HATCHERY DEVELOPMENT OF MARINE
BIVALVE MOLLUSCS
Special project/Internship
Report
by
E. Mark Solon
A Final Report Submitted to
Oregon State University
School of Oceanography
In partial fulfillment for the
Degree of Master of Science
Completed February 16, 1984
Commencement June, 1984
HATCHERY DEVELOPMENT OF MARINE
BIVALVE MOLLUSCS
E. Mark Solon
Special project/Internship
Report
A Final Report Submitted to
Oregon State University
School of Oceanography
In partial fulfillment for the
Degree of Master of Science
Completed February 16, 1984
Commencement June, 1984
PREFACE
The direction of my Marine Resource Management program has been to
develop an expertise in a particular area of aquaculture, complemented by
a general knowledge of oceanographic principles. Specifically, my area of
interest is the culture of marine bivalve molluscs. Prior to entering the
MRM program, I had worked with Systemculture Corporation in Hawaii on the
research and development of large-scale phytoplankton cultures for the
closed system production of the Pacific oyster, Crassostrea gigas. As
part of my program at O.S.U., I interned with the Whiskey Creek Oyster
Farm in Tillamook, Oregon. This hatchery is among the largest and most
efficiently operated hatcheries in the world and has become the primary
seed supplier to commercial oyster growers along the west coast of North
America.
Aquaculture-oriented coursework in my program included Fish and Molluscan Culture, Fish Diseases, and Seafood Processing. In a special projects course under Wilbur Breese at the Marine Science Center, I learned
the histological microtechinque for the preparation and interpretation of
bivalve gonadal samples and worked with the Pacific weathervane scallop,
Patinopecten caurinus, to develop an induced spawning method and investigate early larval development parameters. My courses in Agricultural Finance, Statistics and Microcomputers will be useful in developing and managing an aquacultural business.
Given my past experience and current education, I feel I am now
qualified to work towards the development of hatchery production technology of aquaculturally desirable bivalve species.
This report presents an outline for the development of a bivalve
species to hatchery production. In addition, included are aspects of
work with induced spawning and larval rearing of the Pacific weathervane
scallop, Patinopecten caurinus, a species which shows potential for commercial hatchery production.
TABLE OF CONTENTS
HATCHERY DEVELOPMENT OF MARINE BIVALVE MOLLUSCS
E. Mark Solon
I. Hatchery Development of Marine Bivalve Molluscs
a.
Introduction
1
b. Hatchery considerations
3
c. Determination of annual cycle of gonad maturity
5
d.
8
Stripping and induced spawning
e. Larval development through metamorphosis
14
f. Hatchery application
15
g.
algal cultures
17
broodstock and conditioning
21
larval maintenance
22
cleaning
25
handling of eyed larvae
25
Conclusion
26
II. Scallops: Investigations on Spawning and Larval Rearing of Patinopecten
Patinopecten caurinus.
a. Acquiring and maintaining scallops
29
b.
32
Salinity substitutes
c. Treatment of stripped eggs
35
d. Larval salinity and temperature
36
e.
41
Induced spawning
f. Discussion
43
Bibliography
48
Appendix A. - Gonad indices
52
Appendix B. - Induced spawning methods of some scallop species .
54
- 1 -
INTRODUCTION
Culturing of bivalve molluscs has occurred since ancient times.
Early Romans cleared growing areas and planted them with young oysters for
later harvest. In Japan, records dating from 746 A.D. show clams being
transported and planted in bays where the species did not formerly exist.
In France, the blue mussel has been cultured for over 700 years (Magoon,
1981).
Although controlled rearing of young bivalves has long existed,
hatchery production is a more recent advancement. Perhaps the first
step toward hatchery development occurred in 1879 when eggs of the
American oyster, Crassostrea virginica, were observed in early development at Johns Hopkins University (Borgese, 1980). In the 1920's and
1930's, development work concentrated on the rearing of larvae in outdoor
tanks, supplemented by laboratory investigations of larval rearing requirements. During this time, in Conry, England, adult European oysters,
Ostrea edulis, were placed in large concrete tanks which had been constructed for allowing harvested mussels to purge themselves of estuarine
pollutants prior to marketing. Oysters were allowed to spawn naturally
in these tanks, and the young oysters (spat) left to settle on lime and
sandcoated tiles.
In the late 1930's. Galtsoff (1938) found that spawning of the
American oyster could be induced by increasing water temperature. Induced spawning methods allowed great numbers of fertilized eggs to become
available for larval rearing studies. However, it wasn't until the early
1950's that Loosanoff and Davis were able to cussessfully rear
C. virginica from larvae to metamorphosis, fed on cultured phytoplankton,
- 2 -
thus completing the hatchery cycle. Since then, much of the hatchery
development effort has been directed towards meeting the nutritional requirements of large numbers of bivalve larvae, with a general emphasis on
monospecific high-density cultures of unicellular algae.
Perhaps the most recent development in bivalve hatcheries has been
the marketing of eyed larvae to shellfish growers. Just prior to settling out of the water column onto a substrate, the larvae develop a
darkened area, referred to as an eyespot. Eyed larvae can be bagged in
moist towels and shipped in small, iced containers to oyster growers
throughout the world. The eyed-larvae method reflects the state of the
art in terms of shellfish supply and marketing and is rapidly replacing
the former hatchery method in which spat already attached to shell or
some other substrate (cultch) must be shipped, commonly requiring heavy
duty machinery and many additional hours of labor.
Upon arrival at the oyster farm, eyed larvae are released into prepared tanks of warm, well-aerated brackish water where mesh bags of clean
cultch, commonly oyster shell, have been placed. Within 24-48 hours, the
larvae will settle onto the cultch and after a few additional days will be
ready to transfer to growing areas.
- 3 -
HATCHERY CONSIDERATIONS
A major stumbling block for successful bivalve culture can be the
sufficient availability of seed. A hatchery can meet these demand requirements and provide additional benefits including:
- Elimination of seasonal dependency on natural seed.
- Production of clean, disease-free monospecific seed.
- Elimination of labor costs associated with natural seed collection.
- Affords the potential capacity of genetic selection.
- Allows an option of producing individual (cultchless) vs. substrateattached spat.
Before considering the aquacultural development of a bivalve species,
a need for hatchery seed must be evident. It is assumed that the technology for rearing young to market size has been established and that a
market for the product exists. If these criteria are met, investigations
to produce hatchery seed may be initiated.
Hatchery-produced seed has several advantages but their production
may be unnecessary if naturally occurring seed are abundantly available.
In Japan, for example, several years were spent to develop a hatchery
technology for the scallop Patinopecten yessoensis. However, populations
of the bivalve were found sufficient to supply more than adequate amounts
of seed and that only an efficient means of seed collection was needed to
accelerate the industry. The problem was solved with the onion bag collector. Japan's scallops now account for more than half of the world's
total harvest, largely due to this seed collection method (Magoon, 1981).
A scallop species under investigation for commercial development would
4
be the North American weathervane scallop. This species is similar in appearance to the Japanese scallop, but its populations are small and scattered, as in Puget Sound (Olson, 1982). Seed collection attempts of this
species in the Canadian Puget Sound have, to date, been unsuccessful
(Bourne, 1983). Other known scallop populations are found in deep offshore areas, as off the Oregon Coast. Because of the rigors of open
ocean seed collection, collection attempts might prove to be impractical
if at all fruitful. Results of continued investigations may indicate
that the development of hatchery methods for this species would be the
most suited means of achieving commercial production.
Other bivalve species with hatchery production potential include the
tropical oysters. Various types of mangrove oysters have large, firm
meats which could be desirable commodities for the international market.
One such oyster species is Crassostrea belcheri, occurring in estuaries of
Eastern Borneo. Although seed of C. belcheri can be obtained by setting
collectors at the appropriate time of year, another small, undesirable
oyster, C. cucullata, also spawns at the same time. The result is that
collectors have 90% C. cucullata and only 10% C. belcheri (Lim, 1980).
A hatchery may eliminate this problem, and perhaps produce an oyster
species well suited to tropical aquaculture. An alternative approach to
develop a desirable species for tropical aquaculture may be through genetic selection of the commonly cultured C. gigas, a temperate region
oyster species. Along these lines the College of Oceanography and Fisheries Science of the University of Washington (Beatie, 1983) has been
working with the Pacific Oyster to develop, through selective breeding, a
strain resistant to summertime conditions which result in heavy oyster
-5-
mortalities in areas of Puget Sound.
Development of the necessary technology to produce bivalve hatchery
seed may follow a sequence of steps, the first of which, identification of
the need for hatchery seed, has already been discussed. Other steps are
outlined in Figure 1.
DETERMINATION OF ANNUAL CYCLE OF GONAD MATURITY
The bivalve gonad is a large organ of ciliated tubules which surround
the intestine (Barnes, 1974). In oyster species, it is composed of two
gonads so close to one another that the paired condition is difficult to
detect. The interconnected tubules have many sacs or follicles. From
the walls of these follicles, sex products grow and during spawning move
to the mantle cavity through one of two gonadopores. Alternatively, in
some bivalves, the nephridia will provide exit for the sperm and eggs.
Although the majority of bivalves are dioecious, some (e.g. Pecten
maximus) are true hermaphrodites and others are protrandric hermaphroditic
(e.g. Ostrea edulis), alternating sex products after each spawning.
Crassostrea gigas is sexually similar to 0. edulis, but Walne (1979) believes that C. gigas first develop as described then the population divides
into those which remain prodominantly male and those which are predominantly female.
Gonad condition is determined by microscopic examination of the
stages of gametogenesis within the gonad follicles. However, once familiarity has been established for a given species, macroscopic or gonad
weight indices may be used to determine gonad condition (see Appendix A
for application of these methods on the scallop Patinopecten caurinus).
At Oregon State University's Marine Science Center, the method em-
Identify a need for
Hatchery Seed
Determine seasonal cycle
of gonad maturity
Induced Spawning / Strip Gametes
Obtain viable eggs
Determine larval rearing
technology for growth to
metamorphosis
Prove post larval growth
to marketable adult size
Apply Hatchery Methods
Figure 1. Development of a bivalve species to hatchery production.
- 7 -
ployed for determining seasonal sexual maturity in all bivalve molluscs
is through microscopic examination of samples of at least twenty live
specimens. From these samples, gonads are preserved and brought to the
laboratory where approximately 1 cm
3
of tissue is taken from a designated
area of each gonad. Tissue samples are dehydrated in alcohol, cleared
in toluene, embedded in paraffin, and sectioned to a thickness of 6 microns with a microtome. The sections are then mounted and stained using
the standard hemotoxylene and eosin method.
Female gonadal development is monitored by determining the percentage
of oocytes in different growth stages present in follicle sections. Complete follicles are counted until a minimum of 100 oocytes have been re1
corded. The stages of oocyte maturation are identified as follows :
I. , SMALL OOCYTES: dark staining synaptic cells.
II. GROWING OOCYTES: light staining, often club-shaped cells
extending from the wall of the follicle.
III. PRIMARY OOCYTES: fully developed oocytes in the follicle
lumen which take on a polygonal shape due to pressure of
adjacent oocytes.
Male histological samples are rated according to a predetermined index.
The stages of development are:
I. EARLY DEVELOPING: Spermatogonia form a layer on the follicle walls. Inside this layer is another layer of spermatocytes. The lumen contains spermatozoa arranged radially.
II. LATE DEVELOPING: Few spermatogonia and spermatocytes remain
near the follicle wall. Lumen becoming closely packed with
spermatozoa.
1
Definitions are based on verbal descriptions by Robinson (1983) with
reference to Mason's (1958) microscopic observations.
-8-
III. FULLY MATURE: Spermatozoa very dense and arranged radially from the lumen of the follicle. A few spermatogonia
visible along follicle lining though little connective
tissue remaining.
IV. MOSTLY SPAWNED: Center of follicle mostly empty. Few
residual spermatozoa in periphery of the follicle but
appear clumped.
V.
SPENT - RECOVERING: Center of follicle empty. Spermatogonia around follicle wall, among them some spermatocytes.
