Also there is the possibility that in-
troduction of predators and diseases may have had some influence. Cer-
tainly, in the case of 0. lurida, the introduction of the flatworm
Fseudostylochus has played a part. Such a species wo\ild certainly be
more effective against a small, slow-growing species such as 0. liirida
than against the large, fast-growing C. gigas . In Australia it has
been reported that 0. angasl was decimated by the "worm" disease (Poly-
dora ciliata or P. ligni ) .

REFERENCES

Andrews, Jay D. and W. G. Hewatt. 1957. Oyster mortality studies in
Virginia, II. The fungus disease caused by Dermocystidium
marinum in oysters of Chesapeake Bay. Ecol. Monogr. 27:1-26.

Beaven, G. Francis. 19^+6. Effect of Susquehanna River stream flow
on Chesapeake Bay salinities and history of past oyster mor-
talities on upper Bay bars. Third Annual Report, Maryland Bd.
Nat. Res., pp. 123-133-

Burkenroad, Martin D. I946. Fluctuations in ab\mdance of marine ani-
mals. Science 103(2684) : 6^-686.

Carriker, M. R. 1955. Critical review of biology and control of oys-
ter drills, Urosalplnx and Eupleura. U. S. Fish and Wildlife
Serv., Spec. Sci. Rept. - Fish. No. l^fS, 1^9 pp.

DollfuB, Robert Ph. 1922. Resume ^e nos principales connaissances

pratiques sur les maladies et le ennemis de I'huitre. Office
Scientifiques et Techniques de Peches Maritimes, Notes et
memoires. No. "J, 58 PP-

Gaarder, T. and E. Alvsaker. 19^+1. Biologie und Chemie der Auster in
den Norwegishen Pollen. Bergens Mus . Aarb., Naturv. Rekke
236 pp.

Gross, F. and J. C. Smyth. 19^+6. The decline of oyster populations.
Nature 157:540-5^2.

-38-

Gunter, G. 1950. The relationship of the Bonnet Carre Spillway to
oyster beds in Mississippi Sound and the "Louisiana Marsh",
etc. Mimeographed Report, Corps of Engineers, U. S. Army,
60 pp.

Hoek, P. P. C. 1902. Rapport over de Oorsaken van den Acteruitgang
in Hoedanigheid van de Zeeuwsche Oester. Ministerie van
Waterstaat, Handel en Mzverheid, s ' Gravenhage .

Hopkins, S. H. 19^9 • Preliminary survey of the literature on Stylo-
chus and other flatworms associated with oysters. Texas A
& M Research Foundation, Project 9 Report, April I8, 19^+5.

Korringa, P. I9U7. Les vicissitudes de 1 'ostreiculture Hollandaise
elucidees par la science ostreicole moderne. Ostreiciilture
ciiltures Marines, 16 (juillet I9U7) :3-9

Korringa, P. 1952. Recent advances in oyster biology. Quart, rev.
biol. 27:266-308, 339-365.

Lambert, L. 19^6. Les huitres des cotes f rancaises . Peche Marit.
29:31-33.

Loosanoff, V. L. ajid James B. Engle. 19^4. Feeding and fattening of
oysters. Natl. Shellfish. Assoc, Convention addresses, 19^4.

Lucas, C. E. 1955- External metabolites in the sea. In: Papers in
Marine Biology and Oceanography, Deep-Sea Research, Suppl. to
Vol. 3, Pergamon Press, London and N. Y., pp. 139-147'

Lunz, G. Robert. I936. The effects of the flooding of the Santee
River in April, 1936, on oysters in the Cape Romain area of
South Carolina. Mimeographed Report, Corps, of Engineers,
U. S. Army, 2k pp.

Mackin, J. G. and A. K. Sparks. 1959- A study of the effects on oys-
ters of crude oil loss from a wild well. Texas A & M Research
Foundation, Project 23 Report.

Mackin, J. G., P. Korringa, and S. H. Hopkins. 1952. Hexamitiasis

of Ostrea edulis L. and Crassostrea virginica (Gmelin) . Bull.
Mar. Sci. Gulf Carib. l(4) : 266-277.

Menzel, R. W. and S. H. Hopkins. 195'+- Studies on oyster predators
in Terrebonne Parish, Louisiana. Texas A & M Research Foimd-
ation. Project 9 Report, l^tO pp., I8 figs.

Moore, H. F. and T. E. B. Pope. I9IO. Oyster culture experiments

and Investigations in Louisiana. Report, U. S. Comm. Fish.
1908, Doc. 731, 52 pp.

-39-

Orton, J. H. I92U. An accoimt of investigations into the caiose or

causes of the unusual mortality among oysters in English oys-
ter beds during I920 and I921. Gt. Brit. Min. Agr. Fish.,
Fish. Invest., Ser. II, 6(3):1-199, 1923; 6(i+) :l-69, 1921^.

Orton, J. H. 1937- Oyster biology and oyster cultvire. Buckland
Lectxires, Wo. Ill, Edward Arnold, London, 211 pp.

Owen, H. Malcoljii and L. L. Walters. 1950. Report of the investigation
to determine the effect of the 1950 opening of Bonnet Carre
Spillway on Mississippi Sound. Louisiana Conserv., March-
April, 1950, pp. 16-19 j 26-27.

Oyster Commission of Louisiana. I906. Second biennial report. Oyster
Commission of Louisiana, Secretaries' Report, 18 pp.

Rose, S. Meryl. 1959- Failure to survive of slowly growing members
of a population. Science 129 (3355):1026.

Roughley, T. C. I926. An investigation of the cause of an oyster
mortality on the George's River, New South Wales, 1924-25.-
Proc. Lliin. Soc. N. S. Wales 51:^1^+6-491.

Steinhaus, E. A. 1946. Insect microbiology. Comstock, Ithaca, N. Y.
767 pp.

Steinhaus, E. A. 1949. Principles of insect pathology. McGraw-Hill,
New York. 757 pp.

Topley, W. W. C. and G. S. Wilson. I936. The principles of bacterio-
logy and immimity. William Wood, Baltimore. l654 pp.

Von Brand, Theodore. 1946. Anaerobiosis in invertebrates. Biodynamica
Monograph, No. 4, Biodynamica, Normandy 21, Missoiiri. 328 pp.

Woelke, C. E. 1957- Flatworm Pseudostylochus ostreophagus Hyman, a
predator of oysters. Proc. Natl. Shellfish. Assoc, I956:
62-67.

-40-

A METHOD OF ESTIMATION OF MORTALITY RATES IN OYSTERS

J. G. Mackin^

In Louisiana, and sometimes elsewhere, it has been customary
to use the "box count" method of estimation of mortality. It has been
widely used in short-term studies of planted beds and natural growths
of oysters. Where more elaborate studies are possible, with sufficient
periods of time, the tray method is highly accurate, and has been ex-
tensively used by the author and others (Hewatt and Andrews 195^^, Bea-
vin 19^9) . Otherwise productivity studies may be based on analysis of
mortality and growth as interacting factors (Hopkins and Menzel 1952,
McHugh and Andrews 1955, Andrews and McHugh 1957) • Basically this
method compares the number of oysters planted per unit measure with
the number harvested per unit measiire taking into consideration also
the number of units harvested per unit planted. This also is a highly
efficient method.

Estimation of mortality rates on natural reefs and planted beds
when it must be done quickly and without knowledge of planting and har-
vesting data, and without long-term use of trays, has been mostly based
on the "box count" method. In using the method, random samples of oys-
ters are collected and percentage of hinged shells in the total of live
oysters and hinged shell is usually reported as "mortality" . It has
been my contention that this method is highly inaccurate, misleading,
and, as a method of population investigation, unacceptable. There are
legitimate uses for box counts and studies of condition of boxes and
shells when it is necessary to make rough calculations of the extent
of very recent and cataclysmic mortality. But box counts are a source
of gross error in any study of oyster populations or productivity. To
show the inadequacy of the system, two illustrations are given. Nine
samples of oysters from natural reefs in one limited area of Louisiana
were pooled to make a total of 3252 oysters, of which lU.l per cent
were boxes. This normally would be reported as "mortality." But there
is no hint as to the time required to produce the ik.l per cent of
boxes foiindj neither is there any method of determining Just how many
"boxes" lose their right valves in a given length of time and hence
cease to be boxes. The 1^.1 per cent boxes cannot in any sense be a
rate, and only rates are useful in productivity or population calcul-
ations. In the case cited above, a careful analysis indicated that
the population did not contain any oysters older than four and one-
half years. This being true, the mean annual loss could not be less
than 50 per cent and actually must have been greater. Menzel and Hop-
kins (1952), in studies of bottom plantings made very near the area
from which these samples were taken, also found that boxes were a poor
indicator of mortality rates and demonstrated, using the tray method.

1 Contribution from the Department of Oceanography and Meteorology,
Agricultural and Mechanical College of Texas, Oceanography and Mete-
orology Series.

-41-

that mortality rates ranged from 25 to 31 per cent annually in oysters
two months old (at start) to 96 to 100 per cent annually in oysters 16
months old or older. This indicates that the l4.1 per cent boxes in
the nine samples mentioned above gave a grossly unrealistic indication
of mortality rates .

A second check was made using samples of South Carolina oysters
from Wadmalaw Island's We Creek. Through the coiirtesy of Dr. Robert
Lunz^ a sample of I29I oysters was analysed. This sample contained
eight per cent boxes, but the oldest oysters were roughly about four
years of age. The mean annual mortality was, therefore, in excess of
60 per cent.

Because increasing data indicate that oyster mortalities are
generally much higher than the estimates fo\md in the literature, a
study of the matter using statistical methods borrowed from fin fish-
eries Investigations, has been projected. This study has only begun,
but it is believed that the approach may be helpful in population
analyses . It is remarkable that so few studies of natural oyster
populations have been made. This is more to be wondered at since it
has been assumed by most researchers that oysters are ecological do-
minants in the oyster reef community, both as to numbers and as to
community control.

STUDIES ON MORTALITIES IN LOUISIAm OYSTERS USING THE TRAY METHOD

Prior to beginning this study, all available data beajring on
mortality rates in Louisiana oysters were studied. A considerable
number of studies using the tray method were examined. In these,
oysters were held in trays or cages for varying lengths of time and
in different areas. Results of these studies are presented here as
backgroimd material. These tray (or cage) studies showed unexpectedly
high rates of mortality at widely scattered places in Louisiana. In
Table 1, the data are summarized and reduced to an annual basis. This,
in some cases, was difficult to do because of different rates which
pertain to different seasons, but they are believed to be within +5
per cent of the true rates. Most studies reported for Menzel and
Hopkins are round-number approximations of means for several studies
in each area, as is true also for some studies by Mackin and Wray.
The data from Owen (1955) were difficult to assess, since he ran his
tray studies only during the summer, for seven and one-half months.
However, winter mortalities are generally light, so Owen's figures
were only raised to the next higher figure which roxonded to the near-
est 5 per cent. The same applies to figures from Mackin and Sparks
(1959)' These authors reported on tray check of mortality from March
to September In 1957. St. Amant et al. carried one group of caged
oysters (equivalent to tray) for six months, with a mortality of kk
per cent. This one would have been about 60 per cent if extended to
one year, but the data are reported for the six months only in this
case, because of uncertainty of the locale of the study.

-U2-

Table 1. Annual rates of mortality of oysters in Louisiana as shown
by tray studies (Oysters one year old or older) .

Period of Study

Author

Place

Per cent
Mortality

1947-48

Mackin and
Wray I949

Bayou Rigaud

80

191+7-1+8

Mackin and
Wray I949

Sugar House Bend,
Baratarla Bay

80

19^7-48

Mackin and
Wray I949

Bassa Bassa Bay

70

19^7-48

Mackin and
Wray I949

Chene Fleur Bay

30

19I+8-49

Mackin and
Wray I95O

Bayou Rigaud

80

19I+8-I+9

Mackin and
Wray I95O

Lower Baratarla
Bay

75

1948-49

Mackin and
Wray I95O

Bassa Bassa Bay

60

1948-49

Mackin and
Wray I95O

Chene Fleur Bay

30

19I+8-49

Mackin and
Wray I95O

Lake Grande
Ecaille

70

1947 to

1949

Menzel and
Hopkins 1952

Bayou Bas Bleu

65

1947 to

1949

Menzel and
Hopkins 1953

Bay Ste. Elaine

60

1947 to

1949

Menzel and
Hopkins 1951

Lake Barre

70

1947 to

1949

Menzel I95O

Lower Terrebonne
Bay

75

1947 to

1949

Menzel I95I

Lake Pelto

80

1949

Owen 1955

Bayou Pierre

60

1949

Owen 1955

Quarantine Bay

45

-43-

Table 1 (Continued)

Period of Study

Author

Place

Per cent
Mortality

1949

Owen 1955

Grand Bay

50

191^9

Owen 1955

Sandy Point Bay

50

19^9

Owen 1955

Bayou Scofield

60

19^9

Owen 1955

Bay Adam

65

19^9

Owen 1955

Lake Grande
Ecaille

70

19if9

Owen 1955

Northern
Baratarla Bay

i^5

19^+9

Owen 1955

Bassa Bassa Bay

40

1949

Owen 1955

Lower
Baratarla Bay

75

19*19

Owen 1955

Lake Felicity

45

19^9

Owen 1955

Sister Lake

30

19I+8-49

Mackln, Welsh
and Kent I95O

Bay Adam

85

191+8-1^9

Mackin, Welsh
and Kent I95O

Bayou Cook

75

19I+8-49

Mackin, Welsh
and Kent I95O

Bastian Island

80

19li8-l^9

Mackin, Welsh
and Kent I95O

English Bay

90

1948-49

Mackln, Welsh
and Kent I95O

Bay Pomme D'or

80

19^8-49

Mackin, Welsh
and Kent 195O

Skipjack Bay

75

1948-49

Mackin, Welsh
and Kent I95O

Bay Jacque

65

1948-49

Mackln, Welsh
and Kent 1950

Bay Tambour

55

^k-

Table 1 (Continued)

Period of Study Author

Place

Per cent
Mortality

1957

Mackin and

Sparks

1957

Mackin and

Sparks

1957

Mackin and

-Sparks

1957

Mackin and

Sparks

1957

Mackin and

Sparks

1957

Mackin and

Sparks

1957

Mackin and

Sparks

1956-57

St. Amant,

at al.

Billet Bay

65

Northern

55

Grande Ecaille

Southern

65

Grande Ecaille

Dredged cut

30

Freeport S\ilph\ir

Southern

45

Baratarla Bay

Southern

80-

Baratarla Bay

Sugarhouse Bayou

55

(not stated)

kk*

* Six months only; estimated about 60 per cent for a year.

Data in Table 1 indicate that annual mortality rates of oysters
more than a year old are usually in excess of 60 per cent annually.
St. Amant et al. (1958) believed that 20 per cent is a "normal" rate
for bedded oysters "over the growing period." This latter, in Louis-
iana, could be any period from three months to tvo years . It was in-
dicated that in most bedded oysters, the growing period might be about
nine to ten months . If that is so, 20 per cent is much too low an
estimate . It is believed that there has never been a mortality rate
consistently that low in Louisiana. St. Amant et al. (1958)^ in sev-
eral mortality studies, failed to find any that low, and one study by
these authors showed about 80 per cent anniial mortality. It may be
that these authors consider all mortality rates in Louisiana to be
abnormal, and that the "normal" is a desired low rate of mortality,
never to be actually attained.

A5-

MORTALITY RATES BY YEAR-CLASS ANALYSIS

Because of the objections to box counts stated above, it was
decided to apply the method of year-class analysis to determine whether
or not such a method used with natural oyster populations would cor-
roborate figures derived from the tray method as shown in Table 1.
It was believed that if a natural population of oysters could be made
to yield data from which the number of year-classes represented in the
population could be determined, and also the approximate nvimber of
oysters in each year-class, the problem could be resolved with the
simplest kind of calciilation.

As a beginning, calculations were made to determine the time
necessary to bring any year-class population to practical extinction,
using various rates of mortality. "Practical extinction" was arbit-
rarily decided to mean that less than 5 per cent of the original popu-
lation would remain. This procedure was adopted because the time re-
quired to eliminate a population completely, when the percentages of
annual loss are very low, is a very long time. For example, even at
50 per cent annual mortality, a population may be reduced to less than
5 per cent of its original size in four to five years, but to eliminate
the remnant 5 per cent requires another six to seven years. Since
sampling methods cannot be depended on to detect remnant populations
of very small size, and because mortality rates become excessively
erratic when dealing with small nimibers of oysters, the compromise re-
duction to 5 per cent of original size was adopted, and found to be
satisfactory.

In Table 2 are figures showing the time required to reduce a
population of oysters to less than 5 pei" cent of its original size at
various annual mortality rates .

Table 2. Time required to reduce a population of oysters to less than
5 per cent of its original size with different mean annual
rates of mortality.

Annual mortality rate,
percent

Years required to reduce
to less than ^ per cent

10
20
30

ko

50

60
70
80
90

28-29
about 13
8-9

about 6
U-5
3-i+
2-3

nearly 2
1 plus

-46-

This table shows that in any natural undisturbed population
of oysters, there should be about 28 recognizable year-classes if the
mean annual mortality rate is 10 per cent, and a small part of the
population would be 28 to 29 years old. If the rate was 20 per cent,
one should find oysters about 13 years old, etc. These preliminary
calculations emphasized the fact that commonly- quoted low annxial mor-
talities of 10 to 20 per cent in Louisiana oysters have no validity,
since oysters attaining an age of five years are certainly rare, if
they exist at all.

Analyses were made of a considerable number of natural popu-
lations to determine whether or not it could be determined with a fair
degree of accuracy how many year-classes were represented in a sample
and approximately how many oysters made up each year-class. If this
could be done, it would greatly simplify determination of annual death
rates. Oysters in large samples were carefully measured for length of
the right valve, and size distribution diagrams were made. Most of
these showed clearly that the approximate age of the oldest oysters
could be determined, and that fused modes of the year-classes could
be recognized. This can be done only if recruitment is reasonably
constant. One of the diagrams in block form is shown in Figure 1.
The process of separation of year-classes was aided because oysters
in their first year could be recognized by fonn and color. Other year
classes were clearly indicated by sub-modes, and corroborated by grow-
th data. The histogram in Figiwe 1 showed that the 195^ year-class
was the oldest in the population and made up only about one per cent
of the total.

Where difficulty in recognition of year-class modes is encoun-
tered, aid is found in a knowledge of normal growth rates . Menzel and
Hopkins have made extensive studies of growth in Louisiana oysters
(see any of several references given in the bibliography). These data
indicate that oysters in their first year have a modal length of about
30 mm in three months, 55 "to 6o mm in one year, 85 to 95 nm in two
years, and I05 to 115 n™ in three years. Depending on the area, these
growth rates are greater or less. Oysters which are older than four
years are rare and usually form a remnant population on the right ex-
tremity of a size-distribution diagrajn.

Estimates of numbers in each year-class may be made by recon-
structing the normal curves for each year-class. Accuracy in this is
not necessary, so long as the numbers in the youngest and oldest year-
class are approximately correct. An error in estimation of niimbers
in the second, third, or fourth year-classes must necessarily be com-
pensated for by opposite error in the next older or younger class.
After numbers are estimated, the approximate mortality from one year-
class to the next is easily computed. It is convenient to plot the
numbers or per cent in each year-class as the logarithm of the per-
centage of the population in each, as shown in Figure 1. This type
of plot is the same as the fishery statisticians' "catch curve" (Ricker
19^8). Considerable information can be derived from these curves.

-47-

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First, the mean slope of the right hand limh of the curve represents
the survival rate for the period represented by the year-classes plot-
ted, and hence also the mortality rate. To some degree also, if the
slope is not a straight line, the various segments of it indicate
those year-classes in which most mortality occurs, and those in which
the least mortality occurs. In Figure 2 (l959 curve) the youngest
oysters in two South Carolina samples are shown to have the highest
mortality rate, and rates in older year-classes are less. In Louisiana
(Figure l) just the opposite is true.

Since size is a function of age, it was believed that the ne-
cessity for determination of year-class numbers could be eliminated
by plotting the logarithm of the percentage numbers in size-classes
rather than in estimated year-classes. It was found that this method
was reasonably accurate. In using such curves it is convenient (l)
to use a standard graph scale for all plots and (2) make up a group
of comparison graphs based on ass\amed mortality rates in populations
with constant recruitment rate. These comparison graphs, plotted to
the same scale as are the natural populations, give a ready estimation
of mortality rates by comparing slopes. The use of graphs facilitates
comparison with limited parts of a "survival curve," Two samples of
oysters from South Carolina were plotted in this manner (Figure 2) .
These two samples came from about the same area, but were taken nearly
a year apart.

It is believed that, with refinement, this method of approach
to mortality estimations has promise. It is suggested that the mea-
svirement of shell length may not be the best available variate. Volume
might be better, or total weight, or product of length, width, and
thickness. It is recognized that in areas of slow growth and erratic
set the method may be difficult to vae. Nevertheless, the need for
improvement in methods of estimating mortality rates warrants a trial.

SUMMARY

(1) Tray studies of mortalities made by various authors show
that in Louisiana the usual annual loss in oysters one year old and
older is between 50 ajid 70 psr cent.

(2) These same studies show that annual mortality rates of
from 70 to 90 per cent are not unusual.

(3) A few very favorable areas have showed low annual rates
of around 30 per cent. This is believed to be about the minimum in
Louisiana.

(k) A method of rough calculation of mortality rates in oyster
populations has been tried in Louisiana with promising results. This
method involves identification of the number of year-classes in the
population, and calculation of the mean annual rate necessary to reduce

-k9-

a population to near extinction in the Indicated nimiber of years re-
presented by year-classes in the popiilation.

ACKNOWLEDGEMENTS

The author is indebted to Dr. G. Robert Limz of the Bears Blirff
Laboratory and his staff at Wadmalaw Island, South Carolina, for col-
lecting and measuring oyster samples shown in Figure 2, which aided
the study considerably. Many other samples of oysters were taken and
measured by the staff of the Texas A & M Research Foundation Laboratory
at Grand Isle, Louisiana. To all these the author makes grateful ac-
knowledgement .

REFERENCES

Andrews, J. D. and J. L. McHugh. 1957* 1^^ siorvival and growth of
South Carolina seed oysters in Virginia waters. Proc . Natl.
Shellfish. Assoc. 1+7:3-1?.

Beavin, G. F. 19^9. Growth observations of oysters held on trays at
Solomons, Maryland. Conv. Addresses Natl. Shellfish Assoc.
19^9:^3-^9.

Hewatt, Willis, G. ajid Jay D. Andrews. 195^. Oyster mortality studies
in Virginia. I. Mortalities of oysters in trays at Gloucester
Point, York River. Texas J. Sci. 6(2) :121-133.

Hopkins, Sewell H. and R. Winston Menzel. 1952. How best to decide
best time to harvest oyster crops. Atlantic Fisherman 33(9):
36-37-

McHugh, J. L. and J. D. Andrews. 1955. Computation of oyster yields
in Virginia. Proc. Natl. Shellfish. Assoc, k^il^'^k) -.21^-239'

Mackin, J. G. and A. K. Sparks. 1959. A study of the effect on oys-
ters of crude oil loss from a wild well. Texas A & M Research
Foundation, Project 23 Report, 68 pp.

, Billy Welsh, and Charles Kent. 1950. A study of mor-

tality of oysters in the Buras area of Louisiana. Texas A &
M Research Foundation, Project 9 Report, kl pp.

and D. A, Wray. ±9^9 . A study of mortality and mortality-

producing agencies in Barataria Bay, Louisiana. Texas A & M
Research Foundation, Project 9 Report, 39 pp.

1950. Report on the second study of mor-

tality of oysters in Barataria Bay, Louisiana and adjacent
areas. Pt. 1. Texas A & M Research Foundation, Project 9 Re-
port, 39 pp.

-50-

Menzel, R. Winston. 1950. Report on oyster studies in Caillou Island
oil field, Terrebonne Parish, Louisiana. Texas A & M Research
Foundation, Project 9 Report, 6o pp.

1951 • Report on oyster studies at Lake Pelto oil field.

Terrebonne Parish, Louisiana. Texas A & M Research Foundation,
Project 9 Report, 60 pp.

Menzel, R. W. and S. H. Hopkins. 1951. Report on experiments to test
the effects of oil well brine or "bleedwater" on oysters at
Lake Barre oil field. Texas A & M Research Foundation, Pro-
ject 9 Report, 130 pp.

. 1952. Report on commercial-scale oyster

planting experiments in Bayou Bas Bleu and in Bay Ste. Elaine
oil field. Texas A & M Research Foundation, Project 9 Report,
1U6 pp.

. 1953' Report on oyster experiments at Bay

Ste. Elaine oil field. Texas A & M Research Foundation, Pro-
ject 9 Report, 170 pp.

Owen, H. Malcolm. 1955' Oyster industry of Louisiana. Special report
to the Division of Water Bottoms, State of Louisiana, 13 pp.

Ricker, William E. 19i|8. Methods of estimating vital statistics of

fish populations. Indiana Univ. Pub. Sci. Ser. No. 15, Bloom-
ington, Indiana, 101 pp.

St. Amant, Lyle S., A. V. Friedrichs, and Emory Hajdu. 1958- Report
of the Biological Section. Division of Oysters, Water Bottoms
and Seafood, 7"th Biennial Report, Wildlife eind Fisheries Com-
mission, State of Louisiana, pp. 71-92.

-51-

PACIFIC OYSTER CRASSOSTREA GIGAS MORTALITIES
WITH NOTES ON COMMON OYSTER PREDATORS IN WASHINGTON WATERS

Charles E. Woelke

Washington Department of Fisheries
Shellfish Laboratory, Quilcene, Washington

ABSTRACT

Laboratory studies are reported giving approximate pre-
datlon rates of oyster predators found in Washington waters.
Data on seasonal and annual mortality rates of experimental
oyster plots are presented. Mortalities on an area eind state-
wide basis by age are reported as determined through annual
sxorveys of the commercial beds in 1956, I957 and I958. Syn-
dromes of mass mortalities encountered are outlined.

INTRODUCTION

Mortalities and predator-oyster relationships of most species
of oysters have been widely studied and reported. Reference to pre-
dation on the Pacific oyster, Crassostrea glgas, has been made by El-
sey (1933), Glud (19^7), Gaits off (193^) and Kincaid (l95l). In his
survey of the Japanese oyster literature, Cahn (1950) refers to pre-
dation and mortality. None of these authors present data on mortali-
ties or predation rates. Thomson (1952) on the other hand reports
approximately 60 per cent mortality of this species in the first year
after planting and 55 per cent during the second in Australian waters.
From his report it is assumed that predation was not a factor in the
losses recorded. Chew and Eisler (1958)^ in a report on the feeding
habits of the Japanese oyster drill (Ocinebra Japonica), make reference
to oyster predation. Woelke (1957) noted Pseudostylochiis ostreophagus
predation on juvenile Pacific oysters .

Review of the literature provides no scientific information on
Pacific oyster mortalities or predation in Washington waters . This
report deals with predators, mortality by age, seasonal mortality and
annual mortality as a continuous interrelationship.

Extensive studies have been conducted by this laboratory on
predation rates, predator habits and predator control; however, only
one series of predation experiments is discussed in this report. Data
from a series of field experimental plots are presented to demonstrate
seasonal and annual mortalities experienced in the first and second
years after planting (Pacific oyster seed caught in Japan in the sumirier
is imported and planted during the following spring when 5-15 nim in

-53-

Drayton Harbor

I . samish Bay

South
Puget
Sound

Fig. 1. Principal Oystering Areas in Washington State.

-5^^-

length - less than 1 cc in volume).! Pacific oyster cultiire is re-
stricted almost entirely to the intertidal zone. The intertidal nature
of the plantings makes identification of age groups and representative
sampling of the beds much simpler than where oysters are subt'idally
cultivated. Oystering areas are shown in Figure 1.

HIEMTIOW STUDIES

To determine which of the potential predators would attack
Pacific oysters, aquaria studies were conducted. Limited numbers of
seven species of suspected predators were placed in separate aquaria
with four-month-old oysters for 20 days. During the study, temperature
of the flowing water ranged from lU-20°C and the salinity from 2k-26
o/oo. No potential macroscopic food other than oysters was available
in the aquaria.

ResiAlts of these studies are summarized in Table 1. Number
of "predators," number of oysters (kd-^O) , mortality at 8 and 20 days
as well as "20-day predation rates per specimen" are shown. While
these data have many readily recognized limitations they demonstrate
the ability of the species considered to attack and destroy young oys-
ters in the absence of other food. The eighth day mortalities may
Indicate that neither Cancer gracilis nor Cancer magister (the com-
mercial crab of Washington) readily attacks oysters, while Cancer ore-
gonensis and Cancer productus probably do. After 20 days' exposure
all species except Cancer magister had caused appreciable mortalities .
Except for Cancer magister, crabs caused the greatest losses followed
in order by starfish, Japanese oyster drill and flatworm. Observations
in the field have confirmed these findings in all instances except
Cancer gracilis.

SEASOML AND ANNUAL MORTALITY RATES

Field studies were conducted during 1952-53 in three of the
four principal oyster-growing areas to ascertain the variation in
mortality rates by age, area and season. Lots of 20 or kO individually
marked spat and yearling oysters of common stocks taken from our ex-
perimental oyster beds were utilized in this study. Replicate lots
of spat and oysters were placed inside the boundaries of commercial
planxings. All marked oysters were checked each time a mortality ob-
servation was made.

1 As a result of the unique method by which the Pacific Coast oyster
industry replenishes the bulk of its oyster stocks, i.e., annual im-
portation of seed from Japan, both industry and researchers designate
successive year classes by year of planting. Both age and year class
as referred to in this report follows this precedent.

-55-

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-58-

Table 5« Sumnary of North Puget Sound Pacific oyster mortality data

Year of

Planting
year

Total
oyatera
checked

Dead
oysters

Causes of

ieath

Mortality

In per cent

observation

Drill

Crab

Unknovm

Total

Drill

Crab

Unknown

1951

1951

1950
Totals
Per cent

187
129

21

25

u
11

15
lit

11.2
19. !(

3.2
8.5

8.0
10.9

31^

1.6
ll»,6

17

29
9.2

1952

1952

1951
Totals
Per cent

22lt
19't

a
16

3
11

5
5

3.6

8.2

1.3
5.7

2.2
2.6

1.18

2k

5.7

ll*
3.3

10
2.1*

1953

1952

1951
Totals
Per cent

118
103

18

8

2

1

16
7

15.3

7.'»

1.7
0.9

13.6

6.5

226

26
11.5

3
1.3

23
10.2

195l«

195'»

1951
Totals
Per cent

l8i»
lt53

36

75

7

22

27
?9

2
11*

19.6
16.6

3.8
k.9

lit. 7
8.6

1.1
3.1

637

111
17.5

29
k.6

10.1*

x6

2.5

1956

1956
1955
195't
1953
1952

Totals

Per cent

i.ioU
666
936
363
338

62
122
186

56
II.2

26
ll*

37
10
3k

36
108
1U9

1*6
108

5.6
18.3
19.8
15.1.

