d-

1963 RkoCEEDINGS

Marine Biological laboratory
library

DEC 2 2 1967

WOODS HOLE, MASS.

NATIONAL

SHELLFISHERIES

ASSOCIATION

Volume 54

PROCEEDINGS

of the

NATIONAL SHELLFISHERIES ASSOCIATION

Official Publication of the National Shellfisheries

Association; an Annual Journal Devoted to

Shellfishery Biology

Volume 54
August 1963

Published for the National Shellfisheries Association by
Bi-City Ink, Bryan, Texas

1966

TABLE OF CONTENTS

Introduction of Honorary Members DAVID H. WALLACE 1

VICTOR L. LOOSANOFF 4

CONTRIBUTED PAPERS

The European oyster, Ostrea edulis , in Maine

WALTER R. WELCH 7

A bacterial basis for the growth of antibiotic-treated bivalve

larvae. . . . HERBERT HIDU and HASKELL S. TUBIASH 25

A method for increasing survival of locally-caught Pacific

oyster seed in Willapa Bay, Washington

CLYDE S. SAYCE 41

Notes on the occurrence of Dermocystidium marinum on the

Gulf of Mexico coast during 1961 and 1962

SAMMY M. RAY 4 5

A review of the culture method for detecting Dermocystidium
marinum with suggested modifications and pre-
cautions SAMMY M. RAY 55

Radiation pasteurization of oysters ARTHUR F. NOVAK 71

Differentiation of effects of two pesticides upon Urosalpinx

cinerea Say from the Eastern shore of Virginia

LANGLEY WOOD and BEVERLY A. ROBERTS 75

Mortality rates and the life span of the bay scallop,

Aequipecten irradians NELSON MARSHALL 87

Serological studies on the bay scallop, Aequipecten

irradians JUDITH A. PENDLETON 93

ASSOCIATION AFFAIRS

Annual Convention 101

Special Notices 102

Information for Contributors 103

OTHER TECHNICAL PAPERS PRESENTED AT THE
1963 CONVENTION

Oyster mortalities on the Pacific Coast ALBERT K. SPARKS

Oyster mortality trends along the Atlantic Coast and status of

research HAROLD H. HASKIN

Recent progress in Malpeque disease studies in Canada

R. E. DRINNAN
Progress on oyster disease studies at Bureau of Commercial Fisheries

Biological Laboratory, Oxford, Maryland

AARON ROSENFIELD
Preliminary studies on the acute inflammatory reaction in the Pacific

oyster, Crassostrea gigas

GILBERT PAULEY and ALBERT K. SPARKS
Note on a microsporidian hyperparasite of Bucephalus cuculus in

Crassostrea virginica VICTOR SPRAGUE

Infectious necrosis— a disease of larval and juvenile bivalve

mollusks HASKELL S. TUBIASH and PAUL E. CHANLEY

Epithelial lesions of the oyster, Crassostrea virginica , and the

associated "MSX" stages , JOHN L. MYHRE

Irradiation sterilization of Urosalpinx cinerea

KENNETH A. LEON and WILLIAM J. HARGIS

Uptake and retention of DDT by shellfish .

PHILIP A. BUTLER, JACK I. LOWE, and ALFRED J. WILSONJR.
Forest spraying in Washington in relation to the oyster industry of

Willapa Bay CLYDE S. SAYCE

A review of the oyster drill control programs in Long Island Sound,

1963 JAMES E . HANKS

Pesticide problems on a national scale LOUIS D. STRINGER

Use of lindane as an agent in control of the green crab, Carcinides

maenas DONALD M . HARRIMAN

Histological observations on the response of oysters to tissue

implants WALTER J. CANZONIER

Some cytological and chemical characteristics of the Ostreidae ....

AARON ROSENFIELD

Effects of suspended silt on oyster growth

PHILIP A. BUTLER and ALFRED J. WILSON, JR.

Oyster hexamitiasis and the winter mortality AUSTIN FARLEY

A technique for estimating rates of biodeposition of the oyster

(Crassostrea virginica) DEXTER HAVEN

Fate of the southern oyster drill, Thais haemastoma .accidentally

planted in Chincoteague Bay GEORGE GRIFFITH

Manner of exit of "MSX" and similar organisms from oysters: Fecal

string studies of selected oyster stocks suspended in

Chincoteague Bay, Virginia THOMAS C. CARVER, JR.

Survival time of oysters after burial at various temperatures

ELGIN A. DUNNINGTON, JR.
Effects of synthetic surfactants (detergents) on the larvae of clams

(M . mercenaria) and oysters (C . virginica)

HERBERT HIDU
Activity of the hard clam, Mercenaria mercenaria, as a function of

temperature SUNG YEN FENG

The blood circulation in the posterior half of the American oyster,

Crassostrea virginica ALBERT F. EBLE

Gonadal development of the soft-shelled clam, Mya arenaria , prior

to spring and autumn spawning at Solomons, Maryland

HAYES T. PFITZENMEYER
Spermatogenesis in the Maryland soft-shell clam, Mya arenaria ....

WILLIAM N . SHAW

INTRODUCTION OF HONORARY MEMBERS

DAVID H. WALLACE

Director, Bureau of Marine Fisheries,
New York Conservation Department
Former Directoi , Oyster Institute of North America

David Wallace was born 17 February 1916 at Barclay, Mary-
land, where his father was postmaster. At the age of 15 he entered
Washington College, Chestertown, Maryland. There Professor Kathleen
Carpenter, the famous Welsh fresh-water biologist, inspired him to
choose biology as a career. After taking a B.S. degree at Washington
in 193 5 David entered the University of Maryland, where he studied
ichthyology under Professors V. Vladykov and R. V. Truitt and won the
M.S. degree in zoology in 1937 . During the summers of 1936 and 1937
he did field and laboratory work on the rock (striped bass) and shad at
Chesapeake Biological Laboratory, Solomons, Maryland. In 1938 he
was promoted from assistant biologist to biologist in charge of fisheries
research, and extended his investigations to croakers. Eight publica-
tions were based on his researches during this period .

In 194 0 Dave moved to Annapolis to become administrative
assistant in the Maryland Department of Tidewater Fisheries, rising to
executive secretary in 1941 and director in 1946. In 1949 he became
chairman of the Maryland Board of Natural Resources .

Upon the death of Dr. Lewis Radcliffe, the Oyster Institute of
North America chose David Wallace to replace him. From 1951 to 1962
he served with distinction as director of the Oyster Institute and as
executive secretary of the Sponge and Chamois Institute.

Since 1962 he has been Director of the Bureau of Marine
Fisheries of New York's Conservation Department at Oakdale, N. Y.,
with responsibility for research, management, shellfish sanitation,
and law enforcement programs . As a member of the staff of the Univer-
sity of New York at Stony Brook, he is planning the permanent offices
of the Conservation Department and developing a graduate marine
science program for the University.

While mainly an administrator, Dave Wallace has found time
to do research, and to serve on several state and federal committees
concerned with research. He has also served a term as secretary-

-1-

treasurer of the National Shellfisheries Association. Furthermore, he
has found time for leadership in his church and PTA, the Civitan Club,
and community activities so important that he was named Man of the
Year for Anne Arundel County (1951). He is admired by all who know
him for his ability to get along with people, for his tact and ready
sympathy, and for his integrity as well as for his ability.

-2-

DAVID H. WALLACE

-3-

VICTOR L. LOOSANOFF

Bureau of Commercial Fisheries
U .S . Fish and Wildlife Service

Dr. Loosanoff has held offices including the Presidency in this
Association and has always been active in its affairs. Since 1931, he
has vigorously pursued the study of the oyster and other shellfish,
advancing our knowledge in the economic as well as in the academic
and scientific fields . Because of his eagerness to present his useful
findings to science and industry, his research is well documented in
our literature. Those of us who had the privilege of working with him
caught the spirit of his intensive research and made his influence felt
in this country and abroad as we separated to work elsewhere.

Victor Loosanoff was born in Kiev, Russia, 3 October 1899, into
a military family. He received his early schooling in Russian military
academies . During World War I, he served as an artillery officer in
the Royal Russian Army. He managed to escape the purge of the 1917
revolution and the next few years found him on the fluid battlefront
against the Red armies . The fortunes of this war brought him to Siberia,
China, Japan, and finally the United States. These experiences form
an exciting period in his life which only he can tell about.

What prompted him to switch from the military to the life
sciences is not known, but in 192 7 he graduated with honors from the
University of Washington with a B.S. degree. His professional career
started with the State of Washington and in 1931 he came east as Chief
Marine Biologist of Virginia. The U.S. Bureau of Fisheries claimed
him in 1932 as an aquatic biologist when he began his distinguished
career at Milford, Connecticut. From 193 5 to 1963 he served as
Director of the Milford laboratory. He earned a Ph.D. degree from
Yale University in 1936 .

The development of control measures for one of Long Island
Sound's principal shellfish predators, the starfish, was among his first
objectives on the east coast. This led to a vigorous warfare on other
enemies of shellfish. His work with larval shellfish and their artificial
propagation is monumental and shows a way to controlled mollusks .
The energetic, persistent, and often ingenious way he approached the
problems of our industry has made him one of our most distinguished
and honored shellfish scientists .

-4 -

VICTOR L. LOOSANOFF

-5-

THE EUROPEAN OYSTER, OSTREA EPULIS, IN MAINE

Walter R . Welch

Bureau of Commercial Fisheries Biological Laboratory
Boothbay Harbor, Maine

ABSTRACT

Favorable temperature and salinity characteristics of the Maine
coast encouraged several introductions of Ostrea edulis from Holland.
The most successful transplant was in Boothbay Harbor and surveys
indicated that oysters have set annually on suitable bottom below low
tide level, in and near this area, persistently increasing in abundance
and range. Spat set also in the intertidal zone, but most did not survive
the winters. Using shell bags on bottom, and raft- suspended shell
strings, initial set was found to be greatest in shell bags, but survival
through winter was greater on suspended shell strings. Not all trans-
plants were successful, and recruitment rate is low, but the persistent
expansion of the oyster population in Boothbay Harbor is encouraging.

INTRODUCTION

The decline of soft-shell clam stocks in northern New England
from 1946 to 1959 emphasized the economic need for an additional
fishery in intertidal or shallow subtidal environments . The successful
introduction of the European oyster, Ostrea edulis L., into northern
New England appeared theoretically possible. Although the coastal
waters are often too cold to allow spawning, the temperature and
salinity characteristics of many inshore areas are superficially similar
to those in parts of the natural European habitat. According to Korringa
(194 0) this species thrives in the Oosterschelde of Holland, with
salinity varying from 25.1 to 32.9 o/oo; also, spawning can occur at
temperatures of 15 to 16 C with more successful spatfall resulting from
more prolonged higher temperatures. Detailed information on tempera-
ture and salinity was not available prior to introduction of the species,
but data for 1959 will serve as an example of conditions occurring in
Boothbay Harbor. Daily salinity readings at the Bureau of Commercial
Fisheries Biological Laboratory varied from 16.7 to 34.2 o/oo, but
monthly averages of daily readings varied only from 29.0 to 32.8 o/oo.
Salinities below 22.5 o/oo, a minimum for normal growth and setting of
Ostrea edulis larvae (Davis and Ansell, 1962), were recorded on only

7-

three separate days. All variations were well above the 15.0 o/oo mini-
mum found by Chanley (1958) to be suitable for Ostrea edulis juveniles.
Boothbay Harbor water temperatures during the spawning season of 1959
rose well above the 15 C minimum for spawning of this oyster.

The first European oysters were introduced in 1949 and Loosanoff
(1951, 1955, 1962) reported on their initial survival, gametogenesis ,
and spawning . There has never been a coordinated study made of the
first and subsequent introductions, but this report brings together all
known additional information on the existence of the European oyster
in Maine . Since there has been little effort made to improve oyster
setting or growing conditions in Maine, the natural increase and spread
of this introduced species is of considerable ecological interest. The
data have been obtained principally through observations of personnel
of the Bureau of Commercial Fisheries Biological Laboratory at Booth-
bay Harbor, Maine, and the Maine Department of Sea and Shore Fish-
eries . Age or year class was determined by a combination of relative
size and counts of annual rings. Unless otherwise indicated, all
references to oysters in this paper are to the European species, Ostrea
edulis .

TRANSPLANTATION OF OYSTERS

Over a period of 12 years, several attempts were made to intro-
duce oysters from Holland into Maine waters . These attempts and other
related transplants were as follows, with locations shown in Figs. 1
and 2 .

Group 1, Basin Cove, Harpswell

This group and the two following made up the original introduc-
tion in October 1949 (Loosanoff, 1951, 1955, 1962). The oysters had
been shipped from the Oosterschelde in Holland to the U.S. Fish and
Wildlife Service Biological Laboratory in Milford, Connecticut, and had
been carefully screened for diseases, parasites, and other potentially
dangerous organisms. The lot included representatives of the 1947,
1948, and 1949 year classes and was distributed in Maine to determine
survival and ability to mature sexually and spawn. In Basin Cove,
1 , 060 were held in cages .

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Group 2, Boothbay Harbor

Part of the same lot as Group 1; 3 ,600 were held in cages.

Group 3, Taunton River, Franklin

Part of the same lot as Groups 1 and 2; 1, 06 0 were held in
cages .

Group 4, Boothbay Harbor

In June 1954, the Maine Department of Sea and Shore Fisheries
imported 3 90 lbs of adult oysters from Holland . They were held at the
laboratory wharf in an effort to augment the local spawning population
of oysters (progeny of the original introductions).

Group 5, Small Point, Phippsburg

In December 1955, an estimated 4, 000 Dutch oysters were
privately imported in cooperation with the Maine Department of Sea and
Shore Fisheries . These oysters were placed in a lobster pound as the
nucleus ot a spawning population.

Group 6, Peters Island, Bristol

In April 1959, personnel of the Maine Department of Sea and
Shore Fisheries transferred 50 adult Boothbay Harbor oysters (progeny
of the original introductions) to Peters Island to test the possibility of
extending the local range more rapidly by dispersing groups of mature
oysters .

Group 7, Merepoint Bay, Harpswell

In May 1961, approximately 2 bushels of oysters of various
sizes from the Milford laboratory were placed in cages in shallow water,
These oysters had been propagated at the Milford laboratory from Dutch
stock and were placed in Merepoint Bay to serve as spawning stock .

11-

FATES OF TRANSPLANTED OYSTERS

Group 1, Basin Cove, Harpswell

A large proportion of the oysters survived and spawned during
the first 2 years when they were under close observation. By July 1954,
there were 12 0 of the original stock alive in the cages; and in July 1962
SCUBA divers reported 55 in the immediate area of the original cages.
Seven of these, examined at the laboratory, appeared to be very old
and are believed to be of the original stock .

Group 2, Boothbay Harbor

A large proportion of this group survived, spawned, and pro-
duced set during its first 2 years . In May 1952, all survivors of the
group, except 15 retained in laboratory tanks, were returned to the
Milford laboratory. The remaining 15 were also returned in May 1953 .

Group 3, Taunton River, Franklin

This group suffered heavy mortality and by November 1953 no
live oysters could be found .

Group 4 , Boothbay Harbor

These oysters spawned as expected during their first summer,
but during the following winter and spring all were lost because of
damage to the holding cages .

Group 5, Small Point, Phippsburg

Few observations were made on this group and the oysters were
reported as all dead or missing the following spring.

Group 6, Peters Island, Bristol

No observations were made on this group until August 1961, when
only dead shells were found .

