AN ABSTRACT OF THE THESIS OF
Bruce Alan Henderson for the degree of
Master of Science
in Fisheries and Wildlife presented on
February 21, 1983
Title:
Handling and Remote Setting Techniques for Pacific Oyster
Larvae Crassostreaigs.
Abstract approved:
Redacted for privacy
Prof. W. P. Breese
Some biological and operational parameters for the refinement of
commercial
practices in
the handling and remote setting of hatchery
reared Pacific oyster larvae Crassostrea giqa.s
re empirically
evaluated.
Larval settlement in factorial experiments of
temperature
(15 to
35°C) and salinity (15 to 35 O/o) was maximized at 30°C with a
salinity of 30
selected ranges
°/c,.
Interaction
between these
was non-significant.
variables within the
Larval response to the factor
combinations was observed to be limited more by changes in temperature
than salinity.
The provision of select concentrations
the chrysomonad
Pseudoisochrysis paradoxa
(0 to 100,000 cells/mi) of
for larval nutrition during
48 hours of settlement was not found to increase setting
success.
Mature C. agas larvae have the capacity to undergo non-aqueous
refrigerated storage (5°C) with retained setting competency for a
maximum of 10 days.
Observations of post-settlement survival for 90
days reduced this maximum storage retention period to 8 days for
practical commercial application.
Handling and Remote Setting Techniques for
Pacific Oyster Larvae Crassostrea gigas.
by
Bruce Alan Henderson
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Completed February 21, 1983
Commencement June 1983
APPROVED:
Redacted for privacy
Professor of Fisheries and Wildlife in charge of major
Redacted for privacy
(
--.-------
Head of Department of Fisheries and Wildlife
Redacted for privacy
Dean of Graduathool
Date thesis is presented
Typed by Carman McBride for
February 21, 1983
Bruce Alan Henderson
1
DEDICATION
I offer my sincerest gratitude and appreciation to my family
for their constructive and caring support throughout all the ups
and downs they have guided me through.
What I have learned most,
and acted upon the least, is how much my family means to me.
11
ACKNOWLEDGEMENT S
Dr. James E. Lannan is to be gratefully acknowledged for his
critical and practical assistance in bringing this work to fruition.
I wish to also thank the other members of my committee, Dr. Lavern
Weber and Dr. Fredrick Smith, for their encouragement and support.
Marilyn Gum
has always been and continues to be an invaluable
source in acquiring references of the most obscure origin.
Thanks go
especially to Carman McBride for her patience in typing this thesis
and to Anja Robinson for her willingness to lend an ear.
I feel privileged to have worked with Professor W. P. Breese, my
major professor, who has been exceptional in his helpful guidance and
friendship.
His concern with the practicality and application of
research has influenced me greatly.
Mr. Lee Hansen, owner and operator of the Whiskey Creek Oyster
Hatchery, is to be most sincerely acknowledged for his constant and
generous aid, without which this study could never have been completed.
This work is a result of research sponsored by the NOAA Office of
Sea Grant, Department of Commerce under Grant No. NA81AA-D-00086
(Project No. R/Aq-45).
111
TABLE OF CONTENTS
I.
Introduction
1
II.
Materials and Methods
4
III.
Results and Discussion
Temperature and Salinity
Feeding During Settlement
Larval Storage
9
16
20
IV.
Summary
28
V.
Bibliography
29
VI.
Appendix
34
9
iv
LIST OF FIGURES
Figure
Page
1.
Cumulative mean percent settlement of C. gigas larvae,
O/)
inclusive of all experimental salinities (15 to 35
Vertical
at temperatures of 15, 20, 25, 30 and 35°C.
bars indicate Standard Error (S.E.) of means.
10
2.
Cumulative mean percent settlement of C. gigas larvae,
inclusive of all experimental temperatures (15 to 35°C)
Vertical
at salinities of 15, 20, 25, 30 and 35 0/00.
bars indicate Standard Error (S.E.) of means.
11
3.
Cumulative mean percent larval C. gigas settlement for
five temperature levels (15 to 35°C) at five salinities
Points of intersection indicate factor
(15 to 35 °/.,,).
interaction at the given co-ordinates.
13
4.
Response surface of mean percent larval C. gigas set
(settlement) for the temperature x salinity factorial in
Isopleths delineate 5% intervals in the
Experiment 1.
settlement response.
14
5.
Mean percent larval C. gigas settlement at five feeding
Vertical bars indicate
levels of P. paradoxa cells/ml.
Standard Error (S.E.) of means.
17
6.
Mean percent larval C. gigas settlement after 0 to 8 days
Vertical bars indicate Standard Error
of 5°C storage.
21
(S.E.) of means.
7.
Mean percent larval C. gigas settlement after 0 to 12 days
The comparative mean spat survival after
in 5°C storage.
90 days for each 48 hour storage interval is displayed.
Vertical bars indicate Standard Error (S.E.) of means.
23
LIST OF TABLES
Page
Table
A.
Cumulative mean numerical settlement of C. gigas
larvae in temperature by salinity factorial
The standard deviation for each
(Experiment 1).