It2.0

2. It
2.1
3.9
2.8
10.0

3.3

16.2

15.9
12.6
32.0

3>7

568
16.7

121
3.6

IA7
13.1

1957

1957

1956

1955

195'*

1953
Totals
Per cent

661

l,ll(0

560

1.58

Ik
67
35
82
19

1
16
1
2
6

1
3
3

1
k

12
1»8
31
79
9

2.1
5.9

6.3
17.9
13.1

0.2
l.ll
0.2
O.lt
It.l

0.2
0.3

0.6

0.2
2.8

1.8
It. 2
5.5
17.3
6.2

2,96l*

217
7.3

26
0.9

12
0.1*

179
6.0

1958

1958

1957

1956

1955

195'*
Totals
Per cent

291
1,897
2,978
1,562

579

13
kkk
1.26

222
112

3

^k

8

5

15

1
1

5

9

389

1»18

212

97

i*.5
23.1*
ik.Z
lit. 2
19. •♦

1.1
2.8

0.3
0.3
2.6

0.3 .
0.1

0.3

3.1
20.5
llt.O
13.6
16.8

7,307

1,217
16.7

85
1.3

7
0.1

1,125
15.3

-59-

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CO O CO

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(1)

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en ft

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-61-

Table 8. SiJmmary of statewide Pacific oyster mortalities

Per

cent total mor"

bality

Per c

snt unknown ..mortality

Year of

North

South

Grays

Wil-

North

South

Grays

Wil-

obser-

Age in

Puget

Puget

Har-

lapa

Puget

Puget

Har-

lapa

vation

years

Sovind

Sound

bor

Harbor

Sound

Sound

bor

Harbor

1956

0+

5.6

2.9

2.4

3.3

2.0

2.4

1+

18.3

10.3

6.9

16.2

10.0

6.9'

2+

19.8

9.7

15.5

7.5

15.9

9.7

11.6

7.5

3+

15.4

1^.3

12.6

14.3

1++
Average

i+2.0

32.0

all ages

16.7

7.2

15.5

5.5

13.1

6.8

11.6

5.5

1957

0+

2.1

3.0

4.2

2.0

1.8

1.2

2.0

1.0

1+

5.9

9.1

8.8

4.2

4.3

7.6

6.9

4.2

2+

6.3

6.5

7.4

5.0

5.5

4.4

5.9

5.0

3+

17.9

12.1

17.2

11.5

Average

13.1

6.2

all ages

7.3

7.0

7.5

4.1

6.0

5.2

5.7

3.9

1958

0+

h.^

4.8

7.7

3.6

3.1

3.0

4.0

3.6

1+

23. i^

18.0

12.5

6.7

20.5

16.0

10.1

6.7

2+

lif.3

17.2

15.8

8.1

l4.0

15.7

15.0

8.1

3+

1U.2

53.0

15.9

13.6

53.0

15.4

k+
Average

19.3

16.8

all ages

16.6

16.4

13.6

6.1

15.3

14.6

11.8

6.1

Table 9. Summary of mortality by cause from annioal statewide surveys

Oysters
checked

Dead

Crab

Drill

Mass mortalities

Unknown

Totals
Per cent

48,370

272
0.6

348
0.7

1.155
2.4

3,734
7.7

-62-

Data collected are siimmarized by age of oysters (O-l year and
1-2 years) in Table 2 and 3. In view of the small niomber of oysters
per area (6O-80) no intra-area comparisons are attempted. Total mor-
tality during the first year of life was 48.6 per cent with drills
causing 22.3 per cent, crabs 3.2 per cent and "-unknown causes" (neither
valve of dead oysters damaged) 23.2 per cent. Over 20 per cent of the
total loss and over 50 per cent of the annual loss from unknown causes
occurred during the first month after planting. Losses were largest
between May and November. From November to April slightly over 3 per
cent mortality was recorded.

Considerably lower loss was recorded on second year oysters
with a total annual mortality of 21.2 per cent. Drills caused 5-9
per cent, crab 2.k per cent, and 12.9 per cent died from caioses not
determined. Loss of 6.0 per cent was recorded diiring June with all
other observations less than 3.0 per cent per time period except for
7.0 per cent during the four months from January to April. In general,
decreased mortality with increased age, increased mortality d\;iring
summer, and a predominance of losses from undetennined causes summarize
this study.

ANNUAL MORTALITY SURVEYS

Statewide mortality surveys begun in 195^ cover approximately
70 per cent of the major oyster beds in the state and 90 per cent of
the general oyster producing areas. The surveys are conducted on the
last two or three daylight low tide series of the year (August and
September). All age groups present on the beds are sampled. Mortality
vp,lues are generally derived from random samples of 200 or more oysters
per planting. Mortalities are the shells of dead oysters held together
by the hinge ligament at the time of sampling. These samples have been
demonstrated to be statistically valid at the 5 per cent level. In col-
lection of data, mortality causes are assigned as drill, crab, "mass
mortalities" or unknown. Mass mortalities, i.e., sudden, excessive
losses (over 20 per cent) are recorded only when observed in action,
since starfish, sea weed and siltation can also cause heavy losses
but may not be operating at the time of sampling. This method of re-
cording results in a large proportion of the losses being classified
as unknown. On occasion, losses are due to burying of oysters (espe-
cially spat) by mud shrimp (Callianassa and/or Upogebia) . Decomposition
of large "mats" of Enteromorpha and other sea weeds which settle on
oyster beds cause moderate to serious mortalities .

Tables k through 7 present summaries of mortality data from
the four principal oyster areas by year and age. In the northern Pu-
get Sound sxmmary, data from 1951-195^ (primarily from Samish Bay) are
also shown. Mortalities by cause are given in percentages. In Table
8, percentage mortalities only are shown by area, age, and year. To
demonstrate the relative importance of the categories into which the
mortalities fall. Table 9 siommarizes all data collected in the annual

-63-

surveys. In the absence of information on decomposition rate of the
hinge ligament of Pacific oysters^ these data in no sense can be in-
terpreted as true annual mortalities but rather relative from year to
year and area to area at the time surveyed. A second limitation of
the annual survey data is its failure to measure losses of any given
age group when they are greatest, i.e., the first month or two after
planting.

No extensive analysis of these data is presented; however, the
following tentative conclusions are drawn:

1. Mortality from unknown causes generally increases with age,
especially after the third year on the beds .

2. Based on ^8,730 oysters checked in the three annual sur-
veys, 88.7 per cent were live at the time evaluated.

3. Willapa Bay has best survival, southern Puget Sound second.
Grays Harbor third, and northern Puget Soimd poorest.

k. Except in localized instances, none of the known predators
appear to have a major influence on the industry.

5. From the annual survey data, on an industry-wide basis,
unknown mortalities account for the bulk of the losses followed by
mass mortalities, drills and crabs.

MASS MORTALITIES

Pacific oysters, on occasion, have heavy losses for which no
causative agent has been found. Nearly every year at least one mass
mortality may be encountered and in some years they may be quite common.
In terms of overall industry plantings, they do not seem to be serious;
however, to the individual grower they are often important. These mor-
talities usually follow a common pattern. In most cases they are lo-
calized to a single bed or planting and may result in over 50 per cent
loss in a 2-4 week time period. The loss is nearly always on the
yearling (l+) oysters which are in their second growing season after
planting, though both seed and older oysters are occasionally affected.
They are without exception fast-growing, fat (or spawny) oysters .
Usually the areas of loss will be in coves, at the head of a lagoon,
or other type of relatively "dead water" area (in terms of new water
replacement only - often good localized curi'ents exist with rise and
fall of the tide). Heaviest losses are in the sununer when water tem-
peratures are in excess of 18" C. Type of bottom, tidal level of
planting, presence or absence of pollution, salinity (20-30 0/00) and
number of degrees above 18° C seem to have little relationship to the
losses. Occasionally "red tide" is associated with the losses. Los-
ses occur in both diked and open bed plantings. Level of planting

.6k~

relative to tidal height seems to have no bearing on the occurrence
of the mortalities. Fidalgo Bay, Case Inlet, Henderson Inlet, Eld
Inlet, Totten Inlet (Gallaghers Cove and Burns Point), Grays Harbor
(Damon and Alder Points) and Willapa Bay (Stackpole area) seem to have
these losses quite regularly. Neither Dermocystidium nor Hexajnita are
associated with the mortalities. Limited work by this laboratory has
uncovered no potential pathogens. Usually these mortalities are as-
cribed to either "red tide" or "heat kill." Neither of these suggest-
ed causes appear to fit the circimistances associated with the mortal-
ities .

SUMMAEY

1. At least two species of crab, one species of starfish,
the flatworm and the Japanese oyster drill are predators of the Pacif-
ic oysters.

2. Mud shrimp and seaweed, while not predators, will cause
Pacific oyster mortalities.

3. Annual oyster mortality rates decrease with age during the
first two years after planting.

k. Summer is the period of greatest mortality.

5. In control plot studies, mortalities from unknown causes
made up the largest portion of oyster losses .

6. Annual survey data indicates increasing mortality from un-
known causes after the third year on the beds .

7. Based on ^8,730 oysters checked in the three annual sur-
veys, 88.7 per cent were live at the time evaluated.

8. In comparing annual survey oyster mortality data from the
four major oyster ing areas, Willapa Bay has the lowest mortality fol-
lowed by southern Pi:iget Sound, Grays Harbor and northern Puget Sound.

9. Except in localized instances none of the known predators
appear to have a major influence on the industry.

10. From the annual siorvey data, in order of magnitude, un-
known mortalities account for the bulk of the losses followed by mass
mortalities, drills and crabs.

11. Certain conditions surrounding mass mortalities are des-
cribed.

12. No causative agent for the mass mortalities has been dis-
covered.

-65-

REFERENCES

Calm, A. R. 1950. Oyster culture in Japan. U.S. Fish Wildl. Serv.,
Fish. Leafl. 383.

Chew, Kenneth K. and Ronald Eisler. 1958 • A preliminary study of the
Japanese oyster drill Ocinebra japonica. J. Fish. Res. Bd.
Canada 15 (i^) : 529-535-

Elsey, C. R. 1933. Oysters in British Columbia. Biol. Bd. Canada,
Bull. 3^ pp.

Galtsoff, Paul S. 1932. Introduction of Japanese oysters into the
United States. U. S. Bur. Fish., Fish. Circ. 12.

Glud, John B. 19^?. Pacific and Olympia oysters. Ann. Rept. Wash-
ington State Dept. Fish.

Kincaid, Trevor. 1951. The oyster industry of Willapa Bay, Washing-
ton. Calliostoma Co., Seattle.

Thomson, J. M. 1952. The acclimatization and growth of the Pacific

oyster (Gryphaea gigas) in Australia. Austr. Jo\ir. Mar. Freshw.
Res. 3 (l):64-74.

Woelke, Charles E. 1957. The flatworm Pseudostylochus ostreophagua
Hyman, a predator of oysters. Proc. Natl. Shellfish. Assoc,
U7: 62-67.

-66-

HEXAMTA SP. AUD M IlfFECTIOUS DISEASE IN THE
COMMERCIAL OYSTER OSTREA LURIDAl

J. E. Stein, J. G. Denison

Rayonler Marine Laboratory, Hoodsport, Washington

J. G. Mackin

Agric\iltural and Mechanical College of Texas

ABSTRACT

Recent mortalities (1958-59) of the Olympla oyster,
Oatrea lurlda, have occurred In southern Puget Soxmd, Washing-
ton. The mortalities were generally associated with cold water
conditions. Examination of the tissues revealed the presence
of Hexamita sp. and hacterla. Consequently, experiments were
set up ■to ascertain if dead oysters Infected with Hexeimlta and
hacterla could trajismit an Infection of Hexajnlta or hacterla
and cause mortalities. Healthy oysters were therefore exposed
to oysters Infected with Hexamita and bacteria. For controls,
healthy oysters were exposed to autoclaved oyster tissues.
Continuously running water was used and the experiments were
carried out at two temperature levels, 6 and 12° C. At 6° C,
70 per cent of the healthy oysters exposed to diseased tissues
died within 76 days, whereas only l^t per cent of the controls
died within the same period of time. All of the dead oysters
had moderate to heavy infection of Hexamita. Bacteria were
not apparent at light levels of Hexamita Infection and were
either present or absent at higher levels of infection. Tissue
dsimage was severe. Hexamita was found in survivors, but no
bacteria were in evidence. In the warm water experiment, there
was no significant mortality difference between experimental
and control aquaria.

INTRODUCTION

HexEunita is a flagellated protozoan having two anterior nuclei,
six anterior flagella and two posterior flagella (Figiire l) .

Members of the order Polymastigina, to which Hexamita belongs,
have been foimd to be parasitic in trout, salmon, turtles, toads, -^

pigeons, and man (Mackin et al. 1952).

In 1950 and 1951, oyster mortalities in Holland, commonly re-
ferred to as "pit disease", were associated with the presence of Hexa-
mita, (Mackin, et al. 1952) . More recently, the protozoan has been

1 Contribution No. 51 from the Research Department of Rayonier Incor-
porated.

-67-

Fig. 1. Semi-diagrammatic sketch of a trophozoite of Hexamita
showing flagella and axostyles (Mackin, et al.^ 1952).

reported present in mortalities of introduced Ostrea edulis in Prince
Edward Island, Canada (Medcof 1959)* Ttie authors have observed similar
mortalities of the small commercial oyster Ostrea lurida in the southern
Puget Sound area of the Pacific Northwest. These mortalities occurred
in Little Skookum Inlet in April of 1958 and. 1959^ and Hexamita was
found In the tissues of dying oysters.

Cold temperature, poor circulation over the "beds, and overcrowd-
ing appear to be optimum conditions for an epizootic associated with
the presence of Hexamita (Mackln et al. 1952).

From external appearances the oyster generally died fat, but
histological exajoinations revealed protozoan-occluded blood vessels,
necrosis of the gastro-intestinal tract, and histolysis of supporting
connective tissues. Specimens dying in the field frequently had massive
concentrations of Hexajnita and bacteria.

Preliminary experiments by the authors (unpublished) involved
the insertion of Hexamita-infected oyster tissue in the mantle cavity.
The Insertions were made by wedging the valves apart approximately 1
to 2 mm. For controls, autoclaved tissues were inserted. No mortali-
ties resulted from the mechanical separation of the valves. After the
tissues were inserted, all oysters were held out of water at 3° C for
approximately 12 hours; this increased the exposure time by preventing
the oyster from immediately ejecting the tissues.

-68-

Within 2k daya, 78 per cent of the experimental oysters died,
while in the control group there was only a 10 per cent mortality.
These experiments were carried out in 2^-gallon glass aquaria^ each
containing k liters of standing, aerated sea water. The water temper-
ature varied from ^4- to lOO C, with an average for the 24 days of 70 C,

The following experiments were designed to test the effect of
Hexamita on healthy oysters under more natural conditions, to explore
the role of temperature, and to determine whether or not Hexajnita can
be transmitted through running water.

METHODS AMD PROCEDURES
Materials

1. Standard 15-gallon aquaria were used. Each aquarlijm had a hole
in one end to facilitate a constant level of running water.
Throughout the course of these experiments, a 3-inch water head
was maintained over the oysters (total volume = 13.8 liters).

2. Running water was supplied by a salt water circulating system
which pumped directly from Hood Canal (chlorlnlty lh-l6 parts
per thousand) .

3. Ostrea lurlda (2 to 3 year class) were used after all barnacles,
spat, and epiphytic plants had been removed. These oysters were
obtained from Little Skookum Inlet on January 22, 1959 and kept
In Hood Canal until the start of the experiment.

k. Proximity tissues, i.e., tissues heavily infected with Hexamita
and bacteria, were obtained by the simple expedient of exposing
0. lurlda to overcrowding in non-circulating 3° C water. Ade-
quate levels of Infection were thus obtained in 30 to kO days .

5. Control proximity tissues were obtained by autoclavlng healthy
0. lurlda for 15 minutes at 120° C and I5 lbs pressure.

Experimental Design

In order to test the possibility of Hexajnita and bacteria trans-
mission through water, proximity experiments were designed in the fol-
lowing fashion;

A. Cold Water Experiment:

1. All test oysters (O. lurlda) were held in running water aquaria
for a one-week period of observation. Each aquarium contained
approximately 100 oysters. In this manner, any weakened oys-
ters would probably be eliminated before the start of the ex-
periment .

-69-

2. Ten oysters heavily Infected with Hexamlta and bacteria were
selected for a source of Infecting elements in the experimental
aquarium. Hereafter, these oysters will he referred to as
proximity-oysters . The right valve of each proximity-oyster
was removed and the tissue held in place with rubher bands.
This was done to assure a more effective circulation of water
over the tissues of the proximity-oysters, and to prevent them
from floating away from the shell and possibly blocking the
aquaria outlets.

3. In the control aquarium, ten autoclaved oysters were used for
proximity-oysters. In each case, one valve was removed as des-
cribed above.

k. In the experimental and control aquaria, the oysters were ar-
ranged so that five healthy oysters surrounded each proximity-
oyster. In this manner, ^O'o oysters were located in each aquar-
ium. '^-

5, The aquaria were set up in a cold room and aeration was provided
for approximately 6 hours until the aquaria water temperatiure
equaled that of the cold running water system (6.0° C). At
that time water circulation was started in each aquarium. Oys-
ters received an average of 5.0^ liters per day at the start

of the experiment. However, as the dead oysters were removed,
the remaining oysters received proportionately increasing a-
mounts of water, e.g., on the "jSth day, the remaining I5 ex-
perimental survivors were receiving l6«8 liters per day.

6, Since this experiment was started in JvuLy, it was necessary to
design a system that would lower the incoming Hood Canal water
to approximately 6° C, Toward this end, six 50-gallon barrels
were placed in a cold room. These barrels were connected by a
series of siphons such that water flowing into one end of this
system would be progressively cooled to 6° C. It was found
that the maximum rate of flow could not exceed 21 liters per
hour. Accordingly, two lines were metered to provide a flow
of 10.5 liters per hour through the experimental and control
aquaria, (Figure 2) .

7, Both experimental and control proximity-oysters were periodi-
cally renewed to prevent fouling of the aquaria.

8, Each aquarium was examined once every 2k hours, at which time
gapers were removed.

9, When a gaping oyster was removed, the tissues were immediately
examined and the levels of Hexamlta trophozoites were determined.
These slides were prepared by lightly smearing the entire oyster,
gills down, on a slide and approximating the level of Hexamlta
infection. When using a wide field 12. 5X eyepiece and lOX
objective, the following criteria were employed:

-70-

Pump

J Hood

Canal

1 Intake

Punfip

Cold Room

////////////////////// ///////J^f ///////////////

Bl= 50 Gallon Barrel With Floof Valve

B2-6=&0 Gollon Barrels

Tl = Control Aquarium

T2 = Experlmental Aquarium

3-

Fig, 2. Schematic diagram Illustrating the experimental set-up
of the cold water experiment.

-71-

Levels of Niimber of trophozoites

trophozoite Infection per mlGroscoplc field

Heavy = over 100

Moderate = 50 to 100

Light = 10 to 50

Very Light = 1 to 10

None = None

The above criteria apply only to freshly prepared, wet slides.

10. After each oyster was examined, sections were made for histo-
logical study. Giemsa, Heidenhain's Iron-hematoxylin, and
periodic acid Schiff (PAS) stains were used. For the PAS stain,
tissues were predigested with diastase ajid, following the Schiff
reaction, were couaterstained with hematoxylin (Lillle 195^).

11. At the termination of this experiment, all remaining survivors
were examined in the manner descrihed above.

B. Warm Water Experiment:

1. On March 26, 1958^ a similar experiment was conducted in which
the running water temperature ranged from 8° C in March, to
170 C in J\me with an average of 12° C for the 76-day period.
In this study, running water was provided directly from a
10,000-gallon storage tank without preliminary cooling. The
rate of flow per oyster averaged "J. 2 liters per day. In all
other respects this experiment was the same as the cold water
investigation.

RESULTS AND DISCUSSION

General Discussion

In both the cold and warm water proximity experiments the same
sovurce of Infected tissues was used in the experimental aquaria. In
the control aquaria, autoclaved tissues were used. The frequent re-
placement of proximity-oysters for both control and experimental aquaria
Increased the probability of having higher levels of Hexamlta and bac-
teria In the experimental aquaria as compared to the controls .

At the end of the cold-water experiment there was a decided
difference between the mortality rates occurring in the control and

-72-

experimental tanks. The mortality for the control aquaria was ll<- per
cent over a period of 76 days and for the experimental tank it was 70
per cent for the same period of time. No difference in rate of mor-
tality was found for the control and experimental aquaria of the warm
water experiment. In the 76-day period of the warm-water atudy, both
the control and experimental tank had a 6 per cent mortality. These
data indicate that in the presence of Hexamita and bacteria there is
an interaction between temperatiu-e and mortality.

Examination of tissues from Giemsa-stained sections revealed
variable levels of bacteria in dead and surviving oyster tissues. Al-
though no counts were made, the level of bacteria in the surviving
oysters from both the experimental and control aquaria was similar to
that observed in tissues of healthy oysters removed from the field.
In most cases extensive searching was required to find evidence of
bacteria in the tissues of surviving oysters - even when moderate
levels of Hexamita, as determined by wet slide preparation, were found.
In the mortalities of the experimental aquarium, 36 per cent of the
oysters having moderate to massive levels of Hexamita also had very
evident bacterial infections . The remaining 6k per cent had no appar-
ent bacterial infections, nor were bacteria obvious in the tissues of
oysters having light to very light infections of Hexamita . Hexamita
was consistently present in the experimental and control mortalities,
while bacteria were frequently absent even at elevated levels of the
protozoan infection. Furthermore, no developing bacterial levels were
observed in the survivors, thereby depreciating the role of bacteria
as a factor in the cause of death. The consistent presence of Hexamita
and the freqxxent absence of bacteria in freshly dead oysters points to
Hexamita as a prime suspect for the cause of death.

Figure 3 shows the level of infection of each oyster at the
time of death in the cold-water st\jdy and the survivor levels of in-
fection at the termination of the experiment, A pooled Chi-square
analysis (Cochran and Cox 1957) was carried out to determine if the
levels of Hexamita infection in the survivors of the experimental and
control aquaria were signlficajitly different. A Chi-square value of
9.35 was obtained ajid found to be significant on the ,05 probability
level.

In order to locate the cause of this significant difference,
the respective levels of infection for the control and experimental
aquaria were compared separately, e,g., moderate level of Hexamita
infection of control survivors against moderate levels of infection
for experimental survivors. None of the separate tests were found to
be significant. As a consequence, a Chi-square test of significance
for linear regression was carried out (Cochran and Cox 1957) • The
linear regression Chi-square was found to be 8.60, which is very sign-
ificant for the one degree of freedom associated with regression.
Therefore, the meaning of the significant "pooled" Chi-square is that
the level of Hex/^m-tta Infection was building up at a greater rate in
the experimental than in the control aquarium.

-73-

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UJ
Q.

40 —

30 —

20 —

10

® « • • ® EXPERIMENTAL MORTALITIES

^ ^ ^ ^ ^ CONTROL MORTALITIES

AVERAGE TEMPERATURE - 6.3 ^C

-'-'— "■*^-^— ■■»>**■- 1-ifi -

0 10 20 30 40 50

DAYS

'eb' ' ' Vo' " bb" ' 'cb' '

Pig, k. Compares per cent cumulative mortalities of the experi-
mental and control oysters.

-75-

V'-i '

;*«.

-.^1

..» ^-j

Fig. 5* Occlusion and inflammation of a "blood vessel. This photo-
graph shows the results of vascular Involvement "by Hexamita. (Approxi-
mately lOOX)

■■■■ *■' ^^ »"^

• -'-'01 t

Fig. 6. Destruction of intestinal mucosa. The photograph on the
left shows the normal intestinal mucosa. The middle and right-hand
photographs represent varying degrees of destruction seen in oysters
having moderate to heavy infections of Hexamita . (Approximately lOOX)

-76-

Tatle 1. X^ for pooled date of survivor level of Infection in cold
rtinnlng water proximity experiment,

Siirvivor Levels of Infection

None Very Light Light Moderate Total

Experimental

1

7

5

2

15

Control

12

26

1^

1

^3

Total

13

33

9

3

58

X2 = 9.3512 A significant difference at the

0.05 probability level. (3 d.f.)

The occurrence of Hexamlta in dead and siirviving oysters of
the cold-water control aquaria indicates that the protozoan is endemic
to southern Puget Sound- Had the experiment continued for another 70
days it is conceivable, \jnder the conditions of this experiment, that
a substantial number of the control oysters would have died due to
hexamitiasis (Figure k) .

Altho\agh there was no difference in the number of dead oysters
in the control and experimental aquaria of the warm-water experiment,
the few that died (6 per cent) were heavily infected with Hexamita.
This indicates that the protozoan, may well be a saprozoite at warmer
temperatures and takes on the role of a parasite in colder temperatvures .
This may be due to the decreased pumping rate and lowered metabolism
of 0. lurida when exposed to water temperatures at or around 6° C. It
could be speculated that decreased pumping and metabolic activity per-
mits the accumiilation of large Hexajnlta populations which, directly
or indirectly, cause extensive tissue damage and death.

Hlstopathology

Whenever moderate to heavy levels of Hexamlta were observed in
wet slide preparations, stained slides prepared from these tissues re-
vealed one or more of the following pathological aspects:

1. Blood vessels — frequently the involvement was characterized by
inflammation of the vascular epithelium and at times trophozoites
were numeroiis eno\:igh to occlude the vessel (Figure 5)«

2. Gastro-intestinal tract — necrosis of the intestinal mucosa was
frequently observed when trophozoites were common in the intes-
tinal tract (Figirre 6),

3. Leydlg cell connective tissue — in the presence of trophozoites,
lysis and/or disarticulation of the connective tissue was often

-77-

evident (Figure "j) ,

h. Gill TDraJichla — gill tissues frecLuently manifested vario\is stages
of decomposition when trophozoites were common In this tissue.
Moreover, wet slide preparations indicated that the gills
appeared to he the favorite site of trophozoites.

Blood vessels occluded hy hypertrophic leucocytes were also en-
countered in many of the stained slides prepared from the cold-water >
experimental mortalities. These leucocytes frequently contained an
intracellular parasite thought to he a reproductive stage of Hexamita
(Mackin et al. 1952) . These Intracellular forms appeared in 20 per
cent of the experimental mortalities, while few were observed in either
the experimental or control survivors of the cold-water investigation.
Figure 8 is a photomicrograph of the parasitized, hypertrophic leuco-
cytes described ahove. While it has heen assumed that parasites found
in leucocytes and free in hlood vessels are Hexamita, it has not been
demonstrated that that interpretation is the correct one and these
parasites may represent an unrelated infectious agent.

1. A lethal disease can be transmitted from Hexamita infected
tissues to healthy oysters .

2. Bacteria were depreciated as a causative factor In the death
of oysters infected with Hexamita . The "intracellular stages"
of Hexamita may be an associated but independent parasite.

3. The accelerated mortality rate of the cold-vater experimental
aquarium (6° C) and the absence of acceleration at warmer tem-
peratures (l2° C) suggest that there is an interaction between
oyster mortality and parasitization at the colder temperature.

k. There was no difference between the mortality rates of the

experimental and control aquaria of the warm-water experiment;
however, the few oysters which did die had heavy levels of the
protozoan and bacteria. This indicates that vinder these tem-
perature conditions, Hexainita may act as a saprozoite.

5. The accelerated mortality rate in the cold water experiment
suggest that under these conditions Hexamita and the associated
intracellular stages act as parasites. This may be due to the
decreased metabolic activity of 0. lurida at low-water temper-
atixres .

6. Severe tissixe damage accompanies infections.

-78-

\ \ ^

*H*r

^*IWI*t.,».^#

5> c^ Si

\ «!

)•

**^ J "^

^-

N.

Fig. 7- Trophozoites in Leydig cell connective tissue. Note the
histolysis and disarticiilation of the connective tissue cells. (Approx-
imately i4-50X)

-79-

Fig. 8. Intracellular parasites found in leucocytes associated
with Hexamita infected tissues. (Approximately 2500X)

-80-

REFEflEWCES CITED

Cochran, W. G. and G. M. Cox. I957. Experimental designs. Second
edition, Wiley, New York, 61I p.

Lillie, R. D. 195!;. Histopathologic technic and practical histo-
chemistry. P. Blakiston, New York, 501 p.

Mackln, J. G., P. Korringa, and S. H, Hopkins. 1952. HexamitiaBls of
Ostrea ed\ilis L. and Crassostrea virglnlca (Gmelln) . Bull.
Mar. Sci. G\n.f Carih. 1(4) : 266-277.

Medcof, J. C. 1959. Address given at the National Shellf Isherles
Assoc, meeting, Washington, D. C, July 26-30, 1959.

Stein, J, E. and J. G. Denlson. I958. Hexamita transmission by the

insertion of infected tissue. Unpublished manuscript, Rayonler
Marine Laboratory, Hoodsport, Washington,

-81-

EFFECTS OF THE FLATWORM STYLOCHUS ELLIPTICUS (GIRAKD)
ON OYSTER SPAT IN TWO SALT WATER PONDS IN MASSACHUSETTS

Anthony J- Provenzano, Jr.

U.S. Fish and Wildlife Service

Bureau of Commercial Fisheries

Biological Laboratory, Milford, Connecticut

ABSTRACT

During the summer of 1957 'the larvae and juveniles of
the flatworm Stylochus elllptlcus occurred in great abundance
in two salt water ponds on Martha's Vineyard Island. Setting
rates of flatworms were as high as one per shell per day.
Data over a period of three years indicate that such larvae
and post-larvae occurred in these ponds only when salinities
were helow 20'-'/oo . Mortality of newly set oysters due to depre-
dation by these flatworms was severe, approaching 100?!i in some
samples. Dipping infected oyster spat in a concentrated salt
solution proved an effective method of ridding the set of flat-
worms, but this is an effective remedy only if the treated spat
are protected from reinfestation. Because of the widespread
distribution of S_. ellipticus in many oyster-growing regions
in New England and elsewhere the need for a more effective con-
trol of its predation on oyster spat is obvious.

The U. S. Fish and Wildlife Service Marine Biological Labora-
tory at Milford, Connecticut, in cooperation with the Oyster Institute
of North America, is engaged in a study designed to develop the prin-
ciples of shellfish cxilture in salt vater ponds. Two of the ponds
under study. West Tishury Great Pond and Oyster Pond on Martha's Vine-
yard Island, Massachusetts, are periodically open to the sea and con-
tain pop\ilations of soft clams and oysters of economic importance.
These ponds are veil suited for oyster seed production since they are
remarkably free of such oyster enemies as drills, starfish and sponges.
However, observations during 3 years of study indicate that the flat-
worm Stylochus ellipticus is an important predator on oyster spat in
these ponds and that the future utilization of such ponds for produc-
tion of seed oysters may depend on effective control of this pest.