Group 7, Merepoint Bay, Harpswell

This group was examined occasionally and dead oysters were
removed . Although there was no initial count, there did not appear to

-12-

be a very heavy mortality. In October 1962, 1,181 remained alive.
These were removed from the cages and scattered on hard bottom about
3 feet below mean low water.

EVIDENCE OF SETTING

Table 1 summarizes the results of all known reports of oysters
other than those accounted for by known introductions . In the few
cases where oysters were reported by persons other than federal or
state biologists, specimens were obtained and identification verified .

During most of the 11 -year period, reports of oysters resulted
from chance observation or from casual searches. Data for 1957, 1958,
1961, and 1962, however, were obtained by means of systematic
search.

The first such systematic search was conducted in Boothbay
Harbor in June 1957 . At low tide, two men in a skiff examined the
clearly visible portion of the bottom from mean low water to a depth of
minus 4 feet (datum is mean low water) in a 2.4-mile band, averaging
3 0 feet in width, extending around the three major coves in the western
and northern portion of the harbor (Fig. 3). Where conditions of visi-
bility permitted, the survey occasionally included depths to approxi-
mately minus 8 feet.

In addition to the numbers of oysters given in Table 1, the
results of the survey showed that: 1) only occasional small dead
oysters were found between mean low water and the minus 2 -foot level
of extreme low tides; 2) most oysters were found in the minus 2- to
minus 4-foot unexposed zone, where gravel, shell, and other firm sub-
strate are commonly available, and 3) oysters at greater depths were
found only in the rare instances where firm substrate existed .

In October 1958, during a period of extremely low tides, a more
thorough survey was conducted using essentially the same methods as
in 1957. In addition to examining the submerged bottom by boat, the
exposed intertidal zone was explored on foot. The areas surveyed
included a 1 .2-mile section of the west shore of Boothbay Harbor, the
area between Southport and Capitol Islands, and the northeastern
portion of Linekin Bay.

This survey was carried out to gain more information on the
relative abundance of oysters in the occasionally exposed zone between
mean low water and minus 2 feet; and in the unexposed zone between

-13-

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minus 2 and minus 4 feet. Observations were also made on the relative
abundance of spat, yearlings, and older oysters in the same two zones.

Total count of oysters in each area are shown in Table 1 . In
addition, Table 2 gives the number of oysters in each of the two bottom
zones and in each of the three-year class groups . The data confirm
the observation made in 1957 that abundance is generally very low in
the 0- to minus 2-foot zone subject to periodic exposure. The Booth-
bay Harbor data, however, indicate that setting does sometimes occur
in this zone .

Table 2 . Results of survey of European oysters in the vicinity of
Boothbay Harbor, Maine, October 1958

Number

of oy

sters

From

From

0 to -2

-2 to -4

Location

Year Class

feet

feet

Boothbay Harbor

1958

3

0

1957

19

16

Older

58

824

Capitol Island

1958

0

0

1957

0

0

Older

0

20

Linekin Bay

1958

0

63

1957

0

128

Older

0

395

Total

80

1,446

Table 1 shows that the 1958 total for Boothbay Harbor is nearly
three times that for the comparable area in 1957. The increase was not
a result of the 1958 setting, since Table 2 shows that the greatest propor-
tion of the oysters had set prior to 1957. Many oysters were undoubt-
edly overlooked in 1957 but counted during the more intensive search in
1958.

-16

All data for 1961 and Little River data for 1962 were obtained
through survey methods similar to those used in 1958, except that only
total counts for each area were obtained . The two young oysters found
in Basin Cove in 1962 were located by a cooperating group of SCUBA
divers who searched for 13 1/2 man-hours in the subtidal zone of the
cove .

EXPERIMENTAL SHELL PLANTING

During 1959 and 196 0 an experiment was conducted in shell
planting in Boothbay Harbor and Linekin Bay to gain information on the
use of cultch and the success of setting . On 19 June and 10 July,
while daily mean water temperatures were still below the minimum for
spawning (Fig. 4), samples of gonads from Boothbay Harbor oysters
were found to be insufficiently developed for spawning . On 22 July,
after daily mean water temperatures had been above 15 C for 5 days, all
six oysters of a Boothbay Harbor sample spawned soon after being
placed in standing water in trays in the laboratory. On 5 August, a
sample of six Boothbay Harbor oysters included three which had spawned
but no longer contained larvae and three which contained black or well-
advanced larvae in the mantle cavity, nearly ready to be released into
the water. With the prospect of additional spawning and swarming, and,
according to Korringa (1940), possible setting in 2 to 3 weeks at the
prevailing temperatures, cultch was placed as follows:

On 1 1 to 13 August in each of two coves, one on the west side
of Boothbay Harbor, the other at the northeast tip of Linekin Bay, 4 0 to
5 0 shell bags filled with soft-shell clam cultch were placed on bottom
between mean low water and minus 3 feet. Approximately 4 bushels of
loose clam shells were scattered just below the zone where the shell
bags were placed. Seven strings, 6 to 8 feet long, of hard clam shells
were suspended from a raft in Boothbay Harbor.

The cultch was examined occasionally to determine when the
first set occurred, but none was found until 9 September. The first
spat, two in number, were from Linekin Bay shell bags and measured
2 . 0 and 3 .5 mm .

The final examination of cultch for the season was made during
October to determine the success of setting. Five shells were removed
from each shell bag for examination, and the shell bags were then
moved into deeper water, well below the extreme low tide level, for
protection from winter air temperatures . All of the shells on the remain-
ing five raft-suspended strings (two had been lost) were examined .
Those shells having spat on them were returned to the raft and left

-17-

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TEMPERATURE

-18-

suspended through the winter. The loose shell on bottom was sampled,
but since no set was found and the shells were heavily silted, no further
examinations were made.

The results of the shell counts are shown in Table 3 . The data
indicate: 1) the success of setting of cultch in shell bags in Linekin
Bay was of the same order of magnitude as that in Boothbay Harbor,
which had been regarded as both the center of the spawning population
and the area of heaviest setting; 2) the success of setting on raft-sus-
pended shell strings was less than half as great as on cultch in shell
bags in the immediate vicinity (shell strings had accumulated far more
silt and fouling organisms than had the shell in bags), and 3) ranges in
size were typical of set occurring over an extended period of time.

Table 3 . Setting of European oysters in the vicinity of
Boothbay Harbor, Maine, October 1959

Type of
cultch and
location

No . of

shells

examined

No. of
spat

Ratio,
spat per
lOOshel

Shell bags in
Boothbay Hai

Shell bags in
Linekin Bay

•bor

210
220

97
115

46.2
52.3

Size (mm)

13

10

Shell strings in
Boothbay Harbor 105 23 21.9 3 15

During the following June and July (196 0), the shell strings and
all the shell bags that could be found were retrieved to obtain data on
survival and size of the spat. As Table 4 indicates, the spat in shell
bags in both Boothbay Harbor and Linekin Bay suffered extremely heavy
mortalities . The spat on the raft-suspended shell strings fared better,
with nearly half surviving . Percentage survival was based on the num-
ber of spat per 100 shells in October 1959 (Table 3).

The shell bags had been placed at a level where, although
totally submerged, they were subjected to wave action at low tide. All
shells were practically devoid of animal or plant growth. The abrasive
action of the shells scouring each other and the turbidity in this zone of
wave disturbance may have contributed to the mortality of spat.

19-

Table 4 . Survival and size of European oyster spat after one winter in
the vicinity of Boothbay Harbor, Maine, June-July, 1960

Type of
cultch and
location

No. of

Ratio,

Sur-

Size (mm)

shells No . of spat per vival

examined spat 100 shells % Min . Max. Mean

Shell bags in
Boothbay Har-
bor

3,972

108

2.7

5.8 3 26

Shell bags in
Linekin Bay

3,045

11

0.4

0.8 4 14

Shell strings in
Boothbay Har-
bor

105

10

9.5

43.4 4 11

DISCUSSION

The successful culture of the European oyster could be economi-
cally important to northern New England . As far as is known, the
American oyster, Crassostrea virginica, exists in New England north of
Cape Cod only in the Piscataqua River between New Hampshire and
Maine; in a single, small bed in the upper Sheepscot River estuary in
Maine; and as rarely-found individuals in the Damariscotta River in
Maine . Ecological conditions apparently do not permit spawning nor
survival of larvae or spat outside these areas . Except for the bay
scallop (Aequipecten irradians) just north of Cape Cod, the soft-shell
clam (Mya arenaria) and the hard clam (Mercenaria mercenaria) are the
most important commercially-used mollusks living in the intertidal or
shallow subtidal environments . Both of the latter species are generally
most abundant in areas not suitable for oysters; therefore, the ecological
competition of the oyster with these species appears to be negligible.

The fact that Ostrea edulis has lived and reproduced success-
fully in the Boothbay Harbor, Maine area since 1949 indicates that the
environment is basically suitable. Water temperature has been suf-
ficiently high to bring about annual spawning, since setting has occurred
each year, and the range of salinities observed in the area does not
seem to constitute a problem. However, since the oyster population has
yet to reach commercial abundance and the rate of recruitment appears to
be low, there is need for a better understanding of the ecological

-2 0-

requirements of the species and its adaptibility in the area. It should
be noted that introductions of Ostrea edulis from Conway, North Wales,
into Canada at St. Andrews, New Brunswick, and Ellerslie, Prince
Edward Island , were made in 1957, 1958, and 1959. This move was
encouraged in part by the success of the Maine introductions, but
extremely heavy mortalities occurred, apparently associated with low
winter water temperatures (Medcof, 1961).

In Maine the results of the 1957 and 1958 surveys indicate the
existence of two conditions which seriously limit the increase in
oyster abundance . First, the relative scarcity of oysters in. the zone
occasionally exposed at extreme low tides indicates that the oysters
cannot withstand winter air temperatures . According to Gaarder and
Bjerkan (1934), Ostrea edulis in sea water can tolerate temperatures
slightly below OC, but those exposed to freezing temperatures in air
will be seriously weakened and damaged, if not killed. In northern
New England, subfreezing air temperatures coincide with extreme low
tides often enough to virtually eliminate the entire intertidal zone from
year-round production of oysters. Second, oysters were found only on
firm substrate. Unfortunately, a large proportion of the bottom examined
during the surveys and most of the bottom at greater depths, was covered
with silt and completely devoid of the shell, rock, or gravel necessary
for setting .

A possible third limiting set of conditions is of hydrographic
nature but no specific information has been obtained. Korringa (1940)
indicated that Ostrea edulis set may not reach commercial quantities
in areas where water temperatures are not higher than 18 C, or where
tidal exchange may reduce the numbers of planktonic larvae. He
pointed out that in such circumstances proper use of cultch is neces-
sary to make maximum use of the available set . Both of these limiting
conditions of temperature and tidal exchange prevail in many New
England areas .

The encouraging results of the trial use of cultch in 1959 indi-
cate that, through employment of improved methods and materials,
stocks of oysters might be increased far beyond those that have accumu-
lated under natural conditions. Suitable subtidal growing ground is
limited so that raft or rack culture, or relaying, might be necessary for
a commercial operation.

Over a period of 13 years, sufficient encouragement has been
gained from observations of the natural growth of the cyster population
in the Boothbay Harbor area to recommend that further research be con-
ducted on the European oyster. A research program should include:

-21-

1) determination of the physiological requirements of the oyster;

2) ecological studies of areas where the oyster has become established;

3) determination of the setting potential and satisfactory use of cultch,
and 4) determination of the degree of physiological adaptation of the
Dutch strain of Ostrea edulis to Maine waters since it was first intro-
duced .

Future growth of the oyster population and any possible future
research will be aided by the protection now afforded the species in the
Boothbay Harbor area . In June 196 0 the Maine Department of Sea and
Shore Fisheries recognized the potential importance of this oyster and
established an extensive closed area (Fig . 3), within which the taking
of European oysters is forbidden by law. The boundaries of the closed
area include all major known populations and the localities where
continued spread and increase in abundance may be expected.

CONCLUSIONS

1 . Temperature and salinity characteristics of parts of the
Maine coast are basically suitable for survival, growth, and reproduc-
tion of Ostrea edulis from Holland .

2. In the Boothbay Harbor, Maine area, oysters have spawned
and set annually since 1949 and numbers and range have shown progres-
sive increases .

3 . The greatest numbers of progeny exist only in the zone
below the range of extreme low tides .

4. Mortality caused by low winter air temperatures is believed
to limit abundance of oysters in the intertidal zone, and setting at
lower levels is limited by a scarcity of suitable substrate.

5 . More set occurred in shell bags on bottom than on raft-
suspended shell strings, but winter and spring survival was better on
the shell strings .

6 . The persistent increases and spread of the Boothbay Harbor
oyster population warrants further research.

- 22-

LITERATURE CITED

Chanley, P. E. 1958. Survival of some juvenile bivalves in water of
low salinity. Proc . Nat. Shellfish. Ass. 48:52-65.

Davis , H . C . , and A . D . Ansell . 1962 . Survival and growth of larvae
of the European oyster, O. edulis , at lowered salinities.
Biol. Bull. 122:33-39.

Gaarder, T., and P. Bjerkan. 1934. 0sters og (zfsterskultur i Norge .
John Griegs Boktrykkeri , Bergen, 96 pp. (Fisheries Research
Board of Canada Translation Series, No. 217: 66 pp.).

Korringa, P. 194 0. Experiments and observations on swarming, pelagic
life and setting in the European flat oyster, Ostrea edulis L.
Contrib. Govt. Inst. Biol. Fish. Res., Extr . Arch. Neerland-
aisesZool. 5:249pp.

Loosanoff, V. L. 1951. European oyster, O. edulis , in the waters of
the United States . Anat . Rec . 111:126 pp.

Loosanoff, V. L. 1955. The European oyster in American waters .
Science 121:119-121.

Loosanoff, V. L. 1962. Gametogenesis and spawning of the European
oyster, O. edulis, in waters of Maine. Biol . Bull . 122:86-94.

Medcof, J. C. 1961. Trial introduction of European oysters (Ostrea
edulis) to Canadian East Coast. Proc. Nat. Shellfish. Ass.
50: 113-124.

-23-

m

A BACTERIAL BASIS FOR THE GROWTH OF
ANTIBIOTIC-TREATED BIVALVE LARVAE

Herbert Hidu and Haskell S. Tubiash

U « S. Bureau of Commercial Fisheries
Biological Laboratory, Milford, Connecticut

ABSTRACT

Routine addition of the proprietary antibiotic formulation "Combi-
strep" (dihydrostreptomycin- streptomycin sulfates) to larval cultures of
clams, Mercenaria mercenaria, or oysters, Crassostrea virginica, usually
results in a significant increase in growth rate of larvae. It had been
assumed that this increase was effected by the elimination or suppres-
sion of bacterial flora, but plate counts show that the total number of
marine bacteria increases in almost direct proportion to the added Combi-
strep up to 2,000 parts per million. Bacteria-free clam larvae showed no
growth when cultured in autoclaved sea water to which Combistrep had
been added. In Combistrep-treated cultures inoculated with a mixed
flora of marine bacteria, the larvae showed significant growth, while
cultures that received the bacterial inoculum but no Combistrep showed
little or no growth. These results suggest that the antibiotic-induced
bacterial flora in the Combistrep-treated cultures may be utilized by
larvae as a food source.