The mean (X)
treatment is seen in parenthesis.
and standard error (SE) of sample means are
exhibited individually for all factor levels.
35
B.
Sample mean (X) and standard deviation (s) of
larval C. gigas settlement at five P. paradoxa
feeding levels (Experiment 2).
36
Cl.
Sample
larval
larvae
8 days
37
C2.
Sample and 90-day survival means with standard
deviations for larval C. gigas settlement in
The larvae were held in refrigerExperiment 3B.
ated storage from 0 to 12 days with samples removed
at 48 hour intervals.
mean (X) and standard deviation (s) of
The
C. gigas settlement in Experiment 3A.
were held in refrigerated storage from 0 to
with samples removed at 48 hour intervals.
37
Handling and Remote Setting Techniques for Pacific Oyster Larvae
Crassostrea gig'as.
INTRODUCT ION
Overharvesting of the native Pacific Northwest oyster, Ostrea
lurida, initiated the experimental introduction of oyster seed from
Japan to Washington State in 1902 (Gaitsoff, 1932; Woelke, 1957).
This seed was the Miyagi variety of the species Crassoetrea gigas
Thunberg (Quayle, 1969).
Commercial quantities of this oyster were
subsequently imported in 1919, and again in 1928 (Steele, 1964).
With the early success of commercial C. gigas cultivation, the
oyster industry soon expanded throughout the Pacific Northwest.
Since
the waters along the western coast of the United States are generally
too cold for reproduction in this species, the industry became
dependent upon imported seed.
In more recent years, the increasing
cost and inconsistent quality of this source has created a need to
develop domestic, locally controlled shellfish hatcheries (Davis, 1969;
Loosanoff, 1971; Clark and Langmo, 1979).
Private commercial hatcheries have developed from experimental
pioneering such as that by Wells (1920) and Prytherch (1934).
Loosanoff and Davis (1963) are largely responsible for the comprehensive
culture techniques which form the operational basis of most modern
hatcheries.
Todays hatcheries are capable of producing predictable
quantities of shellfish seed, opening new grounds to the cultivation of
marketable species where previously no indigenous bivalve population
could sustain commercial harvest (Davis, 1969).
Technological advances
have also increased the efficiency of commeicial hatcheries where they
now approach cost-competitiveness with imported seed (Chew, 1979).
The product sold by these hatcheries is most commonly marketed in
either of two ways (Dupuy, 1970).
The first seed type are larvae
which have been allowed to settle and metamorphose on substrate
(cuitch).
The young oysters are then sold either attached to the
mother shell (as was the seed introduced from Japan) or removed from
the setting substrate and sold as cultchless seed.
The cuitchiess
product also includes larvae which are set and remain attached to
individual particulates, e.g. crushed oyster shell.
The second method of producing seed is known as the 'eyed-larvae
technique'.
The name refers to the development of a dark pigmented
spot within the shell cavity as larvae mature (Cole and Knight-Jones.
1939; Gaitsoff, 1964).
This method differs from the first on two
basic points; 1) mature larvae are packaged free of substrate and
delivered directly to the commercial grower, thus transferring the
choice of substrate and timing of settlement from the hatchery to the
shellfish farmer, and 2) in turn, this considerably reduces the
purchase cost and transportation cost of hatchery seed, while offering
the individual grower greater operational flexibility.
General experimental design for this study focused on the
manipulation of specific operational environmental factors which may
influence settlement of 'eyed' C. gigas larvae.
Specifically, factor
manipulation sought to define functional ranges of such settlement
variables which growers might apply to their individual economic or
geographic circumstances.
Selection of the variables to be examined
was based on the relative practicality of their commercial application.
3
Temperature and salinity represent two vastly important ecological
factors for estuarine organisms.
They are also prime aquatic variables
which are easier to measure and control than other prominent environme'ital considerations (Kinne, 1963; 1964).
Commercial shellfish
growers and hatcheries are equally dependent upon these factors for the
biological, as well as economic success of their operation.
It is
therefore necessary to understand 1) to what extent do temperature and
salinity influence larval settlement, and 2) in which direction might
they be controlled to insure optimum, or possibly maximum, settlement
success.
Whether metamorphosing larvae require some form of nutritional
supplement during the brief commercial settlement period is of current
issue in the oyster industry.
Larval consumption of cultured algal
species resulting in proper growth and morphological formation prior to
and after metamorphosis has been well documented (Bruce et al, 1940;
Davis, 1953; Malouf, 1977).
Should the commercial shellfish farmer in
order to receive a greater larval settlement be obligated to produce
and feed cultured algae, it could prove to be costly.
Conversely,
this operational cost might well be more than justified by a resultant
increase in oyster seed successfully set.
The capability to store eyed larvae, either for extended transport
or the creation of a production inventory, would considerably increase
a hatchery's marketing sphere.
Shellfish growers would benefit by
having healthy stock on hand which could be set on a flexible
operational schedule.