After data have been recorded for a full year, monthly percentages of
oocyte stages can be graphed to reveal seasonal trends (see Fig. 2). Because of the generally extended season of sexual maturity for male bivalves
and the fact that few sexually ripe males are necessary for egg fertilization, emphasis on interpretation of seasonal gonad development trends is
given to oocyte development. Also, prior to fertilization, sperm activity
can and should be verified by stripping of sex products from an apparently
ripe individual and examining the sperm suspension under high power of a
light microscope. Pronounced sperm activity should be obvious.
In addition to seasonal patterns in gonad development, histological
readings indicate the ratio of males to females in a population and the
percentages of ripe individuals of each sex for a given month. This information will be essential for adequate sex representation when conducting
induced spawning experiments.
STRIPPING AND INDUCED SPAWNING
The annual cycle of gonad development for a bivalve species is important because it indicates the time of year of peak sexual maturity. This
is the time when stripping of eggs and sperm will most likely produce fertile eggs, and experiments on induced spawning methods should be undertaken.
Small oocytes
Growing oocytes
Primary oocytes
100 -
80 -
60 .
H
z
rzi
rz4
40
/
/
/
/
20 _
•
J
F
M
A
M
J
J
A
S
0
N
D
MONTH
Figure 2. Annual reproductive cycle of Patinopecten caurinus off the Oregon Coast.
- 10-
Before attempting induced spawning methods, the first step should be
to strip eggs and sperm from mature specimens to obtain fertilized eggs.
If adequate numbers of viable fertilized eggs can be collected with this
method and if sufficient adult stocks are available, there may be no need
to pursue induced spawning.
During the stripping process, sperm should be filtered through a
small-meshed sieve, separating out ruptured tissue debris. Similarly,
stripped eggs should be allowed to pass through a sieve of mesh size
slightly larger than the egg diameter and held on a small mesh sieve to
wash away debris. Adequate rinsing of stripped eggs is essential to minimize bacterial contamination. As a further aid to minimize bacteria,
eggs may be held in rearing water slightly cooler than that for spawned
eggs, until the straight hinge larval stage is attained (Hansen, 1983).
Eggs should be held separately from sperm until stripping is complete.
A sperm suspension is then added to the eggs and allowed to sit for 5 min
to 1 hr, after which the fertilized eggs and water can be poured into the
larval rearing tank. If the eggs are to be reared at high densities in a
relatively small tank, rinsing of excess sperm is recommended prior to
placing in the rearing tank. Similarly, if a high density sperm suspension
is used, rinsing of excess sperm may be done sooner.
Typically, stripping of sex products yields much lower numbers of eggs
than induced spawning. However, with species such as Crassostrea gigas in
which a mature female may have greater than 100 million eggs (Walne, 1979)
stripping may produce adequate amounts of viable eggs. Also, stripping of
C. gigas is readily accomplished because primary oocytes of this species
will complete the maturation process on exposure to seawater. This is not
the case for most bivalves (Yamamoto, 1964). With Patinopecten, Mytilus,
and some clam species, stripping of eggs has limited or no success due to
incompletion of oocyte maturation.
In nature, bivalve species usually exhibit seasonal patterns of gonad
development. However, tropical species might not be so seasonally dependent. For these species, it is particularly important to be aware of other
environmental parameters which may prove to be natural triggers for gonad
ripening. It is by matching these environmental triggers or by substituting these with other spawning initiators that successful induced spawning
may be accomplished.
Spawning begins with the onset of the final oocyte maturation process.
In bivalves, meiosis is not completed until after fertilization. During
oocyte maturation, the germinal vesicle breaks down and chromosomes which
had been at the diakinesis stage of the first meiotic division move to
metaphase, also of the first division (Gruffydd and Beaumont, 1970). Only
then are spawned oocytes ready for fertilization (Fig. 3).
Spawning maturity, unfortunately, does not always correspond with the
time when specimens exhibit peak microscopic ripeness. With Patinopecten
yessoensis, natural spawning typically occurs 30-50 days past initial microscopic maturity (Yamamoto, 1964). Sperm stripped several weeks prior
to the expected natural spawn may show little or no activity. According to
Yamamoto, postponement of spawning may be due to some unfavorable environmental conditions or to the unreadiness of certain physiological mechanisms.
Consequently, prior to attempting induced spawning methods, it is important
to microscopically examine stripped sperm.
Determining an induced spawning method is a trial-and-error process of
d
Figure 3 . Changes in the egg of Pectin maximus at fertilization. a Oocyte on release; g.v. = germinal vesicle. b Sperm penetration. c, d and e Formation of polar bodies (p.b.); male pronucleus (p.n.) migrates towards female pronucleus. f First cleavage metaphase
(from Gruffydd and Beaumont, 1970)
- 13-
tisting past proven, past modified, or new methods. It should be methodical, and a review of the literature, particularly for related species, may
greatly facilitate the process. Some spawning methods applied to scallop
species are listed in Appendix B.
To optimize success in spawning trials, performed when microscopic
evidence suggests peak seasonal maturity, the following guidelines are
recommended:
- Minimize stress to adult specimens. Maintain water at environmental
parameters similar to those from which the specimens have been collected (e.g. temperature and salinity).
- Supplemental feeding of adult specimens is advised, particularly if
duration of holding is prolonged.
- Determine stripped sperm activity.
- Numbers of specimens used in spawning trials should adequately represent ripe individuals of each sex based on microscopic evidence.
- If spawning occurs, employ techniques necessary to avoid excessive
sperm (which could result in polyspermy).
- All spawning trials should be repeated with fresh specimens.
Helm et al. (1973) concluded that healthy adult oysters, supplementally
fed with phytoplankton during conditioning, produced more viable larvae
than those oysters whose diets had not been supplemented. Therefore, it is
advisable to feed adult specimens.
If proven spawning methods fail, continued attempts should concentrate
on physical methods rather than chemical methods since negative effects of
the latter might not become apparent until later development.
One measure of induced spawning success can be taken as the percentage
of fertilized eggs to develop to normal straight hinge stage. Until this
stage of larval development it is generally accepted that supplemental
- 14-
feeding is not necessary (Breese, 1981 and Hansen, 1983), and nutritional
requirements should not influence this early growth.
LARVAL DEVELOPMENT THROUGH METAMORPHOSIS
Unless sufficiently high percentages of larvae can be reared through
metamorphosis, development of hatchery technology should not be undertaken.
For example, rearing success of Hinnites multirugosus, the purple hinged
rock scallop, is currently limited by low percentages of larvae going
through metamorphosis. Consequently, commercial application is not as yet
practical (Leitan and Phlegler, 1981).
As previously mentioned, early hatchery technology advanced greatly
when feeding with cultured phytoplankton was introduced. Meeting nutritional requirements of the developing larvae with unicellular algal cultures continues to play a major role in successful hatchery development.
Hatchery production methods commonly utilize more than one algal species,
emphasizing a smaller-sized plankton diet until larvae are 120 /A ( Dupuy
et al
, 1977) or 150 (Hansen, 1983).
Pillsbury (1983) showed that growth rate and success of metamorphosis
in Strombus gigas, the great conch, can be related to lipid content of the
phytoplankton diet. Six unicellular algal species were analyzed for lipid
content and artifically manipulated, through modification of growth regimes,
to yield differences in lipid content within species. It was found that a
lipid content of approximately 15% yields the greatest food value for the
larvae of this conch.
Helm (1973) suggests that continuous monitoring of total lipid content
of larvae in intensive culture systems can provide valuable information
concerning their general condition. Gallager and Mann (1981) describe a
- 15-
simple, inexpensive lipid staining techinque which can be used for rapidly
assaying the condition of cultured larvae.
Using lipid staining techniques for bivalve larvae, in conjunction
with biochemical lipid analysis of phytoplankton diet, may provide an efficient means of developing species-specific nutritional technology for
larval rearing through metamorphosis.
HATCHERY APPLICATION
Included here is a general discussion of hatchery operational procedures, with some comparisons of preferred algal or larval rearing methods.
The discussion presents the most current hatchery methods. For further
details of bivalve hatchery operation or design specifications, the reader
should refer to Hatchery Manual for the Pacific Oyster (Breese and Malouf,
1975) and Manual for Design and Operation of an Oyster Seed Hatchery (Dupuy
et al., 1977).
Figure 4 illustrates the flow of operational processes within a shellfish hatchery. The type of hatchery products to be marketed will depend on
the bivalve species and the market for the seed.
Although current applications are limited to oysters, many bivalve
species could potentially be marketed as "eyed larvae". "Cultchless" seed
is another alternative particularly popular among some oyster growers.
this process, larvae are allowed to settle on either a smooth substrate
from which they will later be removed, or on small uniform particles of
cultch (commonly ground shell) on which they will grow individually, as
though without cultch. The advantage of cultchless seed is improved survival and more even growth to market size, resulting in a more attractive
product. "Spat", in Figure 4, refers to young bivalve seed sold attached
"Sterile" Cultures
Broodstock,
Selected Strains
Conditioning
Intermediate Cultures
Spawning
Eyed larvae
Cultchless Seed
LARVAL
FEEDING
Set Spat
CULTURES
CULTURES
PHYTOPLANKTON
Additional rearing
BIVALVE REARING
Figure 4. Flow of operational processes in a bivalve hatchery.
BIVALVE PRODUCT
- 17-
to cultch, commonly broken or half pieces of oyster shell. Although bivalve
methods of substrate attachment vary (e.g. scallops have byssal threads),
mussels, scallops and oysters might be marketed this way. "Additional
rearing" is necessary for clam seed which does not set in a fixed place,
but moves with foot and byssus tethers. Some scallop species which live
attached to cultch for only a brief part of their lives might also require
additional rearing before marketing. Manila clam, Tapes japonica, can be
seeded on small-graveled, somewhat protected beaches. To survive, they
must be planted at 1-3 mm size or greater, thus requiring additional hatchery rearing before planting. Rearing may be done in shallow flumes since
larvae become benthic after metamorphosis.
If larval nutritional requirements have been determined for a particular species, and if preliminary culturing of phytoplankton and larvae has
indicated success through metamorphosis, general hatchery methods can be
applied to attempt commercial seed production.
Algal cultures:
Regardless of phytoplankton species or of culture methods to be used,
nutrient solutions for feeding algae are roughly the same (Breese and
Malouf, 1975). Table 1 lists the nutrients for four stock solutions. When
prepared with distilled water and autoclaved, these solutions can be added at 1 m1/1 to culture media. For diatom species (e g. Thalassiosira
pseudonana, (3-H)), a stock solution of 2 gm/ 1 sodium metasilicate should
also be prepared, to be added at 1 ml/ 1 (Hansen, 1983).
A hatchery facility should have a separate algal isolation room to
allow for the maintenance of contaminant-free cultures. These "sterile"
cultures, grown under fluorescent lamps in test tubes or small (e.g. 125 ml)
Nutrient
Formula
Stock Solution
1.
NaN0r,
sodium nitrate, granular,
refined150 gm/1
2.
sodium phosphate, monobasic,
certified
3.
trace metals'
cupric sulfate
zinc sulphate
cobalt (11) chloride
manganese dichloride
sodium molybdate
4.
vitamins
biotin-crystalline
vitamin B-12 crystalline
thiamine hydrochloride
NaII•P0, • 11,0
CuSO, • 5H,0
ZnSO, • 71120
CoC1, • 61L0
MnCI, • 411.0
Na,N1o0 • 21-120
10
gm/I
1.96 gm/1
4.40 gm/1
2.00 gm/1
36.00 gm/I
1.26 gm/1
1 mg/1
1 mg/1
OD
200 mg/1
The concentrations of the trace metals given in the table are for the 5-primary solutions. The single secondary trace
metal solution is made by mixing 1 ml of each primary solution plus 10 gm of ferric sequestrene and filling to 1 liter.
I
Table 1. Nutrient solutions in algal culture, after Matthiessen and Toner. Taken from "Possible methods of improving
the shellfish industry of Martha's Vineyard, Duke's County, Massachusetts," by G. C. Matta iessen and
R. C. Toner, 1966. Marine Research Foundation, Edgartown, MA.
- 19-
stoppered flasks, maintain phytoplankton cell lines. Growth is retarded
in these cultures by storing them under low light intensity and at a cool
temperature. These cultures are used to inoculate working culture lines
in flasks placed under brighter artificial light, also in the isolation
room. These lines will be grown to greater densities for inoculating
intermediate cultures. In the isolation room, all culture flasks and
their contents should be autoclaved prior to inoculation.