INTRODUCTION

Since the development of methods of rearing larvae of clams,
Mercenaria mercenaria , and oysters, Crassostrea virginica , the role of
bacteria and dissolved substances in their nutrition has been a matter
of conjecture. It has been shown that supplemental live algal foods
are primarily responsible for growth and development of these larvae
(Davis and Guillard, 1958). On occasion, however, other factors
have been observed or suspected to play an important part in nutrition
of larvae. Davis and Chanley (1956) reported an increase in the
growth rate of clam and oyster larvae on the addition of several vita-
mins and antibiotics but offered no suggestion on how the increased
growth was achieved. Carriker (1956) reared clam larvae to meta-
morphosis on an extract of cereal, Pablum , and concluded that the good
growth of larvae was the result of increased microbial populations
stimulated by the addition of the Pablum filtrate. Loosanoff, Davis,
and Chanley (195 5) stated that clam larvae seem able to utilize sulfur
bacteria. Coe (1947) and Rodinca (1948) believed that bacteria played
a part in the diet of adult mollusks . On the other hand, Davis (1953)
fed pure cultures of nine species of marine bacteria to larval oysters
with no success. Loosanoff, Davis, and Chanley (1955) stated that

-25-

lack of success in growing oyster larvae on several species of marine
bacteria contradicts the generally accepted view that marine bacteria
constitute an important part of the oyster diet.

The present paper reports experiments which show the growth-
producing effect on clam and oyster larvae of a commercial antibiotic
preparation Combistrep! (dihydrostreptomycin and streptomycin sul-
fates). It is furthermore demonstrated that the increase in growth of
larvae is associated with a stimulation of marine bacterial populations
by the Combistrep. This antibiotic preparation was originally used at
the Milford laboratory in an attempt to prevent mortalities of larvae
resulting from bacterial diseases, and its effect in increasing growth
rates of bivalve larvae was first noted by Chanley (personal communica-
tion).

MATERIALS AND METHODS

Methods of conditioning and spawning adult bivalves and
rearing larvae in the laboratory have been described in detail (Loosanoff
and Davis, 1950). To determine effects of Combistrep on clam and
oyster larvae, fertilized eggs were cultured 48 hours at concentrations
of approximately 3 0 per ml in filtered ultraviolet-light -treated sea
water. Forty-eight-hour veliger larvae were then collected on stain-
less steel screens and diluted to a known volume. After the number of
larvae per unit volume was determined, appropriate volumes were used
to set up experimental cultures with about 10 larvae per ml.

The culture medium, including test materials, was renewed
every second day by collecting the larvae on a stainless steel screen
and transferring them to new media . Temperatures were held to 24 + 1 C
throughout. After 10 days' exposure to the experimental conditions
clam veligers were sampled quantitatively. Oyster veligers were
similarly sampled after 12 days . Effect of experimental treatment on
growth of larvae was determined by measuring the long axis of 5 0 clam
or 100 oyster larvae. The generally uniform size of clam larvae per-
mits good accuracy with the lesser sample size .

Methods used in determining the effects of Combistrep on
marine bacteria and the effects of the stimulated bacterial populations
on bivalve larvae are included briefly within the respective result
sections .

Reg .U.S. Pat . Off . Chas . Pfizer & Co . , Inc

-26-

Combistrep is a proprietary compound of Chas . Pfizer & Co . ,
Inc ., and has the following composition:

dihydrostreptomycin base (as sulfate) 125 mg/cc

streptomycin base (as sulfate) 125 mg/cc

phenol 0.25%

sodium citrate 1 .3%

sodium bisulfate 0.2%

water 77.4%

RESULTS

A compilation of data from experiments over the past several
years demonstrates that both clam and oyster larvae receiving Combi-
strep have consistently shown more rapid growth than larvae in com-
parable cultures that did not receive Combistrep (Figs, land 2). At
concentration ranges of 100 to 3 00 parts per million (ppm), Combistrep
generally increased the rate of growth of clam larvae by more than
100% in cultures not receiving a supplemental feeding of algae (Fig. 1).
Growth of these untreated, unfed larvae varied considerably, i.e.,
the mean length at 10 days varied from about 115 to about 148(j. Such
differences in growth are certainly due to variations in the amount of
food present in the filtered ultraviolet-light-treated sea water. In all
cases, however, above average growth in the untreated, unfed cul-
tures was accompanied by a correspondingly more rapid growth of
larvae in the Combistrep-treated unfed cultures .

Clam larvae receiving 2 00 to 400 ppm of Combistrep and no
supplemental algal feeding when reared beyond the usual 10-day experi-
mental period in all cases grew to metamorphosis with negligible
mortality within 2 0 days at 24 C. In all such instances, of course,
it required a longer time for these larvae to reach metamorphosis than
for larvae receiving algal food supplements, but untreated, unfed
larvae in parallel cultures never progressed beyond 140u in length.

Combistrep also increased growth of clam larvae receiving live
flagellates as food (Fig. 1). The increase in the growth increment
averaged about 25^ at 12 days of age at optimal Combistrep concentra-
tions . This was about a 25% increase in growth over larvae fed live
flagellates without Combistrep. The optimum concentrations of Combi-
strep appeared to be higher in cultures receiving algal foods than in
those not receiving the algae.

Growth of oyster larvae was increased nearly 100% by the
addition of Combistrep at concentrations between 2 00 and 3 00 ppm

-27-

250

290

210

in
z
o

U 190

170

O

no

M. MERCENARIA

FEO LIVE FLAGELLATES

NO SUPPLEMENTAL FEEDING

t

100 200 30C 400 500 600 700 800 900 1000
RPM. COMBISTREP

Fig . 1 . Mean lengths of clam , M . mercenaria , larvae at 12
days of age receiving several dosage rates of Combistrep, with and
without supplemental algal feeding . Data are a composite of several
experiments which included Combistrep-treated cultures, together with
suitable untreated controls . Each point represents a mean length of 50
larvae from a single population of approximately 10,000 larvae.

(Fig. 2). Figure 2 represents a composite of Combistrep-treated
cultures, some of which received algal food supplements while others
did not. Controls not receiving Combistrep are included in all cases,
however. Because of the sensitivity of oyster larvae to variations in
algal food quality, it is often difficult to distinguish larvae receiving
algal food from those not receiving the food. The composite of mean
lengths of fed and unfed larvae, however, clearly shows the value of
Combistrep in increasing growth rates .

28

195

176

2

95

75

C. VIRGINICA

100 200 300 400

P.RM. COMBISTREP

500

600

Fig. 2. Mean lengths of oyster, C. virginica, larvae at 14
days of age receiving several dosage rates of Combistrep. Data are a
composite of several experiments which included Combistrep-treated
cultures, together with suitable untreated controls. Each point
represents a mean length of 100 larvae from a single population of
approximately 10,000 larvae.

Effects of Combistrep on Bacterial Populations

It was noticed in the above experiments that, although Combi-
strep did help to prevent mortalities in cultures of clam and oyster
larvae, the bacterial population of larval cultures treated with Combi-
strep and some other antibiotics became actually higher than bacterial
populations in untreated cultures . Experiments were then designed to
determine the effect of different concentrations of Combistrep on
bacterial populations in sea water.

-29-

One-liter cultures of filtered ultraviolet -light-treated sea water
were set up and given several different concentrations of Combistrep
but without larvae and algal food. After 48 hours at 24 + 1 C samples
for plate counts of bacteria were taken. The 48-hour cultures were
plated at several dilutions using standard plating techniques on
Trypticase Glucose Yeast Extract Agar made up with sea water. Bac-
terial colonies were counted; no attempt was made to determine the
species of bacteria represented .

Increasing dosages of the sterile antibiotic resulted in almost
directly proportional increases in bacterial numbers (Fig. 3). This

120

•

100

J

z eo

~

ce

Id

0.

^ •

<

E 60

~

B

u

^

t-

I

o

<

CD

•

•

A 40

-

g

is

20

iff'

I

'

1

i

i

200

400

600

800 1000

PPM. COMBISTREP

2000

Fig. 3 . Numbers of bacteria per ml of filtered sea water 48
hours after application of several concentrations of Combistrep. Dupli-
cate sea water cultures were used at each test concentration. Points
represent each of duplicate plate counts made of each sea water
culture .

-30-

effect was not noted at the lowest concentrations (50 to 100 ppm). At
concentrations of Combistrep that have been especially beneficial to
larvae (2 00 to 4 00 ppm), there was approximately a two-fold increase
in bacterial numbers over Combistrep-free cultures. Although the
actual number of bacteria differed somewhat from experiment to experi-
ment, the percentage increase with increasing dosages of Combistrep
remained relatively constant .

Effect of Bacteria on Growth of Larvae

At this point it was important to determine whether the bivalve
larvae were deriving benefit from the Combistrep directly or possibly
from the increased bacterial populations that resulted from its addition
to sea water. To do this we were eventually able to compare growth
of larvae in aseptic and in bacterized Combistrep-treated cultures .
Before we had developed methods for obtaining sterile larvae for
aseptic cultures, however, several experiments were run in an attempt
to correlate the rate of growth of larvae with the number of bacteria
present .

It was postulated that if bacteria here were important in growth,
then larvae receiving Combistrep and sea water with a fully developed
bacterial population should grow more rapidly than larvae receiving
Combistrep and sea water which were sterile. Consequently, non-
sterile clam larvae receiving Combistrep were reared in sea water
(1750 larvae in 500 mis) pretreated as follows:

1 . Autoclaved and aged one week with sterile Combistrep
added at the time of addition of water to cultures of
larvae (sterile water supply).

2. Autoclaved, then aged one week after Combistrep plus
a non-sterile sea water inoculum had been added
(fully developed initial bacterial populations).

Larvae in control cultures, not receiving Combistrep, were reared in
sea water with the following pretreatment:

1 . Autoclaved , then aged one week .

2 . Autoclaved, then aged one week after a non-sterile

sea water inoculum had been added.

3 . Autoclaved with live flagellate food added at the time

of feeding to cultures of larvae .

-31-

The water in each culture was completely renewed every 24 hours and
temperatures held to 20 C to minimize buildup of bacteria in treatments
receiving the initially sterile water supplies . Duplicate cultures were
used to test each treatment in each of the three replicate experiments.
Results are shown as mean lengths of larvae after eight days of culture
(Table 1). Mortality of larvae in all cases was negligible and thus is
not included .

In the first experiment larvae grown in the presence of Combi-
strep with a fully developed initial bacterial population reached a mean
length of 149. 65u, while those grown with Combistrep and sea water
which were initially sterile reached only 117.90u. Control cultures,
i.e., those reared in sterile sea water and those kept in aged water
plus the bacterial inoculum, also showed poor growth. Although the
149. 65u mean length was appreciably less than the 175. 15p mean
length attained by larvae receiving live flagellates as food, it repre-
sents a significantly faster rate of growth than was achieved in control
cultures .

The second experiment (Table 1), although conducted in exactly
the same manner as the first, produced quite different results . In this
trial 5 00 ppm Combistrep actually retarded clam growth, both when
given with a sterile water supply and with a fully developed bacterial
population. Since 5 00 ppm Combistrep is above an optimum dosage for
clam larvae (Fig. 1), variable results might be expected. Growth was
not reduced in a control culture within this experiment given only 250
ppm Combistrep. In a third trial Combistrep concentrations were
adjusted to 25 0 ppm, which is a more nearly optimum concentration for
clam larvae .

At 250 ppm (Experiment 3, Table 1) Combistrep-treated sea
water with a fully developed initial bacterial population again pro-
duced good growth of clam larvae . Larvae kept in this water attained
a mean length of 166. 53u, a length not much smaller than the 189. 05u
achieved by larvae receiving live flagellate food . Larvae receiving
sterile Combistrep and sterile sea water showed some growth (13 5t-i
mean length), while larvae kept in the sterile sea water and those kept
in non-sterile sea water without Combistrep showed very little growth
(121.20 and 1 1 6m. mean lengths, respectively).

Plate counts were made immediately after a change of sea
water and again 24 hours later in this experiment to determine the
typical numbers of bacteria at 0 and at 24 hours with each treatment
(Table 2).

-32-

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-33-

Table 2 . Numbers of bacteria present in non-sterile larval clam

cultures receiving different treatments (Experiment 3, Table 1).

Counts were made at times of water renewal (0 hours) and

at the end of each change cycle (24 hours).

Bacteria per ml

Sea water culture supply

0 Hours 24 Hours

Autoclaved

Autoclaved + Combistrep

Autoclaved + non-sterile
inoculum (aged)

Autoclaved + non-sterile
inoculum + Combistrep
(aged)

19,000—21,000 550,000—580,000
33,000 4,000,000

50,000—100,000 65,000—150,000
100,000—180,000 145,000—500,000

Although bacterial numbers were low at 0 hours in the cultures
receiving the sterile water (19,000 to 33,000 bacteria per ml), there
was a rapid buildup in the cultures by 24 hours (550, 000 to 4,000,000
bacteria per ml). On the other hand, numbers of bacteria were initi-
ally high in cultures receiving the non-sterile water treatments
(50, 000 to 180, 000 per ml) and showed a slower rate of increase in
these cultures (65, 000 to 500, 000 per ml) at 24 hours . The aged sea
water that had received the non-sterile inoculum plus Combistrep had
considerably more bacteria per ml, both at 0 and at 24 hours, than the
aged sea water that had the bacterial inoculum only. These results
are in general agreement with experiments in which we determined the
effect of Combistrep on the number of bacteria in sea water as pre-
viously described .

In subsequent experiments bacteria-free larvae were used to
test the effect of Combistrep in aseptic vs . non-sterile cultures .
Fertilized clam eggs collected on sterile stainless steel screens were
washed several times with autoclaved sea water. These eggs were
then permitted to develop into 48-hour veliger larvae in the trivalent
antibiotic solutions described by Guillard (1959). Five

-34-

to ten of these sterile larvae were then transferred aseptically to 10
ml of autoclaved sea water in each of 16 test tubes for each of the
following treatments:

1. No treatment (sterile control)

2. 250 ppm sterile Combistrep (Combistrep only)

3. 0.1 ml non-sterile sea water (bacteria only)

4. 250 ppm sterile Combistrep + 0.1 ml non-sterile sea
water (Combistrep + bacteria) .

After sterile larvae had been added cultures were held 11 days
at room temperature. Sterility tests in sea water-thioglycollate were
run on treatments 1 and 2 (above) at the end of the experimental cul-
ture period . Larvae in all vials were then killed and length measure-
ments were taken of all larvae that had survived the test period .

Of several such experiments to measure growth of clam larvae
receiving Combistrep under septic and aseptic conditions, only one
was successful. In this experiment many of the culture tubes of the
sterile groups remained sterile to the end of the test period, permit-
ting valid measurement of the effect of treatment. Larval survival in
this experiment was variable, but due to the low initial larval density
survival did not appear to affect growth rates . The results expressed
as mean lengths of larvae are listed in Table 3 . In addition to the
four original experimental treatments, two additional categories
developed . These were the mean lengths of larvae from several cul-
tures of the two originally sterile treatments which proved non-sterile
by the end of the culture period . Although all larvae grew poorly,
differences in mean lengths between treatments were highly significant
statistically. An analysis of variance gave an F value of 21.82, indi-
cating an overall difference of means significant far greater than the
99% confidence level (Snedecor, 1962).

Tests of significance of differences between individual treat-
ments within this experiment showed that (a) mean length of sterile
larvae (102.82(a) was not significantly different (at the 99% confidence
level) from sterile larvae receiving Combistrep (103.68u); (b) sterile
larvae (102.82(a) did not differ significantly from those receiving the
non-sterile inoculum but no Combistrep (105. 54u); (c) larvae receiving
Combistrep plus the non-sterile inoculum (110.84(a) were significantly
larger (at the 99% confidence level) than either those receiving Combi-
strep under sterile conditions (102. 82^) or those that had received
only the non-sterile inoculum (105.54|a).