4
MATERIALS AND NEUHODS
Late veliger stage larvae of Crczssostrea gigas were obtained from
the Whiskey Creek Shellfish Hatchery, Netarts, Oregon, for use throughout the duration of these experiments.
Lots of approximately 250,000
competent larvae, of 240 microns in length or greater, were generously
supplied upon request by the hatchery operator.
The term competency
as used here, is best described by Chia (1977); "A larvae is said to
be competent when it has entered a physiological state in which it is
capable of metamorphosis when given proper environmental conditions".
Each lot varied in parental geneology.
Spawning and rearing methods at the Netarts hatchery are in
general those of Loosanoff and Davis (1963).
Commercially reared
larvae were chosen over those produced in the laboratory to more
closely represent the actual supply available to regional oyster
growers.
The larvae arrived at the Oregon State University Marine Science
Center, Newport, Oregon, wrapped in nylon cloth and damp paper towels.
These were kept in a styrofoam chest with a. frozen chemical coolant
lid during transport and immediately placed in refrigerated storage
at 5°C upon arrival.
Setting units common in all experiments consisted of 1000 ml Pyrex
beakers, each containing a 5 cm square of clean
weathered oyster shell.
The squares were cut from the adult oysters flat left valve.
The
smooth inner surface of the shell substrate (cuitch) was oriented
facing up, being employed as the experimental attachment surface for
the pediveliger larvae,
This surface was of roughly equal area for
comparison of settlement between treatments.
Each unit was filled
with 900 nil of seawater of a specified salinity.
Our seawater supply
at the Marine Science Center is taken from Yaquina Bay, passed through
a high flow rate sand filter which removes particulates larger than
5 microns, then through ultraviolet radiation.
A portion of the refrigerated larvae were removed from storage and
suspended in 200 ml of seawater.
The larvae were kept in suspension
by gentle agitation with a perforated plastic plunger to insure uniform
distribution (after Davis, 1953).
Aliquots of 1 nil were taken and
enumerated for larval density by observation through a binocular
dissecting microscope (after Lannan, 1980).
Taking appropriate
volumes, 1000 of the suspended larvae were dispensed volumetrically
into each setting unit.
The error in this method never exceeded 5%.
Beakers with larvae removed directly upon arrival from the hatchery
were designated Day 0, to be distinguished from units seeded later
with larvae from storage.
In these experiments, 48 hours were allowed for larval settlement
as in general commercial practice (Breese and Malouf, 1975; Jones and
Jones, 1982).
During this time, all replicate units were covered with
opaque black plastic to reduce phototactic influence and eliminate
evaporative changes in salinity (Thorson, 1964; Ritchie and Menzel,
1969).
Factorial analyses of variance, polynomial modeling, sample F
tests, sample means, standard deviations (standard errors) of both
sample data and means were calculated according to Snedecor and Cochran
(1980).
Assumptions were made of normal sample distribution with
fixed effects for experimental factors,
Chi-square testing for
homogeneity (Steel and Torrie, 1980) established validity of
non-preferential settlement for all factor treatments.
A correlation
term of 9.2 was also developed through Chi-square analysis for multiplying larval settlement counts from the experimental cultch-square
surface to estimate total C. gigas settlement in all treatments.
This
term was then used to transform numerical mean settlement (refer to
Appendix Tables A-c) into total mean percent set for graphical representation.
Response surface simulation (Figure 4) followed that of
Alderdice (1972).
Each experiment was conducted in triplicate with three replicates,
a total of nine repetitions for each treatment.
Specific Experimental Design
Three experiments were designed in a step-wise fashion to identify
optimum factor ranges, if existent, leading to numerical maximization
of successful larval C. g-igas settlement.
Experiment 1:
Temperature x Saliniy Factorial.
(15, 20, 25, 30 and 35
o/)
of high salinity seawater.
Five saline solutions
were prepared by distilled water dilution
Replicate beakers of each solution were
placed in water baths at five-degree intervals between 15 and 35°C.
Water bath temperatures were attained through use of H-B Red Top thermoregulators coupled with 500 w Vicor glass heaters.
Vigorous aeration
thoroughly mixed and circulated the water in the large polyethylene
water bath trays.
Both the temperature and salinity range widely
bracketed reported settlement cOnditions for C. gigas larvae (Quayle,
1969; Korringa, 1976; Helm and Millican, 1977).
7
Only Day 0 larvae, direct from the commercial hatchery, were used
in this experiment.
ranges.
Early results enabled a refinement of both factor
A temperature of 30°C and a salinity of 30 0/00 were
established as constants in Experiments 2 and 3.
Experiment 2:
Feeding During Larval Settlement.
Pseudoisochrysis
paradoxa, a motile unicellular algae of the order chrysomonadales, is
cultured at the Marine Science Center Algal Laboratories by the method
of Matthiessen and Toner (1966) as modified by Breese and Malouf (1975).
Daily records are kept on culture health and density.
Counts of algal
cells/ml are performed via a Coulter Electronics Model P64 Size
Distribution Analyzer.