The potential of some polyclad flatworms as oyster enemies has
long been known and certain aspects of the biology and predatory nature
of related species have been published by Pearse and Wharton (1938)^
Dawson (1953)> and Woelke (195?) • Hopkins (l955) ascribed some oyster
mortality to Stylochus and Menzel and Hopkins (195^) included Stylochus
ellipticus in their list of predators on Louisiana oysters. Loosanoff

1 Present address! University of Miami Marine Laboratory, Miami, Florida.

-83-

Table 1. Occurrence
Pond, 1957

of larval and

•

post-larval fla-

bworms in Tisbury

Date

In Pln.nkton

Sallnlty
o/oo

18

On biweekly
cultch samples
#/20 shell faces

20 June

no tow

53

21

a few

18

no sample

2k

a few

17

2k

26

present

17

57

28

present

llv-l8

no sample

1 July

present

16-17

22

3

present

16

no sample

k

no tow

16.5

k

5

present

16.5

no sample

8

many

15.5-16.5

18

10

present

13

12

12

present

16.5

no sample

15

present

16.5-17.5

6

IT

present

15.5-16.5

2

l8 Tlsbury
Opened

• Pond

19

present

10.5-13.5

no sample

22

no tow

11.0-11.5

22

25 July -
August

18

absent

21-28

absent

l8 August
closed

Pond

2U

30 September

1 IndlY.

15

-81h

(1956) was the first to publish an account of predatlon on oyster spat
by Stylochus elllptlcus in the laboratory.

G?lie present paper la a record of observations on the numbers
of StylochuB and their predatlon on oyster set^ dxrring I956, 1957, and
19 5S in the tvo ponds on Martha's Vineyard. During the summer of I956
very few S. ellipticus, either adults or recently set individuals, were
seen. Again in 1958 the population was very sparse. In 1957, however,
large numbers of larvae, recently set individuals and adults were ob-
served.

The first Indication that this was to be an unusually large
year class was the discovery on June 20, 1957 of large nxmibers of what
appeared to be recently set flatworms on Tlsbury cultch examined for
oyster spat. Continued observations on the development of these ani-
mals confirmed their Identity. Plankton samples were being taken three
times a week with a #20 silk net. On June 21 large numbers of what
were assumed to be Stylochua larvae or post-larval stages were noted
In the plankton of Tlsbury Pond. These forms appeared as miniature
flatworms, oval in shape, and measured about 82 x 7I micra. During
the first week of July such numbers occurred that they constituted a
major component of the zooplankton. Flatworm larvae were also found
in the plankton of Oyster Pond, but in smaller numbers. In Tlsbury
Pond, flatworm set were found in considerable numbers until July 18
when this pond was opened to the sea. Some flatworm larvae wei-e found
in Tlsbury plankton samples until July 22 but only occasional specimens
were found on the biweekly set bags after the pond was opened.

Data for three stations in Tlsbury Pond were similar with re-
spect to dates of occurrence of the larvae and approximate nimibers of
flatworm set per shell. Values for one of the sampling stations are
presented in Table 1. The disappearance of flatworm larvae and ces-
sation of flatworm setting just after the pond opening was striking.
While it is possible that this would have occurred regardless of whether
or not the pond was opened, it is more likely that the disappearance
was due to large numbers of larvae being swept out of the pond at the
Initial opening, for at least one quarter of the pond water was esti-
mated to have escaped within 2ij- hoxirs of the opening, and additional
losses of plankton and pond water occiirred with each tidal cycle. Oys-
ter Pond, which was closed most of the summer, continued to show a few
flatworms in the plankton but none were found on cultch until July 29
when pond salinity had dropped to 20 0/00. A few flatworms continued
to set diiring August &a salinity continued to fall.

VThether the rapid change in salinity which followed the open-
ing of Tlsbury Pond was detrimental to the yoving flatworms can only
be conjectured at this point. Available data from 3 years' observation
indicate that planktonlc and recently set flatworms occurred in these
ponds only when salinities were below 20 0/00. Menzel and Hopkins
(195^) found adult Stylochus elllptlcus in ab\indance only in waters
of "relatively low salinity." In several cases in the literature other

-85-

species of flatworms were found associated with high rather than low
salinities (Dawson 1953^ Pearse and Wharton 1938) • Apparently each
species has its own requirements which need not be similar to those
of other members of the same or a related genus . Some adaptability to
salinity is to be expected. Woelke (1957) showed that despite a pre-
ference for 28 o/oo by Pseudostylochus ostreophagus^ that species could
tolerate 10 o/oo for at long as a week and even lower salinities for a
shorter time.

>

Predation by Stylochus on oyster set became apparent when on
August 7 some cultch bags from Tisbiory Pond were examined and in half
an hour several hundred flatworms measuring form X/h to l/2 inch in
length were found. In some cases as many as 15 worms were found on
the two sides of a single shell. Dead oyster spat were common, but
at this time there were still some live spat. A number of times flat-
worms were observed entering live oyster spat up to l/2 inch in great-
est diameter. When the top valves were lifted from recently dead spat,
1-3 flatworms usually were found, occasionally one of them in the act
of eating the young oyster. In one instance a small recently set flat-
worm was observed closely appressed to a two-day-old oyster. The flat-
worm was slightly smaller than the spat.

To estimate mortality due to flatworms and other causes, sajnples
of broadcast cultch were secured on August 13. These samples yielded
590 dead spat, 33 live spat and 8 flatworms. Only those spat were
counted which had grown large eno\igh to leave a scar or be seen with
the unaided eye. This meant that a mortality of at least 95^ tiacL oc-
curred since setting. There are no drills, and no starfish in either
pond. There was no evidence of crab damage and no reason to suspect
epidemic disease. Silting was responsible for some of the mortality,
for portions of the cultch with spat scars showed blackening from a
mud coating over the dead spat. In contrast to the mortality on broad-
cast shell, a sample of bagged cultch showed 303 dead spat, 179 live
spat and 9 flatworms, or a total post-setting mortality of at least
50^. In this case there was virtually no evidence of silting and near-
ly all mortality could be ascribed to flatworm predation alone.

In Oyster Pond a sample of spat examined in September 1957 ^^^
again in October, showed a mortality of 62fo for a six-week period.
These spat were bagged and had been washed by wave action, so silting
was not a factor and there was no evidence whatsoever to indicate causes
for mortality except for the presence and behavior of flatworms among
the living and dead spat. Survivors had grown well in the interval be-
tween examinations.

It is obvious from the above that an outbreak of the natvire
described would be very serious in a pond culture operation. In an
effort to find a method for field control in the summer of 1957> the
Milford laboratory began a series of experiments immediately. One
suggested method which had been tried experimentally in earlier years

-86-

vas the use of concentrated salt solution as a dip for Infested seed.
The method vas field tested "by immersing bags of cultch with spat in-
to pond vater saturated with rock salt. Flatworms curled and after
one minute immersion, none recovered. Q?he treatment also killed algae,
polychaetes and other organisms associated vlth the cultch. Eels and
toadflsh trapped In the bags reacted violently and soon died. After
15-30 minutes draining following a one-minute dip, the bags were divid-
ed into two lots. Some bags were placed in a floating raft to keep
them off the worm- infested bottom. Althoiogh there were no longer large
numbers of flatworm larvae in the plankton, a few Stylochua were f ovind
on the suspended bags several weeks later. These may have resulted from
a few pelagic larvae remaining in the water and setting late, from a
few flatworms surviving the dip treatment inside spat boxes or may have
been carried to the suspended cultch by water currents. Mortality on
the treated bags was arrested but bags replaced on the bottom became
reinfested and again began to suffer mortality.

On December 11, 1957 ^ control sample of vmtreated bagged cultch
vas taken from Tisbury Pond, Shells which in August had shown 50/i post-
setting mortality now yielded 60O scars, 7 live spat and 7 flatworms, a
total mortality of at least 99^*

The treatment with salt was effective in killing the flatworms
but to be commercially practical in these ponds a method of handling
the cultch and seed oysters will have to be developed and methods of
preventing reinfestatlon found.

ACKNCWLEDQEMEINTa

I wish to thank Mr, Ralph J. DePonte for assisting in obtaining
the information presented, I am. also grateful to members of the staff
of the Marine Biological Laboratory at Mllford for critical review of
the manuscript.

REF'ERENCES

Dawson, C, W, 1953. A survey of the Tampa Bay area, Florida Board
of Conservation, Tech, Ser. No, 8, 39 PP»

Hopkins, 3. H. 1955' Oyster setting on the Gvilf Coast. Proc. Natl.
Shellfish. Assoc, k^: 52-55.

Loosanoff, V. L, 1956. Two obscure oyster enemies in New England
waters. Science 123(3208) 1 1119-1120,

1957 • New method for control of several oyster ene-

mies and competitors. Bull. Mllford Biol. Lab., U. S. Fish
Wlldl, Serv, 21(5) i 1-6.

-87-

Menzel, R. W. and S. H. Hopkins. 195^* Studies on Oyster predation
in Terrebonne Parish. Louisiana. Project 9^ Texas A & M Re-
search Foundation, 145 PP-

. 1956. Crabs as predators of oysters in

Louisiana. Proc, Ifa,tl. Shellfish. Assoc. 1+6: 177-184.

Pearse, A. S. and G. W. Wharton. I938. The oyster leech, Stylochua
inlTTilcus Palombi, associated with oysters on the coasts of
Florida* Ecol. Monogr. 8: 605-655.

Woelke, C. E. 1957* Flatworm, Pseudostylochtis ostreophagus Hyman,
a predator of oysters. Proc, Natl. Shellfish. Assoc, k^i
62-67

-88-

FIATWORM DISTRIBUTION AND ASSOCIATED OYSTER
MORTALITY IN CHESAPEAKE BAY

John R. Webster and Royston Z. Medford

Fishery Research Biologists^ Bureau of Commercial Fisheries

Annapolis, Maryland

ABSTRACT

The marine flatworm Stylo chus elllptlcu3 (Girard) Is
reported from 73 widely scattered localities In Maryland Chesa-
peake Bay and Its tributaries. It Is the only polyclad turbel-
larlan so far Identified In dredglngs from oyster beds within
that area. Occasionally found In fresh spat boxes, the worm Is
considered to be a predator of oyster spat In the Bay. Evidence
for worm predatlon on small oyster spat Is presented. Of the
567 worms found In 90 seed-oyster samples, about 95 per cent
were recovered from fresh spat boxes. Statistical treatment
of the data concerning worm Incidence and recent spat mortality
Indicated the probability that such mortality resulted from
predatlon by S. elllptlcus.

INTRODUCTION

Although the marine flatworm, Styloehus elllpticus (Girard),
has heen known to occur in Chesapeake Bay for some time, little atten-
tion has "been given to its potential as a predator of oyster spat. In
view of this, a systematic collection of this species was started in
1958 to determine its distribution and possible association with oys-
ter spat mortalities .

DESCRIPTION OF THE WORM

The largest specimens of S. elllpticus collected in Maryland
Chesapeake Bay measure about 25 nnn. The wonn is oval, leaf -like, flat
and thin with undulating margins. The most common color is brown, but
dark pink, orange and olive drab worms have also been observed. The
ventral surface is usiially lighter than the dorsal. There Is commonly
a median dorsal stripe of lighter color athwart which are paired ten-
tacles near the anterior end. There are numerous ocelli (eye spots)
around the anterior margin and on the tentacles. The mouth, with its
short pharynx, is situated about I/3 back from the anterior end of
the body on the ventral side. The pharynx may be everted to a con-
siderable distance. The intestine has many branches and terminates in
a fine network along the edges of the worm. Male and female genital
pores, situated close together near the ventral posterior margin, pro-

-89-

Fig. 1.

-90'

vide for sexual reproduction. Hyman (l9ifO) gives a detailed taxonomlc
description of this species.

DISTRIBUTION OF STaLOCHUS ELLIPTICUS IN MABTCAHD CHESAPEAKE BAY

The geographical range of this species, as listed by Hyman
(19^10), is from Prince Edward Island to Texas. Pearse (1938) records
the presence of S. ellipticus from a number of localities in Chesa-
peake Bay and Engle (unpublished field data) reported this species
fairly abundant on oyster beds in the Bay as early as l^kk.

Our present collection was started in 1958. Most specimens
were foimd in samples of bottom material dredged dioring spring oyster-
popiilation surveys. These surveys included dredgings on some of the
public oyster beds in Maryland Chesapeake Bay and'.most of its tribu-
taries. The actual finding of worms in these dredgings was incidental
to coxonts bearing upon the age composition of oyster popiJlations . Thus,
while the collection data are not quantitative, the survey records show
that flatworms were present at 73 widely scattered locailitles in the
Maryland Chesapeake Bay (Flguxe l).

The total number of worms collected on the baywide surveys
since 1958^ about I50, is small in comparison with their broad distri-
bution. Within limits of our collection methods, however, Stylochus
has been found in greatest numbers on oyster beds in the lower Potomac
River and its Immediate tributaries. Hyman (19^0) reports that the
habitat of this species is littoral and that it is generally found a-
mong oysters and old shells, barnacles, and under rocks. The data pre-
sented in this paper relate only to flatworm distribution on oyster
beds in the Bay.

EYTDENCE OF PREDATION

Sometimes during oyster-bed surveys flatworms were fovmd in
fresh oyster spat boxes (hinged valves with clean inner faces but no
meat). Pearse and Littler (1938) and Hyman (l9^0) consider S. elllp-
ticus to be an oyster predator. Pearse and Wharton. (I938) demonstrated
that a related species, Stylochus frontalis (=inlmicus), is an active
oyster enemy in Florida. Loosanoff (195^) has shown in laboratory ex-
periments that S. ellipticus feeds on oyster spat. In one experiment,
10 worms consumed 21 spat in less than one month. Although other mem-
bers of the genus Stylochus may be present in Chesapeake Bay, S. ellip-
ticus appears to be the dominant species of polyclad turbellarlan
common to oyster beds in the Maryland part of the Bay. It was the only
species present in our collections as Identified by Dr. Libbie H. Hyman
of the American Museum of Natural History. Thus, while attacks by flat-
worms against healthy oysters were not specifically observed, our find-
ing of worms in fresh spat boxes certainly suggests that predatlon of

-91-

oyster spat "by £. elliptlcus does occur in Chesapeake Bay.

The contention that predatlon of oyster spat hy flatworms may
he common in the field is supported hy the relationship of worm dis-
tribution and spat mortality. The data vere obtained in conjunction
with an oyster-seed production experiment conducted in 1958 at Smith
Creek, a tidal estuary of the Potomac River.

Wire hags, each containing one- quarter bushel of oyster shells,
were suspended in vertical strings at 10-foot intervals along a 2^+0-
foot transect running outward from the shore across groiinds planted
with shell. Depths ranged from 3 feet at the inshore end to about
8 1/2 feet at the offshore end of the transect. Most strings accom-
modated k bags arranged at various depths between surface and bottom.
The entire transect was represented by 90 bags.

After removal of bags in the fall, they were examined and
counts were made of living spat (a measure of survival after setting)
and spat boxes (an approximate measvire of recent spat mortality) . A
search was made for flatworms on shells, in spat and in all spat boxes.
Some bags were free of worms j in others the co\mt ranged as high as 19 .
In all, 567 worms were found for an average of about 6 per bag. Nearly
95 per cent of the worms were found in fresh spat boxes, the remainder
occurring elsewhere on culch shells or in the debris on the examining
table. In most cases, worms were not present in spat boxes whose inner
faces were covered with fouling growth or silt. .

RELATIONSHIP BETWEEN SPAT MORTALITY AND WORM INCIDENCE

Spat-box counts were limited to material from 26 bags distri-
buted throiighout the transect. These counts showed a positive cor-
relation between the percentage of boxes in the total of spat and
boxes together and the numbers of worms per bag. The coefficient of
this correlation was + O.58. Linear regression variance was summarized
as follows:

DF SS MS F li Point

Explained by regression 1 15^^ 15^ 12.1 7.82

Unexplained deviations 2k 30 56 I27

Total 25 h6O0

Apparently the relationship between spat mortality and worm incidence
was not a consequence of chance alone. A scatter diagram of the cor-
relation points, with the line of regression determined by the method
of least squares, is shown in Figixre 2.

-92-

• — * — t — •

8^

_ e- 1^ s g; ?. f

g^

en r*» ("^

^ o\ j» o^ ^ e-
N Ov c^ -^ ^J &v

r\ N N (NJ N rH •-<

c

O

•H
O

1^

o

a

•H
H

o "d
^.5

bb-p
•H 03
f^ Pi

ofi wd LJds io tuawriM

3 a

o

•H

u

H

O

o

•H

t^
H

O

o

o

•P

•H

ft
• CQ
CM

."S
bO (U
•H O

-d

jjasxHi snoa ins to wowu

-93-

DF

ss

MS

F

1> Point

1

26)+256

2614-256

28.2

6.96

88

82U883

937^^

89

1089139

KELATIONSHIP BETWEEN SPAT SURVIVAL AMD WCKM INCIDENCE

Counts representing spat survival and worm incidence for all
90 "bags were treated without regard to l)ag position in the transect.
Here, a coefficient of correlation of approximately - 0.^9 was cal-
culated. Despite this low coefficient, a simple analysis of variance
showed high probability that the relationship was not a consequence
of chance. In this instance:

Exrplained by regression
Unexplained deviations
Total

A scatter diagram of the ralationship between spat survival and worm
incidence, with the line of regression calcvilated by the method of
least squares, is shown in Figiire 3«

CONCLUSIONS

Our field collections prove that the turbellarian flatworm,
Stylochus elllpticus, occurs widely in the Maryland Chesapeake Bay and
its tributaries. Recovery of worms from fresh oyster boxes, mostly
spat, suggests a predatory relationship in explanation of many oyster
mortalities. The evidence is circumstantial but corroborates reports
of predation by investigators elsewhere. Returns from a seed-oyster
production experiment in southern Maryland show that the vulnerability
of oyster spat to flatworm predation could be real and markedly detri-
mental to seed production. The possibility is substantiated by the
statistical association between three sets of numbers, one representing
flatworm incidence, another representing spat mortality and the third
representing the rate of spat survival during the summer.

LITERATURE CITED

Hyman, L. H. 19^10. The polyclad flatworms of the Atlantic Coast of

the United States and Canada. Proc. U. S. Nat. Mus. 89(3101)1
1^1^9-^95.

Loosanoff, V. L. 1956. Two obscure oyster enemies of New England
waters. Science 123(3208): 119-1120.

Pearse, A. S. I938. Polyclads of the East Coast of North America.
Proc. of the U. S. Nat. Mus. Q6{30kk)i 67-98.

-9^-

and J. W. Littler. I938. Polyclads of Beaiifort, N. C.

Jour. Elisha Mitchell Scl. Soc . 5^(2): 235-2llJ4-.

and G. W. Wharton. 1938. The oyster "leech," Stylochus

Inlmlcus Palombi, associated with oysters on the coasts of
Florida. Ecol. Monogr. 8t 605-655.

-95-

THE EFFECTS OF SALT SOLUTIONS OF DIFFEIRENT STRENGTHS

ON OYSTER ENEMIES

L. W. Shearer and C, L. MacKenzle, Jr.

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

ABSTRACT

Boring sponges (Cllona celata), starfish (Asterlas
forbesl)^ and tunlcates (Molgula manhattenslB) were treated in
salt solutions of different strengths for 1. 3, 5, 10 and 15
minutes . Oyster drills (Uroaalplnx clnerea) were exposed to a
saturated solution for 30 minutes, one hour and three hours.
Sponges were killed by a 5-niinute exposure in a saturated solu-
tion and by a 10-minute exposure in solutions 90, 70 and 60 per
cent saturated. If sponges were dried following exposures,
greater mortalities occurred. A one-minute exposure, followed
by a one-hour drying period, was sufficient to cause complete
mortality in solutions 100 ajid 90 per cent saturated, three
minutes in 80 per cent saturated, ten minutes in 70 &nd. 6o per
cent saturated, and fifteen minutes in 50 per cent saturated.
Starfish were killed by a 3-ialiiute exposure in solutions 50 per
cent or more saturated. Tunlcates were killed by a 5-niinute
exposure in saturated and 90 per cent saturated solutions, 10
minutes in solutions 80> 70 and 6o per cent saturated, and 15
minutes in solutions 50 and ^40 per cent saturated. Although
drills, Urosalpinx and Eupleura, are not killed readily by salt
solutions, embryos of both can be killed by treating them in a
saturated salt solution. Caution should be exercised in ex-
posing seed oysters (Crassostrea vlrglnica) with damaged bills
to salt solutions, since they are killed by high salt concen-
trations .

INTRODUCTION

Of more than a thoiisand chemical compounds tested at Milford
Laboratory for their effects on shellfish enemies, none killed the
"boring sponge, Cllona celata, at concentrations low enough to be non-
injurioiis to oysters. In the past, certain workers, including Dollfus
(1921), deLaubenfels (19^7) and Warburton (1958), recommended that
sponges be killed by placing infested oysters in fresh or brackish
water. Loosanoff (19^5) reported that starfish can also be killed in
sea water of reduced salinity. With both sponges and starfish, how-
ever, periods of lethal exposure to fresh water are too long to be
practical.

Since death in fresh water is tJie res\ilt of upsetting the iso-
tonic beilance between body fluids of the organism and its environment
(Prosser et al. 1950, and many others), it was suspected that death

-97-

could be achieved much more rapidly in waters of greatly increased
salinity. In a highly hypertonic solution, an aquatic animal's body
fluids might be lost resulting eventually in destruction of cells and
organs ,

Loosanoff (195T) conducted experiments in which he killed sev-
eral oyster enemies by exposing them to saturated salt solutions (300
parts per thousand) for certain periods followed by drying them in air
(Table l) . The present study was undertaken to determine the effects ,
of less than satiarated salt solutions on these enemies.

METHODS

In these experiments a saturated solution of "rock salt" was
prepared and then diluted with sea water to give 90^ 80, ^0, 60, 50,
kO and 30 per cent saturated solutions.

Because it had previously been found that oyster enemies were
killed within 15 minutes in a saturated salt solution (Table l), the
periods employed In this study were 1, 3, 5, 10 and 15 minutes.

Table 1. Minimum exposures in a saturated salt solution and drying

times required to cause 100 per cent mortality of six species
of oyster enemies (Loosanoff 195?) •

Exposure

in
minutes

Drying time in minutes

None

5

10

15

30

1^5

6o

Asterlas forbesi

Molgula manhattensls

Cliona celata

Crepidula fornicata

Urosalplnx clnerea

and
Eupleura caudata egg cases

3
5-10

3-5
5

5

X
X

X

X

X

Drying, which prolongs the contact of animals with salt and concen-
trates unsaturated solutions by water evaporation, was used only where
it had proven necessary before. Starfish and drills were kept for ik
days in running sea water in the laboratory following salt treatments.
Other animals were suspended for the same period in Milford Harbor in
perforated plastic boxes which allowed adequate circulation of water.

-98-

RESULTS

Boring Sponges

Two samples of ten marketable-sized oysters infested with
Cliona celata were exposed to salt solutions for each Immersion period.
One sample from each was dried for one hovir. Drying caused much higher
mortalities (Tables 2 and 3) .

Sponges, extended with their oscula open in normal sea water,
contracted during exposure. Dying and decomposing sponges pass through
several color changes. From a deep, rich yellow they change to tan,
black and then to white.

Starfish

All starfish (lO per sample) were killed by an exposure of one
minute in 100, 90, 80 and 70 pe^ cent satiorated solutions. In 6o and
50 per cent saturated solutions three-minute exposures killed them.
Starfish were not killed when exposed 15 minutes in solutions less than
50 per cent saturated. Water temperatures during this experiment rang-
ed between 13 and I7.20C.

During the drying periods, red pigment was observed to leach
slowly from the starfish's Integument. If starfish were placed in
normal sea water immediately following exposiire, much red pigment was
lost from their ruptured body cells and organs, and their rays shrajik
and curled at the tips. Soon the starfish flattened out, and autotomy
of the rays and rapid disintegration of the animal followed.

Tunicates

Twenty-five lonattached Molgula manhattensis were used in each
sample. Salt solutions 100 and 90 per cent satinrated killed tunicates
within five minutes. Solutions 80, "JO and 60 per cent satijrated killed
them within ten minutes and 50 and kO per cent saturated solutions,
within 15 minutes (Table h) ,

Dead tvinlcates usually remained rigid for several days after
their return to sea water, but then became flabby aiid often eviscerated
themselves .

-99-

Table 2. Per cent mortalities of sponges 1^1 days after their exposure
to salt solutions of different strengths and immediate return
to sea water. Sea water temperatvtre 9 - 11°C

Immersion
in

Per c

;ent saturation

mi nil tea

100

90

80

70

6o

50

iHD

30

1

0

0

■X-

0

0

0

0

0

3

10

0

•X-

0

0

0

0

0

5

100

50

50

0

20

0

0

0

10

100

100

90

100

100

50

0

0

15

100

100

100

100

100

0

0

0

*Sajnple lost

TalDle 3. Per cent mortalities of sponges ik days after their exposure
to salt solutions of different strengths ajid drying for one
hour. Sea water temperature 9 - 11°C

Trmtierslon
in

Per cent saturation

minutes

100

90

8o

70

6o

50

i^o

30

1

100

100

50

6o

75

25

0

0

3

100

100

100

80

100

80

0

0

5

100

100

100

90

50

50

0

0

10

100

100

100

100

100

50

25

0

15

100

100

100

100

100

100

50

0

-100-

Table k. Per cent mortalities of tunicates ik days after exposttre to

salt solutions of different strengths. Sea vater temperature
5.2 - 11.5°C

TTmnersion
in

Per cent

saturation

minutes

100

90

80

TO

60

50

ko

30

1

8

12

8

8

16

1^-

0

0

3

72

ko

20

52

2k

k

8

8

5

100

100

6k

81+

20

12

12

0

10

100

100

100

100

100

,96

80

k

15

100

100

100

100

100

100

100

0

Drills

Urosalpinx cinerea is an extremely hardy animal capable of
Isolating itself from unfavorable environmental conditions by tightly
closing its operculiom (Carriker 1955)' Therefore, it vas decided to
try a saturated solution for periods as long as 30 minutes^ one hour
and three hours before proceeding to weaker solutions and shorter ex-
posures. Groups of 50 drills were exposed to these conditions. Fol-
lowing exposures to salt solutions one-half the drills in each group
were returned to normal sea water and the others were dried for one
hour. Only three of the six treatments caused any mortality. Eight
per cent of the drills died after three hours of exposure and immediate
return to sea water; 12 per cent died after one hour of exposure and
one hoior of drying; and 32 per cent died following three hours of ex-
posure and one hovor of drying.

Although drills are not readily killed by this method, it was
found to be quite successful in killing embryos of U. cinerea and
Eupleura caudata, regardless of their stage of development (Table l) .

Oysters

Eight to 15 Crassostrea virginica, 10 to 20 millimeters in
length, were used in each sample. Few oysters died after exposvire to
any salt treatment, provided the edges of their thin shells were not
chipped (Table 5). Chipped oysters were killed. While exposed to
salt solutions, oysters remained tightly closed, except when they
opened their sheila slightly, probably to test the water. There was
evidence that oysters were adversely affected by salt solutions be-
cause some of them did not produce true feces for several days follow-

-101-

ing a treatment j however, all produced pseudofeces.

Table 5. Per cent mortalities of young oysters, 10 to 20 millimeters
in length, l^J- days after exposure to salt solutions of dif-
ferent strengths. Sea water temperature 9 - 11°C

Immersion
in

Per (

2ent saturation

minutes

100

90

80

70

60

50

ko

30

1

0.0

0.0

0.0

1.9

0.0

0.0

0.0

0.0

3

2.1

11.1

3.0

0.0

0.0

0.0

0.0

0.0

5

0.0

10.2

0.0

0.0

0,0

0.0

3.1

k.o

10

i+.3

3.5

0.0

9.0

2.3

0.0

1.8

0.0

15

0.0

3.3

*

0.0

0.0

0.0

0.0

0.0

•'<5ample lost

DISCUSSION

Several oyster enemies can he killed readily hy exposing them
to partially saturated salt solutions. Effective concentrations can
he easily maintained hy constantly stirring salt crystals into solution.

In practice, if several enemies are involved, the salt treat-
ment shoiild he administered to kill the species most resistajit to it.

Since water temperatures during these experiments ranged from
5 to 17.2°C, repetitions of them are planned for water temperatures
ranging between 15 and. 25°C. At higher temperatures, minljnum exposures
for these enemies may be considerably shorter.

ACKNOWLEDGMEfflS

The authors would like to express their thanks to Dr. V. L.
Loosanoff for s\iggesting the studies and to Mr, H. C. Davis for his
advice in the preparation of this report.

LITERATURE CITED

Carriker, M. R. 1955. Critical review of biology and control of oys-
ter drills. U. S. Fish Wildl. Serv., Spec. Sol. Rept. Fish.
No. ll|8, 150 pp.

-102-

de Laubenfels, M. W. 19^7» Ecology of the sponges of a "brackish water
environment at Beaufort, North Carolina. Ecol. Monogr. 17 (l):
3l-h6,

Dollfvis, R. 1921. Resume de noa princlpales connaissances pratiques
sur les maladies et les ennemls de I'hultre. Office Scl. et
Tech. des Peches Marltlmes, Notes et Memoirs T: 1-^6.

Loosanoff, V. L. 19^5* Effects of sea water of reduced salinities

upon starfish, A. forbesl, of Long Island Sound. Trans. Conn.
Acad. Arts Scl. 36: 813-835.

, 1957. New method for control of several oyster

enemies ajid competitors. Bull. Mllford Biol. Lab., U. S. Fish
Wildl. Serv. 2l(5): 1-6.

Prosser, C. L. , P. A. Brown, D. W. Bishop, T. L. Jahn, and V. J. Wulff.
1950. Comparative animal physiology. Saunders, Philadelphia.
888 pp.

Warburton^ F. E. 1958. Control of the boring sponge on oyster beds.
Progr. Rept. Atlantic Coast Stations, Fish. Res. Bd. Canada.
69: 7-11.

-103-

CHEMICAL CONTROL OF FOLYDORA WEESTERI
AMD OTHER AJTOELIDS INHABITING OYSTER SHELLS

Clyde L. MacKenzie, Jr. and L. W. Shearer

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

ABSTRACT

A number of chemical compounds were tested and found
either to compel Polydora websteri and nereid worms to emerge
from oyster shells or to kill them directly within the shells.
Compounds which caused more than 50 per cent of both groups to
emerge from shells within three hours were considered effective
vermifuges . Compounds which killed more than 50 per cent of
worms within the same time were considered effective vennlcldes.
The most efficient vermifuges for P^ websteri were benzene and
ethylene compounds . Common salt was the most practical vermi-
cide tested because It is simple to use^ inexpensive and kills
worms quickly.

Worms of the genus Polydora, conmonly called miid-blister worms,
are serious pests. They not only form unsightly black areas on the
inner faces of oyster shells, rendering infested oysters undesirable
for half -shell trade, but also make oyster shells brittle. There are
several published reports of attempts to use chemicals to control these
worTiis. Among them is that of Korringa (l95l) who reports that P.
hoplura and P. cillata can be killed by immersing infested oysters for
three hours in sea water containing di-nitro-ortho-cresol at 500 parts
per million or by placing oysters for l6 hours in fresh water; and
Mackln and Cauthron (l952) observed that a solution of phenol at 500
ppm in sea water causes P. websteri to emerge. These authors do not
state the rate at which worms leave the shells.

During the screening of a large number of chemical compounds
for possible use in combatting shellfish enemies, a study carried on
at Milford since 19^6, several compounds have been found which are
more effective against P. websteri, the species which occurs in south-
ern New England waters, than those proposed by earlier workers. Two
categories of compounds coiild be used in control of these worms: (l)
vermifuges, chemicals that cause worms to emerge from their tubes and
leave the oyster shell, and (2) vermicides, chemicals that kill worms
within their tubes, but do not ordinarily cause emergence.