-35-

Table 3 . Mean lengths of clam larvae after 11 days of culture
receiving several different treatments

Treatment Mean length
(u)

1. Sterile 102.82

2. Sterile + 250 ppm Combistrep 103.68

3. Bacterized non-sterile 105.54

4. Non-sterile + 250 ppm Combistrep 110.84

la . Sterile (contaminated) 106.14

2a. Sterile + 250 ppm Combistrep

(contaminated) 108.53

Also notable was the fact that the larvae receiving the
sterile-Combistrep treatment, which accidentally became contaminated
(108.53u ), were significantly larger than the other replicates within
the treatment which remained sterile to the end of the culture period
(103.68u).

DISCUSSION AND CONCLUSIONS

The mechanism by which Combistrep induces greater bacterial
populations in sea water can only be speculated upon at this time .
Experiments thus far have only measured increase in gross numbers of
bacteria without regard to possible species selection. It is possible
that Combistrep is acting to inhibit certain toxin-producing species,
thus allowing greater total numbers. The possibility of the minimal
quantity of citrate present in dilute Combistrep acting as an energy
source seems remote .

The series of experiments in which non-sterile clam larvae
were exposed to Combistrep-treated sea water both sterile and with
fully developed bacterial populations initially present indicated that
the rate of growth of larvae was associated with the number and

-36-

probably the species of bacteria present and suggested that the larvae
were using these bacteria as foods. In the two experiments in which
Com bi strep was beneficial to larvae (Exps . 1 and 3, Table 1), those
larvae cultured in Combistrep-treated water with high initial populations
of bacteria present in each case showed markedly greater growth than
those receiving the initially-sterile water plus Combistrep. Although
clam cultures receiving Combistrep with low initial bacterial numbers
contained appreciable populations of bacteria by the end of the 24-
hour water change cycle, there undoubtedly were significantly fewer
bacteria present in these cultures throughout most of the 24-hour
change cycle than in those receiving Combistrep with initially high
bacterial populations. The fact that larvae in cultures receiving the
bacterized aged water without Combistrep showed poor growth even
though there were considerable bacterial populations present, through-
out, would indicate that Combistrep was selecting and accelerating the
growth of only certain beneficial species of bacteria .

The experiment using bacteria-free larvae showed that clam
larvae in bacterized Combistrep-treated cultures grew, whereas those
kept in sterile Combistrep-treated cultures and those kept in sterile,
non-treated cultures showed little or no growth. This again demonstrated
that it was the bacteria associated with the Combistrep treatment, not
the Combistrep, itself, that caused the more rapid growth of bivalve
larvae. Again, larvae in bacterized cultures that did not receive Comhi—
strep showed less growth than in similar cultures containing Combi-
strep. This supports the above contention that the Combistrep-induced
bacteria are perhaps of preferential utility to the larvae.

It is conceivable that the increased numbers of bacteria may at
times be too great (over 500 ppm Combistrep). Such a biological mass
might nullify beneficial effects by the creation of adverse environ-
mental conditions for the larvae, such as reduction of dissolved oxygen,
creation of toxic metabolites, etc. The variable results measured at
500 ppm Combistrep between experiments (Table 1) may be the result
of this .

These studies, of course, do not show beyond all doubt that
bacterial populations are utilized directly as food by bivalve larvae
but do very definitely associate increased and probably selected
bacterial populations with larval growth. As we learn more about these
relationships, it may be possible to control bacterial populations to
such an extent that they may become generally useful in future shellfish
hatcheries, possible circumventing the presently difficult culture of
relatively fastidious live algal food cells.

-37-

SUMMARY

1 . Routine addition of the proprietary antibiotic formulation,
"Combistrep" (dihydrostreptomycin-streptomycin sulfates) to cultures
of clam or oyster larvae has consistently resulted in 25 to 100 per cent
increases in the rate of growth of the larvae .

2 . The addition of sterile Combistrep to filtered ultraviolet-
light-treated sea water has produced an increase in numbers of bacteria
roughly proportional to the concentration of Combistrep.

3 . Tests with non-sterile Combistrep-treated cultures showed
that clam larvae grew faster in Combistrep-treated cultures with a high
initial bacterial count than in cultures with a low initial count.

4. In bacteria-free cultures the addition of Combistrep did not
increase the rate of growth of clam larvae.

5. All data indicate that the incre-isea rate of growth of larvae
receiving Combistrep treatment is associated with the increase in
numbers of a possibly selected group of bacteria .

LITERATURE CITED

Carriker, M. R. 1956. Biology and propagation of young hard clams,
Mercenaria mercenaria . J . Elisha Mitchell Soc . 72:57-60.

Coe,W.R. 1947. Nutrition in filter-feeding bivalve mollusks . Anat
Rec. 99:112.

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

Davis, H. C. and P. E. Chanley . 1956. Effects of some dissolved
substances on bivalve larvae. Proc . Nat. Shellfish. Ass.
(1955) 46: 59-74.

Davis , H . C . and R . R . Guillard . 1958 . Relative value of ten genera
of microorganisms as foods for oyster and clam larvae. U.S.
Fish Wildl. Sen/., Fish. Bull. 136, 58:293-304.

Guillard, R. R. 1959. Further evidence of the destruction of bivalve
larvae by bacteria . Biol. Bull. 117:258-266.

-38-

Loosanoff, V. L. and H. C. Davis, 1950. Conditioning V. mercen-
aria for spawning in winter and breeding its larvae in the
laboratory. Biol. Bull. 98:60-65.

Loosanoff, V. L., H. C. Davis, and P. E. Chanley . 1955. Food
requirements of some bivalve larvae. Proc . Nat. Shellfish.

Ass. (1955) 45:66-83.

Rodinca, A. G. 1948. Bacteria as a food for fresh-water mollusks.
Mikrobiologiia 17:232.

Snedecor, C.W. 1962. Statistical methods (Fifth ed.). Iowa State
College Press, Ames, Iowa, 534 p.

-39-

A METHOD FOR INCREASING SURVIVAL OF LOCALLY-CAUGHT
PACIFIC OYSTER SEED IN WILLAPA BAY, WASHINGTON

Clyde S . Sayce

State of Washington Department of Fisheries

ABSTRACT

To enable the oyster industry to increase survival of local Pacific
oyster spat through the first winter, it is recommended that shell strings
be piled on hard bottom above low tide level until spring. This technique
is basically that used in Japan. In the first commercial trial in Willapa
Bay, 7,000 local shell strings held 6 ft above mean lower low water
level from September 1957 to April 1958, then crushed and planted,
yielded 3.68 gallons (13.9 liters) of oyster meats per shell string in the
1961—1962 season, substantially above usual production (less than 1
gallon per string).

Seed growers of Miyagi Prefecture, Japan, catch Pacific oyster
spat on hanging cultch in July and August (Imai et al., 1951). The
cultch strings are placed on flat hardening racks high in theintertidal
zone during September after the major summer heat is past but before
spat growth makes them too large for export. These racks are 12 to 18
inches above the bottom, and the strings are laid horizontally on them
in layers two to four strings deep where they remain until selected and
boxed for shipment to Washington State growers during February, March,
and early April of the following year. The hardening process retards
growth of spat and causes them to develop tightly closing valves which
aids their survival during shipment.

In Willapa Bay it is customary to catch oyster seed in July and
August, then remove the cultch strings from racks and put the shells on
growing beds during fall months before winter storms begin. Because
this bay is turbid, much silting occurs during winter, to the detriment
of these small oysters . Since the Pacific oyster is very hardy and can
withstand the harsh Japanese weather during the seed hardening period,
it can survive the milder climate of Willapa Bay without undue mortali-
ties, providing the seed is kept off the bottom and out of silt. Experi-
ments in which seed oysters are held in trays from two to six inches
above the bottom indicated that, in the absence of silt, spat mortalities
during the first winter were due to space competition for growth.

■41-

Quayle (1957) showed that tray culture gave 64.4 per cent sur-
vival against 3 7.1 per cent survival on the ground for Pacific oyster
seed from 28 April 1956 to 17 November 1956, a period of seven months.
In a study at the Willapa laboratory, one tray containing 1,278 Pacific
oyster seed had 513 (40.2 per cent) survival from 25 April 1956 to
18 March 1957. In another tray containing 1,017 oysters (seed), 563
(55.4 per cent) survived from 29 May 1956 to 19 March 1957. These
survival percentages are not as high as Quayle' s, but support his
findings that in the absence of silting, space competition is the most
important cause of spat mortalities, although predation or disease may
be significant in certain areas or in specific cases . Quayle' s experi-
ments and those at Willapa Bay indicate that few mortalities occur after
the first year. Annual growth and mortality surveys of Pacific oysters
in Washington waters have shown that, in Willapa Bay at least, signi-
ficant mortalities in older year classes of oysters do not occur. In his
productivity experiments, Quayle (1954) found no difference between
broken and unbroken seed because greater numbers of oysters in
broken seed cases were offset by higher mortalities suffered in the
first year from burying and fouling . Using numbers of oysters as an
indicator of yield, his experiments showed productivity ranging from
2 , 168 to 6, 295, with an average of about 4, 000 oysters per case . This
is in agreement with commercial production figures and indicates that
maximum survival to harvest for Japanese oyster seed with a minimum
of 12,000 spat per unbroken case and 16,000 spat per broken case is
not very high .

In the Willapa Bay cultch survey (Sayce, 1958), counts of spat
per equivalent case for local seed ranged from 81,000 to 108,000 when
six shell strings per case were used as a basis of comparison. This
may be considered as high-count seed, yet no oyster company operat-
ing in Willapa Bay has reported a production in excess of one gallon of
oysters per shell string. From this, it is apparent that the potential
oyster production from locally-caught seed is not being realized. To
increase production from local seed, more young oysters must survive
their first winter, and of the two important causes of early mortality,
space competition and silting, the latter seems to be the easier to
control .

When good or excellent local Pacific oyster sets occur, com-
petition for space on the mother shell is lessened by breaking,
crushing, or otherwise fragmenting the shell before placing it on oyster
ground . However, if this is done in fall or winter months, silting
mortalities negate the advantage unless the seed is placed directly on
hard, silt-free ground. By holding seed oyster strings, after spatfall,
on suitable silt-free ground until the following spring, then placing

-42-

them broken or unbroken on growing ground, locally-caught Pacific
oyster seed will provide production comparable to that of imported seed .

To insure high survival of seed, the holding area must be care-
fully chosen. In Willapa Bay, this area must lie less than 7.0 feet
above mean lower low water and should be no higher than about 5.5
feet above MLLW level so that shell strings may be piled two to four
strings deep and still be adequately covered with water during high
tides. Any suitable area below this level may be used if uncovered
at low tide. Higher levels will retard spat growth, and if shell strings
are held below mean lower water level, some spat growth will occur.
This will cause undue spat mortalities during movement to oyster beds
in the spring . Small spat withstand shell breaking or crushing opera-
tions with fewer mortalities than do large spat. Therefore, the tide
level at which shell strings are held should be picked with this in mind .

Holding shell strings up out of silt may be accomplished by
placing them on a natural oyster reef, on a graveled area, or on an area
which has been prepared with several inches of oyster shell. Poly-
ethylene sheeting may be used in place of, or under, oyster shell to
prevent shell strings from gradually settling and becoming silted.
These types of silt-free areas have been successfully used to hold
locally-caught Pacific oyster seed through the first winter on a com-
mercial scale .

The first commercial trial of this method was carried through to
harvest with success. In this operation, 7,000 local shell strings
were held at the 6 .0 foot level above mean lower low water from late
September 1957 until April 1958. At this time, the shells were crushed
and planted in the Stackpole Harbor area of Willapa Bay. At comple-
tion of harvest in the 1961-62 production season, the company realized
3 .68 gallons of oysters per shell string . This was a substantial
increase of production over local shell strings handled in the usual
manner .

Too often local seed is treated as bonus seed, planted when
ltast able to survive, never broken and scattered into singles, seldom
moved from growing areas to a good fattening bed, and often placed on
marginal fattening ground resulting in low production. In order to
fully utilize the production potential of locally-caught Pacific oyster
seed, it must be protected from silting mortalities during the first
winter and be placed on good growing ground the following spring . The
increased survival of oysters will be measured by increased production
in gallons of oysters at harvest time .

-43-

LITERATURE CITED

Imai, T., M. Hatanka, R. Sato, and S. Sakai . 1951. Ecology of
Mangoku-Ura Inlet with special reference to the seed oyster
production. Sci . Rep. Res. Inst. TohukuUniv. D. 1-2:
137-156.

Quayle, D. B. 1954. Oyster productivity . British Columbia Dep .
Fish. 5 (3), 20 p.

Quayle, D. B. 1955. Survival of high count Japanese oyster seed.
British Columbia Dep . Fish. 6(1), 9 p.

Quayle, D. B. 1957. Early mortality in oyster seed. British Columbia
Dep. Fish. 8 (3), 35 p.

Sayce, C. S. 1958. Willapa Bay Pacific oyster cultch survey for the
years 1955, 1956, 1957. Dep . Rep . Washington Fish . Dep .
3 p.

Woelke, C. E. 1957. The quality of seed oysters from Japan. Wash-
ington Dep . Fish . Res . Papers 2(1): 135-142.

1959. Pacific oyster Crassostrea gigas mortalities with

notes on common oyster predators in Washington waters . Proc .
Nat. Shellfish. Ass. 50:53-66.

-44-

NOTES ON THE OCCURRENCE OF DERMOCYSTIDIUM MARINUM
ON THE GULF OF MEXICO COAST DURING 1961 AND 1962

Sammy M . Ray

Marine Laboratory

Texas A&M University

Galveston, Texas

ABSTRACT

In October 1961 this parasitic fungus was found in 8 of 9 Florida
samples of oysters from Tampa, Apalachicola, and Pensacola Bays.
During the period October 1961-May 1962 it was found in all of 12
oyster samples from a Louisiana area extending from Bay Denesse
(east of Mississippi River) to Bayou Du Nord Ouest (north of Lake
Chien). During the period January-May 1962 it was found in 15 of 18
samples of Texas oysters from 10 stations in the Galveston area. The
only uninfected samples were one from St. Vincent's Reef in Apalachi-
cola Bay, and three from a low salinity station near Smith's Point in
Galveston Bay.

INTRODUCTION

During 1961 I received reports of very low incidences of the
oyster fungus parasite, Dermocystidium marinum , in the Gulf of
Mexico. Some reports suggested that the parasite had practically
disappeared from areas that in previous years were heavily infested.
In view of the reduced water salinities during 1959, 1960, and 1961
in many Gulf Coast oyster-growing regions, especially in Louisiana,
a drop in the incidence of D . marinum in some oyster populations
would be expected but not to a point approaching "disappearance" in
such areas as lower Barataria Bay.

These reports led me to conduct a survey in Florida, Louisiana,
and Texas to obtain data on the occurrence of D . marinum, and to
test modifications in the original thioglycollate culture technique,
which preliminary studies indicated would improve this method for
detecting D. marinum . This paper presents the results of the survey
conducted during late 1961 and early 1962. The parasite was found
in 35 of 39 samples of live oysters taken in three Gulf states.

-45-

MATERIALS AND METHODS

The thioglycollate culture method (Ray, 1952a and 1952b) for
diagnosing D • marinum was used . In some cultures the original pro-
cedure, which utilizes penicillin G and dihydrostreptomycin to retard
bacterial growth, was modified with regard to antibiotics . In one
modification mycostatin (nystatin) was combined with these two
antibiotics; in the other modification mycostatin and Chloromycetin
(chloramphenicol) were substituted for penicillin and dihydrostrepto-
mycin . The details of antibiotic concentrations employed will be
presented in the paper following this one .