Rapidly growing phytoplankton cultures, such as the intermediate
and feeding cultures, increase the pH of their medium through utilization
of CO 2. Certain algal species must remain within a pH range for optimal
growth. Consequently, aeration of the cultures with CO -enriched air is
2
advised to control pH and thus prolong growth of healthy cultures.
Another aspect of phytoplankton growth is that initial division enters
an exponential growth phase, followed by a stationary phase and subsequent
degeneration. To insure healthy cultures, when inoculating a newly prepared medium, the inoculant should always come from a culture still in exponential growth.
Traditionally, intermediate cultures have been grown in 20 1 glass
carboys. Although it is the practice of some hatcheries to autoclave
these, it is sufficient to chlorinate the filtered seawater by the addition of sodium hypochlorite to 5 ppm. The chlorinated water is allowed to
stand overnight and dechlorinated by addition of sodium thiosuiphate.
Nutrients are then added, followed by phytoplankton inoculation. An alternative to glass carboys are large (80-160 1), upright cylindrical fiber
glass tanks, or plastic bags of similar volume contained in heavy mesh
cylinders (Earnst, 1984). Both artificial and ambient light is recommended
- 20-
for intermediate and large volume cultures. However, during seasons of
greater insolation, ambient sunlight may be sufficient for all outdoor
phytoplankton growth.
Pure intermediate cultures are used to inoculate the larger, openair feeding cultures. Consequently, the desired volume of intermediate
cultures is related to the size of the feeding culture. Based on the
author's experience, when inoculating open-air feeding cultures an initial
dilution no greater than 1/8 - 1/10 is advised. For example, if a 3,200 1
feeding culture is to be inoculated, 80-100 1 of intermediate culture would
be pumped into the feeding culture and diluted to 800 1. The following day,
if the culture has grown (darkened) significantly, dilution by filling to
3,200 1 can ensue, with the culture expected for harvest on the third day.
Ambient light, condition of inoculant, quality of filtered seawater, and
frequency of air-borne contaminants are some of the many factors which may
alter the expected growth schedule.
Three harvest methods are used independently by oyster hatcherymen in
the Pacific Northwest. Batch culture method, which harvests a complete
culture once it has attained its maximum density, is the most consistent
harvest method in yielding clean, monospecific algal cells. Although this
method is commonly used, semi-continuous harvesting can yield a greater
number of algal cells with a minimum amount of maintenance over a fixed
duration of time. With the semi-continuous method, once a culture has attained a harvestable density, rather than use it all at once, a fraction,
commonly 1/2, is harvested and the remainder is filled with filtered seawater for algal purposes.
2
2
The culture will continue to be harvested,
Algal water at Whiskey Creek Oyster Farm is double filtered through a
fine-grained sand filter compared to larval water which has been once filtered, separating out particles > approximately 5j.4.
- 21 -
once or twice daily, until it can no longer regain a harvestable density.
At that time the culture should be drained, cleaned, and reinoculated. It
is common for cultures to be harvestable for 7-10 days before draining and
cleaning. Considering it usually takes a new culture 3 days to become
harvestable, more cells can be grown and with less effort using semi-batch
vs. batch harvesting method.
For open-air feed cultures, this investigator has found that unless
the seawater source is fouled, there is little difference in the duration
of semi-batch harvested cultures whether chlorinated and neutralized seawater or if untreated filtered seawater is used for refilling harvested
cultures.
A third harvest method is continuous, using a chemostat. Although
this would theoretically be an ideal situation, chemostats are very sensitive to changes and only relatively small, more manageable ones are in
use (e.g. 200 1). Consequently, the numbers of cells is also small by
comparison to batch or semi-batch harvested phytoplankton cultures grown
in larger containers.
Broodstock and Conditioning:
An assumption made before attempting hatchery development of a bivalve species is that sexually mature adult specimens (broodstock) are to
be available for spawning purposes. These adult specimens should be maintained where they can remain healthy without spawning. With the Pacific
oyster, growers may bring their own specimens, usually selected for rapid
growth, to the hatchery for spawning purposes.
Mature bivalve specimens are usually capable of spawning viable gametes for only a relatively brief portion of their annual reproductive
- 22-
cycle. Consequently, the time of sexual ripeness, the "window", must be
modified to extend or initiate sexual maturity. Conditioning is the artificial manipulation of the window for the purpose of obtaining viable
sperm and eggs. This is usually accomplished by modification of environmental conditions such as food and temperature.
Although initial work with a bivalve species may require that stripping or induced spawning occur during the natural spawning cycle, continued
work may allow year-round spawning through development of conditioning
methods.
With C. gigas and C. virginica, seasonal sexual maturity can be manipulated by temperature. To initiate gonad ripening in recently spawnedout specimens of C. virginica, a period of cool water temperature should
be provided before increasing the water temperature of the conditioning
tank. This subsequent warm water will stimulate gamete formation. (For
further descriptions of seasonal conditioning methods for C. virginica
refer to
Dupuy et al
, 1977).
If conditioning methods are successful, it may be possible to supply
year-round bivalve seed to interested shellfish growers.
Larval Maintenance:
From fertilization until metamorphosis, similar treatment is given to
larvae of different bivalve species, with the exception of water parameters
such as salinity and temperature. For Crassostrea gigas. although relatively wide salinity tolerances are allowed (20-30°/00), recommended temperatures have a narrowed range, 24-27°C, to insure optimal growth and
development. Evidence from lipid staining tests (Gallager and Mann, 1981)
suggests that fat reserves, necessary for healthy growth, are better stored
- 23-
at lower temperatures, but that at higher temperatures (e g. 30°C) less fat
is stored presumably because of increased metabolic activity.
Frequent water changes are essential for bivalve larval management.
Seawater contains dissolved organic nutrients which are essential to larval
development (Courtright, 1967). Larval water changes serve several purposes: 1) to replenish organic nutrients which may not be sufficient in
larval feed; 2) to avoid build up of metabolic wastes; and 3) to retain and
sort the larvae, through use of sieves, allowing dead, bacteria-ridden
shells to pass and to separate out larger contaminent zooplankton.
The first water change is recommended after the straight hinge stage
has developed then once every third day until the larvae reach 150)A, followed by every second day until harvest (Hansen, 1983). At some hatcheries,
twice weekly water changes are done for all larval tanks. Water flow capacities must allow for the required exchange volume to pass within a
working day (unless nighttime filling is anticipated), in addition to
water needed for algal culture.
Typically, larvae showing fast initial growth will continue to outcompete smaller larvae. By using different mesh sizes, larvae can be
sorted at each water change, allowing larvae of similar size to be grown
together. Larvae of different spawning dates, however, should not be kept
together. In some hatcheries, larvae are refrigerated and held until tanks
containing larvae of similar size become available. It is not known if
this practice has a long-term effect on larval or postlarval growth.
Also during a water change, larvae can be reduced in density, by dividing one tank between two or three others. Breese and Malouf (1975)
suggest a maximum egg or straight hinge stocking density of 200 eggs/ ml.
- 24-
For growth of straight hinge to harvest, a concentration no greater than
5 larvae/ ml is recommended. At the Whiskey Creek Oyster Farm, approximately 10 ripe female C. gigas are induced to spawn with increasing water
temperature. Loosanoff and Davis mention that 10 ripe C. virginica females
can spawn well over 200 million eggs. If 200 million eggs are assumed
spawned for C. gigas (and the 6,000 gal tanks are filled to 5,600 gal),
then the resulting density at Whiskey Creek is 9-10 eggs/ ml. Because of
subsequent splitting of larvae during water changes, a resultant harvest
culture may have an estimated 30-50 million larvae per tank, roughly
1-2 larvae/ ml.
Feeding, which begins after straight hinge development, can follow
the recommended daily schedule:
1st week - 30,000 algal cells/ ml
2nd week - 50,000 algal cells/ ml
3rd week - 80,000 algal cells/ ml
This schedule for C. gigas, is based on 21 day growth to 315)A(eyed stage
or metamorphosis) (Breese and Malouf, 1975). For faster growing or larger
larvae, the feeding schedule should be modified as appropriate.
An alternative to cell counting and measured cell volume feeding,(described above) is to feed the larvae twice daily, visually estimating the
algal density. Although both methods can be effective, taking regular cell
counts and feeding designated water volumes is much more labor intensive
than visual feeding. This additional labor, however, may be compensated
for by faster and more uniform larval growth. Whiskey Creek, which uses
visual feeding, produces eyed larvae in 14-15 days. Coast Hatchery in
Quilcene, Washington, where larval feeding is with calculated water volumes of known density, claims an ability to grow Pacific oyster larvae to
- 25-
the eyed stage in 11 days.
Cleaning:
Intermediate algal culture vessels are cleaned with acid and rinsed
with hot water prior to filling with seawater and chlorinating. During
semi-continuous harvesting, culture quality usually declines because of
increasing concentrations of contaminant algal or zooplankton species.
Also, the bottom of the culture accumulates dead cells and associated
bacteria. Consequently, to maximize culture duration, care should be
given to clean tanks thoroughly with hot water and bleach between use.
Similar to algal culture maintenance, larval cultures should be
cleaned with every water change. Use of hot fresh water and bleach is advised, although extreme care should be taken to rinse adequately before
refilling and adding larvae.
Handling of eyed larvae:
Larvae of a given species typically metamorphose upon reaching a certain size. Newly formed straight hinge larvae of C. gigas are roughly
90 )Ain length. At the eyed stage, just before metamorphosis, these larvae
are approximately 315/kA in length. Size, appearance of an eye spot, appearance of early gills and development of a foot (thus termed pedivelliger
larvae) are criteria which can be used to indicate approaching metamorphosis.
When growing oysters, these clues indicate the product is ready for
harvest. After draining the contents of a larval tank onto a sieve, the
larvae are rinsed with cold water while being held on a sieve of appropriate size to retain only the largest eyed larvae. Smaller larvae
- 26which rinse through the sieve are collected and placed into a fresh tank
for additional growth.
The retained larvae are then passed through a larger screen to sort
out debris and contaminant zooplankton. The filtered larvae are then
rinsed (also with cold water) into a calibrated settling funnel and counted
by volume, knowing the number of larvae per 100 ml. Alternatively, eyed
larvae can be weighed given a predetermined weight per million count.
Oysters and presumably other bivalve larvae, can be refrigerated and
stored for later setting. Henderson (1983) found that eyed larvae of
Crassostrea gigas are able to retain their setting competancy while in
refrigerated storage for up to 8 days at 5°C. The ability to store larvae
ready for setting allows the product to be sold wrapped only in nylon
cloth and moist paper towels and shipped to the oyster growers in small
iced Styrofoam containers. Marketed this way, Whiskey Creek Oyster Farm
guarentees a 20% set for the oyster growers, which generally yields 50%
settlement or better. Marketing of shellfish by this method eliminates
large shipping costs previously associated with the purchasing or collecting of shellfish already set on bulky cultch.
CONCLUSION
Several bivalve species could show potential as marketable products
if grown commercially. In the Pacific Northwest, there is great interest
in the aquacultural planting of the Manila clam, Tapes japonica, and the
the culture of the weathervane scallop, Patinopecten caurinus (Bourne, 1983).
Other bivalves such as the northern razor clam, Siliqua patula, and the
geoduck, Panope generosa, have become depleted in some areas of their
habitat, generating interest in their hatchery development. Reseeding pro-
- 27 -
jects for these two species are currently underway at the Washington
State shellfish laboratories (Olson, 1983).
Through the successful development of spawning and larval rearing
technology and through sound hatchery management, it will become increasingly possible to provide hatchery seed for many aquaculturally desirable
bivalve species.
SCALLOPS
Investigations on Spawning and Larval Rearing
of Patinopecten caurinus
- 29-
ACQUIRING AND MAINTAINING SCALLOPS
Gonadal sampling of Patinopecten caurinus populations off the Oregon
Coast has indicated that the highest percentages of sexually ripe individuals occur from late January through early June (Robinson, 1983). Consequently, investigations on induced spawning and larval rearing were undertaken during the winter academic term. At this time of year, however,
river discharge into Yaquina Bay is at a maximum resulting in salinity
problems for scallops at the Marine Science Center.