Oysters heavily infested with P. websteri were gathered from
uncultivated beds in West Tisbury Great Pond on Martha's Vineyard Is-
land. They were kept in wire baskets in Milford Harbor until 2k hours
prior to their use when they were brought into the laboratory and
allowed to become acclimated gradually to room temperature. For each

-105-

experiment two oysters, each In a separate finger bowl, were submerged
in a nine-liter container of the solution to he tested.

In initial tests each compound was tried at a concentration of
100 ppm. Following each test, oysters were kept in warm running sea
water for five to eight days to allow any living worms to recover from
the effects of the compound tested. They were then placed for 2^4- hours
in a solution of 0-dichlorohenzene at 100 ppm which was fo\md to cause
aJinost 100 per cent emergence of both Folydora and nereid worms. Thus-,
a group of eight oysters which yielded an average of 88.7 P. websteri
and 10.7 nerelds per oyster upon Initial treatment gave only one or
two worms per oyster on the second treatment with this chemical. Any
worms which survived the initial test could be collected and counted
by this method. Because oysters immersed for 2k hoxirs in a 100 ppm
solution of 0-dichlorobenzene may die, this treatment was used merely
to insure 100 per cent emergence of worms. As many as 332 P. websteri
were recorded for a single oyster.

The effectiveness of vermifuges was calculated directly by com-
paring the number of worms emerging in the experimental vermifuge with
the number emerging in the subsequent treatment with 0-dichlorobenzene.
Since no simple method exists for qviantltatively removing from oyster
shells worms that have been killed in situ, it was necessary in evaluat-
ing vermicides to use the average number from the eight oysters mention-
ed above as the total worm population and the nimiber emerging on subse-
quent treatment with 0-dichlorobenzene as survivors of various chemicals,
Obviously, the data for effectiveness of vermicides are less accurate
than those for vermifuges.

Over 30 compounds caused some P. websteri to emerge, but only
those causing 50 per cent of worms to emerge within three hours are
considered effective vermifiiges (Fig. l) . Of these, 0-dichlorobenzene
was selected as the most practical vermifvige because at concentrations
below 100 ppm it is more effective than monochlorobenzene, trichloroe-
thylene ajid tetrachloroethylene . Moreover, the latter two compounds
are quite volatile and may be somewhat dangerous to people handling
them on a large scale. One compoxond more effective than 0-dichloro-
benzene, J|-nitrobenzene-azo-resorcinol, is difficult to dissolve in
sea water and is expensive. Phenol is not an effective vermifuge at
concentrations much below 500 ppm, e.g. at 100 ppm it caused emergence
of only about 33 per cent of the worms. Naphthalene might be effective,
but is difficult to dissolve in sea water. When added to 0-dichloro-
benzene, however, enoiigh of it goes into solution to increase the ef-
fectiveness of the 0-dichlorobenzene. Certain nereid vermifuges, such
as emulsifiable rotenone, Bulan crystalline, and Tris (acetoxy methyl)
nitro methane, caused P. websteri to retract into their tubes but did
not cause emergence.

Usually it reqxiired about five or six minutes for the first P.
websteri to emerge, probably becaiise it takes about that long for the
chemical to penetrate the worm's tube. Worms usually back away from

-106-

TRICHLOROETHYLENE

^^^^^^^^^^^^^^ fii;;:;!;:::;!;:;:

H

MONOCHLOROBENZENE

PER HOUR
i FIRST

mmmmmmmmm x^mm

4-NITR0BENZENE-A20-RES0RCIN0L

mmmmmmmmm^ mmmm

O-DICHLOROBENZENE

m:mf(^f^m^(mm^mm0i \mm

SEVIN

1 1

P-DICHLOROBENZENE

TETRACHLOROETHYLENE ^

vmmmi mmmm r-^

1 1 stCUN U 1

PHENOL (500 PPM)

1 THIRD

^iiiMiiiiiii!iiif!fiiifiifiif!ii!f!iiii!!!iii W^^ tiiiik

Wm6iimm6^^iimmiKm6imKKmi^ l.::::::;:::::|

• • • • • • 1 1_

0 10 20 30 40 50 60 70 80 90

PER CENT

Pig. 1. Rate of emergeace of P. velpsterl from oyster bnells dur-
ing exposure to 100 ppm solutions of various chemicals for three hours,
Only those compovinds that were more than 50 per cent effective are
shown. Rate of emergence was not determined for Savin.

chemicals and emerge posterior end first. Since they do not swim^
worms drop to the bottom of the pans and, if removed to another vessel
containing untreated sea water and some bottom detritus, they soon be-
gin building new mud tubes.

Nereid worms appear to be more sensitive to certain chemicals
than P. websteri and react to them more quickly (Fig. 2) . Since they
do not live in long narrow tubes and contact a chemical with their
whole bodies, nereids usually begin to emerge as soon as oysters are
placed in the solution.

Several compoimds killed P. websteri within their tubes (Fig. 3).
Dl-nltro-ortho-cresol at 100 ppm killed few worms. At 500 ppm, however,
the concentration which Korrlnga (l95l) used, 8l.if and 90.5 per cent
were killed by exposures of one and three hours, respectively. A 500
ppm solution of 2-chloro-l-nltro propane killed 96.6 per cent of the
worms within three hours. Victoria Blue at 200 ppm killed 82 per cent
with only a 5-mlnute immersalj 89 per cent were killed within 10 minutes j
and 97 per cent, within 15 minutes. Actually, the lethal effects of
this dye on annelid worms were discovered at this laboratory before the

-107-

a^

•p

^

W P(

I

il

i

o|

^1
-I

-I

5l
'I

-I

i

K ^

>-^

■i

^

^

^1

SJ

y "^

5

i

I.

; 2}

" o t

£ Id X
U. « H

IDD-

I

^1

>- 8
^1

^

•H nJ

(U >

0) o •

o

0) CO

o o

o

<u X h

OJ bO

a d !h

53 -rH o

o tj a

0)

-p

w ft

o

o

•^

bO

0)

OQ

M
0)
-P
W
t>>
O

a

o

u

-p

-108-

present study, and this compound has been used routinely during the
last four years to exterminate worms In clam hatchery troughs . The
ahove compounds, except 2-chloro-l-nltro propane, were also lethal to
nereids as were other compounds listed in Figiire k.

VICTORIA BLUE

2,4- dichloropmenol

2,4,5 - TRICHLOROPHENOL

^mmm.

PENTABROMOPHENOL

SEVIN

4-CHLOROBENZYL CHLORIDE
DDT

\A

0 " 40

80

60 70

PER CENT

80

90

100

Fig. h. Mortality of nereid worms in oyster shells after exposure
to various chemicals at 100 ppm for three hours.

Perhaps the most practicaJ. compoimd for worm control is common
salt. From 87 to 98 per cent of P. websteri were killed by a 10- to
15-ininute dip in a saturated salt solution followed by 15 or more min-
utes of drying in air. Shorter dips required a longer period of dry-
ing to kill worms, but even a 1-minute dip was 89 per cent effective
when combined with a drying period of at least two hours . These studies
showed that it is almost impossible to kill every P. websteri in an oys-
ter shell within three hours.

At low concentrations several compounds apparently acted as
Inhibitors to P. websteri. For example, in 2-chloro-l-nltro propane
the worms neither extended their tentacles as in feeding nor emerged.
When combined with vermifuge i^-nltrobenzene-azo-resorcinol, worms
emerged only as the concentration of the inhibitor was decreased
(Table l) .

-109-

Table 1. Comparison of numbers of P. vebsteri emerging within three
hours from shells of two oysters in a 10 ppm solution of
vermifiige ^4— nitrobenzene-azo-resorcinol in the presence of
an Inhihltor^ 2-chloro-l-nltro propane, at various concen-
trations .

Concentration of

Number of worms

Number remaining

inhlhltor in ppm

emerging

alive in shells

50

37

103

20

63

kQ

10

77

78

5

112

67

0

158

35

Inasmuch as oysters stay closed while immersed in solutions of
most chemicals, they are -usually not harmed by them. They pump inter-
mittently, however, in benzene and ethylene compounds j hence, these
compounds may be lethal if the Immersal is too long. In 0-dichloro-
benzene at 100 ppm, for instance, an Immersal of three hovirs caused
little mortality of oysters, but one of 2il- hours killed aljnost all of
them. At the end of this period oysters gaped widely and did not re-
cover after being placed in running sea water. They also pump in a
solution of Victoria Blue, which, even if the concentration is low,
accumulates in their tissues and eventually kills them. Consequently,
in these experiments we used Victoria Blue in fresh water which kept
oysters closed and therefore prevented them from absorbing the dye.
Oysters with chipped bills (7 of 4o) could not keep the dye out and
eventually died.

We believe these vermicides can be applied in commercial oyster
culture. Oystermen could use them to kill worms in- shells of oysters
in spring, thus giving oysters a chance to deposit layers of white shell
over the black m\id blisters by fall. This treatment, however, would
not prevent setting of young P. websteri which could form small blis-
ters at the periphery of shells . ' A saturated solution of salt appears
to be most practical for oystermen, because it is simple to use, in-
expensive, requires only a short exposure to kill worms, and kills
some other foiaing organisms as well (Loosanoff 1957). The other com-
po\mds would be difficult to maintain at precise concentrations. Ver-
mifuges could be used in various ways in studies of the biology of
worms and of oysters. To cause all worms to emerge without harming
oysters. Infested oysters could be alternately immersed in O-dichloro-

-110-

benzene for not longer than three hours in which the worms will emerge
and then in sea water for recovery of the oysters, until all worms
have emerged.

ACKNOWLEDGMENTS

The authors express their gratitude to Dr. V. L. Loosanoff for
sioggesting these experiments and for his guidance in planning them, and
to Mr. Harry C. Davis and Miss Rita S. Riccio for editing the manuscript.

LITERATURE CITED

Korringa, P. 1951- The shell of Ostrea edulis as a habitat. Arch.
Neerl. Zool. 10: 32-135-

Mackin, J. G. and Fred Cauthron. 1952. Effect of heavy infestations
of Polydora websteri Hartman on Crassostrea virginica (Gmelin)
in Louisiana. Proc. Natl. Shellfish. Assoc. 1952: ll+-2l+.

Loosanoff, V. L. 1957* New method for control of several oyster ene-
mies and competitors. Bull. Milford Biol. Lab., U. S. Fish
Wildl. Serv. 2l(5): 1-6.

-Ill-

TRIAL INTRODUCTION OF EUROPEAN OYSTERS (OSTREA EPULIS)
TO CANADIAN EAST COAST

J. C. Medcof

Fisheries Research Board of Canada
Biological Station, St. Andrews, W. B.

ABSTE^CT

In the spring of 1957, 1958 and 1959, seed oysters be-
ginning their second and third growing seasons were imported
from the United Kingdom oyster breeding tanks at Conway, North
Wales. They were examined for parasites and extraneous organ-
isms, carefully cleaned ajid planted in screen-bottomed trays
in Passamaquoddy Bay near St. Andrews, New Brunswick. Some were
taken in late 1958 to Ellerslie, Prince Edward Island, and held
in Malpeque Bay water. Growth was good in Sam Orr Pond, a warm
inlet from Passajnaquoddy Bay, but poor in the cool open Bay.
The oysters brought in by steamer in 1957 suffered a 95^ mortal-
ity within a month after arrival. The 1958 lot was brought in
by air freight and showed a post-shipment loss of only 35^-
The 1959 lot, also air-shipped, suffered only 9^ loss up to
July l6, 1959, but mortalities rose to 53^ by Aiigust 6. Over-
winter survival varied greatly and the flagellate Hexamlta was
found in most moribund oysters . It was found in Ellerslie
aquarium stock which died after three weeks ' exposure to below-
zero water temperatures and in St. Andrews aquarium stock which
survived reasonably well at water temperatures that remained
above 2^0 . It was also found in native oysters taken directly
from their beds and in a sea scallop which was held in an aquar-
ium with European oysters but not in quahaugs . The oysters In
trays under the ice In Sam Orr Pond survived the relatively
mild winter of 1957-58 but died during the severe winter of
1958-59 showing heavy Hexamita infestation.

INTRODUCTION

Since I95O there has been a drastic decline in the production
of soft-shell clams (Mya arenarla) from the Canadian east coast,
with a consequent serious economic effect on shore communities. Ef-
forts at clam farming have been discotiraging. Clams can be farmed in
some areas but the expenses are too high to make the operation profit-
able.

In an attempt to replace this loss in clam production, the
approval of the Minister of Fisheries was obtained for a trial intro-
duction of the European oyster (Ostrea edulis) ,

-113-

ACKNOWLEDGMENTS

I wish to acknowledge my dependence on data collected by my
colleagues at this Station, Miss Joan Mortimer (impublished MS reports),
Mr. Peter J, Downer (unpublished MS report), and Dr. Louis Lauzier.
Mr. R. E. Drinnan, of our Ellerslie Sub-Station, has generovisly fur-
nished the information reported here on his studies of oysters we sent
to him. I am indebted to Dr. J. E. Stein and Dr. Marshall Laird, who
examined specimens for Hexamita infection, and to Dr. E. L. Bousfield,
and Dr. J. P. Harding for identification of invertebrates encountered
in this work and for permitting me to refer to their findings .

I would like to thank Dr. H. A. Cole, Dr. N. Reynolds, Mr. B.
T. Hepper and Mr. P. R. Walne, officers of the United Kingdom Ministry
of Food, Agricultiire and Fisheries, for their co-operation in supply-
ing the oysters.

I am also grateful to Dr. J. L. Hart for his encouragement and
assistance, and to Mr. Drinnan and Mr. Downer for assistance in pre-
paring this paper.

CHOICE OF CONWAY OYSTERS

The European oyster was selected for trial introduction be-
cause it is known to thrive in places where summer water temperatures
resemble those recorded in our soft-shell clam areas. It is readily
marketable and practical culture methods for it have been worked out.

This choice was also influenced by the establishment of a
breeding population of this species from the spawning of a relatively
small stock which Dr. Loosanoff brought to Boothbay Harbor, Maine, in
19^9 (personal communication) .

The choice of the Conway hatchery stock was influenced by its
relative freedom from oyster diseases and parasites. This was consider-
ed to be particularly important in view of the danger of introducing
undesirable species. In this case, care was taken to guard against
the Dutch shell disease, the mussel parasite, Mytilicola intestinalis,
which occurs in some European oysters, and the barnacle, Elminius
modestus, a recently- introduced pest of oyster beds in western Europe.

At the SBjne time it was necessary to obtain oysters which
could withstand the low winter water temperatures in eastern Canada.
All things considered, the oysters bred at the Conway hatchery and
subsequently reared in the Menai Straits seemed to be the best prospect.

IMPORTATIONS
In May 1957 about 5,000 oysters of the 1955 set were packed,

-114-

with damp seaweed (Fucus) in wooden boxes and shipped in the chilled
vegetable compartment of a passenger steamer. These oysters were out
of water for 11 days during transit.

In April 1958> approximately 5,000 of the I956 set were placed
in plastic bags, surrounded by "Vermiculite" as an insulating medium,
and packed in cardboard boxes. They were shipped in a pressurized,
temperature-controlled compartment of a freight aircraft. They were
out of water for approximately k days.

In April 1959, approximately 1,000 of the I956 and 5,000 of
the 1957 set were shipped in the same way and were out of water for
k days.

REMOVAL OF UNDESIRABLE ORGANISMS

There were considerable numbers of barnacles on the oysters
imported in 1957* In case these should include specimens of KlTnln^ns
modestus, it was decided to remove them prior to planting the oysters.
Subsequent identification of samples by Dr. E. L. Bousfield of the
National Museijm of Canada, and Dr. J. P. Harding of the British Museum
of Natijral History, showed that Elminius was present.

Trial batches of oysters were immersed in seawater solutions
of Lindane (benzene hexachloride) in an attempt to kill the barnacles.
The solutions were made up from a commercial powder, which contained
25^0 by weight of this poison, to give concentrations of the active
ingredient of li 50,000, 1:225,000, and Ij 500,000. This poison is
known to be toxic to several species of Crustacea (lobsters and green
crabs) but proved ineffective on the barnacles at the concentrations
used. After overnight exposure, many were still active although a
few appeared to be paralyzed. In case prolonged exposure to Lindane
might harm the oysters, it was decided to abandon this method of de-
stroying barnacles.

It was finally decided that Individual cleaning by hand was
the only satisfactory method. Each oyster was therefore scrubbed
clean of mud and debris and then barnacles, tube worms and other en-
crustations removed with a scalpel. This proved effective as no barn-
acles were found on the oysters in subsequent handling. This method
was again used in 1958 sjid. 1959*

Examination for Dutch shell disease and L^ilicola gave nega-
tive results .

During 19 5^ "the protozoan Hexamita appeared in the oyster stock
during the late summer and following winter. Inspection of the 1959
import immediately after arrival showed none of this protozoan. As
Hexamita had been previously reported from our native oysters ( Crass -
ostrea vlrginica) by Mackin et al. (1952) and by Logie (personal

-115-

coimii\mlcation) and was encountered again by Dr. Laird in 1958^ it is
concluded that our Eiiropean oysters were infested after their arrival
in Canada.

PLANTING

The oysters were planted out in trays with 8- inch legs to
raise them off the bottom, and l/i<— inch mesh wire bottoms and lids .
Periwinkles (Llttorina littorea) were placed in the trays to keep
down epiphytic algal growth.

Plantings were made in several locations. Some oysters were
retained in the Station tanks, and some on the beach below the Bio-
logical Station in the cool water of Passamaquoddy Bay, but most of
them were set out in Sam Orr Pond, a warm, salt-^ater inlet of Passa-
maquoddy Bay.

GROWTH

During their first year in Sam Orr Pond, where temperatures
rise at times to 25^0, the oysters showed excellent growth (Table l) .
This was greater in 1957 than in 1958^ presumably because 1957 '^^^
warmer (Table 2). The survivors of the 1957 stock which lived through
1958 grew well in their second siommer in the pond. By August their
mean length was 80 mm.

Table 1. Growth of oysters during their first year in Sam Orr Pond
trays .

Import Shell Annual

Year Length Growth

mm mm

May

35

1957

39

Nov.

Jk

May

3h

1958

26

Nov.

Go

In both years the oysters in Station tanks and in Passajnaquoddy
Bay grew less than a third as much as those in the pond. This is

-116-

attributed to the cooLnass of the Bay (Table 2) where the highest
temperature recorded in the two years vas I5.80C.

Table 2. Two-year comparison of summer water temperatures in Sam Orr
Pond and Passamaquoddy Bay.

Passamaquoddy
Sam Orr Pond Bay

Year

No. (

Df days

No.

of days

temperature

temperature

vas

above

was above

15°C

20OC

15°C

1957

67+

26

k

1958

hh

6

6

SURVIVAL

The oysters imported in 1957 seemed vigorous. When placed in
water Immediately after arrival^ many appeared to begin pumping. How-
ever, next morning gapers appeared and steadily increased. Approxi-
mately 90/0 died during the first week (Fig. l) and by the end of June
95^ were dead. The 5^ remnant of the stock showed no further losses,
grew well during the summer and survived the 1957-58 winter 100^ in
Sam Orr Pond. This encouraged belief that Sana Orr Pond was a safe
wintering place.

The heavy mortality on arrival was attributed to prolonged
air exposure during transit. This conclusion was supported by Korringa's
(1956) experience and by observations on storage of our native oyster,
Crassostrea vlrglnlca, which shows a similar mortality if placed in
water after long air exposure (Medcof 1959)*

Survival and growth after the initial mortality was over, en-
couraged the 1958 Importation by air which reduced air exposure to
approximately k days. This shipment was made in April before com-
mencement of the growing season. As an added precaution, the oysters
were held in the intertldal zone in Menal Strait for several days
prior to shipment to accustom them to prolonged air exposure. The
Japanese reg\ilarly condition spat of Pacific oysters in this way be-
fore shipping them to the west coast of North America (Glude and
Lindsay 19^+7).

The precautions taken in 1958 did not avert a heavy initial
loss but they seem to have delayed It until some 8 weeks after arrival
and to have reduced it. Approximately kofi of the stock survived until
winter (Fig. l) as compared with 5!/^ in 1957 •

-117-

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-119-

In JuLj 1958^ Dr. Marshall Laird, a protozoologlst from the
Institute of Parasitology, McGill University, examined samples of the
1957 and 1958 imports. In the 1958 import he found ciliates on the
mantle and gills of all specimens and one oyster showed trophozoites
of the flagellate Hexamita inflata in the intestine. All these oys-
ters were in poor condition, thin ajid watery. No ciliates and no
Hexamita were found at that time in oysters imported in 1957* Hiese
oysters were in excellent condition, fat and creamy-white.

During their second (1958) summer in the pond the survivors
of the 1957 importation showed almost no mortality. This was encourag-
ing.

On July 11 and November 6 samples of I50 and 200 oysters, res-
pectively, of the 1958 importation were sent to Mr. Drinnan at Ellers-
lie. Prince Edward Island. He held some of these in trays in Bideford
River and some in laboratory tanks. The oysters transferred in July
grew well and were quite fat by autumn. The November transfers grew
little and were still thin when cold weather set in. All oysters in
both lots died in early winter. Drinnan 's records show that the mor-
tality began about 3 weeks after water temperatures dropped to O^C
Eind below, and that it reached lOOfo within 2 months (Fig. 2). After
the mortality began Mr. Drinnan made many examinations of living, mori-
bund and dead oysters. Most but by no means all proved to be heavily
infected by trophozoites of Hexamita.

Mr. Drinnan 's observations at Ellerslie stimulated sampling of
the oysters wintering in Sam Orr Pond. On March 5 "the ice, which was
28 inches thick, was sawed through and a tray containing oysters of
the 1958 importation was located and raised. Only 2'fo were sound.
The remainder were either weak gapers or dead. On examination, all
of them, livixig and dead, proved to be heavily Infected with tropho-
zoites of Hexamita. Samples were sent to Mr. Drinnan, Dr. Laird and
Dr. Stein, all of whom confirmed these findings. On April 22 when
the ice had cleared from Sam Orr Pond, all the oysters from both im-
portations were foimd to be dead.

In contrast, about two-thirds of the oysters held in the labo-
ratory at St. Andrews survived the winter. The water temperatures in
these tajiks are shown in Figure 2. They were consistently higher than
those in Ellerslie tanks and higher than those in Sam Orr Pond for at
least part of the winter of 19 58- 59. This is shown by two readings
at the pond with a reversing thermometer lowered through holes in the
ice which were +O.IOC on February 5 and -O.^OC on March 6, 1959-
These St. Andrews tank temperatures were probably higher than the pond
temperatures in the winter of 1957-58, but there are no direct obser-
vations to support this assumption. Passamaquoddy Bay hydrographic
records supplied by Dr. Laiizier show that the winter of 1958-59 was
more severe than that of 1957-58 (Table 3).

-120-

Table 3» Comparison of monthly means of Passamaqvioddy Bay water tem-
peratures (°C) taken dally during the two winters oysters
were held at St. Andrews, N. B,

Winter Decemher January Febrixary March

1957-58 5.^+ 3.5 2.1 2.7

1958-59 3.^ 1.1 -0.1 0.1+

The oysters Imported in 1959 arrived hy air on May 2. They
were given the same Intertidal hardening treatment in Menai Strait
"before shipment as those Imported in 1958. The sinrvival since plant-
ing seems to he following the regular pattern (Fig. l) . Up to July
16, 1959^ 10 weeks after arrival. 97^0 of the 3-year-old3 (1956 set)
and 91^ of the 2-year-olds (1957) were still alive. By July 2h, how-
ever, these values had fallen to 88fo and 55/" ^^^ tiy August 6 to 86.5^
and 47. 3^". Diiring the peak of the mortality all the moribund and dead
oysters were heavily infected with Hexamita trophozoites. Healthy-
looking oysters have been consistently free.

ADDITIOML OBSERVATIONS ON HEXAMETA

The q^uahaug (Mercenaria mercenarla) is native to Sam Orr Pond
and at no time has Dr. Laird found it to harbour Hexamita . Even when
held in tanks at the Biological Station alongside heavily infested
oysters, the quahaugs have not been conteuninated.

A scallop (Placopecten magellanlcus) which had been living in
a St. Andrews tank with infected oysters was found dea^ on May 30,
1959> aiid as heavily Infected with Hexamlta trophozoites as the oys-
ters.

Dr. Laird found the flagellate in samples of mud taken April
6, 1959^ from the bottom of Sam Orr Pond at the place where the oyster
trays were wintered.

DISCUSSION

Apparently the European oyster may thrive on our coast in
sheltered inlets in summer but may or may not survive our winters de-
pending on their severity. Mr. John Hurst of the Maine Department of
Sea and Shore Fisheries reports (personal communication) that one
winter the Eioropean oysters (Netherlands strain) growing in Maine
showed substantial mortality, when severely cold weather coincided

-121-

with extreme low tides. But only intertidal animals were affected.
Gaarder and BJerkan (193^) report that the Norway strain of European
oyster does not withstand severe winters very well even when submerged.
Korringa (1957) also recognizes this hut suggests that there are rac-
ial groups within the species which show differences in cold-hardiness.
In his opinion the Netherlands strain is quite hardy. Owe laboratory
and field experiments with Conway oysters clearly indicate that at
temperatures about and slightly below 0°C they suffer some kind of
stress. Under present conditions they may or may not survive, depend-*
ing on the severity of the winter.

McLellan and Lauzier (195^) report sea water temperat\ires for
the Bay of Fundy and the outer coast of Nova Scotia and predict more
severe winter conditions on this coast for the next 25 years. Their
statements discourage hope that this species can be established here.
We find that present conditions are marginal — perhaps too risky to
justify fijrther trial introductions of Conway oysters. If conditions
were less favorable they could rule out any chance of success with any
strain of European oyster.

The role of Hexamita in oyster mortalities is by no meajis
clear. It lives freely in the mud in Sam Orr Pond and is probably
present in the water supply to laboratory aquaria at St. Andrews and
Ellerslie. But it does Infect oysters with various effects imder
various conditions. Some of these are clear] others can be only vague-
ly outlined.

Oysters can tolerate Hexamita infection for long periods with-
out unusual mortality at low temperatures, about i4-°C. This is shown
by the St. Andrews tank experiments.

Hexamita may cause death of oysters that are in stasis (hiber-
nating with low metabolic rates) or that are suffering stress from
still lower temperatures (approaching the lower lethal temperature)
Our observations of low-temperature mortalities in Sam Orr Pond and
at Ellerslie illustrate this and parallel those by Stein (1960) for
Olympia oysters .

Hexaml ta will also attack at high temperatures when oysters
are experiencing difficulty (stress) in adjusting to new conditions
or in obtaining enough food. This is illiistrated by Dr. Laird's I958
summer studies of the thin-meated Sam Orr Pond stock.

Intermediate temperatures which prevented normal summer growth
of oysters (e.g. in St. Andrews aquaria) apparently favored chronic
Hexam-t ta infection. Food shortage may have been involved in this
situation.

Hexamita does not ordinarily infect quahaugs, which live well
in aquaria and show great cold-hardiness. But it may attack sea
scallops which do not thrive in aquaria, although they are tolerant

-122-

to low temperatures (Dickie 1958) •

From this variety of evidence oiJr Hexamlta could be considered
a facultative parasite. It seems to have taken the first evolutionary-
step toward parasitism. If it were a well-adjusted parasite, our oys-
ters might have survived "better. On the other hand, the occurrence
of the flage2J.ate in slow-growing, weak or dying oysters and in scallops
living in aquaria may he purely coincidental with approaching death.
Hexamita may he simply an opportunist.

PLANS FOR FUTURE

At the moment we are still convinced that we need more species
of shore molluscs to stabilize our shellfish fisheries. We would like
to establish some exotic species in our depleted clam areas- We are
not quite satisfied that the European oyster is not adaptable to this
coast. We have tested the Conway strain without very encouraging re-
siilts but Dr. Loosanoff 's experience and the experience of the Maine
Department of Sea and Shore Fisheries with the Netherlands strain are
more heartening. There are several strains^ and one or more of these
may have the degree of low-temperat\ire tolerance that would make the
European oyster adaptable to our coast. Even though hydrographers
warn us to expect colder winters in the next few years, search for a
suitable strain may be Justified.

Aside from this possibility there are other species of shore
molluscs which we have considered as possibly adaptable, for example,
the Pacific oyster (Crassostrea gigas), the Japanese littleneck clam
(Tapes philippinarum), and the Atlantic bay scallop, (Aequlpecten
irradians) . This last species has been reported from the Canadian
Atlantic coast emd the second last is already established on the Brit-
ish Columbia coast.

At this stage no decision has been made to go on and make an
exhaustive study of the possibilities of finding an adaptable strain
of European oyster or to try out what seem to be the hardiest strains
of some of these other species.

LITERATURE CITED

Dickie, L. M. 1958- Effects of high temperature on s\irvival of the
giant scallop. J. Fish. Res. Bd. Canada 15(6): II89-I2II.

Gaarder, T. and P. Bjerkan. 193^. jZ^sters og )/flterskultur i Norge.
Bergen.

Glude, J. B. and G. E. Lindsay. 19^T« Japanese oyster seed export
program for 19^7. NRS, SCAP, Tokyo, Prelim. Study No. 13:
1-13.

-123-

Korringa, P. 1956. Oyster culttore in South Africa. Union of South
Africa, Div. Fish. Invest. Rep. 20: I-85.

^^^ 1957 • Water temperature and breeding throughout

the geographical rajige of Ostrea edulis . Coll. Internat. Biol.
Mar., Stat. Biol. Roscoff, Ann. Biol. 33(l/2) 1I-I5.

McLellan, H. J. and L. M. Lauzier. I956. The prediction of water
temperatures. Canadian Fisherman ^3(9)' 11-12.

Mackln, J. G., P. Korringa and S, H. Hopkins. 1952. Hexamitiasis of
Ostrea ediJlie (L.) and Crassostrea virginica (Gmelin). Bull.
Mar. Sci. Giilf Caril). 1(4): 266-277.

Medcof, J. C. 1959. Studies on stored oysters (Crassostrea virginica) .
Proc. Natl. Shellfish. Assoc. k9 (1958): 13-28.

Stein, J. E. I960. Hexamita sp. and an infectious disease in the

commercial oyster Ostrea lurida. Proc. Natl. Shellfish. Assoc.
5O167.

-12it-

PRELIMINARY REPORT ON
GROWTH AMD SURVIVAL OF THE
PACIFIC OYSTER IN WASHINGTON WATERS""-'^

Albert K. Sparks and Kenneth K. Chew

College of Fisheries, University of Washington, Seattle, Washington

INTRODUCTION

The Pacific oyster, Crassostrea glgas, has become increasingly
important as a fishery resource in the Pacific Northwest of America.
Yet, we have relatively little information on its growth and mortaJLity
rates in local waters, information basic to the utilization of this
valuable resource.

Chapman and Es veldt (l9^3)^ in their study of spawning and
Betting of the Pacific oyster in Washington waters, gave a brief ac-
coimt of its growth. Woelke (l955) discussed the growth of a small
group of the Kumamoto variety of this species and graphicaHy compared
its growth with that of the typical £. gigas seed; both were grown on
the same bed. Quayle (1951), working on the Pacific oyster in British
Columbia waters, followed the increase in length, width, and thickness
of a group of 6' to 8-year-olds for one year. We are not aware of other
published acco-unts dealing with growth and mortality of the Pacific
oyster in our local habitat. Clearly, more information is desired and
needed.