Both rectal and mantle tissues were cultured to obtain com-
parative data on the original and modified methods. The rectum (that
portion of the intestine extending over the adductor muscle) was
split longitudinally into approximately two halves; one piece was
cultured in original medium and the other in modified medium. This
difficult and time-consuming procedure was discontinued after October
1961 . During 1962, pieces (approximately 5x10 mm) of the anterior
portion of the right mantle (referred to as mantle "A" in the text and
in Tables 3,4, and 5) just lateral to the palps were cultured for
comparative purposes . Unpublished data gathered by the author had
indicated that often more D . marinum cells are found per unit of
mantle tissue in the region adjacent to palps than in the region near
the adductor muscle. Care was taken to alternate the tubes represent-
ing each method in order that the anteriormost piece of mantle tissue
from about half of the oysters in the sample would be tested by each
method .

Data on the relative infection intensity in mantle A and the
rectum were obtained for several samples . In such cases a piece of
the mantle (approximately 5x10 mm) and the rectum were cultured
in the same tube .

Some samples of oysters were cultured shortly after being
removed from the water and others remained out of water for two or
three days before being processed . All Louisiana oysters cultured
21 October 1961 had been stored in the boat slip of the Louisiana
Department of Wild Life and Fisheries Laboratory on Grand Terre
Island for several days before cultures were started. Since this
storage occurred during October, I do not believe that the levels of
D . marinum infections were increased .

The largest oysters were selected from most samples since
older oysters are more likely to be infected with D . marinum than

-46-

younger ones . Some samples were rather small and most of the
oysters in the sample were cultured in spite of the selectiveness .

RESULTS

The incidence and weighted incidence (Mackin, 1962) of
D. marinum in oysters from widely-scattered areas in Florida,
Louisiana, and Texas are presented in Tables 1-5. Eight of the nine
Florida samples were positive for D. marinum . The negative sample
came from St. Vincent's Reef, Apalachicola Bay. The incidence of
infection in positive samples varied from 2 0 to 100 per cent, with
weighted incidence ranging from 0.60 to 2.85.

Twelve samples taken in Louisiana were positive for this
parasite; the incidence obtained by the original method varied from
19 to 90 per cent and the weighted incidence ranged from 0.23 to 1.95.

All of the Texas samples were infected with D . marinum
except three samples taken at well 48 (near Smith's Point) in Galves-
ton Bay. The salinity is generally lower there than at other Galveston
Bay sampling stations because water from Trinity River tends to flow
along the eastern shore of the bay. Incidence in positive samples
varied from 25 to 100 per cent, and weighted incidence varied from
0.39 to 2.00. Weighted incidence of all positive samples from West
Bay and Galveston Bay exceeded 1.00, even during January. The
average infection level for the oysters in these samples varied between
"light" and "light to moderate," according to Mackin's weighting
system .

EXPLANATION OF TABLES 1-5

The antibiotics used in the culture medium are identified in the
tables by the following abbreviations: "P+S" for penicillin and dihydro-
streptomycin; "M+P+S" for mycostatin, penicillin, and dihydrostrepto-
mycin; and "M+C" for mycostatin and Chloromycetin.

Information on the sources of the oysters and the extremes of
length (right valve) as well as the mean length are also presented in
the tables. Samples taken from leased oyster beds (either planted or
natural populations) are identified by using the name of the lease
owner, except the sample from Bay Denesse, Louisiana, which is a
composite sample from six leases . All other oyster samples were col-
lected directly from natural growing areas, except the East Bay
(Pensacola, Florida) stock, which was transferred to Santa Rosa Sound

-47-

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about August 20, 1961. The oysters taken at Galveston Bay sampling
stations identified by "well numbers" (Humble Oil and Refining Com-
pany) were from natural populations growing on shell pads placed on
the bottom to support drilling equipment. The Galveston Bay stations
are approximately in mid-region of the bay and are arranged in Tables
4 and 5 to indicate their location generally along an east-to-west line.
The eastem-most station is well 48 near Smith's Point and the western-
most station is Todd's Dump just west of the Houston Ship Channel.

DISCUSSION

If the data obtained during this survey are representative, it
appears that D. marinum is still prevalent in many Gulf Coast areas,
especially in moderate to high salinities. Lower Barataria Bay,
Louisiana, was one of the areas from which low incidences of D .
marinum were reported during 1961. The data obtained during this
study indicate that the oyster populations sampled from lower Barataria
Bay were highly infected during October 1961 . High mortality and high
levels of D . marinum infections are characteristic of market-sized
oysters that spend the summer and fall in this area . I have no data on
D. marinum from this area between March 1960 and October 1961.
This period covers much of the time during which the low levels of
D . marinum were reported .

During March 1960 live oysters from three experimental trays
that were maintained at Sugar House Bend, lower Barataria Bay, for
approximately one year were checked for D . marinum . The incidence
of infection varied from 46 to 100 per cent and weighted incidence
varied from 0.56 to 1.33. Live oysters from three experimental trays
maintained in Bayou Rigaud, Louisiana, during the same period showed
incidences of infection ranging from 7 0 to 90 per cent and the weighted
incidence varied from 0.83 to 1.10. The oysters in all six trays had
extremely low levels of D. marinum infection when they were trans-
ferred from Bay Chene Fleur, Louisiana, to Sugar House Bend and
Bayou Rigaud during February and March of 1959. Based on the limited
data presented above, it seems likely that oysters in lower Barataria
Bay had ample opportunity to become infected during the summer of
1959. Furthermore, those oysters surviving through the winter (1959)
and spring (1960) probably carried infections that became elevated
during the summer and fall of 1960. A sharp drop in D. marinum
infection levels may have occurred during the winter (196 0) and spring
(1961), but the high level of infection found in many Louisiana samples
in October 1961 suggests that the parasite should not have been diffi-
cult to detect in market-sized oysters during the summer of 1961 .

-53-

Although I do not have any direct evidence to account for the
reported low incidences of D. marinum during 1961, I believe that
faulty technique may account for some of them . This conclusion is
based on the data presented in this paper and subsequent discussions
with some of the individuals who had originally reported the scarcity
of D . marinum in areas where oysters are often highly infected .

The suspected errors in technique as well as modifications,
based on the comparative data obtained during this survey with regard
to antibiotics, for improving the thioglycollate culture method will
be presented in the following paper.

ACKNOWLEDGMENTS

I wish to thank Dr. Lyle S. St. Amant, Louisiana Wild Life
and Fisheries Department; Dr. Philip A. Butler, Bureau of Commercial
Fisheries; and Mr. Robert M. Ingle, Florida State Board of Conserva-
tion for providing many of the oyster samples examined during this
survey .

LITERATURE CITED

Mackin, J . G . 1962 . Oyster disease caused by Dermocystidium
marinum and other microorganisms in Louisiana. Publ .
Inst. Mar. Sci . Univ. Texas 7 (196 1): 13 2-229 .

Ray, S . M . 1952a . A culture technique for the diagnosis of infec-
tion with P^nnocj^tidjmn marmiirn Mackin, Owen, and Collier
in oysters. Science 116:360-361.

Ray, S . M . 1952b . A culture technique for the diagnosis of infec-
tion with Dermocystidium marinum in oysters . Nat . Shellfish .
Ass. Conv. Add. 1952:9-13 .

-54-

A REVIEW OF THE CULTURE METHOD FOR DETECTING

DERMOCYSTIDIUM MARINUM, WITH SUGGESTED

MODIFICATIONS AND PRECAUTIONS

Sammy M . Ray

Marine Laboratory

Texas A&M University

Galveston, Texas

ABSTRACT

Tissues from Gulf of Mexico oysters cultured with mycostatin (nystat-
in) and Chloromycetin (chloramphenicol) often showed more and larger
cells of the parasitic fungus Dermocystidium marinum than did tissue
cultured with penicillin and dihydrostreptomycin. The difference was
greater in spring than in October. The thioglycollate culture method for
diagnosing D. marinum is reviewed and improvements are suggested.
Mycostatin and Chloromycetin, 200 units and 200 micrograms, respec-
tively, per ml of medium, are recommended instead of penicillin and
dihydrostreptomycin.

INTRODUCTION

The thioglycollate culture method for detecting Dermocystidium
marinum in oysters was developed about 10 years ago (Ray, 1952a and
1952b). Although the method is not complicated, I believe that occa-
sionally the parasite is either overlooked or found in less than actual
abundance because of faulty technique. This belief is due to reports
received during the summer of 1961 (Ray, 196 6) that D. marinum was
almost impossible to detect in some Gulf of Mexico areas, where
previously it had been prevalent.

My first reaction to these reports was that D . marinum had
"disappeared" because it seemed unlikely that errors in technique
could be grave enough to allow moderate and heavy infections to
escape detection with such consistency. During the summer of 1961,
however, I encountered an apparent error in technique associated with
a reported failure to find D. marinum in the Gulf. When the suspected
error was corrected, high levels of infection were found consistently
in oysters from the area in question .

My increasing concern regarding the validity of some D.
marinum incidence data, new data concerning antibiotics, and several
years of additional experience, have prompted me to present modifications

■55-

and precautions that should increase the reliability of the culture
method for detecting D . marinum in oysters.

A step-wise synopsis of the procedure is presented below for
rapid reference . The steps that contain modifications of the original
procedure (Ray, 1952a and 1952b) are indicated by an asterisk.

1. Rehydrate 29.3 grams of Fluid Thioglycollate Medium
(Difco, No. 1.0256-02 or Baltimore Biological Laboratory,
No. 01-140) in 1 liter of distilled water containing 20
grams of NaCl .

2. Dispense rehydrated medium in 10-ml amounts in culture
tubes and autoclave . Store sterile tubes of medium in the
dark at room temperature until needed.

*3 . Fortify each tube of medium with 2 00 units of mycostatin
(nystatin) and either 2 00 micrograms of Chloromycetin
(chloramphenicol) or 500 units of penicillin G and 500
micrograms of dihydrostreptomycin per ml of medium just
prior to use .

*4 . Plant test tissues (gill , mantle, and/or rectum) in the
fortified tubes of medium and incubate in the dark at
room temperature for at least one week .

*5 . Blot the incubated tissue on absorbent paper toweling,

flood it with 2 or 3 drops of diluted Lugol's solution, and
then tease tissues into fine bits .

6. Examine stained tissues microscopically at 25X to 100X
magnification for brown, green, blue, and blue-black
spheres .

CULTURE MEDIUM

The use of thioglycollate media that do not contain dextrose
appears to have been the cause of at least one consistent failure to
detect D . marinum ♦ More than one laboratory had such a medium on
its shelf and had possibly used it to check for D . marinum . Dextrose
is one of the constituents of Fluid Thioglycollate Medium that is
required for consistent enlargement of D . marinum cells in some
oyster tissues (Ray, 1954a).

-56-

A Fluid Thioglycollate Medium without dextrose, such as
Baltimore Biological Laboratory, No. 01-3 94, may be used if 5 grams
of dextrose and 24 .3 grams of dehydrated medium are used to prepare
a liter of medium . Except as noted above and in the synopsis, pre-
pare and store the medium according to the instructions on its con-
tainer .

ANTIBIOTICS

Antibiotics are added to the sterilized tubes of medium to pre-
vent excessive bacterial growth and tissue putrefaction. The enlarge-
ment of D . marinum may be inhibited if the cultures become excessively
putrid during the early stages of incubation. Furthermore, after prolonged
incubation in putrid cultures some tissues may become too badly decom-
posed for proper examination . Since some antibiotics are readily
inactivated at room temperatures and/or by light, these agents should not
be added to the medium until shortly before inoculation and the cultures
should be incubated in the dark .

Recent studies, which will be elaborated upon in this report,
indicated, however, that the culture method may be improved by employ-
ing antibiotics other than penicillin G and dihydrostreptomycin . The
data suggest that mycostatin (nystatin), a broad-spectrum, antifungal
agent and Chloromycetin (chloramphenicol) are more suitable than the
originally recommended antibiotics as antimicrobial agents . In this
paper the abbreviation "P+S" indicates the use of both penicillin
(5 00 units/ml of medium) and dihydrostreptomycin (5 00 micrograms/ml
of medium) in the cultures. The abbreviation "My" indicates either
the combination of mycostatin, penicillin, and dihydrostreptomycin or
the combination of mycostatin and Chloromycetin. In the former combi-
nation, used during October, 1961, about 100 units of mycostatin were
added per ml of medium; and in the later combination, used during
1962, about 200 units of mycostatin and 500 micrograms of Chloromycetin
were added per ml of medium .

In general, Chloromycetin and the combination of penicillin and
dihydrostreptomycin have about the same effect on the enlargement of
D. marinum . Occasionally in tissues from the same oyster, Chloromy-
cetin will inhibit enlargement of the parasite to a greater extent than
the P+S combination; and in some cases the opposite effect occurs . The
inhibition of enlargement by either Chloromycetin or the P+S combination
is "spared" in the presence of mycostatin. The influence of mycostatin
on enlargement of D . marinum is shown in Figure 2 . The author prefers
the use of Chloromycetin since it appears to have a slightly broader
spectrum for the bacterial flora associated with oyster tissues than the
P+S combination .

-57-

The data presented in the previous paper in this volume (Ray,
1966) show a slightly greater incidence and greater weighted incidence
of D. marinum in My cultures than in P+S cultures for several samples.
The differences, especially in weighted incidence, were greater in the
spring samples than in the October samples . In some spring samples
the weighted incidence value for My cultures was about double that of
P+S cultures. The same data (Ray, 1966) are used to prepare Fig. 1
and Table 1, but the data are presented on the basis of individual
oysters to give an idea of how the infection intensity ratings and cell
counts differed with the two treatments . These methods of presenting
the data also show generally that more D . marinum cells were observed
in My cultures than in P+S cultures .

In many cultures, especially during the spring, numerous cells
were observed at 25X to 3 OX magnification in My-treated tissues,
whereas either no cells or very few cells were observed in P+S-treated
tissues . Subsequent examination of the P+S-treated tissues at increased
magnifications (100X to 43 OX), however, frequently revealed very small
D. marinum cells that were visible only after careful search. These
observations led me to make comparative cell counts of My- and P+S-
treated tissues .

The D . marinum cells v/ere actually counted in all tissue prep-
arations that were estimated to have less than 800 cells visible at
100X magnification. This concentration of cells in a piece of oyster
tissue (about 5x10 mm) is about the upper limit of my estimation of a
"light to moderate" infection.

The data obtained from the comparative cell counts were used to
compute the cell-count ratios: No. cells in My culture , which are
presented in Fig . 1 . No • cells in P+S culture

An examination of Fig . 1 reveals generally that more D. marinum
cells were observed per unit of tissue in My cultures than in P+S cul-
tures . The ratios exceeded 1 for about 80 per cent of the October cell
counts and for about 95 per cent of the spring counts. Furthermore, it
is evident that the disparity in the cell counts for the two methods is
considerably greater in the spring samples than in the October samples.
For example, about 75 per cent of the spring cell-count ratios exceed
5, whereas only approximately 25 per cent of the October cell-count
ratios exceed this value. The maximum ratios were 2 0 to 3 0 for the
October oysters in contrast to maximum ratios of 100 to 3 00 for the
srping oysters. In view of this marked seasonal difference, myco-
statin very likely enhances the enlargement of the proliferating stage (s)

-58-

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-60

Fig. 2. Comparison of effects of mycostatin on the enlarge-
ment of D. marinum in thioglycollate culture. A. and E. Penicillin
(500 units/ml) and dihydrostreptomycin (500 micrograms/ml). B.
Mycostatin (200 units/ml), penicillin and dihydrostreptomycin .
C. Chloromycetin (200 micrograms/ml). D and F . Mycostatin and
Chloromycetin. A-D . Mantle tissue from same oyster; photographed
at 100X magnification, value for each scale unit is 6.6 microns.
E-F. Gill tissue from same oyster; photographed at 3 5X magnification,
value for each scale unit is 21.5 microns. All preparations stained
with diluted Lugol's solution.