Scallops have little tolerance to salinity or temperature extremes
and exhibit stress in reduced salinity environments (Yamamoto, 1964 and
Itoh, 1982). Because of these potential problems, scallop specimens were
kept in closed system aquaria fitted with aeration and maintained at salinities and temperatures similar to the areas from which they were harvested. Since there is no current hydrographic data on these locations,
monthly salinity and temperature averages were calculated from previously
existing data (Table 2 and Graph 1).
Table 2.
Month
January
February
March
April
May
June
Salinity
Temperature °C ppt
8.8
9.1
9.2
8.5
8.2
7.7
33.8
33.5
33.5
33.7
33.6
Month
July
August
September
October
November
December
Temperature
7.5
7.8
7.7
8.0
8.6
9.0
0
Salinity
ppt
33.7
33.6
33.7
33.7
33.7
33.6
Hydrographic data off Yaquina Head, 1961-1964, 105 m depth (station NH-25).
(Wyatt, B. and N. Kujala, 1962 and 1963, Wyatt, B. and W. Gilbert, 1967).
Newport fishermen primarily harvest off Yaquina Head north to Three
Arches. Trawlers leave the bay during periods of calm seas and, if successful, return after 2-3 days with iced whole scallops. Unfortunately, there
0
"41113
1
0
P
(-)
O
h.•
0
0
0
0
....:
Ilalesew •
2
ffli'l
r_...
:
1111
sti AV
• Mims
I1 rma el•ima
4
iun •,.
a s
mu
11111111111
smullum
:
Tr- runi...
a.:::::::-.4..
„•• II
m"
Apr
:
riiElqqIPHIP11111111911kusellkgrEIBIErilli:II
-hiihuuusienibilaiii.ddiudiWasiklikilin
n ibsi.
°°°;;;IIIIIIHEIRMIPARJHBAIMMOMMIN11111111111111
I:
:
is •'-1 i II
pi.sqmpq.:
sq....
""""Puimim",""101,111111dbikond.maillg
q
111111111.
"""
ue
lure
sumplu m ilim
. .111:911.
I
1
mmm mm
"Fillq
14111.11
11...1
"1
'm
" " am.
6 !".101
mar ""
1
1.11.1....: :::::::...:
1
11111
11111126
doll. a
al
.1 •1111
1
lel
al
11.1
1:11111.: im
1111.114"11111111111111111111111111111111111111111111
11111111111111111111
r
mg
1
4i.
411:1 11111
dIEd
Vill
loll
MI.
p
3=.. 317. .. • .1 .11. ::
ll!' N"q ",91""PMP"111P1111
:::: mi !HI . • .1:111 is. :
as 7: • ••: sidnimpdri
min: Erarri
ril
::::.:
. :1. s. I! 1 1is mlln
. :1
1 ...:19:181
a
ureqiqqilimarrAmiimmum
1
: .1
ingp111:1111111:1111111.mullts.-.
1
"lil i :
raukslud ..... 821:duithilis:041Milliuidiffidilli
1::::
..... nili
1
11111111111
11;8111111111111111111111111111111111111111111111111111111111
IIISIIII:911111101HOur d'Im m u ditim 1:113"111::::::191HolL,:r
.ituil iiimmiffiffillIMMINIMMEN91111M111111011111111111100
g . ...e :
ii
1::::
1:11:111::::1
/
1::::::......1i s :
garel 11:11://1::11
rimirmr"IMPIMPIONPIPPMINPRIEWHIM
gogmagensw,
pemia
m a ring
ommormssmil% ,
I11111111W 'I
•ms:
re/
Imm:
/
1 :
:
/I mm 1131:112 1 .. mums 1. 1 ,..11111:7 41114111 1 11141 1 1. 611
dffigliglillidliniralq11
H....
i llimagmanamer 1111111IN Jai mar 111331 14 ../1.. 4.1.
111111.
mmatimig
11:1-mmhommull „mum=
namussoll
ummillillimulha 111411111111111 ,111111.111flialln
1:11111ftell lig 1111
4 • A../
il
1 dlligailiffifililiMMOIll
:
p:::::..m... :, mussivf:11.11..
1
■
mop
■ 1
:
1 ..... ....iremaialliiii
614"iim1 11"or"14
railli
fi
al
r illimi9911111111
.
11
:TN ..r...:
MI • WPM' 1141•4 ..... amir_qp
sus
1 11111:14111115
I i ■11111111111111 11 ■
i
WEINI /I II
I/
III
::.....
III
.11111
'11::11
limn
imi morns . um _ fillIIIPM
r
-nrit
.
uhrOrprillirrilip
'
sig4
es
g
/1
swims
2.... '
/
"' 4 s :191 .4ii 4 ..I. 1 1
1 11. I
RZM diENIMM0119
1.1 ....:...::::q„ 1: 110.1.1%.%
normei„.„„:•::::::usu..inuommut
s• um:
1 "ii III!:III118111.111
1 I II
:in: : ::::.:::::..
11. 1....:::::::::::16..1
1
:
i
t
la
P
H
i
ii
INOW
I
PPL
I
MMERuskull
.....
.
..-1.1
[
...t.surrupri
mum
:
nu
111111111111211
1....1•11111:11
I
IP11011141.2r1
1 i .1a "SWAP al 11111111911111111111111
::
:
1 1...1 11111111 11111111
1111111
iii
1-11:::::::::11:1.1..,
all 11:1 1
siglii11111111111111111111;111411iiii
11111
si
u
ulu--11:
e
::
Iii.::
::::::.:11i i .111111;::::ifiliiii.sediiiiniiimissublisildifi
:::::::
I:11111111ln
1
II: 111111.111111111111111911111111WERIIIIIIIIIIIIIIIIIIIIIIIMIgni
o
.. 1
1
: :
1....mmullgl i 1 1 1 I 111 11 11 0: 11111 : 11 1111 :101111 : 11:1111111 11■1
1111117 1191 I ralsmova ll ia......9:: 1,9
1 mi.
H
u i..... 111.4 I DA
ma_ ..... pal I .. ..1 8 111 11111111111 1 11 111.. 1
• 1.1111111
• 1111.44111141111111111111111
.111111 11 .1111111m "
1
I••
g 11111 1 " 1 " 1 " m il ml 0:: 1. sla Ai
1:1:1 .111: 1.: :11:1:1 Hi:: 11:1 I. • .:
...
..
1111:11"11111111111111
44"
mr:r111!"
1111111111911 1111115111 II
MMMM
SOMM 11.. ..___.
UW.
/1 u r -33talttlzalt'
" "IPPFiii olibr
i i el• n e. ' KAT INPVIPPIP1'■
iffirigtmiod.slitvimiprin
mgr. pm ir ilippllirdp
pqrpllim
Mihmh1111111111P LEMPlunalr
uniat L.. .. ....::11: i .... ..I WILII:11 :1:1:1 1111 I Ai: 1 i
..:
■
M unuinireir
rdEVIP.. I lipilii::::1114
11:111:filluills:
,
ramn
im....dippiem
ptippo sitirawomprom
su a
u.I. • Idliguidomisti:::::imm11..1
.
:: ... :
• • • ..1
....:..1. .• mg:: ■• . 1 .g: ::.1: :: sr . i11
....:::::.: as.14:1 :t. 1::: .. :..:
::::::: :111:11111111111:1111111211
1111111141
:111
lulu.
11111
1
p.1.1....1
■IIIIIIIII-III
:p.n.
.1
so
:I 1111....
.... Is
3: = :: 3
oulimmica
illmilimull
Iii:111111:11Milidl"
66"
13111111111"
■111:11111
1:11111""
kmmmi:11:111:1131:111111111
1
. •
.
rimpFiReqriququaiqmp_. Trirsur... i nqquopsumeng quust .13.11
Mr
s
i
ttlE
4
513NOBA
t
I momulloilliq
::I- .. skukhillilibuisiiird ■ ■ iiiiiiiffi:::: ImI
lill
Ig.
IIIII
I
I
I.....
......
s:::simmilgi:gniumpoep
girlie
4.00.11116
1116-4441hipihiliiilluil pillowilmongeliliatiiii: "ra
1111:11 i:::
1... :::: : : 1.1. 11:::::: musur
s: .s..
.s : r:
. :..s.1..
..
1:::
IP
Pi
nis
IlifinigliEgilla:16131::13::1 111111:1:111111 .11.::::111.
1
ei
1
1
ES
::. .
1
MASSIMSMASASMASSIMEMMIA
PUMMIngil litll
ME
MESSIESSIM
I
MMIS
....... NEE f ISMS M I MIIAMMEME1
1111
1.
:•
SO.OM El..O.liSM..111
. IN
me
1 41
AIM ....... .10
•
M
S •
2 %dm MM. • ISM "a .
•••
u : : . dfiddIME
il r:
2 OET J
MU
'A
ri
. -
Ail
.0.111 11 E
•' •
i
11115
.11 • S.....
g : - '
g
-
•
MI I .....
MOM • pi
SIM MM. • IS •
•
:: il.
■ : :
1
11:11 1 •
1
- 31 -
were only 4 periods of calm seas during the 10 weeks of this investigation.
Consequently, total numbers of adult scallops were less than anticipated.
The last scallops harvested are usually the first to be unloaded so
it was desirable to be dockside when a trawler returns. Because the process of unloading to cold storage is hectic and potentially dangerous, when
aquiring specimens, the only allowable sorting is to choose unbroken shells.
Scallops were brought by bucket to the Marine Science Center and
placed in one of two 100 1 fiber glass basins of desired temperature and
salinity. The basins were in a freshwater bath in a large fiber glass circular tank. A refrigeration unit and submersible pump were used to maintain the waterbath temperature. Initial temperature and salinity were
8-9 °C and 33.5 ppt. This salinity was achieved by storing the highest
salinity water available (28 ppt at spring tides) and boosting this by
addition of a rock salt mixture (Courtright, 1967).
Newly arrived scallops contain sand and debris which will be purged
within the first 24 hours. Some individuals, however, may be heavily laden with sand due to the rigors of dredging. These specimens can be assisted in purging by applying thumb and index finger gently to the sides of a
gaping individual, forcing the valves to remain open. The specimen is then
held vertically and dunked repeatedly but smoothly in seawater until voided
of sand. Sample specimens with excessive amounts of sand would preferably
be avoided since these individuals have the greatest amount of mantle damage, usually leading to death within the first 3 days of arrival.
Due to purging and death of some specimens, the holding basins may
need to be changed several times in the first few days of the scallops' arrival. Specimens surviving to the third day generally remain healthy.
-32-
Once adjusted, adult scallops were fed approximately 4 1 of a dense
culture of Tahitian isochrysis daily. This amount could be cleared within
6 hrs by 25-30 specimens. Other than feeding, maintenance included a water
change every other day by transfering scallops to a 100 1 basin with prepared water of the same temperature and salinity.
SALINITY SUBSTITUTES
Salinity posed a problem at the Marine Science Center in that the
available salinity of incoming water was less than desirable for scallops.
Consequently, it was decided that a synthetic salt mixture should be used
to boost available sea water to the desired salinity.
Commercially prepared salt water substitutes lack some organic constituents necessary for developing larvae (Courtright, 1967). To eliminate this problem, Courtright prepared his own salt water mixture, "Bio
Sea", which proved suitable for developing Mytilus edulis larvae (Table 3).
Table 3.
Preparation of "Bio Sea" (Courtright, 1967).
Compound
Concentration (g/l)
NaC1 - "Leslie" coarse hide,
solar evaporated rock salt.
33.0
NaHCO .2
3
KC1
.5
MgSO 43.75
CaC1 1.0
2
(Resultant salinity approximately 35 ppt.)
The primary ingredient of "Bio Sea" is solar dried rock salt which was inexpensive and readily available at the time of Courtright's thesis. Unfortunately, the only rock salt currently available in Newport has been
kiln dried, a process which denatures certain desirable organics. Consequently, various whole or partial salt water substitutes were compared
-33-
using available rock salts. Compared salt mixtures were:
(A) Control: 28 ppt collected seawater (MSC), air evaporated until
32.5 ppt.
(B) Courtright's formula, prepared in distilled water using "leslie"
kiln dried rock salt.