Four algal densities plus an unfed baseline control were selected
(0, 25,000, 50,000, 75,000 and 100,000 cells/mI) to observe the effect
of food availability upon larval setting.
Past hatchery experience
indicates that algal food concentrations above 100,000 cells/nil may
result in reduced larval growth rates (Malouf, 1971; Lund,
1972).
Similar observations of a 'critical cell density' have been noted by
other researchers (Loosanoff, et al. 1955; Walne, 1965;
Schulte, 1975).
Select feeding densities were prepared by diluting enumerated P.
paradoxa cultures into the standard setting unit volume of 900 ml.
Prior removal of water in each unit equal to the displacement of the
algal additive maintained the standard volume.
After 24 hours, an
equivalent volume of the cultured algae was again introduced into each
beaker.
Experiment 3:
Larval Storage.
(A) Beginning with Day 0, C gigas
larvae were removed from 5°C refrigeration and dispensed into replicate
beakers for settlement.
This was repeated every 48 hours through the
tenth day of larval storage.
Observations of mean larval settlement
from each 48 hour interval were made to ascertain the effect of
prolonged storage upon larval setting competency.
(B) A duplicate experiment was initiated, similar to the above in
all aspects, with the following exceptions; 1) the duration of larval
storage was extended until a distinct decline in settlement was
exhibited, and 2) the settled cultch squares were suspended in an open
flow seawater tank (13 to 16°C) for 90 days.
Counts of the remaining
live spat for each square were then compared with the initial settlement counts to quantitatively follow the short-term larval survival of
the stored C. gigas spat.
RESULTS AND DISCUSSION
Temperature and Salinity
Larvae of C. gigas display a broad tolerance to both temperature
and salinity within the scope of the selected experimental ranges.
This is presumed to be an adaptive response to the larvaes early freeswimming existence and changing hydrographic conditions.
In Figure 1,
the cumulative mean percent larval settlement (set) at each experimental temperature is displayed, inclusive for all experimental salinities.
An upward trend in mean larval set occurs as temperature increases from
15 to 30°C, then decreases with increasing temperature to 35°C.
Maximum observed settlement occurs at a temperature of 30°C.
Figure 2 presents a similarly peaked response for salinity.
At
each salinity level within the experimental range, the cumulative mean
larval settlement for all experimental temperatures is depicted with
the standard errors of the sample means.
At 30
Ioo, maximum larval
settlement is found inclusive of all experimental temperatures.
A trend is seen in Figures 1 and 2 indicating that maximum larval
settlement might be achieved at a setting tank temperature of 30°C with
a salinity of 30 °/o.
However, this method of grouping larval
settlement at all factor levels for one variable and comparing the mean
to independent levels of the other does not identify the possible
interaction of these variables at specific points of factor intersection.
Interaction between temperature and salinity may be sufficient to
displace or shift the levels at which the maximum larval settlement
occurs.
Interactive shifts in factor tolerances have been noted in
10
40....
35
ILU
Cl)
j
z
Ui
C)
LU
0
z
20
LU
2
15
I0
I
15
20
25
30
35
TEMPERATURE °C
Figure 1:
Cumulative mean percent settlement of C. gigas larvae,
inclusive of all experimental salinities (15 to 35
at temperatures of 15, 20, 25, 30 and 35°C.
bars indicate Standard Error (S.E.) of means.
O/oo)
Vertical
11
40
35I
IUi
Cl)
-'30
4
>
4
-J
z
Ui
C-)
Lii
C.
z
4
Ui
151.1
L0J
I
.L
1
I
15
20
25
30
35
SALINITY %o
Figure 2:
Cumulative mean percent settlement of C. gigas larvae,
inclusive of all experimental temperatures (15 to 35°C)
at salinities of 15, 20, 25, 30 and 35 0/00.
bars indicate Standard Error (S.E.) of means.
Vertical
12
other studies.
Kinne (1957) and Davis and Calabrese (1964) have
observed that at the extremes of an organism's tolerance for a specific
external stimulus, it will be adversely impaired in its response
despite other environmental entities being near optimum.
In terms of
Experiment 1, significant interactimi between these variables may
dictate a new level of maximum settlement response as one or another of
the variables change.
Figure 3 graphically illustrates mean larval settlement in percent
larval set independently for all temperatures at all experimental
salinities.
Where the plotted response trends for each experimental
temperature intersect, factor interaction between these variables is
indicated (Steele and Torrie, 1980).
As the temperature isopleths
from 15 to 30°C are plotted, no significant interaction is seen with
salinity.
The narrowing of isopleth separation however, at both
salinity extremes (particularly 15 °IOO), is indicative of approaching
conditions for factor interaction and response limitation.
With the
placement of the 35°C isopleth, interaction exists over the entire
salinity range, possibly defining the upper temperature extreme for
larval C. gigas settlement.
A response surface approach to defining the intensity of larval
settlement in relation to temperature and salinity is presented in
Figure 4.
The percent settlement response contours are a result of
the graphical transformation of the standard errors about the mean
response for each variable.