-61-

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-63-

of D. marinum to a greater extent than the non-proliferating stage (s).
Possibly the stage (s) affected by mycostatin is either the "amoeboid"
stage of Mackin (1952; or some stage (s) that has not been associated
with this parasite.

Before discussing the data presented in Table 1, some com-
ments should be made regarding D . marinum infection intensity rating
(IR). The IR system (Ray, 1954a, and 1954b; Mackin , 1962) employs
six categories, ranging from "very light" through "heavy," to indicate
the intensity of D. marinum infections . Infection intensities estimated
to fall in the upper and lower portions of a category were rated as plus
(+) and minus (-) , respectively. These subratings, however, were
treated as one category for data analyses. Each category is arbitrarily
assigned a numerical value ranging from 0.5 for "very light" infections
to 5 . 0 for "heavy" infections. These values are used in determining
the weighted incidence (Mackin, 1962). Since intensity rating is a
subjective procedure and since the D . marinum cells often average a
larger size in My cultures than in P+S cultures, there may be a tendency
to rate an infection higher for tissues with large cells than for tissues
with a similar concentration of small cells. Care was taken to consider
the number of cells rather than total mass of D . marinum cells in esti-
mating intensity. As an additional precaution in rating infection inten-
sities, the entire tissue preparation was systematically searched at 100X
magnification, which is the highest magnification that is practical for
routine detection of D . marinum , to reduce the chances of overlooking
small cells .

Table 1 shows the frequency with which the IR of D . marinum
infections in either My or P+S cultures exceeded the other. In the
October samples the IR differed with the two treatments in 85 (50 per
cent) of the oysters showing D . marinum infection; and in 78 of these
cases (about 90 per cent) the IR for the My cultures exceeded the IR
for P+S cultures. Furthermore, as in the case of cell-count ratios
(Fig . 1), the difference between the two types of treatment was more
pronounced in the spring samples than in the October samples . In the
spring samples the IR of 141 (about 85 per cent) of the positive oysters
differed with the two treatments; and in 13 7 of these cases (about 97
per cent) the IR was greater for My cultures than for P+S cultures. The
cell-count data (Fig . 1) indicate that increased intensity ratings
associated with the use of mycostatin are due, at least in part, to an
increase in the number of cells becoming visible, and is not due simply
to a further enlargement of the visible cells .

The cause for the disparity between My and P+S cultures is not
known. Initially, I assumed that inhibition of fungal growths by
mycostatin accounted for this difference, since many more P+S cultures

-64-

than My cultures were contaminated with molds. However, subsequent
examination of many My and P+S cultures that showed no macroscopic
evidence of mold growth after prolonged incubation did not substantiate
this assumption. In many cases the tissues in My cultures showed
either a greater IR or a higher cell-count than tissues in P+S cultures .

An edematous condition of tissues in cultures containing
mycostatin is quite noticeable when compared with tissues cultured
in its absence . The swelling phenomenon may have either a direct
effect by influencing the permeability of the D_. marinum cell wall
or an indirect effect by distributing the nutrients more uniformly through
the tissues (especially mantle and rectum). Frequently, I have noted
that tissues from My cultures tease and stain more readily than tissues
from P+S cultues; this difference is atrributed to the swelling phenomenon

Although I prefer to use 200 units of mycostatin and 200 micro-
grams of Chloromycetin per ml of medium for routine diagnostic work,
the use of penicillin and dihydrostreptomycin instead of Chloromycetin
is satisfactory. Since both mycostatin and Chloromycetin are practi-
cally insoluble in water, one must be sure that these antibiotics are
properly suspended while they are being dispensed. To avoid great
variations in the concentration of antibiotics, it is advisable to fortify
only two or three tubes at a time because these materials, especially
Chloromycetin, settle rapidly.

SELECTION OF TISSUES

The rectum was originally recommended for routine survey work
because this tissue often appeared to contain more parasites than
three other tissues (heart, gill, and mantle) that were tested extensi-
vely. Biologists in the Cheasapeake Bay area usually culture three

Mycostatin, 500, 000-unit vial (List 5915); E . R. Squibb & Sons

2
Chloromycetin, 1-gram vial (S.V.65); Parke Davis & Co. This

ecommended concentration of Chloromycetin, only 4 0 per cent of that
used in the studies reported herein, is based on data obtained from
extensive studies conducted since the submission of this manuscript.
Enlargement of D . marinum cells is occasionally inhibited by Chloro-
mycetin levels of 25 0 to 5 00 micrograms per ml of medium. Inhibition
occurs much more frequently with Chloromycetin alone than in the
presence of mycostatin.

-65-

tissues (rectum, gill, and mantle) from each oyster. These three
tissues are examined as a composite preparation and probably give a
better picture of the overall infection of each oyster than using a
single tissue. D. marinum is more easily observed in gill tissue than
in any other tissue except heart. Furthermore, in recent studies with
Virginia oysters, extremely light infections, presumed to be newly
established, have been found occasionally in gill tissue but not in
either mantle or rectal tissue. This observation indicates that D.
marinum may on occasions, at least, initially invade oysters byway
of the gills rather than through the digestive epithelium as suggested
by histological studies (Mackin, 1951). In well established infections
mantle and rectal tissues often appear to contain more parasites than
gill tissues .

Rectal tissue has a decided disadvantage when the oysters
have well developed gonads . The presence of large amounts of gonadal
material makes the tissue difficult to stain with iodine; consequently,
the parasites may be overlooked in light infections. Furthermore, other
organisms and artifacts found in rectal preparations may confuse
inexperienced users of this technique. The inclusion of appreciable
amounts of adductor muscle in the preparation of rectal tissue makes
intensity estimating more difficult since this tissue usually contains
fewer cells than the rectum. Also, excess adductor muscle increases
the difficulty of compressing the tissue uniformly.

Recent comparative studies (Ray, 1966) with regard to relative
infection intensities in mantle and rectal tissues showed a slight but
probably insignificant higher incidence and weighted incidence in
rectum than in mantle. Mantle tissue was taken from the most anterior
portion of this organ (mantle "A") just lateral to the palps, because
earlier studies often showed noticeably more parasites in mantle tissue
near the palps than in that near the adductor muscle.

I now favor using "mantle A" instead of rectum for the reasons
stated. Also, mantle tissue is easier to dissert out. The rectum is
often damaged in opening the oyster. The entire rectum, that portion
of intestine passing over the adductor muscle, should be used except
in very large oysters; and in small oysters the expanded portion of the
intestine that passes over the pericardium should be included with
rectum. In culturing gill and mantle tissues, pieces about 5x10 mm
should be used .

-66-

INCUBATION AND EXAMINATION OF TISSUES

Previously, I recommended incubating thioglycollate cultures
for at least 48 to 72 hours at room temperature. However, various
workers, including myself, have noted occasionally that D. marinum
cells do not reach maximum enlargement within this minimum period .
Therefore, I suggest incubating the cultures for at least one week.
Inasmuch as my cultures and probably those of most workers usually
are at least a week old when they are examined, I do not believe that
many infections are overlooked because of insufficient incubation.

Tissues usually remain suitable for examination after several
weeks and even months of incubation unless badly decomposed when
first cultured. Occasionally, tissues from live oysters disintegrate
after several weeks' incubation in cultures that contain heavy growths
of certain molds, yeasts and bacteria. Tissues tend to remain suitable
for examination for much longer periods when mycostatin and Chloromy-
cetin are used instead of penicillin and dihydrostreptomycin . Occasion-
ally tissues from badly decomposed gapers disintegrate more readily in
My cultures than in P+S cultures, despite the absence in My cultures
of the marked microbial growth and putrefaction exhibited by the com-
panion (paired) P+S cultures. Such tissue disintegration in the absence
of culture putrefaction is tentatively attributed to the swelling caused
by mycostatin. When large numbers of cultures accumulate and I anti-
cipate long delays in examining them, tubes that have been incubated
two or three weeks are placed in the refrigerator. This prolongs the
time the tissues will remain suitable for examination. Nevertheless,
best procedure is to examine cultures as soon as conveniently possible
after one week of incubation .

The possibility of overlooking light D . marinum infections may
be reduced by exercising certain precautions in preparing the cultured
tissues for microscopic examination. Unless tissues show signs of
disintegration, place a piece of absorbent paper toweling on a slide to
receive the tissue to be examined; do not remove the toweling until the
excess medium is absorbed . This procedure makes it easier to stain
tissues and D. marinum properly, and reduces the need for restaining
preparations. Tissues from "watery" or poor oysters usually stain
readily and require little teasing for penetration of the iodine solution.
On the other hand, tissues from "fat" oysters (containing either much
glycogen or much gonadal material) should be shredded into very small
pieces to insure proper staining . Such tissues may be very difficult
to stain and often the brown color fades rapidly.

-67-

For easiest examination, all or most of the preparation should
be stained light brown . Wait about five minutes before placing a cover
slip over the preparation. Poorly stained tissues may be restained con-
veniently if the preparation has not been covered . Before tissues are
restained, remove excess fluid with absorbent paper toweling. Confirm
negative findings associated with poorly stained tissues by restaining
the preparations, but avoid over-staining. Over-stained tissues
(extremely dark brown) are difficult to examine.

For microscopic examination of tissue preparations many workers
use low magnifications (2 5X to 3 5X). Although such magnifications are
adequate for detecting most D. marinum infections, negative prepara-
tions should be examined, at least cursorily, at 100X magnification for
unusually small cells .

GENERAL COMMENTS

In my opinion some workers do not adequately record details
of procedural changes that they may have made in the culture method .
Information that indicates when and in what manner the routine pro -
cedures had been altered should be recorded for future reference.
Furthermore, I believe it will be helpful in comparing the data obtained
by various workers if certain details of procedure are presented in the
materials and methods section of publications that contain D. marinum
data obtained by the culture method . Such details should include the
kinds of antibiotics employed, the tissues cultured, and other details
of technique that might be significantly different from those generally
used by workers in the field .

ACKNOWLEDGMENTS

I wish to thank Miss A.M. Sievers for technical assistance.
This investigation was supported in part by Public Health Research
Grant EF 00338 from the Division of Environmental Engineering and
Food Protection and by the Organized Research Fund, Texas A&M
University .

LITERATURE CITED

Mackin, J. G. 1951. Histopathology of infection of Crassostrea

virqinica (Gmelin) by Dermocystidium marinum Mackin, Owen,
and Collier. Bull. Mar. Sci . Gulf & Carib . 1:72-87.

•68-

Mackin , J . G . 1962 . Oyster disease caused by Dermocystidium

marinum and other microorganisms in Louisiana. Publ . Inst.
Inst. Mar. Sci . Univ. Texas 7 (1961): 132-229 .

Mackin, J . G. and D . A. Wray . 1952. Report on the second study
of mortality of oysters in Barataria Bay, Louisiana, and
adjacent areas . Part II . Disease caused by Dermocystidium
marinum . Texas A&M Research Foundation, Project Nine,
mimeographed report, 1-4 0.

Ray, S . M . 1952a . A culture technique for the diagnosis of infec-
tion with Dejrnocysti^ium marinum_ Mackin, Owen, and Collier
in oysters. Science 116:360-361.

Ray, S. M. 1952b. A culture technique for the diagnosis of infection
with Dermocystidium marinum in oysters. Nat. Shellfish
Ass. Conv. Add. 1952:9-13 .

Ray, S. M. 1954a. Biological studies of Dermocystidium marinum,
a fungus parasite of oysters. Rice Inst. Pam . Special Issue,
November, 1954, 113 p.

Ray, S. M. 1954b. Studies on the occurrence of Dermocystidium
marinum in young oysters . Nat . Shellfish Ass . Conv . Add .
1953:80-92.

Ray, S . M . 1966 . Notes on the occurrence of Dermocystidium

marinum on the Gulf of Mexico Coast during 1961 and 1962 .
Proc. Nat. Shellfish Ass . 54:45-54.

-69-

RADIATION PASTEURIZATION OF OYSTERS

Arthur F . Novak

Department of Food Science and Technology-
Louisiana State University, Baton Rouge

ABSTRACT

Gamma (6OC0) irradiation of pint cans of fresh oyster meats (0.2
Mrad) extended by several days the time that acceptable quality was
retained under conditions simulating commercial handling and storage,
as shown by organoleptic tests, trimethylamine content, bacterial counts,
pH, and color.

Many problems have plagued oyster packers during the past
five years . One results from the rejection by state health departments
of oysters in transit from one growing and packing area to another,
which are held for extra days to be repacked before final shipment to
the ultimate distributor and consumer.

Although the excessive bacterial counts in such oysters might
not have posed an actual health hazard, they were above the counts
allowed in the standards. Most rejected samples could have been
retained as acceptable if some simple method had been available to
reduce the original bacterial counts in the oysters, thus giving them
a few days' extension of quality.

Exploratory investigations on gamma radiation of fresh Gulf
oysters suggest that such treatment of oysters can provide advan-
tages for the fisherman, processor, distributor, and consumer. If
such a method of processing were developed to successful commercial
application, fresh oysters could be made available to consumers who
presently are able to obtain only the canned or frozen product. Market
prices of oysters, presently subject to fluctuations due to overabun-
dances or scarcity, would tend to be stabilized.

Low dose gamma irradiation of oysters with Cobalt 60 resulted
in an extension of their storage life of five or more days beyond that
of non-irradiated oysters from the same batch . When the oysters were
shucked under supervision and irradiated within six hours the storage
period was extended for several days longer.

-71-

The oysters used in these experiments were treated as follows:
freshly shucked oysters, which had been brought in from the oyster
beds the previous night, were washed in running tap water (50 F) for
two minutes, and allowed to drain for five minutes . All draining was
done on FDA-approved stainless steel skimmers, which had an area
of not less than 300 square inches per gallon (201 cm2 per 6) of
oysters, drained, and which had perforations of at least 0.25 inch
(6.35 mm) in diameter located not more than 1.25 inches (31.75 mm)
apart. The oysters were distributed evenly over the draining surface
of the skimmer but were not otherwise agitated during the draining
period . After the oysters were washed and drained, they were packed
into pint cans and stored in crushed ice. Within six hours, 24 pint
cans were subjected to gamma radiation (0.2 Mrad) in the Nuclear
Science Center on the Louisiana State University campus . After
irradiation, they were stored in crushed ice, along with an equal
number of non -irradiated pints of oysters which were employed as con-
trols . Samples were removed at intervals for bacterial counts and
trimethylamine , pH, and organoleptic tests.

Results are presented in the following tables.

Similar results were obtained with other batches of oysters
treated and tested in the same manner.

-72-

Table 1. Organoleptic scores of irradiated (0.2 Mrad) and non-
irradiated ice-stored oysters

Score after listed storage period

Sample treatment

Initial

7 days 14 days 21 days

Non-irradiated
Irradiated

9.5 6.0 3.8 Spoiled

9.5 8.0 6.5 5.3

Values are averages for 25 participants on taste panel for the attri-
butes of odor, appearance, flavor, and texture.