(C) Courtright's formula, prepared in distilled water using "salt
from the sea" salt (unknown whether solar evaporated).
(D) Fresh sea water (25 ppt) boosted to 32.5 ppt using appropriate
"B" mixture.
(E) Fresh sea water (25 ppt) boosted to 32.5 ppt using appropriate
"C" mixture.
(F) Fresh sea water (25 ppt) boosted to 32.5 ppt using 7.5 g "Leslie"
kiln dried rock salt.
(G) Fresh sea water (25 ppt) boosted to 32.5 ppt using 7.5 g "salt
from the sea" salt.
Standard bioassays were performed on larvae of both Patinopecten
caurinus and Mytilus californianus. Procedures for bioassay are discussed
in detail by Dimmick and Breese (1965). Criterion of effectiveness in all
tests was taken as the percent normal shelled larvae at the straight hinge
stage. Fertilized mussel eggs were obtained by induced spawning of adult
specimens using the algal method (Appendix B). Scallop eggs were stripped,
treated with ammonium hydroxide solution, and fertilized (see next section).
Bioassay results (Graph 2) indicate that substitution of kiln dried for
solar evaporated rock salt in "Bio Sea" is unsuitable when preparing this
mixture from distilled water. More importantly, however, the results indicate that fresh Marine Science Center water of 25 ppt salinity can effectively be boosted to 32.5 ppt by addition of rock salt alone, and that this
new salinity water is safe for larval scallops or mussels. In fact, the
performances of boosted salinity waters with rock salt (mixtures F and G)
were slightly better than the control 3 which suggests that it is preferred
3
A more appropriate control would have been sea water collected offshore at the necessary depth. However, difficulties involved made this impractical.
0
1
11
sill
1
...... ..... I.. Imams
:up
:I:
1:
:
lilli11111111111111111i1 " " mmi ..... 111:1
ssiiiiIIIII
iiiiii11111111111
111111 1 2 1:1:11::::11:1:111:::::::1111:1:.::::: .1
1
:
....
L.:.
1111:111::..1111.111
um
:
.....111.::::::::::.
:...:
...
::::::
Ili:. latighluslImilt Mar: . • ... 0 kiltitimpu
-4,17_ . 1 :;;;...4:11161:11:1111111111111111111111111 :4 9: •11:1•1 p_ms.
1 : 1 1 111116 11
"111.1 1 1111 111
(;)r
1• 1111111
mm1 111•11"
iii..._,...
11:11:1
1:11111. 1:1:11 :1
6 ■. 11140.7::
... ■ ...
II 1
.1.9imi
...I...
Hal
imon•
fill
ITEEPTIMBElpffilebn
. . ... emirml:::.
0
11:11 1 1:1
:::::1111::
AI:
r,
1
•
1:1:11:11 ::::::::111.1... . no!
::.1 111
•1I.:.
11
..:
11111 1111111111111111111111
4
11111111111111111111111111111111111111" 1■1111 MIL 111111111;alio 16111111111111111111111 111111111111111 111111111111111111111111111111111111
11111111111111
11111111111111111111
11111
111111111 411111111111
r 1111111111191111111111111111111 ,. 1' 11111111111111
1111111111111111111
... MMMMMMMMMM
....111114'.1 "111111111111 1-' 111111111111111111111111111111111111111111111111111111
ri1 •■ MM•MM■ regain
um:
:11 ::::
1, r ,..11
0
''J1?
, ■,,■ ssommiss
MOMMgamM sommimMeIMIO
•
•
11 11111
',;::
Imml
lisa mom
so: isms.
m am mgmg 3,,i4
,:: Illibrillilli111111111
•
1
11
111111111
1:111144.191:011:211703::" II°
.,
„
I , en
,,,,l,
:1:
::11:11::...1
,
1....I
iems Iom
m e g lemmal MMM4.441.444
.Trr 1/11mms
gm 0 1 Memel
444444 ememgmm
IIIIII:
Aa.i
•
:
MOO
:$s
44":
:
r
ual
1
111
111111111
Eli
.,i,
:
.
611:1 MIMI Mem i q I 9$
1
1:111°
1.gm ssss . mos
pso. 6 1
MOOS MOUSSE MMMMMMM Aill NM i :
II/1111111111M
::.:
........
0
OOMMOOMMOMOOMMOO
ern ss•
1:■'
,,...:.......
i
MOOS
al
LOOM • ■
• :::
MOPS
..
S.
4
1,
WI
PIP
•
1
/A
II 11I.
11 11:S
0
11"
i iiii.:11 :7111111111
1
:1.1-11
1
111:11 11.. ■
I '' 11111111111111
11111111111111111
z
BIM
:001
ii 11
14. 4I 6•
, rn
:..:......... iliu
.. ::
!II
li 1 115 1di4,
. .1.i.
P P MO.M.S....
..
MageMeMOSIMMO
....
..11...........
......1
...
111MMO:
: I li ::::::•—:
11a
.1 ::::::::::::11::. ....
1m: 111:::::::::::
1111111111111.11
I I"'
swum
leesepegg:
::!,q
:gall
0
1
, mu
; .1
:',:i
•I
Sagami
................
i
ISMOn
,°,4;:,,;
111 111= $ '!'!
1
:
.::1
r--,•:::::::
Ili:11m will ,atv
..,i.ti 1111111111111111
I IIII111111 11011111111111
7$
0
IIIIIIIIIIIII1 milliir
lI mul p : 111:991 . ::: PR:
f
!If
i
ininnummilliiiiii
mmemMEM
III A . .:111:1:::::!: • ° L I! 11111
.
11 m.o.
i ...
: ...
11111111111111 .1 1111111111 '
111111/111111
111111111111111111
'
,Abe
m
1:1111.4.11:1111::
Id
Ilmi ssmiiiiil
11 0
III
... .. OM f MI
11
:MEM
•
>
0
:
I
to
0
0,
•1
a MMMMMMMMM samessan Aga
mm:1114
MM
U
4J1111111
OMmelegggsageMMIMmsamms
MMMMM glin
m sammeMeammee
im
iiiiiiiiiii
i•
. 4I
na
MN I MMMMM ma •
4,40
::::
• Tu
.11111
IL
111 ill
il4e lininirdliffikr
f,.
JO
1122. me:
Magna'
I
IMM
°" IMMOMMUIMM
II.: •
1.0R
mom
gmeamesea MOM
1
ilillill
•
•••••••••••••
•
1111,0
1
Liter
:::
IMMIgggpg:
11MMM :
,... 0
MUM sums
■
Mal
meg ■SUM@
In
021111
0, erns
elliaMmil
1
!
CUIR
eMOK
■ il
II
ES
W
11111111111111
M
lima
1ISS / ,4 N
ONE • • mem
111.....1:::::111111 1::::::MAIIIIIIIIIII 1110115
am 111
/1a•
1
1 •::::..1.1.... ,i.1r4
On
'1
tin
1111:1 1i61
111
.11:
11 ■
li lee
1311111
11 IiiIIIIIII MMMMMMM MB Jilin "s" 1 A II 4
1111"."
iiiiiii
1 .:::
1
::141
mill
:
s
.
1 itr.
tor
4,KP
:• a Mom:" ..... Alf,
mese. q 11 .
...
Wig
11
1
gmeggeggamemem
1111Q
JO,
mam a
1,r
1
1
K
i''
1111
C4,1
4.
III
IJ4,1:
ii
I
1113 I
111111sIs
111141
1111211111
mead RS mu
insisses
lell
MMMMM
■.
1 EN MAIM 1111111111
mo ul ti
II 1111111111
11111111111
111111111111111
11
,
.11
Mgag igegge Pril.
ern Ogg
smIll liam
l
11.11.ME.M.
11 d .
MIIII
ijillIllIl , -I
A
itt,1111 II
Ns I,
:Ina
111111111
1
;I:'''.
11 ..•■lIl1 1 11 HM I ill •l!las
•••r••••
Melo moms
OHM
"moms MIMI
iiii111111
■ RME mag■
JA, :
1
6"1 1.1A 11 ■■..
mesa
magglemismigam map::
:: M/K1 .r
■ 1
magm IS SEM
W
Me
I
maim
g
eel
M
g
1
W4
mill.mmu:
9
"
i
REIT!
EMI
1014
m
■ d:
g—
111 1 1 ■■
11111121111111
Mmme EOM
ga i
gle 74.1
mina gal
:am■■
::
OH
g
:
MMMMMMM I I "
:::
MEM
111111
1
1
tKit
, El
1111111::11:::11.
■ Immo.
SeMMEMMeg
1
a Mal us
NOM
••••••••11SE
:.■
•
amuse
a
samm..1:11 '
''!'r
11111111111
EM:
I
L
1:
i
1
1
ape
6
og
:
:
Mg
ril
::. Allig411111:1 A111111111111 11111111111111111111111111111111111111
1111
1
:...1,...1.111::::::
•:
:
eiWituudthiCIErtri
liffirik..t.: MMMM MM MM MMMMMMMMM MMMMMM
1.1 Elm
Erni lim
11113
. .. .1... 1
r::•+•••:
.
1
•■•
:: — nu:
:::::::::::Es
1:::
::::::::::::::.:11:::: 1:1 ■
mi: and Hem
:
:
:
::
1:11
pima:
:
ihm::::11::::::111 674 : all•
urn
..
1:::::::::.::
:.: :::::.:::::: MMMM1
tioq
449
adtko
. Aim
1:::.:Eigrami n
1111111111111111111182111
111.911 .. ...rip .ismitio
11111111111111111111111111111111111111111111111111111111111111111111111111111111111
poplimpri .vri:
..
iiiiiiisimlemil. .... .1 i. 1 Illaii----"
1::::::!:
4 1.. :..:
.■■ . .1 ...1 ..■i ]igir.: ' • ' 1..i • 1 1;
MMMMM Mi.
1....1 1::::.d.:::::::" ,
■MO
!...1
um
1..1...u
t
wilinithild.
1:1: 11: sae MM 1 111111111111111111 4111111
1
:1:111111111:11:1101
ag e
Oa ea Me Meg eagall::::::::: ::
Malt 1:1
::: :::::::.11.
1:
lemilumbli..1.1
■
ii:::
1.....::::
:
II":
is
:111
umaril IIIIIIIIiiiiii.... .
sass ;;;;EIT:infilillm::::::1:::.:::•-:
1 11:::::11:::::1 :::::1:1::::::iim
::::1:11
1 iiii JAM i
IIIMI 11111111111111111.
••rr.
:.
. . :“. :r
5411
.::::;Ekhummommillsommouor
FM
11111111111111111 111111111111111111111111111/111111111111111111111111111111111111111111111111111111111 111
111111!
S S 11111111111111/11111
SOSO
Mil
III/II
offium
- 35-
to use freshly pumped, boosted salinity water than to use higher salinity
water which has been stored for extended periods of time.
In view of these results, continued work with adult scallops and larvae utilized boosted salinity sea water.
TREATMENT OF STRIPPED EGGS
Although gonadal sampling indicated peak seasonal maturity at the
time of this investigation, stripping of eggs and sperm from ripe individuals at this time yielded only low percentages of viable ova. In initial
trials, only 6.7% of eggs obtained this way became fertilized (n=412).
Yamamoto (1964) observed similar low fertility rates (10-15%) in stripped
eggs of Patinopecten yessoensis, but noted that fertility rates could be
improved greatly by treating eggs with ammonium hydroxide solution. However, although this compound can stimulate oocyte maturation, the incidence
of abnormal embryos is increased.
Unsatisfied with the low fertilization rates of stripped eggs of
P. caurinus, eggs were treated with various concentrations of ammonium
hydroxide, yielding the following results:
Table 4.
Percent fertilization of NH OH-treated eggs
4
fertilized / normal larvae
Concentration
of NH OH
4
none (control)
-4
1 x 10
N
-4
5 x 10
N
-4
10 x 10
N
Duration of treatment
10 min
20 min
(0.0/0.0)
(.22/.04)
0.0/0.0
40 min
60 min
.06/.06
.17/.08
.14/.05
.52/.52
.85/.70
.87/.68
.72/.41
.83/.70
.96/.42
.76/.64
.71/.36
This data suggest that fertilization can be maximized and abnormalities minimized by treatment of stripped eggs with particular concentrations
-36-
of NH OH solutions for a particular duration of time. Based on this in4
formation, stripped eggs for future larval bioassays were treated with
NH OH at 5 x 10 -4 N for 20 min or 10 x 10 -4 N for 10 min, rinsed with sea
4
water then fertilized. Use of this method increased egg fertility to 87%
in the next fertilization trial.