At individual points of factor inter-
section in the factorial grid corresponding to the percent response
levels indicated, a contour was connected to the positive standard
error for both the temperature and salinity variable.
13
35
I
LU
U)
30°C
30
25°C
3
p...ZS
z
LU
0
LU
a-
z
20
20°C
I6J
15°C
Lii
35°C
10
IS
20
25
30
35
SALINITY %e
Figure 3:
Cumulative mean percent larval C. gigas settlement for five
temperature levels (15 to 35°C) at five salinities (15 to
35°/).
Points of intersection indicate factor inter-
action at the given co-ordinates.
14
.30
30-35%
4
J
25
25-30 %
10-15 /o
I
15
20
1
I
25
30
35
SLIN1TY %-
Figure 4:
Response surface of mean percent larval C. gigas set
(settlement) for the temperature x salinity factorial in
Experiment 1.
Isopleths delineate 5% intervals in the
settlement response.
15
As one or both of the experimental variables involved approaches
the extremity of larval tolerance, this alignment becomes skewed from
the horizontal.
A compaction of the response surface contours,
indicating response limiting conditions with increasing factor interaction, is seen at 35°C as salinities approach the experimental maximum
of 35
O/
The eliptical shape of the settlement response contours
implies that C. gigas larvae are less tolerant to variations in temperature than salinity.
The acceptable minimum of commercial settiment for hatchery reared
C. gigas larvae is 20% of the initial seeding by the commercial grower,
established by guarantee from the commercial hatchery.
This minimum
can be achieved by the grower with a setting tank temperature from 20
to 35°C, over a broad salinity range.
Maximum larval settlement, with
a mean response of 35 to 40% success of the initial seeding, was
attained in the realm of 30°C at a salinity of 30%.
The results of Experiment 1 correspond very well with an earlier
study by Lund (1972) who found maximum settlement of C. gigas larvae to
occur at 28°C in salinities between 22 to 34
°/o.
In application of
of these results, it must be noted here that Figure 4 is suggested as a
working tool for approximating expected larval settlement rather than a
definitive instrument for exacting response.
Variables other than
temperature and salinity may act individually or in concert upon the
larval environment to modify development and behavior.
Kinne (1970)
refers to the geographical component in an organism's thermal tolerance.
Bayne et al. (1977) find that when discussing thermal optima, the test
organismt s thermal history must be taken into account.
Muranaka (1981)
16
demonstrated that the survival of C. gigczs larvae in varied salinities
is directly influenced by the ambient salinity at which the adult
broodstock were conditioned prior to spawning.
This adaptive response to prevailing environmental stimuli is,
over time, likely to develop into site-specific tolerances through
selective broodstock survival (Calabrese and Davis, 1970; Lannan et al,
1980).
The larval settlement performance within the experimental
ranges established for this study therefore is subject to the environmental origins of the parental hatchery broodstock.
Feeding During Settlement
Settlement in C. gigas larvae was not found to be improved by the
presence of P. paradoxa at any of the four feeding levels in Experimerit 2 over the control.
Figure 5 presents the mean percent of C.
gigas larvae which set at each level over the 4S hour commercial
settlement period.
With the origination of Experiment 2 came the assumption that the
chrysomonad P. paradoxa contained sufficient nutritional content for
maintenance of C. gigas larvae during this study.
Although not widely
used in commercial shellfish hatcheries, it has performed well as the
primary nutritional supply here at the Newport facilities.
Davis and
Guilland (1958) in the examination of several phytoplankton genera
found the chrysomonads Isochrysis galbana and Monochrysis lutheri, both
closely related to p paradoxa, to be excellent food for oyster larvae.
In retrospect however, the assurance placed in the sole selection of
P. paradoxa as the sole food source for this work may have limited the
17
Lu
C')
-J
4
-J
I-
z
oZO
Lii
Lu
0
z
4
WI0
LI
I
0
25,000
I
I
75,000 100,000
50,000
CELLS / ml
Figure 5:
Mean percent larval C. gigas settlement at five feeding
levels of P. paradoxa cells/mi.
Standard Error (S.E.) of means.
Vertical bars indicate
usefulness of applications of these results.
A stronger approach to
the question of food importance during settlement would include a
variety of algal feeds which are more commonly used in commercial
shellfish hatcheries.
Despite this drawback in experimental design, the results from
Experiment 2 do have some relevance towards commercial applications.
Under acceptable conditions of temperature and salinity, growth in
larval oysters is thought to be primarily a function of food supply
(Helm and Millican, 1977).
The conditional requirements for
maintaining the vigor of the larval food supply however, are often not
consistent with those for obtaining maximum settlement.
Walne (1965)
found that larval food consumption increased rapidly with corresponding
increases in the ambient water temperature, until an upper thermal
tolerance limit is reached for that particular algal food.
Ukeles
(1961) and Brenko and Calabrese (1969) individually reached the
conclusion that for many of the algal species selected as larval food,
this upper tolerance limit is near 32.5°C, perhaps as low as 30°C.