In March 1959^ we started a field study, with emphasis on grow-
th and mortality, of some 3^000 yearling Pacific oysters (1957 year
class) originally Imported from Japan. Results through January I960
on growth, condition index, and mortaility rates at three field stations
are presented in this report.

MATERIALS, METHODS, AM) FIELD STATIONS

The Western Oyster Company at Purdy, Washington, provided more
than 3^000 yearling Pacific oysters, which were imported as seed from
Miyagi Prefecture, Japan in the spring of 1958* These oysters were
ciilled to singles in the laboratory of the College of Fisheries, and
length, height, and thickness of each individual were measmred to the
nearest millimeter. The condition index was determined from 30 oysters,
and 10 specimens were fixed for histological examination.

■^This research was supported by "State of Washington, Initiative 17I
Funds for Research in Biology ajad Medicine".

^Contribution No. 65 from the College of Fisheries, University of Wash-
ington,

-125-

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-126-

Tlaen 1,000 oysters were distrilDuted during the first veek of
March, 1959^ to each of three field stations: Oyster Bay in southern
Puget Sound, Pt. Whitney in Hood Canal, and Wahcotta in Willapa Bay
(Fig. l). Each station consisted of a float (Fig. 2) much like that
used hy Collier (1953)- Four woven-wire baskets, each measuring 35«5
hy 17.0 by 7-0 inches, were contained in the float, and in each basket
were placed 250 oysters.

Three baskets at each station were designated as experimental]
with a few exceptions, they were examined each week from March until
September and every other week thereafter. During these weekly and
biweekly examinations, measurements of length, height_, and thickness
were taken on 100 randomly selected oysters, 33 each from two baskets
and 3^ from the third. The random sample was obtained by thoroughly
stirring all oysters in each tray, then removing a portion of the pile
estimated to contain approximately 33 oysters ajad measuring from this
randomly selected group. Dead ajid dying oysters were also measured;
the latter were fixed in Zenker's solution for subsequent histological
study. Boxes were measured and discarded. Associated fauna was re-
moved, enumerated, and preserved. Every other week one live specimen
from each basket was fixed for histological study and three specimens
from each basket were measured for condition index, making a total of
three normal oysters fixed from each station at each sampling, and a
pooled total of nine oysters for condition at each sampling. Condition
index (C. I.) of oysters was determined by a method outlined by Medcof
and Needier (l9^l) who used the ratio of the dry weight of oyster meat
to the volume of shell cavity, multiplied by 1000. In this study the
ratio is multiplied by 100.

The fourth basket at each station was designated as a control.
Oysters contained therein were left undisturbed except that in July
and October 1959^ aJid. January I960 they were measured once along with
those in experimental baskets. This was to determine if frequent hand-
ling of oysters in experimental baskets had any effect on growth and
survival.

TT

Water temperature, salinity, dissolved oxygen, and p were
determined by standard methods at each station on each visit.

RESULTS

Growth of oysters was measijred In terms of increases in length,
height, and thickness of shells. Data on growth in shell length from
the beginning of the experiment (first week of March) through January
i960, are shown in Table 1. Only the mean values of each set of mea-
surements are given, and the final cumiilative percentage increases
are calculated.

-127-

Table 1. The mean length In ram. of oysters in experimental and control

baskets at each station from March 1959 through Janiiary I960

Hood Canal Oyster Bay Willapa Bay

Exp. Control Exp. Control Exp. Control

Beginning of

Experiment 59.8 55-0 56.6 58.2 60.I 59.3

March 61.O 57-6 60.5

April 6h.9 61.2 62.0

May 65.5 6k. Q 61*^.8

June 70.9 73.7 73.7

July 76.6 91.2 82. If 99.2 80.1 90.2

August 78.0 85.6 82. 1+

September 8I.5 92.1 89.2

October 82.0 98.^ 95-1 117.3 89.5 107.if

November 82.2 97. 0 86.2

December 8l.2 96.2 88.6

January 82.8 97.6 92.7 125-6 87.6 10^^.1

Cumulative percentage increase 38.5^ 77-5^ 63.8^ 115.8^ J+5.8^ 75*5^

As shown in this table, the experimental oysters at the Oyster
Bay Station revealed the greatest percentages of increase in shell
length. The percentage of shell growth of experimental oysters was
next highest at Willapa Bay, followed closely by Hood Canal. At all
stations growth shown by increase in shell was greater in the control
trays than in the experimental ones. This was probably due to break-
ing off of fragile new extensions of shell margins by handling.

At all stations, growth ceased by the end of October, after
decelerating greatly during that month. From the middle of September
until the end of October water temperatures had gradually dropped from
approximately 16.OOC to about 12.0^0 (Table 3).

Condition indices of the experimental and control oysters are
presented in Table 2. Condition indices of oysters at the three sta-
tions were virtually the same in March when the study began; Oyster
Bay, however, was soon superior to the other two stations. By April
30, the condition index of Oyster Bay oysters reached 13-9 compared
to 8.8 at Willapa Bay and 8.7 at the Hood Canal station. During the
summer months the C. I. of Oyster Bay oysters remained high, between
13.1 and 15.5, while the Hood Canal and Willapa Bay oysters improved
to a level of 10.0 to 11.0. In October the condition indices of the
oysters at Oyster Bay and Willapa Bay began to decline while those at
Hood Canal remained high, with the year's highest values for this sta-
tion, 13.1 and 13.0, occurring at the end of October and the middle of
December. Temperature and other hydrographic data showed no obvious
reason for the C. I. remaining high at Hood Canal.

-128-

Table 2. Condition index (C.I.) of experimental and control oysters

from three stations

Hood Canal Oyster Bay Willapa Bay

Date Exp. Control Date Exp. Control Date Exp. Control

3-18-59 5.2 3-18-59 5.^ 3-18-59 5-5

h- 4-59 6.3 h- 5-59 7.6 k- 5-59 8.7

i+-l6-59 7.5 4-16-59 9.6 4-18-59 7.8

4-30-59 8.7 5- 3-59 13.9 5- 3-59 8.8

5-28-59 11.1 5-30-59 1^.6 5-30-59 9-8

6-18-59 10.9 6-18-59 15.5 6-16-59 11.0

7- 2-59 10.8 7- 2-59 13.6 6-30-59 10.5

7- 7-59 9.1 7- 7-59 15-2 7- 9-59 8.3

7-29-59 9.8 7-29-59 13.4 7-30-59 9-5

8-12-59 11.3 8-12-59 1^.6 8-14-59 10.5

8-25-59 9.8 8-25-59 13.5 8-26-59 11.0

9-10-59 11.5 9-10-59 13.1 9-11-59 10. 0

10- 3-59 11.8 8.7 10- 3-59 11-5 9.0 10- I-59 9.0 6.2

10-15-59 13.1 10-15-59 10.6 10-17-59 7.6

10-29-59 11.9 10-29-59 10.8 11- 1-59 7.1

11-11-59 12.3 11-11-59 12.6 11-14-59 8.1

11-29-59 12.1 12- 1-59 10.0 11-29-59 7.5

12-17-59 13.0 12-16-59 11.4 12-20-59 9.8

1- 8-60 11.7 8.5 1- 9-60 10.1 7.3 1-12-60 7.1 5.6

1-22-60 11.0 1-22-60 9.5 1-25-60 7.3

-129-

Table 3- Temperatiire data for thi'ee experimental atationsl from March
to December 1959

Date 1959

Temperature

OC

Date 1959

Temperature '

3C

H C

0 B

W B

H C

0 B

W B

March

Avigust

>

1- k- 9

8.9

9.h

9.h

5- 5- 7

19.^

20.0

18.8

18-18-21

10.0

8.9

9.h

12-12-iU

20.0

17.7

18.3

27-27-28

8.3

8.9

9.h

18-18-20

18.3

17-2

18.3

25-25-26

19.7

18.3

17.7

April

k- 5- 5

9.^

9.h

10.3

September

9-11-11

12.2

11 .1

11.1

10-10-11

16. U

16. U

16.1

16-16-18

10.6

10.6

11.7

19-18-17

13.1

16.1

16.1

23-23-26

12.8

11.7

12.2

30- 0- 0

11.7

October

3- 3- 1

13.6

15.3

15.3

May

15-15-17

12.2

II+.2

Ik.k

0- 3- 3

12.2

12.2

29-29- 0

11.1

12.8

7- 7- 9

13.3

ik.k

Ik.k

21-21-23

li^.I^

13.9

18.3

November

28-30-30

12.8

17.2

16,7

0- 0- 1

12.8

11-11-lit

10.0

10.6

8.6

June

28- 0-29

9.k

9^k

10-10- 8

15.6

15.6

16.7

18-18-16

15.6

15.6

17.2

December

2i+-2l4-25

15.6

16.7

18.3

0- 1- 0

10.0

0- 0-30

18.8

17-16-20

8.3

8.6

8.9

July

Janiiary

2- 2- 0

19.^

16.7

8- 9-12

6.k

6.7

5.0

7- 7- 9

17.7

16.1

18.8

21-21-0

23.3

20.6

29-29-30

18.8

16.9

20.6

1 H C, Hood Canalj 0 B, Oyster Bay; W B, Willapa Bay

-130-

The C. I. of controls was approximately the same as that of
the experimental oysters in Jiily. In subsequent measurements, however,
control oysters showed much lower condition index than the experimental
ones . This was probably due to overcrowding of oysters in the control
baskets, as a result of rapid growth, low mortality, and lack of re-
moval of oysters for condition index and histological study. Sixty
oysters were removed from the control basket at each station during
the first week of October to alleviate crowding. These were placed
on the bottom in nearby areas and will be compared in subsequent per-
iods to the float oysters for growth, condition, and survival.

Mortalities of both experimental and control oysters were s\ir-
prisingly low at all stations. A Chl-square test indicated that the
survival of the experimental oysters between stations was not signi-
ficantly different at the 5 per cent level. The same result was ob-
tained for the control oysters between stations, but a further Chi-
square test revealed that the mortality was significantly greater at
the 5 per cent level in the control tray at the Oyster Bay Station
than in the experimental trays at the same location. No significant
difference was found when the total survival data for the experimental
oysters were pooled and tested by Chi-square against the pooled data
for the controls. Adjusted cumulative mortalities, based on mortalities
less those oysters removed from the trays for condition index deter-
mination and histological studies, in the experimental trays were 2.6
per cent at Willapa Bay, and k.l per cent at both the Oyster Bay and
Hood Canal stations. The pooled cumulative mortality of the experi-
mental oysters at all three stations was 3.5 per cent. Mortalities
in the control trays were 2.8, h.^, and 'J .1 per cent at the Willapa
Bay, Hood Canal, and Oyster Bay stations, respectively.

DISCUSSIONS AND CONCLUSIONS

From the first year of this study it has been demonstrated
that growth in Crassostrea gigas in Washington waters occurs largely
diiring the sijmmer months; mortality rates are extremely low under the
conditions of the present study. The growth data contrasts with that
reported by Woelke (l959) who found growth to be more or less continu-
ous during the year at Case Inlet. Of the three locations studied,
growth was best at Oyster Bay and next best in Willapa Bayj survival
was greatest in the Willapa Bay station. Woelke (1959) found growth
to be better in Willapa Bay than in southern Puget Sound, but condition
index at Hood Canal has been superior to the C. I. in the Willapa Bay
station throughout most of the study and better than in Oyster Bay
from October through December. It should be pointed out, however,
that parts of Willapa Bay are suitable for growth, and other areas
for fattening, with the Nahcotta station selected as Intermediate bet-
ween the two extremes. Because of this, these data probably do not
reflect either best condition or best growth for Willapa Bay.

The relationship between growth and handling is of particular

-131-

Interest because of possible use to the industry. Althovigli handling
vas shown to affect growth as measxired by increases in shell length,
it did not appear to increase mortality.

LITERATURE CITED

Chapman, W. M. and Esveldt, G. D. 19^3. The spawning and setting
of the Pacific oyster. Wash. Dept. Fish., Biol. Rept. No.
i|3Asl-25.

CoU-ier, Albert. 1953. Oysters, their growth rate and survival when
retained under crude petroleum. I-I56. (Unpub. Rept. to Gulf
Refining Co.)

Medcof, J. C. and Needier, A. W. H. I9J+I. The influence of tempera-
ture and salinity on the condition of oysters (Ostrea virginica) .
J. Fish. Res. Bd. Canada 5(3): 253-257-

Quayle, D. B. 1951. The seasonal growth of the Pacific oyster ( Ostrea
gigas) in Ladysmith Harboior. Ann. Rept. British Colimbia Dept.
Fish. 1950 : L85-L9O,

Woelke, C. E. 1955. Introduction of the Kumajnoto oyster Ostrea

(Crassostrea) gigas to the Pacific Coast. Wash. Dept. Fish
Res. Paper 1 (3)1 1-10.

. 1959. Growth of the Pacific oyster Crassostrea gigas
in the waters of Washington State. ( Unpub, Mas., 8 pp.)

-132-

GROWTH OF Tim PACIFIC OYSTER CRASS OSTREA GIGAS
IN TEE WATERS OF WASHINGTON STATE

Charles E. Woelke

Washington Department of Fisheries
Shellfish Laboratory, Quilcene, Washington

ABSTRA.CT

Seasonal growth^ year to year growth variation in a
single area, growth relative to tidal height, and growth between
areas of Crassostrea gigas as determined from experimental plant-
ings are reported. The possibility of predicting the market
size of oysters after six months growth is siiggested. Growth
on commercial beds is described by area from data collected in
annual industry-wide growth surveys.

INTRODUCTION

Growth of the Pacific oyster, Crassostrea gigas, in Washington
state has been systematically studied on an industry-wide basis only
since 1956. Seki (1937) followed monthly growth of Crassostrea gigas
in three areas of Japan. Quayle (1951) reported seasonal growth of
stunted specimens of Crassostrea gigas moved to a more favorable en-
vironment. Thomson (l952) followed the growth of this oyster in ex-
perimental plantings in Australia. Woelke (l955) described growth of
a variety of Crassostrea gigas (the Kumamoto oyster) in Washington
waters in a report on the introduction of this oyster to the Pacific
Coast.

This report summarizes data collected from two sources on
growth of Pacific oysters in Washington. First is a series of studies
designed to evaluate seasonal growth, year to year growth variations
in one area, growth relative to tidal level, and growth variation be-
tween areas. The second source is data collected in annual indvistry-
wide s\irveys measuring growth, mortality and "fatness" of oysters on
commercial beds.

METHODS

Measurement of oyster size is made by two methods, linear and
voltimetric. Quayle (I95I) measiu-ed length, width, depth and volume
in studying oyster growth. He observed, "Because of irregular shape
of the Pacific oyster and the high degree of shell fluting, consistent-
ly accurate linear measurements are difficult to make." Beaven (l952)
recognizes the simplicity of volume measurements but objects to their
use because of problems arising from small oysters, spat, mussels etc.

-133-

Drayton Harbor

^ 4 s\iiish Bay

Fidalgo— ^nis^^dilla Bay

Simllk
Beach

Pig. 1. Principal oystering areas in Washington state,

-13^-

attached to the oysters. Hopkins and Menzel (l952) stress the value
of a volumetric measurement which can be related to net yield. Butler
(1952a, 1952h, 1953) uses volume measurements in growth studies and
points out (195213) the need of using oysters of common heredity axid
known age and avoiding mixed year classes.

All data in this report are from volume measurements . After
removal of aJLl organisms, volume is meas\ired in the laboratory by
subtracting weight in water from weight in air and in the field by
water displacement. Increased speed and reduced measuring error (less
than 0.5 cc per oyster in samples of 10 or more) are achieved by pro-
cessing an entire sample in one measurement rather than measirring indi-
vidual oysters.

Intertidal ciiltivation and limited reproduction in most Wash-
ington oystering areas eliminate the serious fouling problems found
objectionable by Beaven, As a result of the unique method by which
the Pacific Coast oyster industry replenishes most of its oyster stocks,
i.e., annual importation of seed from a common stock in Japan, the
problems of known heredity or mixed year classes are absent.

The data reported have been collected from oysters grown on
the bottom in the intertidal zone. The diurnal tide range in most
Puget Sound areas is about l4 feet. In coastal areas (Grays Harbor
and Willapa Bay) diurnal tide range is about 10 feet. Experimental
plantings, as well as most commercial beds, are located between one
foot below and five feet above mean lower low water. All sampling is
done at low tide when the beds are exposed and are easily accessible.

EXPERIMENTAL PLANTINGS

Seasonal growth as determined by repeated measiirement of the
same oysters (less mortalities) over a 2+ year time period in Case
Inlet is presented in Table 1. Average temperatures and salinities
for the time period preceding each growth observation are also in-
cluded in the table. While the time intervals between data collection
were variable (generally 2-3 months), growth occurred within each in-
terval. The greatest per cent growth occurred during periods of war-
mer water (average of about l8°C), and higher salinities (27-5 - 30.6
0/00). Less growth occurred during lower temperatures (down to 6.U'-'C)
and reduced salinities (20.2 0/00). While these data disagree in
part with Quayle (l95l)> who indicates cessation of shell growth at
about 9°C, the same general trends are evident in both studies. From
Australia, Thomson (1952) reports growth during all seasons (temper-
ature range 1.1 - 25.6°c) though at a reduced rate dviring periods of
low temperature.

To measure growth variations on a long-term basis, represent-
ative samples are collected quarterly from our experimental beds and
brought to the laboratory for volume measurement. Growth at the same

-135-

Table 1. Seasonal gro\rth of the Pacific oyster in Case Inlet - 1952
planting

Growth
Age in Ave. size increment Per cent Ave. temp Ave. sal.
Date months in cc (^) in cc Increase ^ C"'^ o/oo **

V22/52

0

1.2 (100)

9/12/52

5

8.5 (91)

7.26

590.2

18.5

27.9

11/2/52

7

lk,k (89)

5.89

69.3

12.5

30.6

1/1V53

9

16.6 (87)

2.22

15.i+

9.6

20.2

V30/53

12

20.6 (85)

4.03

2lf,2

Q.k

20.5

7/29/53

15

38.8 (if9)

18.16

89.1

18.6

27.5

11/22/53

18

61^.0 (61)

25.22

65.0

12.6

30.2

1/31/5^

21

77.2 (57)

13.16

20.5

6.k

22.9

6/30/5^^

26

92.5 (40)

15.33

19.8

13.6

22.1

* Ntmiber of oysters measured.

•** Average of samples collected during time period preceding date of
growth observation.

-136-

tide level in Case Inlet from I952 to I958 "by year and year class
shows vide fluctuations (Table 2). In the seven years covered, the
average size of oysters at I5 months has varied from 27.3 cc to 8O.7
CG. During excellent growth years the size at I5 months has "been
greater than that of other years' plantings at 27 months of age.

This point is further emphasized by comparing the per cent
growth of the various plantings. First year growth has ranged from
192^ to 2,126/0, second year from llhio to l6itfo. Limited data collected
on 2- to 3-^ and 3- to 4-year-olds do not permit comparison of per
cent growth on the older oysters .

It is demonstrated that a poor growth year is reflected by
per cent growth of all year classes on the beds; therefore, routine
sampling of a single age class adequately detects changes that occur,
younger oysters being the more sensitive measure of change. Annual
growth declined between 1952 and 1955^ and increased markedly from
1955 to 1958.

140

01

a

■H

S 120

rH

u
a

rt 100

A
C

o

a

o
>

80

S 60

40

20

Y = 5.9x + a. 7
r = 0.09

12

16

20

24

28

32

Volume in cc. 6 months after planting

Fig. 2. Six month vs. 26 month size of Pacific oysters,

-137-

Table 2. Annual growth variation of Pacific oysters in Case Inlet
1952 thru 1958

Year of
observation

Year
class

Age

Ave. size
cc (*)

Per cent
growth

1952

1952
1951

0 +

1 +

8.5 (91)
80.7 (20)

— __

1953

1953
1952
1951

0 +

1 +

2 +

15.7 (^2)

38.8 (I19)

357

195^

195^

0 +

—

1953

1 +

^5.7 (31)

192

1952

2 +

90.0 (25)

132

1951

3 +

166.8 (20)

1955

1955

0 +

5.3 (

^^

195^

1 +

52.0 (

22

1953

2 +

103.5 (

:35)

127

1956

1956

0 +

2.0 (

;75)

1955

1 +

29.3 I

^5

iH3

I95i^

2 +

111.3 (

!35

114

1957

1957

0 +

3.0 (

;59)

1956

1 +

27.3 <

20)

1,263

1955

2 +

1^.1 <

15)

155

195^

3 +

179.1 (

!i7)

61

1958

0 +

6.5 (

;3i)

2,126

1957

1 +

65.9 <

22)

i6if

1956

2 +

71.9 (

21)

77

1955

3 +

132.1 (

:i9)

23

195^

k +

221.1 (

1958

* Number of oysters measured.

-138-

From industry's standpoint a very practical application of
growth data is indicated in Figure 2. In this figure the 6-month size
is plotted against the 26-month size of oyster seed planted on our ex-
perimental beds for six successive years. These data suggest the pos-
sibility of using the size at six months to predict the size of oysters
at harvest age. With an r value of O.89, a strong correlation is indi-
cated at the 5 pel" cent level. Within general parameters these data
may provide a useful tool for oyster farmers. Whether this pattern is
peculiar to our experimental beds has not yet been determined.

It seems reasonable to assume that where oysters are intertid-
ally cultured the feeding time of the population is limited by the
tidal level at which they are planted, which in turn might be expected
to materially affect growth of the oysters. Data in Table 3 were col-
lected from seed of a common stock planted at the +2.8, +1.3 and -O.9
foot tide levels in Case Inlet. At 26 months after planting, the
average sizes were 80.4 cc, 10^4-. 7 cc and II8.8 cc in order of decrea.s-
ing tidal height. Oysters planted only 3-7 feet lower in the inter-
tidal zone were nearly 50 per cent larger at 26 months.

Table 3* Pacific oyster growth relative to tidal height in Case Inlet

Tide

Date

Date

Age

in

Ave. size

level*

planted

harvested

months

in cc (**)

+2.8

V23/52

6/30/5^

26

80. 1| (17)

+1.3

V23/52

6/30/5^^

26

ioi^.7 {ih)

-0.9

V23/52

6/30/54

26

118.8 (31)

* Reff erred to mean lower low water
** Number of oysters measvired

In 1952 a study was begun to measure the growth of a common
seed stock in several of the principal oyster growing areas of Wash-
ington at about the same tidal level (plus 2.5 feet). In Table k the
accumulated growth at 28 months of age is presented for the various
areas. Sizes range from 57 "^c to 131 cc. The poorest growth was in
Oakland Bay and the best in Willapa Bay

-139-

Table k. Growth of 1952 year class Pacific oysters in five different
areas

Area

Size

in

cc at 28 mos. (2+ years)

Case Inlet

90 (62)*

Oakland Bay

57 (22)

Oyster Bay

109 (17)

Samish Bay

79 (26)

Wlllapa Bay

131 (23)

* Number of oysters measured.

GROWTH ON COMMERCIAL BEDS

Extension of growth data collection from experimental plots
to industry-wide measurements was begun in 195^. These data were col-
lected on an annual survey basis covering about 70 per cent of the
major oyster beds and 90 pe^r cent of the general oyster-producing areas
of the state. Surveys have been conducted on the last two or three
daylight lower low tide series of the year (August and September).
All year classes present on the beds in each bay were sampled and eval-
uated. Between ^5 and 50 separate beds in the four principal oyster-
growing areas of the state (norther Puget Sound, southern Puget Sound,
Grays Harbor and Willapa Bay) have been sampled each year. The same
areas on the same beds were sampled in each survey to provide continu-
ity of comparable data.

Each sajnple consisted of 20 or more randomly selected oysters
of each year class. In areas of natioral reproduction, "wild oysters"
were excluded from the samples. Volume measvirements were made in the
laboratory .

The results of these surveys are summarized by year, year class
and area in Table 5« Grouping of data by areas in this table masks the
internal variations; however, the average values in general (with the
exception of Drayton Harbor in northern P\iget Sound and Oakland Bay in
southern Puget Sound)* demonstrate oyster growth in the fovir areas.

•^Because of reduced growth and fattening, many northern P\jget Sound
growers did not plant seed in 1957 and 1958- The absence of plantings
on much of the poorer ground gives an apparent Improvement in growth
in these years. Oakland Bay, in southern Puget Sound, has been a poor
growing area throiighout the period covered by this study.

-livO-

Table 5. Growth of the Pacific oyster as determined by annual state-
wide siii'veys

Average vol. in cc

statewide average

North

South

Year

Puget

Puget

Grays

Willapa

Ave. Size

observed

Age

Sound

Sound

Harbor

Bay

N

cc

1956

0+

6.2

h.Q

h.k

8.9

2,125

5.3

1+

22,9

37.0

36.0

71.7

985

38.1

2+

70.2

104.7

89.0

120.8

7if5

97.9

3+

105.5

129.9

173

116.8

W

127.8

103

12i^.3

1957

0+

5.6

5.8

6.3

21.0

l,2i+l|

11.9

1+

33.1

h^.k

kk.2

77.6

633

kT.k

2+

66.2

80. i^

95.6

13^^.6

3i^5

103.2

3+

105.9

179.1

6k

118.5

k+

112.5

3h

112.5

1958

0+

6.8*

5.8

8.1

12.9

1,350

8.9

1+

58.9*

52.5

81.2

75.6

719

61+. 0

2+

80.6

ioif.3

122.8

133.1

816

ioi+.9

3+

122.3

132.1

216

108.2

h+

1^3.1

221.1

78

15^^.9

* These data reflect the planting of 1957 and I958 Japanese seed on
optimum ground available in the area.

Using the 2+ age oysters for comparison, Willapa Bay has con-
sistently had the best growth and northern Puget Sound the poorest.
Grays Harbor and southern Puget Sound have been alternating at second
place. Using 1+ oysters for comparison, since they seem to provide
the best short-term measure of growth, Willapa Bay seems to be staying
about the same, with northern Puget Sound, southern Puget Sound and
Grays Harbor improving. It moat be remembered that improvement in
northern P\aget So\ind reflects the cessation of seed plemtings on a.1 1
except the two best beds. During three years of observation there
appears to be a general increase in growth in all areas except northern
Puget Sound from I956 to 19 58.

In Table 6, data on size of oysters in all surveys to date
are summarized by year class. Since the Pacific oyster is generally
harvested between the second emd third year after planting, the aver-
age market size lies between 102 and 113 cc.

-l4l-

Table 6. Pacific oyster average size by age as determined in state-
wide surveys^ 1956-1958

Age

Average size in cc

0+

8.1

1+

48.6

2+

101.8

3+

113.0

k+

133.5

SUMMARY

1. Growth of the Pacific oyster in Case Inlet appears to he
a continuous process with no evidence of winter hibernation.

2. Between 1952 and 1958, year to year growth fluctuations
have been such that the size at 15 months has varied from 27.3 cc to
80.7 cc in Case Inlet.

3. Percentage growth for the first year after planting has
ranged from 192/0 to 2,126fo, and from UUfo to iCkf in the second year.

k. In our Case Inlet study area growth declined between 1952
and 1955 and has been steadily increasing during the I955-I958 period,

5. There are Indications that it may be possible to predict
size of the Pacific oyster at 26 months from their size 6 months after
planting .

6. Growth of oysters planted intertidally between +2.8 and
-0,9 feet increases inversely with tidal level.

7. Accimiulated growth on oysters 28 months of age at about
the same tide level in different areas has varied between 57 cc and
131 CO.

8. Based on annual indxistry-wlde oyster growth sxirveys, Willapa
Bay is the area of best growth, northern Puget Soimd the poorest, with
Grays Harbor ajad. southern Puget Sound alternating between second and
third.

9. With the exception of northern Puget Sound, growth gener-
ally increased between 1956 and 1958.

10. Based on the age-size relationship, the Pacific oyster is
usually harvested when 102 cc to 113 cc in size.

-ll^2-

REFEEIENCES

Beaven, G. Francis. 1952. Some otservations on rate of growth of

oysters in the Maryland area. Natl. Shellfish. Assoc. Conv.
Addresses 1952: 9O-98.

Butler, Philip A, 1952a. Shell growth versus meat yield in the oys-
ter £. virginica. Natl. Shellfish. Assoc. Conv. Addresses
1952: 157-162.

. 19521). Seasonal growth of oysters C. virginica

in Florida. Natl. Shellfish, Assoc. Conv. Addresses 1952:
188-191.

1953. Oyster growth as affected by latitudinal

temperature gradients. Comm. Fish. Rev. 15 (6): 7-12.

Hopkins, Sewell H. and R. Winston Menzel. 1952. Methods for the
study of oyster plantings. Natl. Shellfish. Assoc. Conv.
Addresses 1952: 108-112.

Quayle, D. B. 1951* The seasonal growth of the Pacific oyster (Ostrea
gigas) in Ladysmlth Harbour. Ann. Rept. British Columbia Dept.
Fish. 1950 1 185-190.

Sekl, H. 1937* On the difference between Ostrea gigas from Hiroshima
and Sendal Bay. Fish. Invest. Imp. Fish. Exp. Sta. Suppl.
Rept., No. kf pp U5-5O (in Japanese). Reported in Cahn,A.R.
(1950) Oyster culture In Japan. U.S. Fish Wildl. Serv., Fish.
Leafl. 383.

Tliomson, J. M. 1952. The acclimatization and growth of the Pacific
oyster ^G. gigas ) in Australia. Aust. Jour. Mar. Freshw.
Res. 3 (1) 1952: 6k-^k.

Woelke, C. E. 1955* Introduction of the Kumamoto oyster (Ostrea

(Crassostrea) gigas ) to the Pacific Coast. Wash. Dept. Fish.
Res. Papers 1 (3): 4l-50.

-li^3-

SELECTION AM) EVA1UA.TI0W OF A METHOD FOR QUAUTITATIVE
MEASUREMENT OF OYSTER CONDITION

Ronald E. Westley

Washington Department of Fisheries
Shellfish Laboratory, Qullcene, Washington

ABSTRACT

Fatness is one of the most Important considerations of
oyster culture. The "Grave method of condition factor index
determination" was used initially but found to lack reprodu-
cibility. Two Improved measuring techniques are presented plus
an evaluation of the modified procedure as used by the Washing-
ton State Department of Fisheries.

Fatness Is one of the most Important considerations in evalua-
tion of oyster culture "by either commercial operators or hlologistB .
Researchers, recognizing the need, have devised various methods for
evaluation of oyster fatness. This paper presents and evaluates the
method found most suitable by the Washington Department of Fisheries .

Grave (1912) describes a method for measuring oyster condition.
This method consists of dropping uaopened oysters into an overflow
container and measuring the water displaced. The same procedure is
followed with the empty shells and the volume of the shell cavity is
then calculated. Volume of the drained meats is measured by water
displacement. "Condition factor index" is the ratio of meat volume
to volume of the shell cavity:

C.F. = Vol Meat X 100

Vol. Shell Cavity

In monitoring oyster condition in Washington state, the "Grave
method" was initially used. Subsequently it was found to lack repro-
ducibility in volumetric measurement and in estimation of meat quantity,

Quayle (1950), measuring volumetric growth of the Pacific oys-
ter (Crassostrea gigas), utilized a method borrowed from J, C. Medcof
involving an application of Archimedes' principle. Oysters are weighed
first in air, then in water. The difference between the two weights
equals volume. To eliminate errors present in wet-meat volume measure-
ments, Higgins (1938) suggests oven drying of meats at 100°C, and Galts-
off et al. (19U7) propose initial drying at 50°C and final drying at
100°C. Haydu (personal coramvmicatlon) found variation in drying at
100°C due to removal of varying amounts of chemically-bound water that

-llf5-

did not occin* with Initial drying at 500C. More recently, Korringa
(1955) suggests further improvement in drying meats ty use of toluene
distillation.