LARVAL SALINITY AND TEMPERATURE
Once an adequate method of obtaining normal fertilized eggs was available, salinity and temperature bioassays were undertaken. For each bioassay, eggs of a visibly mature individual were stripped, filtered through
a 120)1 mesh nytex screen, and held on a 384 mesh screen (egg diameter of
P. caurinus is 72)4 ). This was followed by NH 4OH treatment, rinsing, and
distribution among the bioassay beakers. To each beaker, .1-.3 ml sperm
suspension was added. Sperm was obtained from at least 2 stripped males,
passed through a 38)4 mesh screen, and examined microscopically for activity.
In the first bioassay, 24, 250 ml beakers were used. For each of 3
temperature ranges, 8°, 10-11° and 13-14°C, 4 different salinity levels
were tested. Each parameter was done in duplicate. Results of the bioassay emphasizing temperature are presented in Graph 3. In Graph 4, salinity differences are emphasized.
Unfortunately, larval counts in this first salinity-temperature bioassay were very low. Consequently, results are inconclusive although perhaps indicative of trends. Considering all salinity data, a rough indication is that salinities of 30.0 and 32.5 were almost consistently better
than the low or high salinities of 27.5 or 35.0 ppt.
Age to straight hinge development was probably the most relevant temperature information from this bioassay. At 8°C, larvae reach straight hinge
HhoE
di. iiumii
Hhimilin
jormu llitiobbillopm imillmill
a limplo p momili g ing
'''' lopriliPPREMINIIIIPHim
i
lin
6" •2
10111111101111phpli
II rilliniticri
11
Bull
10119111011poilwill illy m i r 1
111111111 omrimo111111141 11111111E Eli
rumpling
11. 1101
1
11111 1 1 1011alipliprin1 1111111111 01111 111111011 111111111111111111111111111
111110,110111101111110111111111
billmANIMINI111111
011111111111111WPIIII
IMMINEVilill
■
t r il P ilildhlihni dim
lun ILI mi lun ildim l h
.1.0 limirr ...
1.
9 °°°°°°° ihr muuPvl ii m9111,1, 11111bollibliirrupliplink
f ld lmpgibihm l m p
Hill if RHIN
mil11111
:""""'""'"Nur"koriii ilormitroolooril .
imam ibilliii pi I,
dLiniiii iii r: ii lifil 10 li
IllihhiMili
!pre
al bu l m r P vi..
Trili i 1 '
ipmhilhiimillehil b 11 4 ji
imilliiiiii
ii
iiii
.o
Pi l lol
1111.10.911EN
1111111111111111111111,
II ME
VI
'1,611,911111,11
III il
ih
l
mummuirmilo di 1 J.
iiiimbillill.pumm oll Hp iiii
imphombilimill111111111111
1 e ll e s 1
J'Ildid■
ii
I, t 1111
lill'Illitili1111111101f1111111111
ii
!III 011111
1111
II I 1 11061111111111161111
1111 il l 11111111111111111 111110.1
wpm
riniminummoilli lowigm
rum imillonpi
1 millibli111111111110111 1
1.2 1 . .. p111 IF 111111 IffiliguillIMMIM0
.41111111111PIRPHIPPI
11111111ffiblifOR
1111111M1111111101
1110111
mi 0
r ddi i HIIIMIFIl
HUM lihillillill iiiriptiourri
pi lii ll:11;;;114 1";1101111111Millirt
i i i hilli 1 mom p
oilliilli 1 . .
■
i.hininoll •
IringbhilmimdbHPENItt ili g h T iiiiiimbp.r
I
MOIlliffiffilli
110 101 blorli
"' .91171
ililiiiiiiiii ld ilib il old.i
,i, 1 II g
11111116111
'"'"! ill .1111111 1111.1111 ..
:1 r
l ihi gniiii
i immismoilviiiiimi
Ihi h
11...yli.ki i
lid ■len
uk
111111111111011111
%
.
IlliiiiiiiiiiiibilliiiiiiiiiiiiiiiiiiiiiazikuilliiIIIIIIIMIIII
ol
A iloommor
ommordimi 11E9091
11111011111111011111111111 HMI II I
11 JIM
si p
11111111111111
I Mil
ow
it'd ill
111
II 1
I
111111111lliiiiiii Hill Niglio!
it, a
numimullid
rd ftt IS
11
iii •' E ■
■
11"
rar"InTj:
-P7
1:11
14711!
•
■■■MS
MI II
MMMM
MMMM■
M ■■■
Ma OS
./ SO
SO a
1111
6-4.
ifili1
8 1111111 111
'
II
dl
11 1.1
tH-1- I + ,- TJ. -1-t
H
r17:1'
Tr i- l' rtfr 1:„._"
di iillITIMPIMIRM SIMI '
1 11111 1 111
4 11111. 1111•1 11: e.:.
11
urualppi:
r
. 11
19100111116110111111111111111111,1
ad_...
■ ..
.41111611•• •••15111 1118111
!MrdliialPHIPEr.M
11111
s"
+ NM
14
+1
11111 MIERH INIMMIN
111111111111111111111111111
11111 i
III 111111111111111111 %11:11111111111 11111111111 11 4 11111111111111111111111111
11
I am:11111 1411 11111111111111111111111111111 111E1111 1
I
III
11111
si
I
fi t
I 111
liIl IIIII 11111110111
mummoomMINOW
H
0 111 11111 9 11111"
ttt
111111111111111111111 Fill
Li
1
11
t
4
4
1l;
4-
4
it
11111111EIRHOMMilii .
ct
.im
tIi't 11.-ii OE .
n
g
01101111 lifidlillighiliiffirlillS••11 Ali
14#.;
ilipqrqum
Ili i
1 MAW WV 'NM
,
••••
■
I-
■
101111001111111111
o I .1
•
•P'..milloollohimmil
l
1
1
mos
1' use
m il ifin EPA.
111:1141: '11:14" 1111"1111
III'
11■61
1
4•
„
•
■
■
• 1 '4
.1110
; E:
t I
;1 ' •
4t,1,11:
1101101111
1
13
:
1
1 ... 1 .. Le 1111 1 . AI
II 111
1 111111111111111111111111111111 11
mompumumff
111111111111111111111
: i
' I
:
■
1
I
11
1
■
111111111111111
0,
21
null iinlIMPINI9
1 U Unimp .mimur.
132 :5 1t
I iiiipPiril
MN_
R
.1111111111111111f:his
4
11:1:1
:F :1.
R
4'
L.12
:14 ;;,:
-7-
■
1111111111 11111111111111111
i
111111111111iiiiiilli
pug
are
t um
Nil
ti 1 T
Mil I
ii
1 1111111:m
Iv
„H
1
1
A 4
...
tt
1
■4
_q14,
io'as'g'ay,-j-1.7017ii dry :49
BrdhinBillhal
a 1
f
;1!:,:•:
1111111111111111111111111111
1-4
04
-44-4 1 - 1 •,- ,
•
illiheF(iciyer:the
i ;Lp
1.TA uesil l h4av" 'eerii ;
,
1
. 4 . 44,
•
-39-
in 5-6 days, 3 and a half days at 10-11°C, and less than 3 days at 13-14°C.
Straight hinge size was measured at 84/u x 100).k.
The second salinity bioassay was maintained at one temperature range,
10-11°C. This was set up such that the amount of rock salt added to achieve
the desired salinity was the same percent amount for each salinity level.
For example; for a salinity of 30 ppt, bay water at 24 ppt is collected
to which 6 g/1 rock salt was added, yielding a test water 4/5 bay salt and
1/5 rock salt. Similiarly, for 35 ppt, bay water at 28 ppt is collected
to which 7 g/1 rock salt is added, also yielding the 4/5 : 1/5 mixture.
Salinities were checked by refractometer prior to placing in eggs. (In the
previous bioassay it was learned temperature corrected hydrometer readings
are not consistent with refractometer values.) Larger, 1 liter bioassay
beakers were utilized, to which .3 ml sperm suspension was added. Salinities compared were; 30.0, 31.0, 32.5, and 33.5. One set of beakers was prepared using raw, MSC seawater which was bag-filtered. All other beakers
were prepared with the standard, filtered and U.V. treated "P" water.
Results indicated that of the tested salinities, 30 ppt was preferred,
with the exception of the raw sea water in which the percent normal larvae
had their highest numbers, The raw sea water was at 31.5 ppt salinity
(Graph 5).
Summarizing these results, the best available information indicates
30 ppt salinity and raw, bag-filtered sea water are preferred for rearing
P. caurinus to the straight hinge stage. If held at 10-11 °C, this stage
should be reached in 31/2 days. It should be noted, to strengthen the
statistical significance of this data, it is recommended that additional
bioassay replicates be performed with scallop larvae.
O
r
U
z
0
MOO OEM
IMMIMIMMIMMI:
....1.1
.
• ..
1:11: .:::::::::::
us... ....
11:::
:::111:111 " '"11111 111:::::::::::
;
siourl
11:
11111 :
:1-1P ur:M111111immouriludisminiumhummulkokimminill
i
Imu i : 1....:...
:
ti........1..1
:::
...1:
111111111""1111121111 1111:::::1:01111111111:11:1111111111111111111111911111
11111111111111:1111111111111....m .smallaulamil
1111111111
1111111111111111111111111111111M 111111111111111111111111111111111111111111111111111111111111 1111111111111111111 11111111111111111i
11111
IfIllim
P
It:. ::::::.:::::: •
T
1
.- • -
.
11. .. .2 7111111::::111:: 1.11
1
i
.1K.:
M : MM : -..::::::11:!!!::: :. •
:: .i 111. .
H
%NT """"
rat
Er
1 1:11111::fig•
Mil
MEM
nom
11111
1111111111
IIIIIIIIIIIIIIIIIIIIIIIIIIIIII : till IIIIImmIMIIIIIIIIIIIIIIIIIIIIPPF"911111111111111111111111111111111111111111111111
..
• I,
1..11111111112121111 1
MUM:
Hippraidi rigun i
irmiiiiphilidglIPPRIENTinfflnnirliAl
1
iiiiiiiiii
:: MMMMM 1111 ::::::..::::11 :111:::.:1.11 iiiiiiiiiiiiiilliiiiii
:11:11-":1111111 1:
111:11:::::::::::11:::1
i
OM OM MU i
•
01111111111111111111111111111111111111111111
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII1 11m1111:11:1 . 41111111111111111111111111
RI■MI
1111111111111111111:1111111111111111111191
10111111111111111111 11111111111111. 111111111111111111111 11111111111111111 III"
■ ■:
II
:
• -1 - 4.. ....: . 1.. :1:
t. : •::: mum: :111
aullu • • :.:.1 1::::11044
11:1111111111111 IIIIIIIIMMIIIIIMM Mil
' '"' "' Ili 111111111M111
11111111111"1::
"'"uliiiiiniiiiikilli
r.---muumu
: u . .. . ..: '111
...
1: 1:::11:::
"'"
111::::::
1..:::::: 11.
1:11:1
PI1:'ll.:'III 111111111111111
" "'II 111:11I .:1111
i
1
" 1 auel
11"1;0111111111111= m11:-1•11 um
1
m
aquilliiiiilil
1 1:1:::::
■11
"m l:Hu 1 : MP P I
IN
11111
Mall
il
13331311131111■
iiiii11111111
IIIIIIMINNSMINI1101 11111101110,111
04011111
ril .......momrserd .:41111:::::
11111 I
i 11111111111111 111111111111
... 1
""11111111
....1:41
-nu lugussallan . • °II ..
ilm
333 33
11 111111111 1
1111111111111
1
11 1
11 11
:
0
1111111:19 11 "11111111111:
1 118. Huh
AZ ..1.111
u1:1111:111::■
1:::::=1:1 Ina •
• ::::::::1
•••:•••r•••i•
eine
annsearestar:
11::::aunp
iinp. ...m MMMMM i Nheag
MI
11111111iiiiii MEE
Mealdlild
u.....1111111
11 11111111:11" "
1:1111=111111 ' '
11111112111111
:mum
I:Jill
19
iiiiiiiiiiiiiili
.