At
temperatures in excess of the practical boundary, poor growth occurred
in the experimental larvae even though the algal supply was actively
consumed.
This was attributed to the inability of the algal species
in surviving the high temperatures, resulting in cellular decomposition.
The velum (the larval feeding organ) of the experimental larvae then
became fouled with the algal debris.
Secondary toxic bacterial blooms
developed with detrimental effect to the larvae's growth and general
health.
Thus the temperature of 30°C found to maximize larvae settlement
in Experiment 1 (Figure 4) used as a constant throughout this study,
19
may not be the most efficient temperature for a commercial shellfish
grower feeding concentrated algae as the larval food supply.
Setting
tank temperatures of less than 30°C, being perhaps more equitable in
the maintenance of both larval and algal culture health, may prove to
deliver more commercially advantageous results.
In Figure 5, no significant difference was found in mean larval
settlement between the experimental controls absence of algal feed and
treatment levels to 75,000 cells/mi.
apparent decrease in settlement occurs.
At 100,000 cells/mi however, an
A hypothesis might be formu-
lated that the previously mentioned disincorporation of the algae's
cellular composition, occurring in all t'atments with algae present, is
densest in fouling debris at the highest algal concentration.
At this
concentration, the larval velum is then most seriously impaired by the
algal debris, resulting in reduced larval vigor and setting competency.
Another possibility exists, although not necessarily completely
distinct from the above.
Some molluscan researchers, notably Walne
(1965) and Schulte (1975), have indicated that larval food concentrations past some critical density result in decreased larval filtration
rates.
Usually reported in either cells/mi or cells/larvae, this
It
critical level is variable, dependent upon the species involved.
is equally dependent upon existing environmental conditions, such as
temperature and salinity, which also affect larval filtration rates.
Schulte (1975) found that at 20°C, the critical cell density for mature
C. virginica
larvae (200-300 microns in length) feeding on
closterium was 70,000 cells/mi.
Nitschia
Walne (1965) in similar studies also
at 20°C, observed that substantial increases in density of the algae I.
galbana
larvae.
caused decreasingly smaller increases in the growth of 0.
edulis
Although these pairings of larval oyster species and algal food
are not identical to those in this study, a similarity in feeding
behavior does appear to exist.
Whether the decline in larval C. gigas
settlement at 100,000 cells/mi of P. paradoa is due to a behavioral
response to a saturation food level (critical cell density) or to a
mechanical impairment of the larvae's ciliated velum, or both, from the
commercial grower's viewpoint the possibility of overfeeding is
indicated and should be avoided.
Larval Storage
Mean set of C. gigas larvae held in storage over the initial 8
days of Experiment 3A is illustrated in Figure 6.
Although no
significant difference exists between the treatment levels, increased
mean larval set from Day 2 to Day 4 was sustained through Day 8.
A
hypothesis may be formed that this slight rise in mean set is
indicative of a stress induced quickening of the larvaes settlement
behavior.
Since a time limit of 48 hours is placed on comparative
larval settlement for this study, a faster rate of settlement in the
latter storage periods would account for the increased mean larval set.
The important conclusion to be drawn here is not that refrigerated
storage will increase larval C. gigas settlement, but that storage of
competent larvae at 5°C does not negatively impair their ability to set.
An extension of these results is to ascertain 1) at what point
will the storage effect adversely influence the larvae's setting
ability, 2) do the stored larvae metamorphose properly, surviving as
full normal spat, and 3) what physiological mechanism allows the larvae
to retain setting competency when held in storage.
21
100
Ui
Cl)
60
J
I::
0
I
o
a
DAYS
Figure 6:
I
I
I
4
6
8
IN 5°C STORAGE
Mean percent larval C. gigas settlement after 0 to 8 days
of 5°C storage.
(S.E.) of means.
Vertical bars indicate Standard Error
22
The ability of larvae of many marine bivalve species to delay
metamorphosis has been well documented (Lynch 1947, 1949; Wilson, 1958;
Gaitsoff, 1964).
Primarily cited as a response to unsuitable substrate
or environmental conditions, the larvae are able to continue developmental growth up to the point of metamorphosis and maintain this status
until more favorable conditions prevail.
Bayne (1965) observed mature
larvae of the mussel Mytilus edulis delaying settlement and metamorphosis for 30 days in the absence of suitable setting substrate.
Crisp
(1974) found that the selective discrimination for substrate by most
pelagic larval invertebrates was inversely correlated with the length
of metamorphic delay.
This parallels observations in this study that
C. gigas larvae, once metabolism is fully recovered from lengthy
storage, set rapidly with a higher incidence of attachment to the walls
of the glass setting beaker.
The prolongment of larval storage time in Experiment 38, coupled
with subsequent observations of spat survival on the individual cuitch
squares provided some answers to questions posed.
In Figure 7, the
mean percent of larval C. gigas set over an extended storage retention
period is seen.
The trend in larval settlement is similar in both
Experiment 3A and 3B; a slight increase in mean percent larval set
occurring with larval storage, sustained through Day 8, then declining.