By combining the weight-in-alr, weight-in-water method with
oven drying of the meat, we have evolved a procedure for measurement
of fatness of Craesostrea gigaa and Ostrea lurida In Washington state.
Seunple size is based on size of adult oysters, reproducibility of
measurements, and variation in meat qviality in oysters. In the case .
of the large Pacific oyster (average adiilt size 125 cc volume) 20 oys-
ters are used, giving a total sample size of 25OO cc. In the case of
the small Olympia oysters (average adult size h.O cc volume), 50 oys-
ters were used giving a total sample size of 200 cc. In both cases,
amount of variation is only about +0.6 condition index unit at the
95^ confidence level (Table l) .

The following procedures ■*<■ are followed!

1. Oysters are carefully cleaned. Those gaping or having chipped
edges are rejected,

2. The oysters are held in running sea water for at least one hour
prior to weighing to insure that no air is trapped between the
valves .

3. Volume is determined and oysters are opened, care being taken not
to break the shells or leave meat attached to the shells. Then
the volume of the shell cavity is determined by subtracting volume
of shell from total volume.

h. The meats are placed in a tarred aluminum foil tray and dried in
a forced air oven for two days at 50°C and two days at 100^0
(producing constant weight) and weighed.

5. Calculations I

A. Dry weight of meats in grams ^^. _ ,.,. _,

=7-^ ^ — r— rrj T-7 — 7 X 100 = Condition Index

Volume of shell cavity in cc

In measuring oysters, considerable error can be Introduced if
there is air between the valves of the whole oysters (reason for step
2 in the procedure). The weight of the whole oysters in water should
be equal to, or slightly greater thaji, the shells in water if there
is no air trapped in the whole oyster.

* When routinely sampling the same age oysters from an established
station, this procedure provides data which can be used for growth
evaluation. The procedure and calculations can be shortened if growth
data are not desired.

-146-

Table 1. Results of replicate analyses of multiple lots of oysters

Crassostrea gigas

Area:

OflWancJ Ba.y

Date:

July 25, 1956

Ave. vol. per

oyster:

C.I.

7.1

7.7

S.D. =

= 0.66

6.3

t for

N-i 95?^

7.8

7.0 +

1.0

Area: Oakland Bay
Date: July 5, I956
Ave. vol. per oyster: 100 cc
C.I.

7.8 S.D. = 0.40

7.2 t for N-1 95$ C.I.

8.0 7.8 + 0.6

8.1

100 cc

C.I.

Area J Oakland Bay

Area: Worth Bay

Date: July 12, 1956

Date: August 8, 1957

Ave. vol. per oyster: 100 cc

Ave. vol. per oyster:

125 cc

C.I.

C.I.

7.6

13.3

7.5

15.0 S.D. = 1.10

7.1 S.D. = 0.61+

12.3 t for N-1 9%

C.I.

7.1 t for W-1 95^ C.I.

13.if 13.5 + 1.7

6.2 7.1 + 0.7

Area: Oakland Bay
Date: July 19, 1956
Ave. vol. per oyster:
C.I.

100 cc

7.3

7.0

7.1
6.7

S.D. = 0.20

t for N-1 95^ C.I.

7.0 + 0.3

Ostrea

lurida

Area: Oyster Bay

Area: Oyster Bay

Date: July 11, 1957

Date: August 8, I957

Ave. vol. per oyster:

Ij-.O cc

Ave, vol. per oyster:

k.O cc

C.I.

C.I.

7.2 S.D. = 0.30

17.3

6.7 t for N-1 9'?$

C.I.

17.4 S.D. = 0.60

6.6 6.8 + 0.5

16.9 t for N-1 95^

C.I.

6.6

16.1 16.8 + 0.6

17.2

16.0

-147-

RelialDility of the method Is affected by two factors^ acciaracy
of techniques of measurement and sampling error^ and variation of oys-
ters on the heds . To check the accuracy of the measuring method re-
peated measurements of the same oysters and ohjects of known volume
were made^ which demonstrated that measurements are hoth accurate and
reproduciiale. The problem of sampling error^ or adequacy of sampling.
Is more complex and has no easy solution. To minimize this, field
samples are collected at set locations. Table 1 presents data from
several sets of Pacific and Olympia oyster samples. Each set was col-
lected from the same area (bed) on the same date. The various sets
were collected from known areas of good and poor oysters . These data
demonstrate the combined effect of accuracy of method and sajiipling
error.

The standard deviations of Crassostrea gigas samples indicate
consistency of results when oyster condition is poor with increased
variation in better oysters. In the case of Ostrea lurida the devia-
tion is low for both poor and good oysters. The failure of the method
to distinguish whether a high condition factor index is due to fatness
or spawn poses no serious problem as presence of spawn is easily deter-
mined by eye. To date the relationship between condition factor index
and commercial yield has not been determined.

The method provides a rapid and reproducible way of evaliiating
oyster condition. The actual time required to process a sample is
less than any method using the overflow technique, although the intro-
duction of oven drying creates a four-day waiting period. The pro-
cediire has been utilized for five years by the Washington Department
of Fisheries for measurement of oyster condition and has proven to be
a valuable tool (Westley 1959). Information collected permits de-
tection of seasonal variation, long range changes, and effects of ex-
treme weather conditions. The method is also being used in assessing
oyster condition in laboratory and field bio-assays.

The writer wishes to gratefully acknowledge the help and £id-
vice of Dr. D. B. Quayle in setting up this procedure.

REPEIIENCES

Galtsoff, Paul S., Walter A. Chipman, James B, Engle and Howard N.
Calderwood. 19^7, Ecological and physiological studies of
the effect of siilfate piilp mill waste on oysters in the York
River, Virginia. U. S. Bur. Fish., Fish. Bull. 43:59-186.

Grave, Caswell. 1912. A manual of oyster culture in Maryland.

Fourth Kept. Board of Shellfish Commissioners of Maryland,
376 pp.

Higgins, Elmer. 1938. Progress in biological inquiries. U. S. Dept.
Commerce Adm, Kept. No. 30, page 50.

-Ilf8-

Korringa, P. 1955. Quality determination of musBels (Mytilus edulis)

and oysters. Q,ualitat8be3timniiingen an Miesmuscheln \md Austem.
Arch. f. Fischereiwiss. 6(3/i^) :l89-193.

Medcof, J. C. and A. W. H. Needier. 19i^l. The influence of temperature
and salinity on condition of oysters (Oetrea virginica) . J.
Fish. Res. Bd. Canada ^ (3): 253-257.

Quayle, D. B. 1950. The seasonal growth of the Pacific oysters

(Ostrea gigas ) in Ladysmith Hartour. Kept. British Columbia
Dept. Fish. I95O185-9O.

Westley, R. E. 1959. Olympia and Pacific oyster condition factor

data. State of Washington 195^^-1958. Mimeographed report of
Wash. Dept. Fish. Shellfish Lab.

-Ilt9-

EFFECT OF HYDRAULIC ESCALATOR PIAEVESTER
ON WIDER-SIZE SOFT-SHELL CLAMS

J. C. Medcof

Fisheries Research Board of Canada
Biological Station, St. Andrews, N. B.

ABSTMCT

A modified Maryland-type hydraulic escalator shellfish
harvester was used at high tide on Intertldal beaches In Nova
Scotia to determine Its effect on soft-shell clams less than 2
Inches long. The boat was equipped with a propeller guard to
prevent bottom scouring aaid three types of experiments were
carried out with small, marked clams: (l) Dead clams were re-
leased in the scoop to determine their scatter pattern after
passing through the harvester. (2) Plots of pleinted dead clams
were dug through to discover breakage rates ajid distribution.
(3) Plots of planted living clams were dug through to see how
the harvester affects their distribution and ability to re-
establish themselves. Results were observed on the dry beach
at low tides .

Most of the small clams sifted through the mesh of the
escalator belt before they reached the surface of the water.
In spite of strong currents from harvester jets and the boat
propeller, 90?^ of the clams were returned to the harvester
track within 75 or 100 feet of the place where they entered
the harvester. Soil is heavy and settled first in the track.
Clams are lighter and were deposited on the soil surface, not
burled and smothered. The harvester broke 7 to 10^ of the
living clams but the rest dug In again quickly. Because damage
was so small compared with that caused by conventional clam
hoes we think production would be improved if hydraulic esca-
lator harvesters were used.

INTRODUCTION

Since 195^ the Fisheries Research Board of Canada, with support
from Industrial Development Service of the Department of Fisheries,
has been modifying the Maryland-type hydraulic escalator clam harvester
(Manning 1957) • This machine works vmder water from a "boat containing
a pump supplying powerful jets of water to wash shellfish from the soil
into the harvester's digging head, or scoop, and then onto the escalator
belt which brings them up to the boat at the surface. We were looking
for ways of increasing productivity of stocks of soft-shell clams (My a
arenaria) which are depressed by Intensive fishing and by wasteful
methods of fishing.

Our early work with the harvester was described by Dickie and
MacPhall (l957)' Fiirther modifications have been made in design of

-151-

the scoop, and a larger pump installed (75O gallons per minute at ^^^0
pounds pressure per square inch). Harvester performance has greatly-
improved. Now it takes more than 95^ of marketahle-size, soft-shell
clams in its path and. when fine-mesh escalator "belts are used, it
takes small clama (3/V' to 1") with this same efficiency. It has al-
so been used successfully in harvesting quahaugs and "bar clams from
"beds up to, 6 feet deep. In 1959 a new- type head, designed by Mr. J.
S. MacPhail and Mr. H. Y. Brownrigg of our Station, proved highly
efficient for fishing bedding-size and adult oysters from shallow
grounds and for cleaning oyster beds .

The Department of Fisheries has approved use of harvesters for
fishing quahaugs and bar clams from public beds and for fishing oysters
from leased grounds, but has hesitated to legalize it for fishing soft-
shell clams. We knew too little about how it might affect productivity
of clam grounds and economics of industry. The Fisheries Research
Board carried out experiments described here to provide a partial basis
for regulatory policy.

The work was done in May I958 with M. B. Cyprina (Skipper Earl
Durkee). I am grateful to Mr. Durkee and Mr, MacPhail for co-operation,
to Dr. L. M. Dickie who initiated work with the harvester, to my Direc-
tor, Dr. Hart, who fostered the project, and to Mr, L. S, Bradbury,
Director of Industrial Development Service, Department of Fisheries,
for providing funds.

PROCEDURE

GeneraJ.

Three types of experiments were carried out with sub-legal size
clams — release of dead clams in the harvester scoop to determine scatter
patterns of clams and guide designing of other experiments, diggings
through planted plots of dead clams to discover breakage rates and
distributions of under-size clams that might be expected in ordinary
digging and, finally, diggings through planted plots of live clams to
see how harvesters affect ability of under-size clams to re-establish
themselves ,

Clam Harbour, Nova Scotia, was chosen as a work site because
it has wide expanses of nearly level, uniform-textured, sandy flats.
In 1958 these flats supported only sparse populations of clams, which
was an important advantage because it reduced catch-culling efforts.

Minimum legal length for market clams in eastern Canada is 2
inches. Our interest was focused on smaller sizes because harvester
effects on them would influence recruitment of marketable stocks.
Accordingly, we used only clams 1 l/h to 1 'i/k inches long in all ex-
periments. It is difficult to do marking experiments with smaller
animals and this is the sole reason for choice of this size. Shells
were marked with Volger's ink for identification.

-152-

The harvester was equipped with an escalator belt vlth 1-inch-
square mesh, which is commonly used for commercial harvesting, and the
scoop lip was adjusted to dig I5 inches into bottom as in regular fish-
ing for commercial-size clams. A propeller guard (Manning 1957) was
used to prevent bottom scouring and in other ways the harvester was
run as in commercial fishing. M. B. Cyprina draws 2 feet of water
and the shallowest depth she will work in satisfactorily is 28 inches.
This limits periods of operation on intertidal flats.

Scatter Patterns

Clams were killed by soaking them overnight in a 10^ solution
of formaldehyde. Next day at high tide the harvester was set on a
straight coiirse and run for 200 feet. Then 110 clams were released
deep in the harvester scoop jtist above and behind the lip by dumping
them through a 6-foot pipe (diameter 5 inches) whose upper end was at
gunwale level. A buoy was cast overboard when clams were dumped to
mark the release point and the harvester was kept in operation for
another 100 feet. Experimental clams probably experienced full effects
of propeller blast and hydraulic jet currents as do under-size clams
in normal commercial fishing. This same release operation was carried
cut at three water depths (30, kO and 50 inches) over intertidal flats.

About a third of the animals csune up on the escalator belt and
dropped off over the end. The rest apparently passed through belt
meshes and dropped back to bottom before reaching the water sxirface.
In contrast, many large native clams were brought up.

At the next low tide, plots were visited and distribution of
marked clams on the dry beach was simply recorded because, being dead,
they did not burrow in. Gulls were everywhere and some were feeding
on damaged native clams left exposed in shallow trenches or tracks
which marked harvester paths. So far as could be judged they had not
touched experimental animals. These retained a strong odour of form-
aldehyde but it is doubtful that gulls would detect this.

• Recovered marked clams were classified as buried or exposed
on beach surface and as occurring in harvester track or on shoulders
of undisturbed flat beside tracks. Besides this, clams were classified
as having broken or intact shells. They were also classified accord-
ing to distance transported from point of release. For this classi-
fication, 25-foot-long zones were marked off along digger tracks in
front of and behind release points which were identified by buoys.

If any part of a clam's shell was showing above sand that ani-
mal was classed as surfacing. We searched for buried clams only in
harvester tracks where soil wa^ loose. Elsewhere the surface was firm
and londisturbed and without soil deposits that could bury clams. Track
soil was usually soft eno\igh to probe with the hands for the first two
hours after tidal exposure. When it was too firm it was turned with
conventional clam hoes. Both methods of search co\ild have damaged

-153-

Table 1. Distribution coimts of small, dead clams released throiigh a
shoot into harvester scoop and recovered on dry beach at
next low tide. "T" indicates clams found in harvester track.
"S" indicates clams found on the shoulders beside the track.

Water depth when
clams were released
(inches)

30

50

Disposition of clams

Surface Buried
T S T S

Surface Buried
T S T S

Distance behind -50-0
point of release

(feet) 0-25

25-50

50-75
75-100
100-125

2

0

0

0

11

0

0

0

7

1

0

0

15

3

0

0

22

10

0

0

30

1

0

0

3

0

0

0

10

5

0

0

6k

11

0

0

1

3

0

0

0

0

0

0

0

0

0

0

JNo. Recovered

Distribution of
recoveries

No. recovered
Ko. released

No. broken
No, recovered

87 15 0 0

85^ 15^ 05^ oi)
(102) ^^

78 19 00

80^ 20^ oio oio

( 97)

(Ho)
( 97)

88^

2$

-15^-

Table 2, Distribution coiJJits of small, dead^ planted clams recovered
on beach at next low tide after harvester cut through plots .
"T" indicates clams found in harvester track,
clams found on shoulders beside track.

Water depth when
plot was dug
(inches)

1^0

i^O

Disposition of clams

Surface Buried
T S T S

Surface Buried
T S T S

Distance behind -50-0
plot (feet)

0-25

25-50

50-7^

3 0 0 0

&k Q 10

J+1 10 ho

111 0 10

10 0 0

Ik 7 10

163 7 10 0

05 00

No. recovered

Distribution of
recoveries

No. recovered

No. in path of digger

Washouts (difference)

No. broken
No. recovered

239 18 60

9lfo Ji 2<f> 0$

263) 1660^
I5H) ^^^

105

( iM
(253)

178 19 11 0

(208) ,^2^
(155) ^32^

150

( 18)
(20H)

32?i

-155-

some of the recovered clams "but very few were burled and most were so
close to surface that numbers broken in searching must have been neg-
ligible. Looseness of track soil persisted for more than a week.

Results of tests conducted at 30 and 50 inches appear in Table
1. Those for kO inches were essentially like those for 50.

Digging Planted Dead Clams

Fo27malin-killed clams were planted, 9 per square foot, in
plots measuring 7 by 15 feet. Long axes of plots were set at right
angles to prevailing directions of tidal currents across the flats .
For precision in density of planting we used a grid frame, about 3 by
5 feet, strung lengthwise and crosswise with cod-line to give U-inch
squares. In planting, this frame was laid on the beach, a hole was
made for each square with a wooden spike and a clam placed, siphon-end
up, in the hole. Upper ends of clams were 2 to 3 inches below surface,
which is normal for clams of this size (Medcof 1950)« Corners of
plots were marked with stakes.

Next day at high tide the harvester was set in operation on a
straight course 200 feet from each plot. Digging was continued up to
and through each plot and 100 feet beyond. Few planted clams were
brought to water surface. At next low tide clams that could be found
on the dry flat were classified as before.

Harvester tracks measured 50 to 75 inches wide and their sur-
faces averaged il- to 6 inches below levels of adjacent beach. Apparent-
ly crumbling of track shoulders and erosion by ebb-tide currents ex-
tended track widths sometimes to twice or more the original widths
(digger scoop 30 inches wide). Where tracks had cut through plots,
several clams were found in various stages of being washed from plant-
ed positions along shoulder edges. Approximate numbers of washouts
were estimated roughly from differences between the expected number
of clams (158) in direct path of harvester and the numbers actually
recovered (Table 2) . In spite of erosion there were few buried clams
in the track.

Results of two diggings at intermediate depths (^0 inches)

appear in Table 2. Meats were missing from four of the I8 broken

clams found in the second plot (Table 2) as though they had been eat-
en by gulls.

Digging Planted Live Clams

Procedures were as in previous tests except that living clams
were used. These were dug manually at low tide, marked, and kept in
flowing water until next day's planting. Beach soils were firmer in
some plots than in others. Depths of water over plots, when they were
dug, varied but were mostly less than kO inches. Few marked clams
were bro\ight to water surface by the escalator belt,

-156-

After digging one plot we rowed a small boat over the track.
Tidal currents cleared the water soon after harvester operation ceased
and marked clams could "be seen lying on sandy tottom. All had closed
shells but In a few minutes many extended siphon and foot and within
a quarter of an hour from the time they had been disturbed most had
burled or partly burled themselves . This observation agrees with
others, for example Mead's (19OI), that clams of this size re-establiah
themselves quickly.

We visited the flats at following low tides before gulls could
attack the experimental clams .

Table 3 reports results of four tests. By surface-picking and
digging it was Impossible to recover all marked clams distiorbed by the
digger. Sometimes more than I58 (the number directly in harvester
path) were recovered, sometimes fewer. This is understandable because
we know from experience that careful digging of a plot with hand tools
seldom recovers more than 80^ of market-size clams in it.

DISCUSSION

The propeller guard prevented scouring of bottom which was a
problem in early trials (Dickie and MacPhail 1957) •

We assume that observed effects on clams 1 l/4 to 1 3/^ inches
long are typical of effects on all sub-legal sizes. In digging in 30
inches of water they were seldom carried more than 100 feet (Table l)
and in 50-lnch depths seldom more than 75 feet. All tables show that
most clams are carried backward by water currents set up by the har-
vester and deposited "behind" the place where they entered the har-
vester. There are exceptions. After the harvester passes, tidal
currents in digger tracks, which form shallow channels, apparently
carry some clams "ahead" as much as 50 feet. Such distributions are
Indicated by negative values in the tables. The general res\ilt of all
disturbeinces is that most clams come to rest near their former homes
where they should find equally good conditions .

The harvester deposited about SQ^o of test clams on the loose
track soil and live ones burrowed in quickly. Ebb-tide currents in
tracks washed some clams out of their plots. Sometimes we visited
the flats before they were completely exposed and when this washing-
out process was still going on. The flats dried off before some of
the washouts had a chance to dig in and oounts of surfacing clams
were accordingly high (Table 3)- This leaves opportunity for attacks
by strictly surface-feeding clam enemies. And looseness of track soil
might leave buried clajns more than ordinarily s-usceptible to attack
by gulls (Medcof I9U9), flounders (Medcof and MacPhail 1952a) and clam
drills (Medcof and Thurber 1958). It la not believed that this risk
is greater than that Involved in manual harvesting even though track
erosion sometimes affects almost as many more clams as lie in the

-157-

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-158-

harvester's direct path (Tables 2 and 3). Unpublished studies indicate
that disturbance enliances clam growth. This effect would offset dis-
advantages of greater exposures to enemies .

It is obvious, even without making allowance for clam breakage
caused by probing in search for distiorbed animals^ that the total break-
age was slight. Table 1 suggests that after clams enter the scoop
breakage increases by only 2 to 3/o. An eq.ual or slightly higher break-
age is caused by the scoop cutting through the soil. This is not easy
to estimate becaiise its frequency is masked by the numbers of washouts
which do not pass through the harvester. If no allowance is made for
these, total breakage works out to about 7fo (Table 2). If it were
worked out from the number of clams directly in the harvester path
(158) and we assumed complete recovery of these, then the breakage
would work out to about lO/o. Breakages exceeding lO/o, like those re-
ported in Table 3, are attributable to damage done experimentally in
recovering live clams that burrowed in. However, even Table 3 values
seem low compared with breakages caused by hand tools (Medcof and Mac-
Phail 1952b).

Table 2 shows that the escalator harvester buries few clams
(less than ^fo) and biiries these shallowly in soft soil where \mbroken
live clams can quickly establish themselves. This low b\irial freq-
uency is attributed to differences in the settlement rates for soil
and clams . Apparently almost all bottom soil disturbed by the har-
vester settles out first (Manning et al. I959) ajad in the track.
Clams are not much heavier than water and settle out last, landing on
top of the soil. Thus, in accounting for damage done by the harvester,
biurlaJ. and smothering seem far less important than shell breakage. The
reverse is true for damage caused by hand tools (Needier and Ingalls
19^) where burial and smothering are chiefly responsible for 50/° mor-
talities at every digging. From this it is deduced that if hydraulic
harvesting were adopted, damage incidental to digging would be reduced
to about 20'fo of that now regularly inflicted by clam hoes.

The ordinary clam digger with his conventional hoe seldom har-
vests more thaji 6ofo of the marketable clams from the soil he tijrns
(Medcof, unpublished MS report). The other kO'fo are hidden in soil
clods ;-ind encourage him to return soon and redig the same grovtnd.
Every digging is attended by 50/0 mortality of the clams left behind.
Experiments (MacPhail, unpublished MS report) have shown that the
hydraulic digger harvests more than 90^ of marketable-size clams in
its path. This means that grounds exploited by hydraulic harvesters
are likely to be redug at longer intervals than those dug with hoes.
This might reduce digger damage to even less thein 20'fo of that now be-
ing caused by hoes. Conservationwlse oirr results agree in all es-
sential respects with those reported from Maryland (Manning 195?)
where harvesters have been used since 1950 ♦

It is generally accepted (Glude 195l) that clam fishing is
the chief of controllable factors regulating clam abiondajice in ex-

-159-

ploited areas. Other data (Needier and Ingalls 19^^) show that in-
direct effects of fishing (damage Incidental to digging with clam
hoes) are more Important in contributing to fishing mortality than
direct effects (removal of marketahle clams). Our experiments indi-
cate that present indirect fishing mortality rates would "be reduced
by 80^ or more if escalator harvesters were adopted. Such a reduction
should substantially Increase per-acre yields of clam grounds.

It must not be assumed that such benefits could be brought a- '
bout in all clam areas . The whole Bay of Fundy region has such high
tides that ordinary escalator harvesters could not work profitably.
In many outer-coast Nova Scotia harbours tidal amplitudes are suitable
but clam beds are too rocky^ too small or too intricately shaped to
make escalator harvesting practicable. In these areas conventional
harvesting methods, or some other type of harvesting yet to be develop-
ed, will have to serve. There are many places, however, both on the
outer coast of Nova Scotia, and in the Gulf of St. Lawrence, where
tides are right, where soil texture is right and where extent and
topography of well-stocked clam beds are right for successful esceilator
harvester operations. The majority of these beds are Intertidal and
more or less regularly fished, although wastefully, with clam hoes.
But there are beds, such as those around Heron Island in Bay of
Chaleur, that are permanently submerged and seldom fished. Use of
escalator harvesters woiild be expected to bring about the most con-
spicuous per-acre yield benefits in these areas because they would
then be efficiently fished for the first time. The harvester would
be expected, to effect smaller per-acre yield Improvements in inter-
tidal beds that are now dug. But, because of their vaster area, their
Improvement would likely mean more to the clam Industry than develop-
ment of the few currently unexploited areas like Heron Island.

REFEREKCES

Dickie, L- M. and J. S. MacPhail. 1957- An experimental mechanical

shellfish-digger. Fish. Res. Bd. Canada, Progr. Kept. (Atlantic)
• 66j 3-9.

Glude, Jolin B. 1951. The effect of man on shellfish populations.
Trans. l6th N. Aaier. Wlldl. Conf., pp. 397-^03-

Manning, J. H. 1957. The Maryland soft shell clam industry and Its
effects on tidewater resources. Maryland Dept. Res. Educ,
Resources Study Rept. 11, 25 pp.

, Carol B. Whltesell and H. T. Pf itzenmeyer . 1959.

Some effects of intensive hydraiilic clam dredging on the
biotlc and physical structure of a Maryland clam flat. Proc.
Natl. Shellfish. Assoc. (Unpublished.)

-160-

Mead, A. D. 190l(l900). Observations on the soft-shelled clam.
Ann. Kept. Rhode Island Comm. Inl. Fish. 31: 21-4i<-.

Medcof, J. C. 19^9- "Puddling", a method of feeding by herring gulls.
Auk. 66 I 204-205.

1950 ♦ Bxirrowing habits and movements of soft-shelled

clams. Fish. Res, Bd. Canada, Progr. Rept. (Atlantic) 50'
17-22.

and J. ST MacPhail. 1952a. The winter flounder —

a clam enemy. Ibid. 52: 3-8.

1952b. Breakage — the bug -bear in clEun

handling. Ibid. 5^: 19-25 .

and L. W. Thurber. 1958« TriaJL control of the great-

er clam drill (Lunatla heros) by manual collection. J. Fish.
Res. Bd. Canada 15(6): 1355-I369.

Needier, A. ¥. H. and R. A. Ingalls. 19^4. Experiments in the pro-
duction of soft-shelled clams (Mya) . Fish, Res. Bd. Canada,
Progr. Rept. (Atlantic) 35 J 3-8.

-161-

INHERITANCE OF SHELL MARKINGS AM) GROWTH IN
THE HARD CLAM, VEMUS MERCENARIA

Paul E. Chanley

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

ABSOEACT

Inheritance of Venus mercenaria (Mercenaria mercenaria)
notata shell markings was followed throiogh two generations .
Offspring from three crosses of unmarked "white" clams were
unmarked, while three crosses of white clams with clams having
the "notata" marking, produced about half unmarked offspring
and about half marked with the reddish-brown zigzag lines, typ-
ical of notata subspecies. When two clams with notata shell
markings were crossed, about one-fourth of the offspring were
unmarked and about half were marked with zigzag lines . The re-
maining one-fourth were almost solidly reddish-brown, with a
light band from the umbo around the lunule, and two other light
bands from the umbo to the margin of the shell, dividing it
roughly into thirds. This marking is considered typical of
clams homozygous for the color factor. The zigzag lines, which
are commonly used to identify the notata subspecies, are con-
sidered the phenotyplc markings of heterozygous clams.

Offspring from fast-growing sibling clams were 6o per
cent larger, after 15 months, than offspring from clams ran-
domly selected from wild stock. This suggests that only a few
generations would be required to develop fast-growing races of
clams.

INTRODUCTION

Advances in the laboratory culture of lamelinDranch larvae
have made possible the rearing of shellfish of known parentage. Be-
cause of these advances, studies of inheritance in shellfish are now
possible. Knowledge of the principles of inheritance has permitted
great improvements in agrictilture in the past and knowledge of the
principles of molluscan Inheritance may be as beneficial to modern
shellfish cultivation in the future.

Because of length of time required and difficulty in rearing
larval stages, genetic studies of commercial bivalves are scarce. In-
terspecific crosses have been reported by Bouchon-Brandely (1883) and
Davis (1950). Survival and growth of V. mercenaria, V. campechiensls
and their hybrids have been studied in Virginia by Haven and Andrews
(1957) and in North Carolina by Chestnut, Fahy and Porter (l957).
Chanley (1955) reported that clean larvae from one set of parents grew
more rapidly than those from other parents, but dealt only with larvae.
Davis (1955) reared five generations of Olympla oysters (Ostrea lurlda)

-163-

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in an unsuccessful attempt to develop a strain that would survive New
England winters, but there are few reports involving more than one
generation.

The purposes of these experiments were (l) to study inheritance
of the typical reddish-brown shell markings of V- mercenaria notata,
and (2) to determine the feasibility of developing fast-growing races
of hard clams by selective breeding.

RESULTS

Inheritance of Notata Markings

The subspecies V. mercenaria notata is easily identified by
typical reddish-brown zigzag lines on both valves of the shell (Abbott
195^). It occurs along the east coast from Maine to Florida but is
rather uncommon particularly over the northern part of its range (Miner
1950, Morris 195^) . This form is rarely seen in Long Island Sound not
only because it is uncommon but also because the shell markings are
frequently obscured by shell discoloration caused by the substrate.

In studies of the Inheritance of notata markings, two original
crosses of Long Island Sound clams were involved. In each case female
A, displaying notata markings, was used. In the first cross this fe-
male was crossed with vinmarked male A. The ratio of notata- marked to
unmarked offspring was close to the 1 to 1 ratio expected when a
heterozygous individual is crossed with a homozygous recessive one
(Table l). When the same female, A, was crossed with male B, also
bearing the notata markings, the ratio of marked to unmarked offspring
was essentially 3 'to !• This is the expected phenotypic ratio in a
cross of two individuals heterozygous for a simple Mendellan character.

Counts were not made until marked and unmarked clams had reach-
ed a minimum length of about 5 inm^ since the notata markings were not
always apparent in smaller clams. Even at this size markings were
sometimes faint and difficult to detect. Subsequent counts, however,
failed to show any appreciable change from the original ratios.

Offspring from the cross of marked female A with unmarked male
A were raised to matiirity and used in another series of crosses. Prog-
eny from the female A by male B cross were not yet large enough to
give adequate amoionts of spawn at this time. Improved techniques in
culture methods provided more clams than could be conveniently examined
in these crosses and, therefore, random samples of 1,000 were counted.

The first three crosses of the Fi generation involved unmarked
clams (Table l) . Although a few marked clams were found among the off-
spring, we believe that these crosses actually produced no marked clams.
It is possible that they were introduced accidentally from other groups
during the handling that was necessary to rear the clams from eggs to
more than a year old.

-165-

Both Fi crosses involving a marked clam with an unmarked clam
produced offspring roughly in a ratio of 1 marked to 1 unmarked. This
is again the expected ratio for a cross of a heterozygous Individual
with a homozygous recessive.