1 Imams ...... ma •
MOM MEI ma OMifMOMA
••••"11111111 II 1111
ingrdirMi
III
aammallailimi_._,__,.,---.--,
m•
id HEN& lE HO Eihmi 'PI 1 1111111 111111111
II Iffillilaii
:-....11.121:1:11 111166111:
Urn ii EM
. unpp.::::::
tunur orarnurnum:
1m:11111111
1 "MP"'
um•in ..... : Pi ■
unur
Njuffnarkw:::: MIMI
MIMPINMEMEll . NH IN
......
....111
;
P
ni: kit nu
........ MM
a
uul
-3;
1
........1...:::::11111;111111.12111111
1:1111111
:
1 1
.11
., .
11 1""-"":1 55 ,i,..„.
moo
g
E,
.....
•
•1 •
l
i
pd•••• i
• i
•
•
m
il:
: : I 111111101111111MMINI MINIM
1
MI
... 41,1
ih
.
.m
1 An
;;;;0 ::.1IDIMMIIII RIM 11010110111111011MOM
-------:MO
1 uusur
m .... 1 SOMLIE
!Hi
....
IIIIIIIIIIIIII
UM:
I.Lial .......... ut.innirds••••
rim
-ummhgag:un 1 1 ell h Tr 011.1111111111111 RIM
1111:11
•
ea:: 111111
tin
.
111ItanUr
1111i1111111111111111111111 1111111111 1 INmi...
11"11111.111
111 z:;1 mi'm
i ■em I:11111111s
a 1 . wamm.: g1 ...
IIIIII:14.11. 1tqpiihdhalnain
1..WILIMPINIMPVINNI
01
• 11.1
um ma
ago
...II....
41
•
iii 111111116.
.. ....AI.
pliTILIII::::: :h4qp::::;:::::himmollimmoommoommia :dmmaim
. mg1111111611i111111111
...
llinlipqlp.rg
•
, 1 1i ea .1. • 1
mum uume.16::::: IMIIIIIMIMAIIIIIIIIIIIIIIII r inirpro mi p i l" . h iu ghommulimmuffilloom .1.polpmempill
1
HU
1
ulu.:61:111:1011:11 :II- u.... 14'
a3lu t.u
1 1111 1:1111111 11: ..
11;;;11111111111111111111111111 11111:
i
:
.
I
11 i .1. •
sus
al........ 1..11
1.1111:11uudu 111: :um" m:
lullipulu11-4:::::11:1:12"
:1
::::...:::
111:1:1:11:11:111:1 1111::11Husuu
Iffitliwkihdi
1.:1:1:11:11:11:11: :111111:1::1111111: ililiiiiillifill111111111/111
MMMMMMMMMMM .
11111Um q.•5
p
lin. Insrad: a
4
f4, L4
MIME IMNIK
1111111111111111111111111
9lipprrUpriip
ell
MO • di • M •
11111; m
Lia_at 0_ _ecte
RIONIMBEI 11111111
1111111111:: :
L
::::.:11T. : 111
muumFur
mompollmmoummuomondomommillimmll
1.:1:9:
1
1
r rilqininlin::tgulbh111111111111hEll MI irlillipilifill IMMO
J
i 9 ;la 1-',kihrdoll
751. egusrogi
l:'il'-'.,' -
riffinnyur
OM MIM MOM .. .
11111118:1::::
....1511
.. t
:.
uxintt
111.1 011......
•
-
't t --- tilliniMbEilithh LI I
$111111131 11 011111•111111111111110110111
•
--T1TH -t.T
■
MI
Et,
• ,- -
T
1
MI 11111
- 41 -
INDUCED SPAWNING
One of the primary goals in working with P. caurinus was to develop an
induced spawning method. A dependable method would be a major step towards
realizing hatchery potential of this species. At the time of this investigation no published information regarding induced spawning of weathervane scallops was located.. However, Neil Bourne (1983) recently reported
some success with the use of thermal shock and ultraviolet light treated
water as spawning methods for this species.
It is important to acquire sexually ripe scallop specimens for spawning trials. Sex of individual scallops is determined easily and safely by
visual inspection while gently forcing the valves to remain open after a
specimen is lifted out of the holding tank. Size and color intensity of the
gonad and a knowledge of microscopic examinations of gonadal tissue of aimiIar
animals are factors which allow the selection of the most gravid scallops.
Hence, for each spawning trial, ripe individuals were selected and placed
in round plastic spawning basins (15 cm diameter) and covered with 5 1 of
water. Two females and 1-2 males were placed in each basin without aeration, to avoid agitation.
Since spawning attempts were done in duplicate,
another treatment basin was prepared and two similar basins for controls.
Although several spawning replicates would have been desired for each
spawning method or concentration, replicates were subject to the availability of ripe scallops.
The established method for spawning P. yessoensis in Japan is to
raise water temperature by about 5 °C. In fact, temperature alone is sufficient to induce the maturation process in oocytes of extracted gonadal
tissue of this species (Yamamoto, 1964). Unfortunately, temperature in-
- 42 -
crease was not successful to induce P. caurinus to spawn.
Generally successful spawning methods attempted without success were:
(1) Temperature increase, from 9°C to 13°C and 9°C to 16°C.
(2) Temperature decrease, from 11-12°C to 8°C.
(3) Algae method. Algal culture grown without CO 0 (higher pH) is
added to the spawning water to bring algal cell density to
6
2-5 x 10 cells per ml. Algal species: VA-12.
(4) Algae, with temperature increase of 5°C.
(5) H 0 adjusted to pH 9.1 with 1 x 10 -3 molar NaOH. Concentra2 2'
-3
tions of 2-4 x 10 were suggested for all bivalves. Concentrations tested were: 1.5 x 10
M
-3
2.5 x 10
M
3
3.5 x 10- M
-3
4.5 x 10
M.
(Morse, Hooker and Morse, 1978).
(6) KC1 at 2 g/ 1. Scallop response to this method was the most adverse. Valves remained closed for the duration (3 hr) of treatment.
Considering the relative success of NH 4OH used for treating eggs, this
compound was tried as a spawning stimulus. Yamamoto had mentioned NH 4OH as
one of several induced spawning methods used for bivalve molluscs. It was
used in 1936 to stimulate Crassostrea gigas. In the initial attempt,
NH OH was added to achieve a 2 x 10 -3 N concentration, adjusting pH with
4
NaOH as described for the H 0 treatment. The scallops appeared very re2 2
laxed with tenticles extended. Within an hour strands of mucous were visible in the water. After 3 more hours, scallops were inspected, and
3.4 x 10
5
eggs were found. Specimens were transferred to fresh water and
allowed to stay overnight. By the next morning, 1.7 x 10
6
more eggs were
spawned. Since 2 females were in the spawning basin, it was not known
whether both or one spawned.
Subsequent investigations were directed to determine the minimum con-
-43centration of NH OH necessary to induce spawning and whether adjustment of
4
pH was necessary.
It was determined that 1.5 x 10
-3
N NH 0H is effective to induce
4
spawning in ripe female weathervane scallops. No pH adjustment is necessary
and addition of NH OH at this concentration causes no measurable pH change.
4
Concentrations tested for induced spawning with NH 4OH were:
.5 x 10
-3
N
(no spawning)
3 N
(no spawning)
-3
x 10
N
-3
x 10
N
-3
x 10
N
-3
x 10 N with and without pH 9.1.
1.0 x 10
1.2
1.3
1.5
2.0
With the 1.5 x 10 -3 N treatment solution, spawning occurred in 2-3
hours. At lower concentrations, less eggs are spawned (although this may
be related to ripeness of the individual) and the onset of spawning is
later. In all cases where spawning was induced, only female scallops
spawned. To obtain fertilized eggs, 1-2 males were stripped and a sperm suspension was added.
Due to the scarcity of ripe individuals, in some spawning trials, females used in earlier unsuccessful spawning attempts were used again. In
these circumstances, however, there was always at least one fresh (untested)
female. After success with NH OH was determined, most visually ripe female
4
scallops were spawned regardless of previous unsuccessful attempts, implying that the earlier attempts had little effect on their spawnability.
DISCUSSION
Patinopecten caurinus shows potential for hatchery applications. It was
found that spawning of mature female scallops can be induced with 1.5 x 10
-3
ammonium hydroxide solution to yield viable ova. Spawning was induced with
N
- 44 -
solutions as low as 1.2 x 10
-3
N but numbers of eggs were less and the time
to initiate spawning extended. Although male scallops have not been induced to spawn, this is not a problem since viable sperm are readily obtained from stripped male gonads.
Larval rearing studies indicated a preference for fresh (raw) sea water
of 30 ppt salinity. Bioassays on larval temperature preference were not
considered conclusive, although a temperature of 10-11°C yielding healthy
straight hinge in 31/2 days may be recommended.
Although icreasing water temperature was not successful as an induced
spawning method in P. caurinus, water temperature may be related to gonadal
development. Average temperature values for seasonal bottom water (Graph 1)
reveal that gonad maturity in this species occurs after several months of
increasing temperatures. Spawning occurs during months of decreasing temperatures. A knowledge of bottom temperature, where scallops are harvested,
would contribute to understanding environmental factors affecting spawnings
and populations. Kruse and Huyer (1982) suggest that hydrographic data,
which include bottom temperature and salinity, can be determined from calculations of sea level and upwelling indices.
Manipulating temperature might prove to be a tool for conditioning
scallops or for extending their spawning season. To test this, newly arrived scallops of of March 23, 1983 along with 12 tagged scallops of earlier
collections were held at 10°C and fed 4 1 of algal culture daily. On March
23, a description of the gonad condition of each tagged scallop was written for later comparison. On May 20, the tagged scallops were examined, and
very little change was found in the color or texture of the gonads. Females
that had been very ripe were still in the same condition. Spawning with
- 45 -
NH OH was successful with these specimens despite having been held for
4
2 months.
Physiological response to NH4OH:
NH OH appears to induce primary oocytes in the ripe ovary of P. caurinus
4
to mature as temperature does for oocytes of P. yessoensis. Changes that
occur include a rounding of the cells, a breakdown of the germinal vesicle,
and the assumed continuation of meiosis to metaphase of the first meiotic
division. At this time the ova are ready to be fertilized.
Ova are enveloped by a vitelline membrane and a transparent jelly-like
layer. In Mytilus edulis the vitelline membrane is about 1ptand the jelly
layer 7-10)4 (Reverbi, 1971). The membrane is thought to play an important
role in maintaining the integrity of the developing embryo through early
cleavage (Dan, 1964). Although blastomeres of early cleavage exhibit a
considerable degree of mutual contact, they are held together primarily by
restraint of the vitelline membrane. Dan isolated lysin derived from sperm
acrosomes to dissolve away the vitelline membrane (Figure 5), which resulted in blastomeres that are only loosely connected. Embryos of the same
appearance were often seen when eggs of P. caurinus were treated with
strong solutions of ammonium hydroxide, or when spawning was induced using
2 x 10
-3
N NH4OH.
Stripped eggs treated with NH 4OH show varying degrees of vitelline membrane disruption. Where concentration and duration of treatment resulted
in the highest percentages of abnormal larvae, the vitelline membrane was
often greatly expanded around the ova or was ruptured. It is the belief of
this investigator that minimizing the exposure of the egg to NH OH can allow
4
optimal development of normal larvae due primarily to the ability to retain
Figure 5.
a. - c. Normal cleavage.
d. - f. Cleavage when hyaline material of the vitelline membrane has been
dissolved.
(adapted from J. Dan, 1964)
- 47 -
a healthy vitelline membrane. Also, visual inspection of the egg membrane
may be useful to indicate success of induced spawning.
Although increased numbers of fertilized eggs have been made available
through induced spawning of the weathervane scallop, larvae have not been
reared beyond 150 /
LA
size. Larval rearing was not an objective in this in-
vestigation (other than larval parameters). Consequently, this would be one
of the next major steps towards developing the hatchery potential for
P. caurinus. It is hopeful that future studies will emphasize this problem area.
- 48 Bibliography
Barnes, R. D. 1974. Invertebrate zoology. Third edition. W. B. Saunders
Co., Philadelphia, London, Toronto.