Settlement was negligible at Day 12, implying an upper limit for
acceptable commercial application of stored C. gigas larvae from 8 to
10 days at 5°C.
A comparison is made in Figure 7 of initial larval set from
Experiment 3B with post-settlement survival.
The term survival here
is limited to the percentage of C. gigas larvae from the initial
23
60
f1201
I00
I-
80
-J
4
>
6Q
-j
I-
z
40
Ui
3.
z
42
Iii
I
I
0
2
4
DAYS
Figure 7:
6
IN 5°C
6
JO
12
STORAGE
Mean percent larval C. gigas settlement after 0 to 12 days
in 5°C storage.
The comparative mean spat survival after
90 days for each 48 hour storage interval is displayed
above.
Vertical bars indicate Standard Error (S.E.) of means.
24
seeding which have successfully metamorphosed as spat and survived
after 90 days of being suspended in flowing, unfiltered seawater.
Mean larval survival for the storage treatments from Day 0 through
Day 8 appeared to increase slightlywith storage retention.
Survival
of the Day 10 and Day 12 larvae was considerably reduced, perhaps
reflecting the decreased initial settlement competency.
As to the
apparent trend in increased larval survival with duration of storage,
it is difficult to assign causitive agents.
Unlike the increased
initial larval settlement with storage in which the larvae are able to
delay settlement and metamorphosis due to stress of some nature,
increased survival would seem to dictate a positive influence acting
upon the larvae.
If continuing research finds that this apparent
increase in survival is more than an experimental anomaly, it may
benefit the commercial industry.
At this point however, it would be
irresponsible to positively correlate storage retention of C. gigas
larvae with long-term survival.
It is sufficient to correctly
identify prolonged larval storage at 5°C as having no detrimental
effect upon the survival of hatchery reared larvae.
The physiological mechanism which enables bivalve larvae to retain
setting competency during periods of reduced food abundance, as in
storage, has been proposed to be stored lipid energy reserves (Holland,
1978; Gallager and Mann, 1981).
These same energy reserves may also
allow hatchery reared C. gigas larvae to successfully complete
metamorphosis when unable to feed during commercial settlement.
Bayne
(1965) suggests that in Mytilus edulis, stored lipid reserves present
an important source of energy in times of starvation and stress as
as during larval metamorphosis.
e11
Walne (1966) observed that larval shell
25
growth in the European oyster Ostra edulis is able to contInue for a
minimum of 48 hours in the absence of food.
The limiting factor in
larval utilization of these reserves is presumed to be the quantity
and nutritional quality of prior food consumption.
A direct relation-
ship between the lipid content of newly released Ostrea lurida larvae
and their subsequent viability and setting competency has been
described by Helm et al. (1973).
What is evident from
cperiments 3A and 3B is that mature C. giga.s
larvae are able to sustain themselves for some time in the absence of
food and total water immersion without serious effect upon their
ability to later function as normal, healthy bivalve spat.
Applications
This study was designed with a goal of determining specific
methods in which the oyster industry might be able to increase
commercial handling and remote-setting efficiency of hatchery reared
Pacific oyster larvae.
Through manipulation of operational and
biological factors, increased larval C. gigczs settlement can be
attained.
Application of these methods can readily be made to the
commercial shellfish industry.
In this study, emphasis was placed on biological rather than
economic efficiency in consideration of the factor levels which
resulted in the maximization of larval settlement.
The commercial
grower should therefore weigh all costs to be incurred in increasing
the percent success of larval settlement with that of simply purchasing
more larvae initially from the hatchery.
26
Larval settlement can be improved by raising the temperature of
the commercial setting tank to a maximum of 30°C.
To go beyond this
temperature will result in decreasing larval set.
A cost trade-off
exists for the oyster grower ho intends to increase expected larval
settlement with temperature and the cost of heating the settlement tank.
It will be important then that cost be most carefully assessed for
individual operations and weighed against the commercial benefit of
increased larval settlement.
Should the cost of attaining maximum
settlement at 30°C be greater than the worth of the increased seed, a
lower tank temperature may prove to be economically more efficient.
The findings of Experiment 2 indicate that settlement of C. giga.
larvae will not be enhanced by the presence of P. parad.oxa in concentrations up to 100,000 cells/ml.
Assuming that the larvae are well
fed at the commercial hatchery, healthy and properly cared for during
shipment, larval setting competency will not be impaired by the absence
of algal food during the normal 48 hours of commercial settlement.
This ability of larvae to sustain settlement and metamorphosis
utilizing stored energy reserves relieves the shellfish grower of the
burden of larval nutrition.
Should the individual grower choose to lengthen the settlement
period further than the 48 hours used here as the experimental standard
it may then be advisable to implement some form of nutritional supply.
For the commercial grower to culture select algal strains on site at
the setting facility, in similar manner to that of the commercial
hatcheries, considerable cost would be incurred.
A far more practical
and economical method would be for the grower, after the initial 48
hours settlement, to mix new water from the estuarine source with the
27
water already held in the settlement tank.