When two notata-marked clams from the Fi generation were crossed
the offspring bore notata markings in a 3 to 1 ratio as expected in a
cross involving heterozygous individuals. In this last cross two types
of markings coxjld he recognized (Fig. l) . The most common markings

BT^^

HETEROZYGOUS

^^hI

HOMOZYGOUS ^^1

Fig. 1. Juvenile V. mercenaria notata showing the difference
between homozygoiis and heterozygous markings.

were the typical reddish-brown zigzag lines. This marking was quite
variable and ranged from faint fragments of lines to dark, heavy lines
so concentrated that they produced almost a solid color. The other
type of marking also varied, but typically the shells were almost
solidly colored reddish-brown with a light band from the umbo around
the lunule. Two other broad light bands extended from the umbo to the
margin of the shell, dividing it roughly into thirds. The zigzag lines
occiirred only in lighter areas of the shell. Althovigh this marking has
not been described for the notata subspecies, it is probably the color-
ation characteristic of clams homozygous for the color factor, since
it occurred only when two notata-marked clams were crossed. Apparently
the more familar zigzag lines, accepted as characteristic of the notata
subspecies, are actually a phenotypic blend exhibited by heterozygous
clams.

-166-

A count "based on these different markings gave 233 unmarked,
507 marked with zigzag lines, and 260 solidly colored with light bands.
This is roughly the expected 1 to 2 to 1 genotypic ratio resulting when
two heterozygous individuals are crossed. Chi square in this case is
equal to 1.65^, a value that woiild occur "by chance in over 30 per cent
of such crosses. Unfortunately, time and space limitations did not per-
mit raising these clams so that homozygous marked clams could be crossed.

If we assume that the notata marking is a simple Mendelian
character, either with complete dominance (marked vs. unmarked) or more
probably with incomplete dominance in which the heterozygous individuals
can be separated from the homozygous dominants by careful examination,
then the chi square test shows no evidence that observed ratios are
significantly different from the expected Mendelian ratios, except in
three Fi crosses of unmarked clams where a few marked individuals were
fovmd at the end of one year (Table l) .

Selective Breeding

The second phase of these studies was an attempt to demonstrate
the feasibility of deve2.oping a fast-growing race of clams through
selective breeding. The original plan involved rearing the fastest
and the slowest growing offspring from a single pair of clams. Hered-
itary influence on growth would then be determined by comparing the
rate of growth of offspring from each group. However, the slow-grow-
ing clams did not respond to spawning stimuli and parents from wild
stock had to be substituted.

The experiments were begun by crossing one female hard clam
with one male and rearing the surviving offspring. In several instances
individual clams grew well during the first growing season and then
poorly or not at all the second season. Undoubtedly, some variation
in the rate of growth was caiised by environmental factors, even though
clams were reared londer as nearly identical environmental conditions
as possible.

At the age of 28 months these clams ranged from ll4-.5 to 42.0
mm in length. Of these, the 120 largest clams were selected as brood
stock. When these clams were about kk months old a large male was
crossed with a large female. At the same time, one female and 2 male
clajns, chosen randomly from wild stock, were spawned and their larvae
reared as controls.

Soon after setting it became apparent that the selectively-
bred clams were growing more rapidly than the controls.

At the age of 10 weeks approximately 10,000 control clams had
a total volume of 8.6 cc, while 10,000 selectively-bred clams were
about 70 per cent larger with a total volume of ik cc. When the clams
were about I5 months old the total volume of 200 randomly-selected
control clams was 53 cc, while the total volume of 200 randomly-select-
ed selectively-bred clams was 86 cc or, roughly, 60 per cent greater
(Fig. 2).

-167-

SELECTIVELY -BRED

CONTROL 1

1

I^^HH

Fig, 2. Ftfteen-month-old V. mercenarla, showing the largest
clams from a group of selectively-bred clams compared with the largest
clams from a group of clams that were not selectively hred.

At 15 months the selectively-hred clams ranged in length from
5.5 "to 23.0 mm, while the control clams ranged from 6.6 to I9.O mm.
The average length of the selectively-bred clams was 13.2 mm, or 11.3
per cent greater than the average length of the controls, which was
11.8 mm.

Further selective breeding should resvilt in even more rapid
average growth of progeny, as the selection of brood stock becomes
more stringent. These studies suggest that a rapid-growing strain of
clams could be developed in only a few generations since in one gener-
ation of selective breeding, clams attained a 60 per cent larger size
in 15 months than control clams.

ACKNOWLEDGMENTS

The author expresses his gratitude to Mr. Spofford Woodruff
for his assistance with this work and to Dr. V. L. Loosanoff and Mr.
H. C. Davis for their valuable advice and assistsmce in all phases of
these experiments, from counting and measuring clams to the critical
review of this report.

-168-

LITERATURE CITED

Abtott, R. Tucker, 195^. Americaa aeashells. Van Nostrand, New York.
5^1 pp.

Bouchon-Brandely, G. I883. Report relative to the generation and
artificial fecxmdatlon of oysters^ addressed to the Minister
of the Marine and the Colonies. Bull. U. S. Fish Comm. 2;
319-338.

Chanley, P. E. 1955. Possible causes of growth variations in clam
larvae. Proc. Katl. Shellfish. Assoc. J+5 (l95^)j 8^4-9^^.

Chestnut, A. F., W. E. Fahy and H. J. Porter. 1957. Growth of yoimg
Venus mercenarla, Venus campechiensis, and their hybrids .
Proc. Katl. Shellfish. Assoc. 47 (195&)i 50-56.

Davis, H. C. 1950. On interspecific hybridization in Ostrea. Science
111:522.

. 1955. Mortality of Olympia oysters at low temper-

atures . Biol. Bull. IO91 l+0l+-if06.

Haven, D. and J. D. Andrews. 1957. Survival and growth of VenuB
mercenarla, Venus campechiensis^^ and their hybrids in sus-
pended trays and on natural bottoms. Proc. Natl. Shellfish.
Assoc. k-J (1956): ll3-^9.

Miner, Roy W. 1950. Field book of seashore life. Putnam, New York.
888 pp.

Morris, Percy A. I956. A field guide to the shells of our Atlantic
and Gulf coasts. 3d ed. Houghton Mifflin, Boston. 236 pp.

-169-

PROGRESS IN THE COOPERATIVE STATE-PUBLIC HEALTH SERVICE- INDUSTRY
PROGRAM FOR THE CERTIFICATION OF INTERSTATE SHELLFISH SHIPPERS

Eugene T. Jensen

Senior Sanitary Engineer
U. S. Public Health Service

ABSTRACT

The Cooperative State-PHS-Industry Program for the Cer-
tification of Interstate Shellfish Shippers has been effective
in preventing the interstate spread of disease due to shell-
fish, and Is, therefore, of importance to health agencies.
This program la also Important to the shellfish Industry since
it Insures public acceptance of their product. However, the
program also has a pronounced Impact on Industry operations
by regulating available sources of supply and by Influencing
costs of operations .

During 1958-1959 the program made considerable admini-
strative and technical progress . Important administrative ad-
vances Include: Adoption of new sanitation standards; a de-
cision to develop equipment construction guides; the adoption
of a new interim bacteriological standard for shucked oysters
at the market level; the formation of regional shellfish sani-
tation seminars In the New York-New England, Gulf and West
Coast areas; and the adoption of quarantine levels for paralytic
shellfish poison in shellfish growing areas . In the technical
field, progress was made In establishing bacteriological re-
lationships for eastern shelli"lsh; in development of a simple
colorimetrlc test for the freshness of shucked oysters; ajid,
in the perfection of better methods for assaying for paralytic
shellfish poison. The National Institutes of Health have also
supported a number of research projects which have application
in the shellfish sanitation field. Training has been accom-
plished through a one week shellfish sanitation course held at
a West Coast location.

The shellfish Industry requires large estuarine areas
which are essentially free of sewage pollution. Federal grants
to communities for the construction of sewage treatment plants
apparently have accelerated construction of such facilities.
In 1958 contract awards reached $389 million versus $222 million
in the period preceding the grants program. However, It is es-
timated that construction must be at a rate of $575 million
yearly to meet the Nation's requirements.

The Cooperative State-Public Health Service-Industry Program
for the Certification of the Interstate Shellfish Shippers Is an unu-
sual control program In which the regvilated industry has an Important
administrative role. This cooperative program has been most success-
ful in the prevention of disease transmission through fresh or frozen

-171-

shellfish. In almost 35 years of operations, there have "been no major
disease outbreaks attributed to commercially distributed oysters, clams
and mussels. This is in sharp contrast to the pre-certification period
when shellfish were frequently associated with disease. Since the
economic health of the shellfish industry is almost entirely dependent
upon the acceptability of its product by the public, it is obvious that
this program is of great importance to them. In addition, the certifi-
cation program has a significant impact on management through its In-
fluence on the availability of shells tock and on the unit cost of pro-'
cesslng and marketing. Regulation of this degree — and the shellfish
industry is probably one of the most strictly regulated Industries in
the food industry — could be extremely oppressive to management if the
sanitary requirements adopted by the official agencies were unrealistic
or were not well understood by industry. Industry therefore has both
economic and moral reasons to take an active part in the administration
of this program.

The informal organization of the certification program has posed
many difficult administrative problems. It is essential, for example,
that we avoid overlaps and conflicts between the Federal agencies which
have adjoining responsibilities. The Public Health Service Act directs
the Public Health Service to cooperate with the States in preventing
the interstate spread of commimicable disease. The Federal Food, Drug
and Cosmetic Act gives the Food and Drug Administration, also of the
Department of Health, Education, and Welfare, legal responsibility for
safety and purity of foods shipped in interstate commerce. Also, by
law, the Fish and Wildlife Service of the Department of Interior has
primary responsibilities in the fishery field. In both cases, bilateral
agreements have been developed which define each agency's responsibilities.
These intragovernmental agreements are fulfilled throiigh almost daily
contacts at the working level and through more formal liaison meetings.
By these means, we have been able to occasionally pool research resour-
ces, to avoid overlaps in inspectional operations, and to avoid con-
flicts of interest in which one Federal agency directs industry to fol-
low one set of instructions while, at the same time, another agency is
advising a quite different course. In the past we have also encountered
many problems in maintaining adequate communication channels with State
regulatory agencies and industry. Adequate communications are, of
coiorse, essential if these groups are to have an adequate voice in the
direction of this program, and if they are to be kept acquainted with
the technical and administrative problems that the Public HeaJLth Ser-
vice encounters in its position as trustee for this program.

Real progress has been made in the administration of the pro-
gram during the past year which will ultimately place it on a sounder
technical basis. This will improve its public-health effectiveness
without placing a substantially greater burden on the major part of
the shellfish industry. This paper is essentially a progress report
for the past year.

-1Y2-

Very satisfying progress was made at the 3rd biennial Shellfish
Sanitation Workshop held in Washington on August 26-27, 1958. This
workshop — and it was a workshop in the truest sense of the word — was
attended by about 93 persons representing some U3 State and Federal
agencies. The shellfish industry had 9 representatives at this meet-
ing. From the standpoint of the regulatory agencies, the most impor-
tant accomplishment of the workshop was the completion of Part 1 of
the Shellfish Sanitation Manual. This manual, which has been under
development for the past two years, is an essential step toward the
adoption of uniform appraisal methods which can be used by the Public
Health Service in the evaluation of the adequacy of States' shellfish
sanitation programs.

From the standpoint of industry, the most important change in
the program is probably the adoption of the so-called 80^ rule which
is stated in the manual as follows:

"Effective September 1, 1959, interstate shellfish shipper
certificates are Issued only to those establishments sub-
stantially meeting the construction requirements of Part
II of this maniial ajid which maintain a plant sanitation
rating of at least 90 per cent during periods of operations.
(The State shellfish control agency shall suspend or re-
voke certificates if a plant sanitation rating drops be-
low So per cent or if significant Individual sanitation
item is violated repeatedJ.y . ) Ratings will be determined
on the basis of compliance with the applicable provisions
of Part II of this manual as measiired by an inspection
report comparable to that contained in appendix B of this
manual , "

Under this rule those shippers who have substandard plants or
who fail to maintain reasonably sajiitary conditions in their operations
will not be included on the Public Health Service list of State certi-
fied shippers even though they may have valid operating permits from
the State in which they are located. This new plant inspection system
and the uniform scoring procedvire has been in use for two years by our
regional shellfish sanitation consultants in most parts of the country.
Their experiences indicate that most shippers should have little dlffi-
culty in meeting these requirements if they give a reasonable ajnount
of attention to plant operating procedures. The importance of good
operating procedures should be emphasized since previous scoring pro-
cedures attached less importance to this aspect of shellfish sanitation.
Plant inspections will, as in the past, be made by State officials with
occasional check inspection by Public Health Service officers.

The problems which the shellfish industry has experienced in
obtaining equipment which will meet the needs of the packer and yet
be acceptable to the inspecting agency were also discussed at the
workshop. This problem is not, of course, peculiar to the shellfish
industry, and, in fact, has been faced by most of the food processing

-173-

industries. It was agreed that equipment construction and fabrication
guides would help the shellfish industry secure equipment which would
meet their needs, which could he manufactured economically, and which
would also meet the sanitary requirements of the official agencies.
Since the shellfish industry is quite small, and since there are only
three or four equipment suppliers, it was decided that a formal organi-
zation such as the milk industries 3-A group was not necessary. The
Public Health Service agreed to develop preliminary construction guides
for review by the shellfish packers, the equipment fabricators, ajid
official agencies. Such guides would not have any legal standing ajid
would not constitute official agency approval of equipment.

Real progress has been made in the organization of regional
shellfish sanitation seminars which will meet annually to discuss
specific problems in the local administration of the cooperative pro-
gram. Shellfish sanitation seminars have been organized in the New
England-Worth Atlantic, Gulf and West Coast areas. These groups pro-
bably follow the same general pattern of operation as that of the
Chesapeake Bay Seafood Seminar. These regional meetings, coupled with
a biennial national shellfish sanitation meeting should insure that
both States and the shellfish ind\istry will have an ample voice in the
organization and administration of the cooperative certification pro-
gram.

The effort to develop workable bacteriological standards for
shucked oysters at the market level was continued. The project is
now nine years old. For many years a coliform MPN (Most Probable Wiim-
ber) or coliform score (also a statistical estimate of bacterial den-
sities) and Standard Plate Coimt have been used as rough guides to the
sanitary quality of oysters as marketed. More specifically, these
tests have been used as an Index of sanitary conditions in the packing
plant and the adequacy of refrigeration since it has long been recog-
nized that such laboratory tests cannot always distinguish between
shellfish from polluted and non-polluted sources. To gain additional
information on the validity of these tests for oysters as marketed,
several of the Eastern and Southern States, the government of Canada,
the New York and Chicago City Health Departments and the Public Health
Service in 1955 undertook a cooperative study of the changes in the
bacteriological quality of oysters diiring shucking and shipment to
market .

The 1958 Workshop spent almost a full day in an evaluation of
the results which had been obtained in this study and concluded that
the interim standards adopted at the I956 Shellfish Sanitation Work-
shop were of questionable value. Accordingly, the following interim
standards were adopted for use during the 1958-59 and I959-60 merket-
Ing season}

Satisfactory

A fecal coliform density (MPN) of not more than 78 per 100 ml

-171^-

of sample as Indicated by production of gas in E.G. liquid
broth media and a Plate Count of not more than 100,000 per
ml of sample, except that a fecal coliform density (MPN) up
to and including 230 per 100 ml of sample and/or a Plate Count
up to and including 500^000 per ml of sample will be accept-
able in occasional samples. (For convenience these will be
referred to as Class 1-A and 1-B samples, respectively) .

The official agency in the receiving area should notify the
shipper and shellfish control agency at the point of product-
ion of any Class 1-B results. If two consecutive Class 1-B
shipments are fo\md, the receiving area is justified in ex-
cluding the shipper from the market area until a satisfactory
report on the shipper is received from the State control agency
in the producing area.

Unsatisfactory

A fecal conform density (MPN) of more than 230 par 100 ml of
sample or a Plate Coirnt of more theua 500,000 per ml of sample.
(For convenience these will be referred to as Class II sample3).

The official agency in the receiving area should immediately
notify both the shipper and shelQjfish control agency in the
producing area of a Class II result. A single Class II result
is Justification for excluding the shipper from the market
pending receipt of a satisfactory eKplanation from the official
control agency in the producing area.

The same groups of agencies continued their studies of these
bacteriological relationships in the I958-59 marketing season. The
resiilts have not yet been subjected to statistical analysis or to
evalimtion by the bacteriologists. However, it appears that these
new Interim standards are realistic. The study will be continued for
at least another year.

The real significance of this change in standards lies in the
use of the Eijkman test. It la believed that these organisms are a
more concliisive indicator of fecal pollution and should be much better
adapted for use with shellfish sanitation work. Unfortunately, the
use of the Eijkman positive coliform means discarding years of exper-
ience with the coliform group — an action which many reg\ilatory agencies
ajre reluctant to take.

The Workshop agreed that if these interim standards were to
have real value, they should, if possible, be related to sanitary con-
ditions in the growing areas. Accordingly, several States have agreed
to vindertake comprehensive studies of these organisms in their shell-
fish growing areas. The results of these studies over the next two
years should do much to test the validity of the presently recommended

-175-

market standards. If proven successful, these new 'bacteriological
procedures should make it possible for the regulatory agencies to
make much more exacting sanitary siorveys of shellfish growing areas.

Paralytic sheULflsh poison was also discussed at the 195^
Workshop, and, for the first time, definite quarantine levels were
established for domestic production areas. This problem is not of
immediate concern to the oyster industry; however, because of the
trade inter-relationships, the oyster industry cannot ignore any such
problem which affects other segments of the industry.

The Workshop agenda included a limited discussion of the re-
lationships between shellfish production and radioactive waste disposal
practices. It has been amply demonstrated that shellfish, like other
marine animals and plants, can accumulate radioactive materials from
their environment. It is, therefore, quite Important that this char-
acteristic be considered in any proposals for disposal of radioactive
wastes in marine or estuarine areas. The use of nuclear reactors in
merchant and naval vessels and the increasing tempo of construction
of nuclear-powered electric generating stations may make this a pro-
blem which the control agencies and the shellfish industry will have
to face in the quite near future.

The Public Health Service Shellfish Sanitation Laboratory,
under the direction of Mr. C. B. Kelly, has been moved from Florida
to the State of Washington to undertake long-ran.ge studies on the
bacteriological relationships in Western shellfish. This is also a
cooperative undertaking in which the Washington State Department of
Health is providing the research facility and the Public Health Ser-
vice the staff and equipment. The organized shellfish industry of the
West Coast has taken an active part in getting this laboratory estab-
lished and in planning the research activities which will be under-
taken during the coming years.

An entirely new research development is found in the color test
for oyster freshness. This simple chemical test, being developed by
the Public Health Service Sanitary Engineering Center is based on a
color change in a chromate solution. It will apparently indicate the
age of shucked oysters and/or the temperatijre at which they have been
stored. This simple colorimetric test is still in an early stage of
development; however, the preliminary work with it has been quite pro-
mising. A traced-lot field trial will probably be undertaken.

During the past year a new brochure on the cooling rates of
oysters was also published. This study, made by the American Can Com-
pany, gives information on the amoiint of time required to cool shucked
oysters in various size cans.

The Public Health Service Sanitary Engineering Center has also
continued experimental work on chemical and laboratory procedui-es for
measuring the amo-unt of paralytic shellfish poison present in shellfish,

-176-

Their collaborative efforts have resulted in the refinement of the
bioassay procedure to a point at which it has heen accepted as an
Official Method by the Association of Official Agriculture Chemists.
A second AOAC collaborative study of a chemical method for measure-
ment of the poison has also been initiated.

The Public Health Service, through the National Institutes of
Health, has also supported shellfish research by a number of Univer-
sites, and State and private research agencies. For example, the
Maryland Department of Research and Education has undertaken a long-
range study of the bacteriological relationships between the soft-
shell clam and its aquatic environment, and of the effects of the hy-
draulic dredge on the bacteriological quality of the clam. At the
Haskins Institute in New York City a research grant has made possible
some very significant advances in the shellfish poisons field and
which may greatly facilitate control of this problem. In another NIH
supported study, which incidentally was carried on in Japaja, a research
group has apparently found that certain types of industrial wastes may
be concentrated by shellfish and cause illnesses in consumers.

A specific sanitation program, if it is to be successful, must
also include training activities. In this respect, we have been parti-
cularly successful in the shellfish program this past year. For the
first time the training branch at the Sanitary Engineering Center ar-
ranged to present the one-week Shellfish Sanitation Course on the West
Coast. Consequently, this course was very well attended by both re-
presentatives of the West Coast regulatory agencies and by plant man-
agement. In our opinion it was a great success. Detailed plans have
been made with the States of Virginia and Maryland for two three-day
courses in shellfish sajiltation. These courses will emphasize plant
sanitation practices.

The activities of the Public Health Service in the administra-
tion of the certification program are important to the economic health
of the shellfish industry. However, the Water Pollution Control acti-
vities of the Service are also very important.

As presently operated in the United States, the shellfish in-
dustry requires large estuarine areas which are aJLmost entirely free
of sewage pollution. In years past it has not been difficult to find
such areas because of our relatively low population density; however,
this has changed rapidly in recent years I Our population has been
expanding at a very rapid rate and gives every evidence of continuing
do do so in the foreseeable future. The impact on the sanitary quality
of our surface waters has been unmistakable.

The control of pollution depends on the construction and ade-
quate operation of treatment facilities by both communities and industry.
Many sewage treatment plants have been constructed in the United States
over the years j however, the population has continued to increase at a
rate faster than new treatment plants could be constructed. As a conse-

-177-

quence of this deficit in sewage treatment plant construction and the
wearing out of existing facilities, the problem of water polution has
become very acute in the United States. The Congress has recognized
this problem and for the past several years has made Federal grajats
available to communities to assist in the construction of sewage treat-
ment plants. During the five-year period from 1952 through 1956 immed-
iately preceding the Federal grants program, contract awards for sewage
treatment works construction averaged $222 million annually. In the
first full year of the program, 1957^ construction expanded 58 per cent
over the previous annual average to reach $351 million. The second year
of the program brought an even greater increase in construction, with
contract awards reaching $3^9 million — 75 per cent over the earlier
five-year average. The lion's share of the Increase in construction
during 1957 and 195^ came from projects receiving Federal aid. This
amounted to $ll8 million in 1957 and $ll<-3 million in I958. These facts
point strongly to the conclusion that had it not been for Federal
grants, sewage treatment works construction would have remained at a-
bout the average level experienced during the five-year period preced-
ing the grants program. They also indicated that a doubling of grant
fiinda would further Increase construction to about the required level
of $575 million, the rate which has been necessary to eliminate our
high backlog of construction needs and to compensate for pleint obso-
lescence and population growth.

It should be quite apparent that without this sxirge in construc-
tion many additional coastal areas would have been lost to the shell-
fish industry. It should be equally apparent that this construction
pace must be at least maintained If the industry is to avoid a further
loss of growing areas. On the positive side of this, it should be
pointed out that we now have a few instances in which growing areas
have again become available for shellfish culture as a result of the
construction of sewage treatment facilities.

From this discussion of our activities during the past year,
it should be apparent that the Cooperative Program for the Certifica-
tion of Interstate Shellfish Shippers is on reasonably solid ground,
both from the administrative and research standpoints although there
are some pressing administrative problems which have not been discussed
in this paper. It is hoped that the program can continue to provide
adequate public-health protection to shellfish consumers at minimum
cost both to the taxpayer and to industry. However, this progress
will require the wholehearted cooperation and support of industry,
both as individuals and as an association, and of the interested State
regulatory agencies.

REFERENCES

Journal of the Association of Official Agricultural Chemists,
February 1959.

-178-

Proceedings^ I958 Shellfish Sanitation Workshop, U. S. Public
Health Service.

Sanitary control of the shellfish Industry, Public Health Ser-
vice, Pub. No. 33, Gov't Printing Office, 1959.

-179-

A BACTERIOLOGICAL STUDY OF THE NATURAL FLORA OF PACIFIC OYSTERS
(CRASSOSTREA GIGAS) WHEN TRAJISPLANTED TO VARIOUS AREAS
IN WASHINGTON 1> 2

R. R. Colvell and J. Listen., College of Fisheries,
University of Washington, Seattle, Washington

ABSTRACT

Total viable bacterial populations on oysters held ex-
perimentally on floating trays in areas of different water con-
ditions fluctuated between 10^ - lo5 organisms per ml oyster
liquid. ColifomiB were never higher than 0.5^ of the total
viable bacterial flora. Study of 152 randomly Isolated cul-
tures (taken from gill, flesh, Intestine and oyster liquid)
indicated that Gram negative, asporogenous , rod-like bacteria
of the Psevidomonas, Vibrio, Flavobacterium and Achromobacter
groups predominated. The bacterial flora of fin fishes is
overwhelmingly oxidative in nature, but 50^ of the oyster bac-
teria tested were able to ferment glucose anaerobic ally.

INTRODUCTION

Most of the bacteriological work on oysters, from Cameron
(l88o) to the present day, has been concerned with the detection of
human enteric bacteria. The studies of Fabre-Domergue (l912), Dodgson
(1928) and Kelly and Arcisz (l95^) revealed that molliiscs contaminated
with enteric bacteria will cleanse themselves in a few days when placed
in clean, uncontaminated sea water. As a result of application of pro-
cedures based on these and similar findings, and of the very close con-
trol exercised on shellfish growing areas, the oyster is now the safe
food product that it is.

Little information is available concerning the other types of
bacteria which are present in oysters, the so-called saprophytic bac-
teria, harmless in the main to man, but known to be potent agents of
spoilage in foodstuffs. Earlier workers have noted such groups in
oysters as Splrochaeta (Dimitroff I926), Proteus, Alcaligenes and
Pseudomonas fliiorescens (Geiger, Ward and Jacobson I926) without actu-
ally analyzing the flora to determine the relative importance of each.
Two reports concerning the bacterial types involved in the spoilage
of oysters have been made. Eliot (I926) observed that during spoilage

■^ This work was supported in part by Grant No. 'E,-2kl'J, National Insti-
tutes of Health, and in part by the Initiative I7I Fund, University
of Washington.

2 Contribution No. 67, College of Fisheries, University of Washington,
Seattle 5> Washington.

-181-

of shucked oysters at 20°C (68°F) "water" forms Including green fluo-
rescent, pigmented and non-pigmented bacteria^ and "vibrio" types in-
creased steadily in number while coliform types increased only during
the first two days of storage. Tanikawa (1937) characterized bacteria
from the groups which he considered of greatest importance in the
spoilage of shucked oysters at 0°C (32°F), namely, Achromobacter,
Pseudomonas, Flavobacterium and Micrococcus.

Similar types of bacteria are known to be Lmportajit spoilage
agents in the deterioration of fin fishes, post-mortem, and have been
shown to be derived directly from the normal bacterial flora of the
living fish (Shewan and Llston I956) .

The purpose of our study was to determine the natioral flora
within the oyster as a first step towards providing information which
will enable shellfish technologists to take rational measures to deal
with potential spoilage agents at an early stage in storage. The in-
formation was also sought to fill a considerable gap in oin: knowledge
of the bacteriology of marine animals.

MATERIALS AOT) METHODS

Yearling Pacific oysters were maintained in floats in three
areas, Willapa Bay, Oyster Bay and Hood Canal, and a control was kept
in the seawater aquarixm at the College of Fisheries (Sparks and Chew
i960). Most Probable Number (MPN) of coliforms and total viable
counts were carried out on fluid extracted aseptically* from three
oysters and also from seawater, taken from the aquari\im weekly and
from the floats at three-week intervals, according to procedures out-
lined in Standard Methods for the Examination of Water, Sewage and
Industrial Wastes (19^5) . The mediiam chosen for the plate counts con-
tained 0.8fo nutrient broth, 0.5/0 yeast extract and 1.5^ Bacto-agar in
seawater (MacLeod, Onofrey and Morris 195^4-). From experimental data
obtained in our laboratory, this medium appears to yield a maximum
plate co\mt, in that it affords good growth of non-marine and also
marine types which are otherwise not picked up.

Cultures of microorganisms were obtained by random selection
from the count plates Inoculated from gill> intestine, body flesh and
liquid, purified by routine methods, and maintained as pure cultures
in seawater peptone broth (l^ peptone in seawater) or in the basal
agar medium described, depending on how fastidious the organism was.

The microbiological procedures applied in the tests for classi-
fication and identification are those described in the Manual of Micro-
biological Methods (l957).

\rlal counts on gill, intestine, whole oyster (flesh and liquid),
and oyster liquid indicated that oyster liquid provided the most con-
sistent, high total viable coiints.

-182-

Difco dehydrated media, including the Enterococcl Presumptive
Broth, Ethyl Azide Violet Broth (foi* enteroccocci). Brilliant Green
Bile Broth and Eosin-Methylene Blue Agar (for coliforms and E. coli),
S S Agar and Triple Sugar Iron Agar (for Salmonella, Shigella) were
used to assist in the identification of possible memhers of the En-
terobacterlaceae. Pure cultures of all the organisms isolated In this
study were streaked on basal agar plates for colonial morphology and
for tests of sensitivity to 0/129 vibriostat compoxmd and to 2, ^,
10 \init Difco penicillin discs (Shewan, Hodgkiss and Liston 195^)'
Tests and media used were as follows: litmus milk, seawater nutrient
gelatin, lead acetate agar slopes, methyl red, Voges-Proskauer, nitrate
broth, indole, urea agar slopes, Koser's citrate broth, Hugh and Leif-
son oxidative and fermentative medium (Hugh and Leifson 1953), lactose,
dextrose, maltose, majinitol, and sucrose fermentation tubes, axid am-
monia production. Temperature growth tests at 0°C, 25°C and 37°C were
carried out in Vfo peptone water containing 0.5/" WaCl. Routine tests
and identification media were inoculated and incubated at 25°C (RT),
but selective media for enterobacteria were incubated at 37°C. One
hundred fifty-two cultures were thus studied.

RESULTS AJtD DISCUSSION

The MPN of coliforms present in oysters and seawater during
the course of the study (February- July 1959) is given in Table 1.

Table 1. Coliform content of oysters and seawater examined at three-
week intervals (expressed as MPK coliforms per 100 ml sample)

Tim

s (Weeks)

Source

0

3

6

9

12

Aquarium Oysters

0

0

0

0

0

Aquarium Seawater (Control)

0

0

0

0

0

Hood Canal Oysters

-

450

k^O

200

200

Hood Canal Seawater

-

0

0

2

2

Oyster Bay Oysters

-

0

200

0

0

Oyster Bay Seawater

-

0

0

0

0

Willapa Bay Oysters

-

450

1100

20

20

Willapa Bay Seawater

-

0

200

2

2

-183-

TOTAL VIABLE BACTERIAL COUNT OF OYSTERS a SEAWATER

I

-aye

Aquarium Oysters( Control)

10

1-

1 1 1 1 1 1 1 1

8-

6-1

Z^'C

KfV.

4-
3-

'---- —

--j-_-T.~ • 25°C

2-

Hood Canal Oysters
Hood Canal Seawater

(

D 1

23456789
TIME (Weeks)

Fig. 1. Total viable count of bacteria per ml oyster fluid and
per ml seawater for aquarium-held oysters (control) and Hood Canal.