Beattie, J. A. 1983. Personal communication. Selective breeding for improved
meat quality in the Pacific oyster, Crassostrea gigas, in Washington
State Perdue. Report presented at the 37th annual convention, Pacific
Coast Oyster Growers Association. Sept. 9-10. Tumwater, Washington.
Borgese, E. M. 1980. Seafarm: The story of aquaculture. Harry N. Abrams,
Inc., Publishers, New York.
Bourne, N. 1983. Personal communication. Canadian scallop research. Report
presented at Aquaculture Northwest. Nov. 3-4. Seattle, Washington.
Breese, W. P. 1981. Personal communication. Molluscan Aquaculture. Oregon
State University. Newport, Oregon.
Breese, W. P. and R. E. Malouf. 1975. Hatchery manual for-the Pacific oyster.
Oregon State University. Sea Grant Program. Publication No.
ORESU-H-75-002.
Courtright, R. C. 1967. Formulation of a synthetic seawater for bioassays
with Mytilus edulis. Masters thesis. Dept. of Fisheries and Wildlife.
Oregon State University.
Dan, J. C. 1962. Vitelline coat of the Mytilus egg. I. Normal structure and
effect of acrosomal lysin. Biol. Bul. 123: 531-541.
Dupuy, J. L., N. T. Windsor and C. E. Sutton. 1977. Manual for design and
operation of an oyster seed hatchery for the American oyster, Crassostrea
virginica. Virginia Institute of Marine Science. Sea Grant Program.
Dimick, R. E. and W. P. Breese. 1965. Bay mussel embryo bioassay. Proceedings of the 12th-Pacific Northwest Industrial Waste Conferance. University of Washington, College of Engineering, Dept. of Civil Engineering.
Earnst,, D. 1984. Personal communication. Oregon State University, Dept. of
Agricultural Engineering.
Gallager, S. M. and R. Mann. 1981. Use of lipid-specific staining techniques
for assaying condition in cultured bivalve larvae. J. Shellfish Research.
Vol. 1, No. 1, 69-73.
Galtsoff, P. S. 1938. Physiology of reproduction of Ostrea virginica. II.
Stimulation of spawning in the female oyster. Biol. Bul. 75(2): 286-307:
Gruffydd, L. D. and A. R. Beaumont. 1970. Determination of the optimum concentration of eggs and spermatozoa for the production of normal larvae
in Pecten maximus. Helgolander wiss. Meeresunters. 20: 486-497.
Hansen, L. 1983. Personal communication. Whiskey Creek Oyster Farm.
Tillamook, Oregon.
-49-
Helm, M. M., D. L. Holland and R. R. Stephenson. 1973. The effect of supplementary algal feeding of a hatchery breeding stock of Ostrea edulis L. on
larval vigour. J. Mar. Biol. Assoc. U. K. 53: 673-684.
Henderson, B. A. 1983. Handling and remote setting techniques for Pacific
oyster larvae, Crassostrea gigas. Masters thesis. Dept. of Fisheries
and Wildlife. Oregon State University.
Itoh, S. 1982. Personal communication. Director, Aquaculture Center, Aomori
Prefecture, Japan.
Kruse, G. H. and A. Huyer. 1982. The relationships between shelf temperatures,
coastal sea level and the coastal upwelling index off Newport, Oregon.
Oregon Agricultural Experiment Station. Technical paper: 6505. Oregon
State University.
Leighton, D. L., and C. F. Phleger. 1981. The suitability of the purple hinge
rock scallop to marine aquaculture. Dept. of Natural Science. Report No.
T-CSGP-001. San Diego State University.
Lim. 1980. Personal communication. Bureau of Fisheries. Tawau, Sabah, (East)
Malaysia.
Magoon, C. 1981. Introduction to shellfish aquaculture in the Puget. Sound
region. Division of Land Management, State of Washington, Dept. of
Natural Resources.
Mason, J. 1958. The breeding of the scallop, Pecten Maximus (L.), in Manx
waters. J. Mar. Biol. Asoc. U. K. 37: 653-671.
Morse, D. E., N. Hooker and A. Morse. 1978. Chemical control of reproduction
in bivalve and gastropod molluscs. III. An inexpensive technique for
mariculture of many species. Proc. World Mariculture Soc. 9: 453.
Motoda, S. 1977. Biology and artificial propagation of Japanese scallop, general review. Proc. 2nd Soviet-Japanese Joint Symp. Aquaculture. Nov.,
1973. Moscow (1977).
Olson, S. J. 1982. Shellfish enhancement project. Part II. Invertebrate
aquaculture. State of Washington, Dept. of Fisheries. Point Whitney
Shellfish Laboratory. Annual Progress Report.
Olson, S. J. 1983. Personal communication.
Pillsbury, K. 1983. Lipid requirements for Strombus gigas larvae. Report
presented 37th annual Convention Pacific Coast Oyster Growers Asoc.
Sept. 9-10. Tumwater, Washington.
Chapter 6 in G. Reverbi (ed.) Experimental
Reverbi, G. 1971. Mytilus.
embryology of marine and freshwater invertebrates, North-Holland, Publ. Co.
Robinson, A. 1983. Personal communication. Oregon State University, Marine
Science Center, Newport, Oregon.
-50-
Walne, P. R. 1979. 2nd edition. Culture of bivalve molluscs: 50 years at
Conwy. The Whitefriars Press Ltd., London, and Tonbridge.
Wyatt, B. and W. Gilbert. 1967. Hydrographic data from Oregon waters. 1962
through 1964. Dept. of Oceanography, School of Science, Oregon State
University. Data Report No. 24. Ref: 67-1.
Wyatt, B. and N. Kujala. 1962. Hydrographic data from Oregon waters. June,
1960 through May, 1961. Dept. of Oceanography, School of Oceanography,
Oregon State University. Data Report No. 7. Ref: 62-6.
Wyatt, B. and N. Kujala. 1963. Hydrographic data from Oregon waters. June
through December, 1961. Dept. of Oceanography, School of Oceanography,
Oregon State University. Data Report No. 12. Ref: 63-33.
Yamamoto, g. 1964. Studies on the propagation of the scallop, Patinopecten
,Vessoensis (Jay), in Mitsu Bay. Fisheries Research Board of Canada.
Biological Station, Nanaimo, B. C. 1968. Translation series No. 1054.
APPENDICES
- 52 Gonad Indices for Patinopecten sps.
APPENDIX A.
I. NUMERICAL
gonad index =
gonad weight x 100
wt. of soft body parts
This method was developed in Japan for Patinopecten yessoensis but has
been used in British Columbia for P. caurinus (Bourne, 1983).
For P. yessoensis, numerical indices are interpreted as:
<5%
5%
7-8%
8-9%
20%
>20%
-
spent
resting
early developing
late developing
maturing
breeding
II. MACROSCOPIC
Daniel Hennick (1970) developed the following gonad index for P. caurinus.
This index was originally described by Mason (1958) for Pecten maximus but was
adapted for use with the weathervane scallop.
(1) Empty or spawned out: Gonad reduced in size and collapsed, contains
free water throughout. Transparent loop of alimentary canal clearly
visible. Testes nearly colorless, ovaries dull amber to nearly colorless.
(2) Initial recovery: Gonad increasing in size, free water exists in portions. Loop of alimentary canal visible but fading. Portion of testes
containing spermatogonia cloudy white, rest is transparent. Ovaries
amber to dull pink or orange.
(3) Filling: Gonad near maximum size, contains 3/4 or more of the estimated capacity of sex products. Free water exists only as small
canals or close to the surface. Testes pasty white; ovaries bright
orange.
(4) Full or ripe: Gonad relatively large in relation to other body parts,
completely full and rounded, contains no free water. Loop of alimentary canal not usually visible except for distal portion. Texture
appears granular. Testes flat white; ovaries bright orange.
(5) Immature or juvenile: Gonad relatively small in relation to other
body parts, angular and flattened, transparent and colorless. Loop
of alimentary canal clearly visible.
III. MICROSCOPIC
The following gonad index was developed at the Marine Science Center in
Newport, Oregon and is used for P. caurinus as well as other bivalve molluscs
(Robinson, 1983).
In this method, preserved tissue samples taken from the distal portion of
each gonad are preserved, stained, sectioned and slide-mounted for interpretation. Descriptions refer to observed stages of gametogenesis within gonad
follicles. Definitions were based on verbal descriptions by Robinson with
referance to Mason's (1958) microscopic observations.
53
Male bivalve molluscs:
(1) Early developing: Spermatogonia form a layer on walls of follicles.
Inside them, a layer of spermatocytes. Lumen contains spermatozoa
arranged radially.
(2) Late developing: A few spermatogonia and spermatocytes remain near
the follicle wall. Lumen becomming closely packed with spermatozoa.
(3) Fully mature: Spermatozoa very dense and arranged radially from the
lumen of the follicle. A few spermatogonia visible along lining.
Little connective tissue.
(4) Mostly spawned out: Center of follicle mostly empty. Few residual
spermatozoa in periphery of the follicle appear clumped.
(5) Spent-recovering: Center of follicle empty. Spermatogonia around
follicle wall, among them some spermatocytes.
Female bivalve molluscs:
With female samples, numbers of oocytes at particular stages of oogenesis
are recorded. Complete follicles are counted until a minimum of 100 oocytes
have been recorded. Stages observed are:
(1) Young synaptic oocytes: Small, rounded cells, attached to follicle
walls and darker staining than other oocytes.
(2) Growing oocytes: Elongated, often club-shaped cells, also attached to
follicle wall.
(3) Primary oocytes: Fully developed oocytes in the lumen of follicles.
Round when few in numbers, polygonal when packed with many of same.
Percentages of each of these stages is calculated and used to indicate
gonad maturity (e.g. if % primary oocytes is > 50, spawning potential is indicated). If sampling is monthly, seasonal trends of gonad maturity can be
determined.
- 54 Induced spawning method of some scallop species.
APPENDIX B.
Species
Method
Referance
Argopecten irradians
Thermal stimulation
Sastry (1963) as cited in
Yamamoto, 1964.
Hinnites multirugosus
H 0
2 2
Morse, D.E., N. Hooker and A.
Morse, 1978.
Leiton and Phlagler, 1981.
U.V. treated water
Patinopecten caurinus
U.V. treated water
Thermal shock
NH OH
4
Patinopecten yessoensis Thermal stimulation
(temperature increase)
Thermal stimulation
(temperature decrease)
U.V. treated water
Seratonin
Pecten maximus
Desiccation and U.V.
treated water.
Bourne, 1983
Bourne, 1983
Solon, 1983 (also in this report).
Yamamoto, 1964 and Motada, 1973.
Motada, 1973
Yamamoto, 1964
Matsutani, T. and T. Nomura, 1982.
Gruffydd, LL.D. and A.R.
Beaumont, 1970.
Method descriptions:
Thermal stimulation - Usually accomplished by a sudden increase in water
temperature of about 5°C. Spawning expected to begin 1-3 hours after temperature adjusted.
Thermal stimulation (temperature decrease) - Sudden decrease in water temperature of about 5°C. This method was used in conjunction with addition of
a sperm suspension.
H 0 - Scallops set in a bath of 7.5 x 10 -3 molar H2 0 2 which has been pH
2 2
adjusted to 9.1 by addition of 2 molar tris-hygroxymethyl aminomethane
("Tris"). Tris can be substituted with 1 x 10 molar NaOH.
U.V. treated water - Water which has been treated with ultraviolet light
has been known to induce spawning in some species and is often used in conjunction with sperm suspension. U.V. light is known to form peroxide free
radicles and it is believed that this may be the stimulus.
NH 4OH - Scallops set in a bath of 1.5 x 10 -3 molar NH4OH. Spawning expected
to begin in 2-3 hours.
i
Desiccation and U.V. water - Scallops allowed to stand in air of same temperature as water. After 2 hours, place them in filtered, U.V. treated water.
Spawning expected 2-4 hours after reimersion.
Seratonin - Single intragonadal injection of 0.4 ml Serotonin (5-hydroxytryptamine)
creatinine sulfate.