This would act to create a
food supply for the spat which have already undergone metamorphosis and
are ready to begin feeding again.
A secondary benefit of this method
might be the reduction of temperature shock as a mixing of the heated
and estuarine waters would lower the tank temperature more gradually.
The duration of,the larval settlement period beyond 48 hours however,
is a matter of the relative success of the larval settlement and the
production schedule to be determined by the individual growere
Perhaps of greatest benefit to the commercial shellfish industry
is the finding that hatchery reared C. gigas larvae are able to retain
setting competency while held in refrigerated storage.
This ability
will substantially expand the potential market to which properly
packaged larvae can be successfully shipped with resultant commercially
viable settlement and survival.
It allows the commercial grower, upon
receipt of the hatchery product, to develop a short-term larval
inventory reducing repetitive shipment and handling costs, thus
increasing operational efficiency.
One competent commercial oyster
hatchery will have the capability to serve regional seed demands, with
the possibility of international expansion.
No longer does the Pacific Northwest oyster industry need to
depend upon unreliable and costly sources of oyster seed.
With the
development and refinement of handling and remote-setting techniques
for hatchery reared larvae, the shellfish industry will be able to
compete in the marketplace with a sustainable,
quality product.
AI
SUMI1ARY
Results from the manipulation of specific operational and environmental factors influencing the remote settlement of hatchery reared
C.
gigas larvae are summarized below.
1)
Maximum observed larval C. gigas settlement was attained
with a setting tank temperature of 30°C and a salinity of 30 0/00.
2)
Temperature appears to be a more sensitive factor than does
salinity in limiting larval settlement.
3)
Within the restricted temperature and salinity ranges used in
this work, no significant interaction exists between these factors.
Some factor synergism is found however at the extremes of both ranges,
having a negative influence upon larval settlement.
4)
Providing setting C. gigas larvae with the chrysomonad P.
paradoxa as an algal food source, in concentrations from 0 to 100,000
cells/mi, had no significant effect upon larval settlement numbers.
5)
C. gigas larvae, when held free of total aqueous Immersion
and refrigerated at 5°C, can retain setting competency for commercial
application for a maximum of 10 days.
6)
Commercially acceptable settlement, with subsequent 90-day
survival of C. gigas spat, can be obtained from refrigerated larvae for
a maximum of 8 days in 5°C storage.
29
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34
APPENDIX
35
Table A.
Cumulative mean numerical settlement of C. giga$ larvae in
The
temperature by salinity factorial (Experiment 1).
standard deviation for each treatment is seen in parenthesis.
The mean (X) and standard error (SE) of sample means are
exhibited individually for all factor levels.
Temperature C
Salinity
15
20
25
30
35
X
SE
9.90
10.10
21.50
24.10
24.30
17.98
(7.37)
(11.41)
(10.73)
(21.89)
(16.27)
(20.46)
12.70
24.40
28.40
28.40
26.60
24.08
(6.58)
(11.65)
(22.50)
(27.26)
(26.86)
(19.09)
16.60
25.10
27.70
36.90
29.10
27.10
(7.33)
(13.71)
(19.63)
(25.62)
(38.71)
(18.59)
20.00
27.80
31.80
42.70
27.80
30.02
(8.28)
(18.39)
(23.78)
(40.60)
(32.57)
(28.50)
17.60
22.00
31.10
34.50
15.00
24.04
(8.46)
(17.43)
(17.11)
(25.18)
(24.95)
(13.39)
X
15.36
21.88
28.10
33.32
24.56
SE
(4.03)
(6.90)
(4.08)
(7.27)
(5.63)
°I
15
20
25
30
35
36
Table B.
Sample mean (X) and standard deviation (s) of larval
C. gigas settlement at five P. paradoxa feeding levels
(Experiment 2).
Pseudoisochrysis paradoxa cells/ml
0
25,000
50,000
75,000
100,000
Sample
Mean
35.84
32.84
37.92
35.42
18.50
Standard
Deviation
13.59
14.89
10.87
13.67
8.83
37
Table Cl.
Sample mean (X) and standard deviation (s) of larval
The larvae were
C. gigas settlement in Experiment 3A.
held in refrigerated storage from 0 to 8 days with samples
removed at 48 hour intervals.
Days in 5°C Storage
0
2
4
6
8
34.33
38.89
52.78
53.11
16.11
12.88
16.71
17.98
14.04
35.59
Sample
Mean
Standard
Deviation
Table C2.
Sample and 90-day survival means with standard deviations
The
for larval C. gigas settlement in Experiment 313.
larvae were held in refrigerated storage from 0 to 12 days
with samples removed at 48 hour intervals.
Days in 5°C Storage
10
12
0
2
4
6
8
24.00
67.67
62.67
73.33
88.00
40.33
6.00
Standard
Deviation
10.15
34.59
9.61
28.31
6.24
7.64
2.65
Mean
Survival
13.33
16.67
30.00
38.33
41.67
14.67
3.33
Standard
Deviation
10.10
8.02
16.40
18.78
7.65
8.43
3.02
Sample
Mean