TOTAL VIABLE BACTERIAL COUNT OF OYSTERS 8 SEAWATER

lOq
8-
6-

4
3

C 10

2-

37°C

-Willapo Boy Oysters
•Willapo Boy Seawater

-+-

-+-

-+-

-+-

-Oyster Bay Oysters
-Oyster Bay Seowater
25''C

sr-c

4 5 6
TIME (Weeks)

Fig. 2. Total viable count of bacteria per ml oyster fluid and
per ml seawater for Wlllapa Bay and Oyster Bay samples.

-l81t-

There was a low degree of pollution, indicated "by these tests, In the
three areas except for a brief rise in the Wlllapa Bay area during the
sixth week, and there was no apparent pollution in the aquarium. The
ability of oysters to concentrate sewage bacteria from the surrounding
water is demonstrated by the disparity in the counts for oysters and
seawater at any given time. However, comparison of these results (re-
calculated on a weight basis) with the total 25°C count results given
in Figures 1 and 2 reveals that at no time did the coliforms constitute
more than 0.5^ of the total flora. Numerically, therefore, the con-
form bacteria represent an Insignificant part of the oyster flora.

The total viable count in oysters at 25°C, which may reasonably
be expected to include both mesophilic and psychrophilic bacteria, re-
mained constant in ail cases at about 10^ organisms/ml of body fluid,
while the count in the surrounding seawater was consistently lower and
subject to rather large variation.

The generic distribution of the 152 strains obtained by random
selection from the oyster plate counts is shown in Table 2. There is
a remarkable similarity in the oyster flora in the four test areas. Gram
negative rod-like bacteria, showing the characteristic marine properties

Table 2. Generic distribution of organisms isolated from oysters in
controlled and natural environments.

Salt-

water

Wll-

Oys-

i>

Aquar-

Hood

lapa

ter

Distri-

lixm

Canal

Bay

Bay

Total

bution

Number of Isolates

h3

50

30

29

152

100

Pseudomonas-Vibrio

20

27

18

Ik

79

52.0

Achromobacter

2

5

1

0

8

5.3

Flavobacteri\im

6

7

5

8

26

17.1

Coryneform

1

3

0

1

5

3.3

Alcaligenes

2

1

0

0

3

2.0

Micrococcus

6

5

2

3

16

10.5

Bacillus

k

1

2

0

7

k.6

Enterococci

1

0

1

0

2

1.3

Miscellaneous

1

1

1

3

6

3.9

-185-

of salt dependence and psychrophilic growth, predominated in each
area. Outstanding among them was the Pseudomonas/Vihrio group which
constituted ahout 50^0 of the flora in each case, while Flavobacterium
present to ca. I7/0 were also prominent. Micrococcus alone among the
Gram positive types was isolated in significant numbers, but was pre-
sent only to ca. lO'fo. A corresponding identity of biochemical char-
acteristics was observed among organisms isolated from the various
areas . They were predominantly proteolytic and only weakly saccharoly-
tic in nature. It thus appears that the natural flora of oysters is
similar to the flora of free-swimming fish, the only other marine ani-
mal extensively studied bacteriologically.

The only major point of difference between the two floras is
a biochemical one, since ca. 50^ of the organisms isolated by us from
oysters were observed to ferment glucose anaerohically by the Hugh
and Leifson (1953) test. This is a property which has not been re-
ported for many fish bacteria. The difference may be related to the
composition of oysters and of fin fishes. From the practical point of
view the property may "be very significant in relation to spoilage.
Eliot (1926) showed, and this has been confirmed repeatedly since, that
there is a rapid fall in pH during the early stages of spoilage of oys-
ters. This may be due, at least in part, to the fermentative activity
of these "bacteria.

The types of bacteria identified by Eliot and Tanikawa as being
of primary importance in spoilage are so similar to the microorganisms
found in this study to constitute the natural flora of oysters, that it
seems very likely that we are in fact dealing with the same groups of
organisms .

It appears therefore that as in the case of fin fishes, the
spoilage flora of oysters is derived from the natioral population of the
living animal and the generally proteolytic character of the natural
flora lends verisimilitude to the hypothesis.

SUMMARY

The natural flora of oysters was characterized by study of pure
cultiires of bacteria isolated from plate counts of oysters held in
floating trays in three areas of Washington: Southern Puget Sound,
Hood Canal, and Willapa Bay, and the saltwater aquarium at the College
of Fisheries, Seattle. Degrees of pollution were very low in each of
the three natural environment areas, and there was none present in the
saltwater aquarium, as detected by MPN of coliforms ajid standard plate
•counts at 250C and 370C.

From a total sample of 152 pure cultures extensively studied,
it was concluded that the flora of the oyster consists primarily of
the Pseudomonas /"Vibrio, Flavobacterium and Achromobacter groups, i.e.

-186-

the Gram negative, asporogenous bacilli. Gram positive organisms,
except for Micrococcus, were found to be a very minor fraction of the
total population. These results are discussed in relation to the
known data and theories concerning post-mortem spoilage of free-swim-
ming fishes and of oyster meats .

LITERATURE CITED

Arcisz, W. and C. B. Kelly. 1955. Self -purification of the soft clam,
Mya arenarla. U. S. Pub. Health Kept. 70 (6):605.

Cameron, C. I880. On sewage in oysters. Brit. Med. J. 2:^+71.

Dimitroff, V. T. I926. Spirochaetes in Baltimore market oysters. J.
Eacteriol. 12 (2)il35-177.

Dodgson, R. W. I928. Report on mussel purification. Gt. Brit. Min.
Agric. and Fish., Fish. Invest., Ser. 2, 10 (l):l-i^98.

Eliot, C. 1926. Bacterial flora of the market oyster. Amer. J. Hyg.
6 (6):755-TT6.

Fabre-Domergue, M. I912. Bacterial ptrrlfication of oysters by stand-
ing in filtered artificial seawater. (See Chem. abstr. 6:10ii5,)

Geiger, J. C, W. E. Ward, and M. A. Jacobs on. I926. The bacterial
flora of market oysters. J. Infect. Dis., 38 (3) J 273-280.

Hugh, R. and E. Leifson. 1953. The taxonomic significance of ferment-
ative versus oxidative metabolism of carbohydrates by various
Gram negative bacteria. J. Bacteriol. 66:2^-26.

Kelly, C. B. and W. Arcisz. 195^. Siirvival of enteric organisms in
shellfish. U. S. Pub. Health Rept. 69 (l2) : 1205-1210.

MacLeod, R. A., E. Onofrey, and M. E. Norris. 195^. Nutrition and

metabolism of marine bacteria. I, Survey of nutritional re-
quirements. J. Bacteriol. 68 (6):680-686.

Manual of microbiological methods. Society of American Bacteriologists.
1957.

Shewan, J. M. and J. Listen. 1956. Objective and subjective assess-
ments of fish quality. Bull. Inst. Refrig. Suppl. Annexe.
1956-1:137-

, W. Hodgkiss, and J. Llaton. 195^. A method for the

rapid differentiation of certain non-pathogenic asporogenous
bacilli. Nature (Lond.) 173:208.

-I8T-

Sparks^ A. K. and K. K. Chew. I960. A preliminary report on growth

and mortality of a population of Japanese oysters (Crassostrea
glgas) when transplmited to various areas in Washington. Proc.
Natl. Shellfish. Assoc. 5O:

Standard methods for the examination of water, sewage and lnd\istrial
wastes. A. P. H. A. 1955.

Tanlkawa, E. 1937- Bacteriological examination of oysters stored at ^
low temperatures. Zentr. Bakt. Par., Ser. 2, 97:133-lil-7.

-188-

ASSOCIATION AFFAIRS

ANMJAL CONVENTION

The 1959 Convention of the National Shellflsheries Association
and the Oyster Growers and Dealers Association of North America, Inc.,
was held July 27 to 30, 1959, at the Statler-Hilton Hotel, Washington, D.C.
An interesting feature was a joint seminar at which reports on "Oyster
cult lire in Europe" and "Oyster culture in Japan" were presented "by V. L.
Loosanoff and J. B. Glude. These reports contained observations from
their recent foreign trips. The membership voted overshelmlngly to
increase Association dues from $2.00 to $4.00 to cover the cost of fi-
nancing the Proceedings. Tlie Oyster Institute of North America has
continued to support publication of the Proceedings. Vice President
Cronln distributed a list of titles of all papers presented at annual
conventions since 1930. Most of these papers are out-of-print eind no
longer available.

OFFICERS OF THE NATIONAL SHELLFISHERIES ASSOCIATION - 1959

President - Melboiirne R. Carriker, Department of Zoology, University
of North Carolina, Chapel Hill, North Carolina.

Vice-President - L. Eugene Cronln, Maryland Department of Research and
Education, Solomons, Maryland.

Secretary-Treasurer - Philip A. Butler, Biological Laboratory, Bureau
of Commercial Fisheries, Gulf Breeze, Florida.

Editorial Committee - J. D. Andrews, editor; S. H. Hopkins, associate
editor j Lawrence Pomeroy, associate editor.

Executive Committee - M. R. Carriker, Chalrmanj L. E. Cronln, P. A.
Butler, G. R. Lunz, H. C. Davis, G. F. Beaven.

COMMITTEES FOR 1959 CONVENTION

Program Committee - L. Eugene Cronln, Chairman; J. F. Allen, J. D.
Andrews, D. H. Wallace

Nominating Committee - A. F. Chestnut, Chalrmanj G. Gunter, V. L.
Loosanoff, J. L. McHxogh

-189-

INFORMAa?ION FOR COWTRIBOTQRS

Original papers given at the Annual Association Convention and
other papers on shellfish biology or related subjects submitted by mem-
bers of the Association will be considered for publication. Manuscripts
will be judged by the Editorial Committee or by other competent review-
ers 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 submission to the Editoral
Committee. Papers published or to be published in other journals are
not acceptable.

Manuscripts shoiild be typewritten and double -spaced: original
sheets are required but extra copies will facilitate reviews. Tables,
numbered In arabic_, should be on separate pages with the title at the
top. Scientific names should be underlined. Do not imderline section
headings. Illustrations should be reduced to a size which fits on 8 x 10|-
Inch pages with ample margins. Glossy photographs are preferred to ori-
ginals. Illustrations smaller than a page should be carefully oriented
and loosely attached to plain white paper with rubber cement. Legends
should be typed on separate sheets and numbered in arable .

Use the following style for literature citations: "Smith,
Rebecca Joyce. 1958* Filtering efficiency of hard clams in mixed
suBpensiona of radioactive phytoplankton. Proc. Natl. Shellfish. Assoc«
kQx 115-12^)-." Note In Volume kQ punctuation for literature citations
in text. In abbreviations for names of serial publications, follow
Biological Abstracts (see 29(5) J v-xxxv, 1955)* Abbreviations for
units of weight and measure in the Handbook of Chemistry and Physics,
36th Edition, pages 3108-3134 will be followed. Punctuation will be
strictly limited for abbreviations of common measurements, literature
references in the text, and certain other usages. Note usage in re-
cent PROCEEDINGS. Clarity of meaning and brevity of style are the
keynotes of our policy.

Each paper should be accompanied by an abstract which Is con-
cise yet underetaxidable without reference to the original article. It
ia our policy to publish the abstract at the head of the paper and to
dispense with a summary. A copy of the abstract for submission to
Biological Abstracts will be requested when proofs are sent to authors.

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 re-
prints 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
manxiscripts should reach the Editor prior to October 1. Send manu-
scripts and address all correspondence to the Editor, Dr. Sewell K.
Hopkins, Biology Department, Texas A&M College, College Station, Texas.

-190-

DIRECTORY OF MEMBERS OF THE NATIONAL SHELLPISHERIES ASSOCIATION

(To November I960)

Active Members (Continued)

Honorary Membera

Coe^ Dr. Westley (Deceased)
138 E. Oleander Drive
Chula Vista, California

Coker, Dr. Robert E.
University of North Caxolina
Chapel Hill, North Carolina

Gaits off. Dr. Paul S.
Biological Laboratory
Bureau of Commercial Fisheries
Woods Hole, Massachusetts

Kincaid, Professor Trevor
I90U East 52nd Street
Seattle 5^ Washington

Nelson, Dr. Thurlow C. (Deceased)
8 North Main Street
Cape May Court House
New Jersey

Trultt, Dr. Reginald V.
Great Neck. Farm
Stevensville, Maryland

Life Member

Gunter, Dr. Gordon

Gulf Coast Research Laboratory

Ocean Springs, Mississippi

Active Ifembers

Aldrlch, Dr. F. A.
Academy of Natural Sciences
19th & the Parkway
Philadelphia 3> Pennsylvania

Allen, Dr. J. Prances
5702 Queens Chapel Road
Apartment 3
West Hyattsville, Maryland

Andrews, Dr. Jay D.

Virginia Fisheries Laboratory

Gloucester Point, Virginia

Arcisz, Mr. William
Star Route, Box 576
Gig Harbor, Washington

Arve, Mr. John E.
Snow Hill, Maryland

Atlantic Biological Station
Fisheries Research Board of Canada
St. Andrews, N. B., Canada

Ball, Mr. Eric T.
212 Summit Street
New Haven 13 > Connecticut

Baptist, Mr. John P.
Biological Laboratory
Bureau of Commercial Fisheries
Beaufort, North Carolina

Beaven, Mr. G. Francis
Maryland Department of

Research and Education
Solomons, Maryland

Blake, Mr. John W.
Department of Zoology
University of North Carolina
Chapel Hill, North Carolina

Blovint Seafood Corporation
383-393 Water Street
Warren, Rhode Island

Brittain, Mr, G, J., Jr.
56ii3 Flamingo
Houston 21, Texas

Buchanan, Mr. A. F.
Seabrook, South Carolina

-191-

Bvirton, Mr. Richard W.
Biological Laboratory-
Bureau of Commercial Fisheries
Oxford, Jlaryland

Butler, Dr. Philip A.
Biological Laboratory
Bureau of Commercial Fisheries
Gulf Breeze, Florida

Campbell, Mr. Robert
Narragansett Marine

Laboratory-
Kingston, Rhode Island

Carlson, Mr-. Frank T.

Bioreau of Commercial Fisheries

Mill-vllle, Delaware

Carrlker, Dr. Melbourne R.
Depar-tment of Zoology-
University of North Carolina
Chapel Hill, North Carolina

Carver, Mr. Thomas C, Jr.
Bureau of Commerical Fisheries
Franklin City, Virginia

Castagna, Mr. Michael

Bureau of Commercial Fisheries

Franklin City, Virginia

Chanley, Mr. Paul E.
Biological Laboratory
Bureau of Commercial Fisheries
Milford, Connecticut

Chapman, Mr, Charles R.
River Basin Studies
U. S. Fish anid Wildlife Service
Vicksburg, Mississippi

Chestnut, Dr. A. F.
Institute of Fisheries Research
University- of North Carolina
Morehead City, North Carolina

Chew, Mr. Kenneth K.
College of Fisheries
University of Washington
Seattle 5? Washington

Chlpman, Dr. Walter A.
Washington Biological Laboratory
Bureau of Commercial Fisheries
73I+ Jackson Place, N. W.
Washington 25, D. C.

Collier, Mr. Albert
Galveston Marine Laboratory
A & M College of Texas
Bldg. 311, Fort Crockett
Galveston, Texas

Colwell, Mr. J. H.
1115 East Iflst Street
Seattle 5* Washington

Cooley, Dr. Nelson R.

U. S. Fish and Wildlife Service

G\ilf Breeze, Florida

Cronln, Dr. Eugene
Maryland Department of

Research and Education •
Solomons, Maryland

Currier, Mr. Wendell N.
Research and Development
Campbell Soup Company
Cajnden, New Jersey

Darling, Mr. J. S. 8s Son
P. 0, Box kl2.
Hampton, Virginia

Dassow, Mr. John A.
Technological Laboratory
U. S. Fish and Wildlife Service
Seattle 2, Washington

Davidson, Mr. Ardell E.
16-D Westway South
Baltimore 21, Maryland

-192-

Davis, Mr. Harry C.
Biological Laboratory
Bureau of Commercial Fisheries
Milford, Connecticut

Dawson, Mr. C. E.

iflO Pine Drive

Ocean Springs, Mississippi

Edwards, Mr. Malcolm B.
Coast Oyster Company
South Bend, Washington

Eisler, Mr, Ronald
College of Fisheries
University of Washington
Seattle, Washington

Deiler, Mr. Frederick G.
Freeport Sulphur Company
Port Sulphur, Louisiana

Denison, Mr. John
Rayonier Marine Laboratory
Hoodsport, Washington

Dow, Mr, Robert L.
Department of Sea and

Shore Fisheries
State House
Augusta, Maine

Downes, Dr. Kenneth
Department of Tidewater

Fisheries
State Office Building
Annapolis, MarylEind

Ellis, Mr. Ian E.
College of Fisheries
University of Washington
Seattle 5> Washington

Engle, Mr, James B.
Biological Laboratory
Biireau of Commercial Fisheries
Oxford, Maryland

Esveldt, Mr. George D.
E. H. Bendiksen CompeLny
820 Broadway
South Bend, Washington

Fahy, Dr. William
University of North Carolina
Institute of Fisheries Research
Morehead City, North Carolina

Drinnan, Mr. R. E.
Federal Department of

Fisheries
Prince Edward Island Biological

Station
Ellerslie, P. E. I., Canada

Dumont, Mr. William H.
3896 Porter Street, N, W.
Washington I6, D. C.

Dunker, Dr. Carl F.
Seafood Processing Laboratory
University of Maryland
Crisfield, Maryland

Dunnington, Mr. Elgin A.
Department of Research

and Education
Solomons, Maryland

Falk, Dr. Lloyd L.
Engineering Department
E. I. du Pont de Nemours

and Company, Inc .
Wilmington 98/ Delaware

Fieger, Dr. E. A.
Louisiana State University
Baton Rouge 3, Louisiana

Fishing Gazette

Carroll E. Pellissier, Editor

k6l Eighth Avenue

New York 1, New York

Flower, Frank M. & Sons
Bayville, Long Island, New York

-193-

Found, Mr. H. R.
Department of Fisheries
Prince Edward Island

Biological Station
Ellerslie, P, E, I,, Cajiada

Priedrlchs, Mr. A. V., Jr«
806 W. Michigan Avenue
Hammond, Louisiana

Prisbie, Mr. Charles M.

U. S. Fish & Wildlife Service

Box 65

Franklin City, Virginia

Gaucher, Mr. Thomas A.
Narragansett Marine Laboratory
Kingston, Rhode Island

Girard, Mr. John G.

State Department of Health

Smith Tower

Seattle k, Washington

Glancy, Jr. Joseph B.

Shellfish Inc.

Box 212

West Sayville, L. I., New York

Glude, Mr. John B.
Bureau of Commercial FlsherleB
Department of the Interior
Washington 25, D. C.

Hammer, Mr. Ralph

9 North Cherry Avenue

Annapolis, Maryland

Hanks, Dr. James E.
Biological Laboratory
Bixreau of Commercial Fisheries
Oxford, Maryland

Hanks, Mr. Robert W.
Biological Laboratory
Bureau of Commercial Fisheries
Boothbay Harbor, Maine

Hargls, Dr. William J., Jr.
Virginia Fisheries Laboratory
Gloucester Point, Virginia

Harrlman, Mr. Donald M.
Newagen, Maine

Harrison, Mr. George T.

The Tllghmaji Packing Company

Tilghman, Maryland

Haskln, Dr. Harold H.
Department of Zoology
Rutgers University
New Brunswick, New Jersey

Haven, Mr. Dexter

Virginia Fisheries Laboratory

Gloucester Point, Virginia

Grice, Dr. George D.
Woods Hole Oceanographlc

Institution
Woods Hole, Massachiosetts

Hewatt, Dr. Willis G.
Biology-Geology Department
Texas Christian University
Fort Worth, Texas

Griffith, Mr. George W.

Box 65

Franklin City, Virginia

Gusta.f3on, Dr. A. H.
Department of Biology
Bowdoln College
Brunsvflck, Maine

Heydecker, Mr, Wayne D,
Atlajitlc States Marine

Fisheries Commission
22 West First Street
Mount Vernon, New York

Hofstetter, Mr.
Rt. 1, Box 132
La Porte, Texas

Robert P.

_l9li^

Hoplclns, Dr. Sewell H.
Biology Department
Texas A & M College
College Station, Texas

Hoss, Mr. Donald

Bureau of Commercial Fisheries

Beaufort, North Carolina

Huber, Mr. L Albertson
297 E. Commerce Street
Bridgeton, New Jersey

Hulings, Mr. Neil C.
Biology Department
Texas Christian University
Fort Worth, Texas

Ingle, Mr. Robert M,
Florida State Board of

Conservation
W. V. Knott Building
Tallahassee, Florida

Isaac, Geary

Rayonier Marine Laboratory

Hoodsport, Washington

Jensen, Mr. Eiagene T.
U. S. Public Health Service
Shellfish Branch
Washington 25, D. C.

Kahan, Dr. Archie M,

TexELS A & M Research Foundation

College Station, Texas

Kelley, Mr. R. L
R. L. Kelley Farm
Atlantic, Virginia

Kelly, Mr. C. B.

Shellfish Sanitation Laboratory

U. S. Public Health Service

Star Route, Box 576

Gig Harbor, Washington

Kogenezawa, Mr. A.
Tohoku University
15 Yakushido
Minami Koizumi
Sendai City, Japem

Landers, Mr. Warren S.
Biological Laboratory
Bureau of Commercial Fisheries
Milford, Connecticut

Lednum, Mr. J. M.

Town Engineer

Town of Islip, New York

Lindsay, Mr« Cedric E.
Washington State Shellfish

Laboratory
Quilcene, Washington

Littleford, Dr. Robert A.
Ward's Natural Science
Establishment, Inc.
Rochester 3^ New York

Loesch, Mr, Harold

A & M College of Texas

College Station, Texas

Logie, Dr. R. R.
Fisheries Research Board

of Canada
Ellerslie, Prince Edward

Island, Canada

Loosanoff, Dr. Victor L.
Biological Laboratory
Biireau of Coinmercial Fisheries
Milford, Connecticut

Lunz, Dr. G. Robert
Bears Bliiff Laboratories
Wadmalaw Island, South Carolina

MacKenzie, Mr. Clyde L., Jr.
Bureau of Commercial Fisheries
Biological Laboratory
Milford, Connecticut

-195-

Mackln^ Dr. J. G.
Texas A & M Reseeircli

Foundation.
il-10 West Ifth Street
Thibodanx:, Louisiana

Macomber^ Mr. Ronald G.
11 Prescott Avenue
Montclair, New Jersey

Menzel, Dr. R. Winston
Oceanographic Institute
The Florida State University
Tallahassee, Florida

Merrill, Mr. Arthur S.
Biological Laboratory
Eiireau of Commercial Fisheries
Woods Hole, Massachusetts

Manning, Mr. Joseph H.
Chesapeake Biological

Laboratory
Solomons, Maryland

Mansuetl, Mr. Romeo
Marylemd Department of

Research and Education
Solomons, Maryland

Marshall, Dr. Nelson
University of Rhode Island
Kingston, Rhode Island

McConnell, Mr. James L.
P. 0. Box 103^
Bay Towing & Dredging Co.
Mobile, Alabama

McConnell, Mr. James N.
Department of Wildlife and

Fisheries
1+00 Royal Street
New Orleans l6, Louisiana

McHugh, Dr. J. L.

Bureiiu of Commercial Fisheries

Washington 25, D. C.

McNlcol, Mr. Douglas

Fire Island Sea Clam Co., Inc.

West Sayville, New York

Medcof, Dr. J, C.
Fisheries Research Boajrd

of Cajaada
Biological Station
St. Andrews, N. B., Canada

Messer, Mr. Richard
1205 Dinwiddle Avenue
University Heights
Richmond 29, Virginia

Miles, J. H. & Co., Inc.
Norfolk 1, Virginia

Miller, Mr. George C.

Box 273

Ilwaco, Washington

Mortimer, Miss Joan E.
Biology Department
Memorial University of

Newfoundland
St. Jolin's, Newfoundland

National Fisherman

Goff stown. New Hampshire

Nelson, Mr. J. Richards
The F. Mansfield & Sons Co.
6l0 Quinnlplac Avenue
New Haven, Connecticut

Novak, Dr. A. F.
Louisiana State University
Baton Rouge, Louisiana

Penner, Dr. Lawrence R.
Department of Zoology
University of Connecticut
Storrs, Connecticut

Pereyra, Walter T.
College of Fisheries
University of Washington
Seattle 5, Washington

-196-

Perljnutter, Dr. Alfred
1776 Seaman Drive
Merrick, L. I., New York

Pfitzenmeyer, Mr. Hayes T.
Chesapeake Biological Laboratory
Solomons, Maryland

Pomeroy, Dr. Lawrence
Marine Biology Laboratory
Department of Biology
University of Georgia
Sapelo Island, Georgia

Porter, Mr. Hugh J,
University of North Carolina
Institute of Fisheries Research
Morehead City, North Carolina

Posgay, Julius 0,

Box 431

Woods Hole, Massachusetts

Price, Mr. T. J.
Biological Laboratory
Bureau of Commercial Fisheries
Beaufort, North Carolina

Pritchard, Dr. Donald W.
Chesapeake Bay Institute
The Johns Hopkins Ifriiversity
121 Maryland Hall
Baltimore 18, Maryland

Provasoli, Dr. Lxiigi
Haskins Laboratories
305 East if 3rd Street
New York I7, New York

Provenzano, Mr. A. J«, Jr.
Marine Laboratory
University of Miami
Miami, Florida

Quayle, Dr. Daniel B.
Fisheries Research Board

of Canada
Nanaimo, B. C, Canada

Ray, Dr. Sammy M.
A 85 M College of Texas
Marine Laboratory
Galveston, Texas

Rhode Island Department of

Agriculture & Conservation
Veterans Memorial Building
83 Park Street
Providence 2, Rhode Island

Rice, Dr. Theodore R.
Biological Laboratory
Bureau of Commercial Fisheries
Beaufort, North Carolina

Ropes, Mr, John W.
29 Linden Street
Salem, Massachusetts

Ronholt, Mr. Lael L.
College of Fisheries
University of Washington
Seattle, Washington

Russell, Mr. Henry D.
Springdale Avenue
Dover, Massachusetts

Rust, Mr. John D.
Biological Laboratory
Bureau of Commercial Fisheries
Beaufort, North Carolina

Sangree, Dr. John B., Sr,
Qlassboro State Teachers College
Glassboro, New Jersey

Sayce, Mr. Clyde S.

Box 205

Ocean Park, Washington

Scheltema, Mr. Rudolf 3.
Occajiographic Institute
Woods Hole, Massachusetts

Sellmer, Mr. George
Department of Biology
Upsala College
East Orange, New Jersey

-197-

Shaw, Mr. Williain N.
R. F. D. 1, Box 158
Falmouth, Massachusetts

Shearer, Mr. L. W.
8 Elm Street
Milford, Connecticut

Shuster, Dr. Carl N.
Marine Laboratory-
Department of Biological

Sciences
University of Delaware
Newark, Delaware

Sieling, Mr. Fred W.
Department of Research

and Education
Snow Hill, Maryland

Smith, Dr. P. G. Walton
The Marine Laboratory
University of Miami
1 Rickenbacker Causeway
Virginia Key
Miami k9, Florida

Sellers, Mr. Allen A.
1305 Park Avenue
Baltimore 17, Maryland

Sparks, Dr. Albert K.
College of Fisheries
University of Washington
Seattle, Washington

Sprague, Dr. Victor
Department of Research

and Education
Solomons, Maryland

St, Amant, Dr. Lyle S.

Wildlife and Fisheries Commisalon

ItOO Royal Street

New Orleans I6, LoulBlana

Staples, Mr. George M.

Box 66

Crisfield, Maryland

III

Stauber, Dr. Leslie

Rutgers University

New Brunswick, New Jersey

Stein, Mr. Jerome
Rayonier Marine Laboratory
Hoodsport, Washington

Stern, Mr. Joseph A.
Bothell, Washington

Strawbridge, Mr. Bruce H.
Jekyll Island Packing Co.
P. 0. Box 26k
Brunswick, Georgia

Stringer, Mr. Louis D.
18 Marcia Coixrt
Alexandria, Virginia

Swabey, Miss Yvonne H.
Academy of Natixral Science
19th & the Parkway
Philadelphia 3, Pennsylvania

Thompson, Mr. Frank E., Jr.
4520 Newport Street
Richmond 27, Virginia

Trezise, Williaja R.
318 - 12th Street
Raymond, Washington

Tubman, Mr. Arnold W.
Technological Laboratory
Bureau of Commercial Fisheries
College Park, Maryland

Udell, Mr. Harold F.

State of New York Conservation

Department
Shellfisheries Management Unit
65 West Sunrise Highway
Freeport, L. I., New York

Ukeles, Dr. Ravenna
Biological Laboratory
Bureau of Commercial Fisheries
Milford, Connecticut

-198-

Valaer, Mr. Edward P.

D. C. Department of Public Health

Bureau of Food & Public Health

Engineering
Washington 1, D. C.

Varln, Mr. Clifford V.

Fire Island Sea Clam Co.

95 Cherry Avenue

West Sajrville, L. I., New York

Virginia Commission of Fisheries
Newport News, Virginia

Wallace, Mr. Dana E.

Department of Sea & Shore Fisheries

Vlckery-Hill Biiilding

Augusta, Maine

Wallace, Mr. David H.
Oyster Institute of North America
6 Mayo Avenue, Bay Ridge
Annapolis, Maryland

Webster, Mr. John R.
Biological Laboratory
Bureau of Commercial Fisheries
Oxford, Maryland

Weiss, Dr. Charles M.
Department of Sanitary Engineering
School of Public Health
University of North Carolina
Chapel Hill, North Carolina

Welch, Mr, Walter R.
Biological Laboratory
Bureau of Commercial Fisheries
Boothbay Harbor, Maine

Wells, Mr, Harry W.

Box 162

Buxton, North Carolina

Westley, Mr. Ronald E.
Washington State Department

of Fisheries
Shellfish Laboratory
Qullcene, Washington

Whaley, Mr. Horace H.

Chesapeake Bay Institute

Edgewood Road

R. F. D. 3, Box Ul^A

Back Creek

Annapolis, Maryland

Whitcomb, Mr. James P.
Virginia Fisheries Laboratory
Gloucester Point, Virginia

Wllbour, Mr, Frederick C, Jr.
Division of Marine Fisheries
15 Ashburton Place
Boston 8, Massachusetts

Wilde, Mr. Frank W.

Box 5

Shady Side, Maryland

Wilson, Mr. Alfred J., Jr.
Biological Laboratory
Bureau of Commercial Fisheries
Gulf Breeze, Florida

Woelke, Mr. Charles E.

Box 323

Qullcene, Washington

Wolffian, Dr. Abel

The Johns Hopkins University

Whitehead Hall

Baltimore I8, Maryland

Wood, Dr. John L.

Virginia Fisheries Laboratory

Gloucester Point, Virginia

Woodruff, Mr. Spofford
Apple Tree Lane
Earrlngton, Rhode Island

Wurtz, Dr. Charles B.
32U7 Disston Street
Philadelphia, Pennsylvania

-199-

MBL/WHOI UBRARY

WH lACK

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