Code of Scores:

(10) No change from fresh product of highest quality

(8) First noticeable slight change in attributes

(6) Moderate degree of changed attribute: increased in
intensity and occurrence from score of 8

(4) Definite or strong degree of changed attribute

(2) Extreme degree of changed attribute

-73-

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-74-

DIFFERENTIATION OF EFFECTS OF TWO PESTICIDES UPON
UROSALPINX CINEREA SAY FROM THE EASTERN SHORE

OF VIRGINIA

Langley Wood and Beverly A. Roberts

Virginia Institute of Marine Science
Gloucester Point, Virginia

ABSTRACT

Adult Urosalpinx were exposed to various combinations of two pesti-
cides ("Sevin", a methyl carbamate, and "Polystream", a mixture of
chlorinated benzenes) recommended for oyster predator control by the
Milford Biological Laboratory of the U. S. Fish and Wildlife Service.
Concentrations used were within the recommended range, and the field
procedure suggested was modified by us for application in laboratory
trays. Under controlled laboratory conditions, Polystream used alone
killed half the animals within a period of 5.5 to 6.8 days. The use
of Sevin, which is highly toxic in crustaceans, is therefore questionable.

INTRODUCTION

Several recent publications (Loosanoff, MacKenzie and Davis,
1960; Loosanoff, MacKenzie and Shearer, 1960; Loosanoff, 1960, and
MacKenzie and Gnewuch, 1962) have reported the effectiveness of
toxic chemicals on oyster predators, particularly Urosalpinx . The
chemical agents recommended by these writers are "Polystream" and
"Sevin." The former is an aggregate of chlorinated benzenes pro-
duced by the Hooker Chemical Company; "Sevin" is the trademark of
an established insecticide manufactured by the Union Carbide Cor-
poration .

It has been suggested that these two agents be mixed with
sand and broadcast over oyster grounds, and considerable success
has been reported (Davis et al., 1961) with this procedure in field
tests in Long Island Sound. In light of this it seemed necessary to
obtain additional basic information on these toxic chemicals and their
possible effects upon the marine habitat.

Two immediate questions were proposed. First, we wanted to
know something about the specific effects of the recommended dosage
of the two agents upon the large, Eastern Shore Urosalpinx . For
instance, does the mixture kill them directly, or does it simply cause

Contribution 163 of The Virginia Institute of Marine Science

-75-

them to swell so that they are easy prey for some roving larger animal
which is itself unaffected by the poison? The second question grew
out of the report (Loosanoff et al., 1959) that "Sevin" killed crabs.
Since we were considering using "Sevin" in an area that supports a
blue crab fishery, it seemed vitally necessary to establish the relative
effectiveness of the Polystream-sand treatment with and without 'Sevin ."

MATERIALS AND METHODS

All experimental animals were adult Urosalpinx cinerea Say of
the large variety, collected near V/Gchapreague, Virginia, during the
summer of 1962. They were maintained in running seawater aquaria or
in a recirculating aquarium until 23 January 1963 . Then they were trans-
ported to the Virginia Institute of Marine Science at Gloucester Point,
Virginia, where they were placed in running York Paver water aquaria at
ambient temperature and salinity for several days or longer. Three
animal samples were used in these experiments. For two months prior
to the experiment, the first group (Run A) was supplied with food and
maintained at a controlled temperature of ca_. 20 C. The two groups
of animals used in Runs B and C were maintained without food and at
incoming salinity and temperature prior to the experiments.

Seven enamel trays served as the experimental containers, each
containing 50 animals. Treatments were applied to each of the trays
as follows :

Tray Number Treatment

1.5 Polystream-Sevin

2.6 Polystream

3.7 Sevin

4 Control

The concentrations of Polystream and Sevin were within or near
the range of treatment suggested by the United States Fish and Wildlife
Service Biological Laboratory at Milford, Connecticut (Table 1). In
Runs A and C concentrations of chemicals were computed assuming uni-
form coverage over the whole tray bottom. In Run B approximately 2/3
of the bottom of each tray was covered and concentrations were based
upon this area .

After the animals were placed in the trays, the treated and
untreated sand was spread over the tray bottoms.

-76-

Table 1 . Concentration of chemicals

Run Polystream Sevin

2 2

A 3-4 Hi/cm lOOUg/cm

B 7-9 ml/cm 200-250 ^g/cm

2 2

C 3-4 m/cm 100 ng/cm

Several observations were made the first day, with daily
observations throughout each run. Run A was terminated at 380 hours,
Run B at 168 hours, and Run C at 143 hours .

Each observation included salinity, temperature, and number
of animals dead, retracted, attached or swollen. The criterion used
to determine death in a gastropod was the presence of a "rotting"
odor. Retracted animals included all animals that were not attached,
swollen or dead and had partially or completely withdrawn into their
shells . A gastropod was considered attached when the foot was
extended and attached to a surface or when the animal was mobile.
If the body was distended and white, and the gastropod was unable
to withdraw it completely, the animal was counted as swollen.
There were many cases in which one animal was included in both of
the categories, "swollen" and "attached."

RESULTS

In Run A the first mortality count was made six days after the
application of treatments (Table 2). In this run the number of animals
killed by the Polystream-Sevin treatment was similar to the number
killed by Polystream alone throughout the course of the run (Fig. 1).
At the termination of Run A, 16 days, the total mortality of the Poly-
stream-Sevin group was 77% and that of the Polystream group was
78%. In Runs B and C the total mortality was greater for those animals
treated with Polystream-Sevin than for those treated with Polystream
alone. There was a total mortality of 11% observed among the animals
treated with Sevin alone in Run B.

The observed LDcg (time required to kill half the animals in a
sample) of the Polystream-Sevin and Polystream groups in Run B, 3 .8

-77-

and 5 .4 days respectively, was less than the IX^g's in Runs A and
C (Fig. 1). There was a difference of 1-2 days in the LD5o's of the
Polystream-Sevin group and the Polystream group in Runs B and C,
while the LDcq's of these two groups in Run A were almost identical .

In both the Polystream-Sevin and Polystream treatments the
percentage of animals retracted was high in each of the three runs
(Fig . 2). This high incidence of retraction was usually first observed
shortly after the application of treatments and continued throughout
each run .

Most swelling occurred in the Sevin-treated groups in each of
the three runs (Fig .3). The maximum number of animals was found
swollen in the Sevin-treated groups at the end of 6 or 7 hours; swollen
gastropods were not found after 2 or 3 days . Swelling was also noted
in the Polystream-Sevin and Polystream treatments in each of the runs
and was usually still evident in both of these treatments at the termina-
tion of each run. There was a higher percentage of swelling found in
the Polystream-Sevin treatments than in the Polystream treatments.

The fraction of animals attached in the Polystream-Sevin and
Polystream treatments was low throughout each run . In both of these
treatments in Run A and in the Polystream-Sevin treatment in Run B,
the proportion of animals increased gradually until approximately
one third of the surviving animals was attached.

DISCUSSION
General

The conditions under which these experiments were run, as
contrasted with those obtaining in most field situations, tended to
favor the pesticides against the drills . That is, the flow of dilution
water through the trays was rather low compared to the large volumes
moving across most natural oyster beds, and contact with the drills
by the poisoned sand was maximized by the method of administration
and the lack of topographic relief of the tray bottoms . Despite these
facts, the treatments described here never resulted in the catas-
trophic mortality rates reported for field trials by Loosanoff (1962a,
1962b).

The mean terminal kill for all our "Polystream" treatments was
only 72.2%; this differs from previous field studies in which 90 to
"more than 99%" (Loosanoff, 1962a) were reported as "eliminated."

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-82-

The differences in the mortality rates shown amongst the three
runs of this experiment are not fully understood. However, it is
reasonably safe to conclude that they may be the result of interaction
between two factors: the condition of the drills and the concentrations
of the chemicals .

It will be recalled that the test sample for Run A was conditioned
and fed for two months prior to treatment; those for Runs B and C were
not. Runs A and C employed the lower concentrations, Run B the
higher, by a factor of about 2. On the basis of these facts, the inter-
pretation would proceed as follows: In Run A, animals in good con-
dition were exposed to initial concentrations comparable to those
expected in the field application of the Milford Formula. The LD50
was obtained in about 7 days, and mortality never reached 80% even
by the end of 16 days . In Run B, animals in relatively poor condition
were treated with a concentration about double that of Run A. The
LDrnwas obtained in 4 to 5 days, and the maximum mortality of greater
than 80% was obtained in 7 days . In Run C, animals in perhaps even
poorer condition were treated with the same concentrations used in
Run A, and the LDc q was obtained in about 4 .5 to 6 days . This inter-
pretation of our data leads to a tentative recommendation for field
workers: If Polystream is to be used on an oyster bed, it might best
be administered in the early spring when the drills are just emerging
from their winter "hibernation" and are presumably weakened .
Another investigation (Wood, unpublished) has shown that Urosalpinx
is less resistant to osmotic stress in the spring than in the fall.

Polystream-Sevin or Polystream Alone ?

Using the chi-square test we have determined that in Run A of
our experiment it was not possible to distinguish the effects of the
two treatments; however, in Runs B and C the Polystream-Sevin mix-
ture produced slightly greater cumulative mortality on the following
days :

Run B: Days 4 through 7 (P less than 0.01)

Run C: Days 5 and 6 (P less than 0.01)

Several investigators have reported (Carriker and Blake, 1959;
Loosanoff et al . , 1959) the effect of Sevin in causing drills to swell;
it has been claimed further (Davis et al., 1961) that this swelling
renders the gastropods easy prey to other species such as fish and
sea stars . Since a primary object of most pesticides is to kill only
the selected pest, and that as quickly as possible, it is, in our

-83-

opinion, a poor pesticide whose effectiveness depends upon the
presence of an unaffected second party. We have also been unable
to find many reports of direct observations of predation upon disabled
and swollen gastropods . To the extent that this question applies to
the waters of the Eastern Shore of Virginia, it would appear that the
chief candidate for the job of cleaning up disabled Urosalpinx would
be the blue crab, and the possibility exists that this organism would
itself be rendered inoperative by the inclusion of Sevin in the treat-
ment, at least in the early days .

Therefore it is our conclusion that in light of the failure of our
experiments to indicate the absolute necessity of Sevin in this treat-
ment, the lack of such evidence from other quarters, and finally the
possibility that its inclusion might do harm to another valuable fishery
(blue crab), we cannot justify the employment of Sevin in Virginia's
Seaside waters . We have shown that Polystream alone kills Urosal-
pinx directly, in the laboratory; other investigations at the Virginia
Institute of Marine Science have disclosed (Haven et al., 1964) that
the Polystream-Sevin combination killed up to 85% of the benthic
associates of the oyster when applied in field tests near Wacha-
preague, Virginia. Should it be shown, however, that Polystream
does not permanently damage the bottom communities of which com-
mercial oyster grounds are a part, this pesticide might prove to be a
valuable adjunct to other modern ostreicultural practices .

BIBLIOGRAPHY

Carriker, Melbourne R. and John W. Blake. 1959. A method for full
relaxation of muricids . Nautilus 73(1): 16-21.

Davis, H. C, V. L. Loosanoff, and C. L. MacKenzie, Jr. 1961.
Field tests of a chemical method for the control of marine
gastropods. USDIFWS (Milford, Conn.) Bull. 25(3): 3-9.
(Mimeo: 7 August 1961).

Haven, Dexter, M.,Castagna, J. Whitcomb, and P. Chanley. 1964.
Report to the industry. Oyster drill control studies with formu-
lations of "Polystream" and "Sevin" for 1963. I. Treatment of
planted oyster grounds. Virginia Institute of Marine Science,
Gloucester Point, Va . (Mimeo: February 1964).

Loosanoff, V. L. 1960. Recent advances in the control of shellfish
predators and competitors. Proc . Gulf and Caribbean Fish.
Inst. 13: 113-137. (November 196 0) .

-84-

Loosanoff, V. L. 1962a. Observations in Long Island Sound.

USDIFWS (Milford, Conn.) Bull. 26(2): 2-4. (Mimeo: 26 July
1962).

Loosanoff, V. L. 1962b. Observations in Long Island Sound.

USDIFWS (Milford, Conn.) Bull. 26(4). (Mimeo: 27 August
1962).

Loosanoff, V. L., C. L. MacKenzie, Jr., and L. W. Shearer. 1959.
Use of chemical barriers to protect shellfish beds from preda-
tors . USDIFWS (Milford, Conn.) Bull. 23(6): 2-10. (Mimeo:
12 November 1959).

Loosanoff, V. L., C L. MacKenzie, Jr., and H. C. Davis. 1960.
Progress report on chemical methods of control of molluscan
enemies. USDIFWS (Milford, Conn.) Bull. 24(8): 3-20.
(Mimeo: 16 November 1960).

Loosanoff, V. L., C. L. MacKenzie, Jr., and L. W. Shearer. 1960.
Use of chemicals to control shellfish predators. Science 131:
1522-1523 .

MacKenzie, Jr., C. L., and W. T. Gnewuch . 1962. Effects of

selected quantities of chemicals tried under field conditions
on shellfish-killing gastropods. Nat. Shellfish. Ass. Abstracts
of Formal Papers. (Mimeo: 29 July— 2 August 1962).

-85-

MORTALITY RATES AND THE LIFE SPAN OF THE BAY SCALLOP,
AEQUIPECTEN IRRADIANS l

Nelson Marshall

Graduate School of Oceanography,
University of Rhode Island, Kingston, R.I.

ABSTRACT

When high mortalities cut down scallop populations held in the labo-
ratory and in cages in the field, the younger specimens did not exhibit
greater survival capacities than the older, even when the latter had
reached what has been reported as the age of marked senescence and
death. This supports more recent field observations which suggest that
senescence symptoms are not pronounced. If senescense is of a lesser
severity and mortalities resulting from environmental stresses play a
relatively greater role, the observed variations in the life span of the
scallop are accounted for more readily.

Belding (1910) stated that relatively few scallops live more than
two years . He reported that scallops show a marked period of physical
decline starting at the age of 18 months. Marshall (1960) also stated
that the natural expectancy of the life of the scallop in the southern
New England area is two years or slightly less but he encountered very
high mortalities of both young and older scallops throughout the winter
and early spring months and did not find the marked period of physical
decline in older specimens that was so conspicuous to Belding .
Sastry (1961) did not find scallops beyond an age of 19 months in his
program of observations on the shoals off Alligator Harbor, Florida.
Gutsell (1931), working in the Beaufort, N. C. area, was notable to
determine the life span of the scallop since intense fishing mortality
obscured natural conditions; however, his observations suggested the
possibility of significant survival beyond two years, at least for the
populations of his area of study. My field collections also suggest
that third year specimens are more common in North Carolina than in
New England but that there may be local and even year-class variations
in life span in the latter region.

1
Contribution from the Graduate School of Oceanography of the

University of Rhode Island . This study was supported in part by a

grant, No. G- 12 149, from the National Science Foundation.

-87-

Reported herein are observations on the rate of survival of
scallops from the Niantic River in Connecticut (Figs . 1 & 2), kept in
trays of running sea water at the University of Connecticut Marine
Laboratory at Noank, and comparable observations for scallops from
Bogue Sound, N. C. (Fig. 3), kept in similar trays at the U . S. Bureau
of Commercial Fisheries Laboratory at Beaufort. At both laboratories
the running sea water was pumped continuously without filtration from
a source similar to that from which the scallops were taken. Though it
had been anticipated that the continuous flow of water in and out of the
plastic and hard rubber holding trays might so approximate conditions of
natural circulation that both growth and mortality would be normal, the
growth data from trays showed stunting in the laboratory when compared
with length-frequency shifts from field collections . Apparently for a
variety of reasons, which may include a scarcity of benthic and epi-
benthic components important in the food of scallops (Davis and
Marshall, 1961), the laboratory setups did not offer an adequate
environment. Obviously a December pump shut-down at Noank must
be suspected as the cause for heavy mortalities which followed there.

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-88-

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-89-

In spite of the stunted growth and eventual high mortalities in
the trays, and in the caged scallops (Figs. 4, 5, and 6) previously
reported by Marshall (1960), all the holding observations exhibited a
similar mortality pattern in which the younger and older scallops
seemed to be affected indiscriminately. For the caged specimens in
the Niantic River, starfish predation seemed to strike the seed more
than the adults, and the seed sample from Beaufort was too small to
consider critically, yet the parallel mortality trends of the age groups
is quite apparent and is very noticeable in the Noank set-ups . This may
aid in the interpretation of the life span. Particularly important is the
fact that this parallelism even prevailed during the late spring, the
period for which Belding (1910) reported a pronounced mortality in the
older scallops linked with a complex he referred to as old-age effects.
Though parallel mortalities of younger and older scallops may seem to
contradict Belding's emphasis on the dying off in an old-age period, it
may be interpreted instead as evidence that mortalities from environ-
mental stress, indiscriminate for age groups, may obscure the evidence
of any physiological decline associated with aging . Such a masking
effect may be operative in nature as well and is the probable explana-
tion for my failure to discern senescence while following the popula-
tions closely in the field through the spring and summer of 1955
(Marshall, 1960).

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7/4/55 . For legend see Fig. 4.

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Fig. 6. Scallops surviving in a cage off Saunders Point,
Niantic River, Conn. Average depth about 8 feet, 12/8/54 — 7/4/55
For legend see Fig . 4 .

-91-

These interpretations fit better the observed discrepancies in
life span. Though I continue to point to the possibility of inherent
racial and even year-class differences, it is clear that varying environ-
mental mortality rates, on a species that does not exhibit marked ter-
minal age effects, may contribute to the observed differences in life
span .

ACKNOWLEDGMENTS

The facilities and the cooperation of the staffs of the University
of Connecticut Marine Laboratory and the U.S. Bureau of Commercial
Fisheries Laboratory at Beaufort, N. C, made these observations
possible .

LITERATURE CITED

Belding,D.L. 1910. A report upon the scallop fishery of Massachus-
etts . The Commonwealth of Massachusetts, Boston. 150 p.

Davis, Roberta L. and N. Marshall. 1963. The feeding of the bay

scallop, Aequipecten irradians . Proc . Nat. Shellfish . Assoc .
(1961) . 52:25-29.

Gutsell,J.S. 1931. Natural history of the bay scallop . Bull . U . S .
Bureau of Fish. 45:569-632.

Marshall, N. 1960. Studies of the Niantic River, Connecticut, with
special reference to the bay scallop, Aequipecten irradians .
Limnol. and Oceanogr. 5:86-105.

Sastry, Akella N . 1961 . Studies on the bay scallop, Aequipecten
irradians concentricus Say, in Alligator Harbor, Florida.
Doctorate dissertation (typed), Library of Florida State Uni-
versity, Tallahassee.

-92-

SEROLOGICAL STUDIES ON THE BAY SCALLOP,
AEQUIPECTEN IRRADIANS1

Judith A. Pendleton2

Department of Bacteriology

University of Rhode Island

Kingston, Rhode Island

ABSTRACT

Serological relationships between four distant populations of the bay
scallop, Aequipecten irradians, and samples of the calico scallop,
Aequipecten gibbus, were studied by means of an agar gel diffusion
technique and the ring precipitin test. Also the serological reactivity of
these scallops with the sea scallop, Placopecten magellanicus, was
investigated. Both techniques indicated a close serological similarity
among the four populations of bay scallops and between the bay and the
calico scallops. The serological relationship between the bay and the
calico scallops was closer than the relationship of either to the sea
scallop. Agar gel diffusion studies revealed some bands in common in
all the samples tested.

INTRODUCTION

The taxonomic position of the members of the bay scallop,
Aequipecten irradians , complex has been confused . In a recent review
Abbott (1954) recognizes a northern subspecies around southern New-
England, a middle Atlantic population in the North Carolina area, and
a southern group along the Gulf coast. Life history studies on these
different subspecies near their centers of distribution show differences
which seem to relate to the environment (Belding, 1910; Gutsell, 1931;
Marshall, 1960; Sastry, 1961). Thus these subpopulations may not be
true genetic races. Serological studies might help in determining the
extent of racial difference among the populations which would then
aid in evaluating the extent of environmental response.

Aequipecten gibbus , the calico scallop of the southern off-
shore waters, differs from Aequipecten irradians in its habitat and in
minor characteristics, yet it is very closely related. Serological
comparisons with this species provide a frame of references to judge

Contribution from the Graduate School of Oceanography of the University

of Rhode Island . This work is supported in part by a grant from the

National Science Foundation.
2
The author is now Judith A. Hoitink and is at the Wisconsin State

Laboratory of Medicine , Madison, Wisconsin.

-93-

the significance, taxonomically , of minor differences within the
irradians group . The frame of reference is broadened by comparison
with a more distinct species, the sea scallop, Placopecten magellani-
cus .

METHODS AND MATERIALS

The scallops used in this study were collected from various
areas along the Gulf and Atlantic coasts of the United States as shown
below:

Scientific Name
Aequipecten irradians

Aequipecten gibbus

Aequipecten irradians

Aequipecten irradians

Aequipecten irradians

Aequipecten irradians

Aequipecten irradians

Aequipecten irradians

Date Received
3/24/61

3/24/61

5/2 9/61

7/13/61

8/10/61

5/7/62

7/13/62

7/18/62

Placopecten magellanicus 7/2 0/62

Source
Beaufort, North
Carolina

Off North Carolina
Coast

Niantic, Connecti-
cut

Alligator Harbor,
Florida

Tarpon Springs ,
Florida

Beaufort, North
Carolina

Alligator Harbor,
Florida

Niantic, Connecti-
cut

Off southern New
England coast

Scallop specimens from Niantic were received alive; specimens from
other locations consisted of only the adductor muscle which had been
removed from the scallop, frozen, and shipped to this laboratory after
collection .

Two methods of antigen preparation were used; both were
phosphate buffered saline extracts of the scallop adductor muscle. In
one method the lipid material was removed from the muscle prior to
extraction .

-94-

Antisera were produced in rabbits by subcutaneous injection
of a one to one mixture of Freund's complete adjuvant and the antigen
once a week for three or four weeks .

Ring precipitin tests were done with serial doubling dilutions
of the antigen layered over undiluted antisera in small tubes . A white
line at the interface of the antigen dilution and the antiserum was a
positive test . The titer was indicated by the reciprocal of the highest
antigen dilution giving a detectable reaction. Each test was run in
duplicate .

Agar diffusion tests were run in 1% Oxoid agar medium. Cut-
tings were made in the agar to provide a central well into which the
antiserum was placed and six peripheral wells into which the antigens
were deposited. When additional wells were needed on one plate, an
extra cutting was made. Thus each well containing heterologous
antigen had on either side of it a well containing homologous antigen
so that cross reactions might be observed . Each test here was also
done in duplicate.

DISCUSSION

In terms of the particular serological observations presented
herein, the subspecies of the bay scallop as well as the calico scal-
lop are closely related as their morphology suggests . The sea
scallop, which is easily distinguished from the other two types of
scallops by morphological criteria, is slightly different from the
other two types of scallops serologically, although it possesses anti-
genic components in common with them .

These serological comparisons may be interpreted in support
of the link, now recognized through shell characteristics, between
Aequipecten irradians and Aequipecten gibbus . They do not support
the breakdown of irradians into three readily recognized subspecies .
It is possible that differences might be found among these scallops
by the use of more sensitive serological techniques, i.e., adsorption
tests or immunoelectrophoretic techniques . Whether or not the dif-
ferences would correlate with the subspecies as now recognized
remains to be seen. Meanwhile, there is good reason to focus atten-
tion on the likelihood that the bay scallop, throughout its range, may
exhibit differences both in environmental responses and in genetic
strains that may be highly localized and may even occur, as Marshall
(1960) has suggested, in separate year classes in the same locale.

-95-

Table 1 . Results of ring precipitin tests with scallop antigens pre-
pared by method A^ and their antisera

Antigen^

Antiserum A.i.(N) A.i.(B) A. I. (AH) A.G.(NC)

A.i. (N) 1280 1280 320 640

1280 2560 160 640

A.i. (B) 1280 1280 640 640

1280 1280 320 640

A.i. (AH) 320 160 160 80

160 160 160 160

A.g.(NC) 1280 2560 640 2560

1280 2560 640 2560

Antigens prepared by method A were phosphate buffered saline ex-
tracts of the scallop muscle from which lipids were extracted .

2
A.i. (N) = Aequipecten irradians from Niantic , Conn.

A.i. (B) = Aequipecten irradians from Beaufort, N. C.

A.i. (AH) = Aequipecten irradians from Alligator Harbor, Fla .

A.g. (NC)= Aequipecten gibbus from off the North Carolina coast

■96-

Table 2 . Results of ring precipitin tests with scallop antigens pre-
pared by method B and their antisera

Antigi

2
=n

Anti-
serum

A.i.(N)

A.i.(B)

A.i.CTS) A

.i.(AH)

A.g.(NC)

P.m.(SNE)

A.i.(N)

640
640

1280
1280

1280
1280

320
320

1280
1280

160
160

A.i.(B)

1280
640

1280
2560

1280
2560

1280
640

2560
2560

640
320

A.i.fTS)

1280
1280

1280
1280

2560
2560

640
64 0

2560
2560

640
640

A.i.(AH)

640
640

1280
1280

2560
1280

640
640

2560
2560

320
320

A.g.(NC)

640
640

1280
1280

1280
1280

320
320

5120
Z560

320
160

P.m.CSNE)

1280
2560

1280
640

1280
1280

320
640

2560
5120

1280
2560

Antigens prepared by method B were phosphate buffered saline
extracts of scallop adductor muscle from which lipids were not
extracted .

2 , »

A.i.(N) = Aequipecten irradians from Niantic , Conn.

A.i.(B) = Aequipecten irradians from Beaufort, N.C.

A.i.tTS) = Aequipecten irradians from Tarpon Springs, Fla .

A.i.(AH) = Aequipecten irradians from Alligator Harbor, Fla.

A.g . (NC) = Aequipecten gibbus from off the North Carolina coast .

P.m.(SNE) = Placopecten magellanicus from off the southern New

England coast .

-97-

Table 3 . Results of agar diffusion studies indicating bands formed
between scallop antigens prepared by method A^ and the
various antisera produced against them

2

Antigen

Antiserum

A.i . (N)

A.i .(B)

A.i. (AH)

A.g.(NC)

+3

C

P

C

A.i.(N)

+
+

C

P

C
C

+

P

P

C

P

+

P

P

A.i . (B)

C

P

+
+

P

c
c

c

+

—

c

c

C

+

c

A.i.(AH)

c

-

+

p

p

P

+

p

c

c

P

+

A.g.(NC)

E

■ c

p

P

+

c

c

P

+

Antigens prepared by method A were phosphate buffered saline
extracts of scallop adductor muscle from which liquids were
extracted .

A.i

.(N)

=

A.i

• (B)

=

A.i

.(AH)

=

A.g

.(NC)

=

Symbols :

+

C

Aeguipecten irradians from Niantic , Conn .
Aequipecten irradians from Beaufort , N . C .
Aeguipecten irradians from Alligator Harbor, Fla .
Aequipecten gibbus from off the North Carolina coast .

= band present with homologous antigen and antiserum
= band present at same location and connecting with
corresponding band
P = band present at approximately same distance from

central well as + band
E = extra band not present with homologous antigen
- = band not present

■98-

Table 4 . Results of agar diffusion studies indicating bands formed
between scallop antigens prepared by method B1 and the
various antisera produced against them

Antigen^

Antiserum

A

.i.(N)

A,

,i.(B)

A,

.i.(TS)

A,

,i.(AH)

A

.g.(NC)

P.m.(SNE)

+ 3

P

P

P

P

P

A.i.(N)

+

P

P

P

P

P

+

P

P
E

P

P

-

-

+

-

-

-

-

P

+

-

-

-

-

A.i.(B)

C
P

+
+

P
P

P
P

P
E

P

c

+

C

C

C

-

-

P

+

P

-

-

A.i.Crs)

c

p

C

P

+
+

P
P

c

p

P

c

P

+

c

p

C

A.i.(AH)

P
P
P
C

C
P
C

c

p
p
c
c

E

+
+
+
+

P
P

P
P

C

c

c

c

+

-

A.g.(NC)

C

c

p

p

+

-

P

p

p

p

+

p

-

p

-

-

-

+

P.m.(SNE)

C

p

p

c

p

+

P

p

p

p

p

+

Antigens prepared by method B were phosphate buffered saline extracts
of the adductor muscle of the scallop from which lipids were not ex-
tracted .

I

A.i.(N) = Aequipecten irradians from Niantic, Conn.

A .i . (B) = Aequipecten irradians from Beaufort, N . C .

A.i.CTS) = Aequipecten irradians from Tarpon Springs, Fla .

A.i.(AH) = Aequipecten irradians from Alligator Harbor, Fla.

A.g.(NC) = Aequipecten gibbus from off the North Carolina coast.

P .m . (SNE) = Placopecten magellanicus from off the southern New

3 England coast .

Symbols:

+ = band present with homologous antigen and antiserum

C= band present at same location and connecting with cor-
responding band

P = band present at approximately same distance from central
well as + band

E = extra band not present with homologous antigen

- = band not present

-99-

ACKNOWLEDGMENTS

I wish to thank Dr. Nelson Marshall of the Graduate School
of Oceanography of the University of Rhode Island for his assistance
in the collection of scallop specimens and for his suggestions on the
interpretation of this work with reference to the scallop classification
problem. I would also like to express appreciation to Dr. Chester W .
Houston for help in preparing the manuscript and for valuable assist-
ance throughout the work .

REFERENCES CITED

Abbott, R.T. 1954. American Seashells . D . Van Nostrand Co . , Inc.,
Princeton, xiv + 541 p.

Belding, D. L. 1910. A report upon the scallop fishery of Massachu-
setts. The Commonwealth of Massachusetts, Boston. 150 p.

Gutsell,J.S. 1931. Natural history of the bay scallop . Bull . U . S .
Bureau of Fish., 45:569-632.

Marshall, N. 1960. Studies of the Niantic River, Connecticut with
special reference to the bay scallop, Aeguipecten irradians .
Limnol . and Oceanogr . , 5:86-105.

Sastry, A.N. 1961. Studies on the bay scallop, Aeguipecten
irradians concentricus Say, in Alligator Harbor, Fla .
Doctorate dissertation (typed). Library, Fla. State Univ .

100-

ASSOCIATION AFFAIRS

ANNUAL CONVENTION

The 1963 convention was held jointly with the Oyster Institute
of North America and the Oyster Growers and Dealers Association on
July 21-25 at the Hotel Mayflower. Washington, D. C.

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A new slate of officers was elected, as shown below:

President JOHN B. GLUDE

Bureau of Commercial Fisheries,
Seattle, Wash .

Vice-President JAY D . ANDREWS

Virginia Institute of Marine Science,
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Texas A&M University,
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101-

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-102-

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