Ž .
Langdon in press in that abalone were fed on the different algal types separately from the co-culture system; therefore, culture conditions for dulse did not have a direct
influence on abalone growth. A diet of wild-harvested bull kelp, N. luetkeana, was used as a control diet.
2. Materials and methods
2.1. Experimental organisms Ž
. Juvenile red abalone 11 mm SL 0.3 mm were supplied by The Cultured Abalone,
Santa Barbara, California, where they were reared on a diet consisting of kelp, Macrocystis pyrifera. P. mollis was originally obtained from Fidalgo Island, Washing-
Ž ton, for culture at HMSC, Newport, Oregon. Dulse was cultured in 110-l tanks 0.155
2
.
y1
m surface area at a stocking density of 9 g wet wt. l . Dulse culture conditions varied
Ž
y1
. by seawater exchange rate 1, 6, or 35 seawater volume exchanges d
and light Ž
y1
. exposure 0 or 24 h supplemental illumination d
. Supplemental illumination was supplied by 1000-W metal halide bulbs, which provided 21–24 mol photon m
y2
d
y1
of Ž
. photosynthetically active radiation PAR at the water surface. Average daily PAR
supplied by sunlight alone ranged from 29 mol m
y2
d
y1
in July to 7 mol m
y2
d
y1
in September. Seawater for the dulse cultures was sand-filtered and UV-disinfected. All
Ž dulse cultures were fertilized to excess on a continual basis with nitrogen added as
. Ž
. Ž
NaNO and phosphate added as NaH PO
as described by Evans and Langdon in
3 2
4
. press . Cultures had been established for approximately 1 year before the start of this
Ž .
study, during which time morphological differences became apparent Fig. 1 . Tempera- tures of dulse cultures during the experimental period ranged from 17.28C to 19.08C.
Ž .
The control diet of bull kelp N. luetkeana , was collected weekly from the north jetty at Yaquina Bay, Newport, Oregon.
2.2. Experimental design Ž
. The abalone feeding experiment was conducted over an 8-week 56 days period
from July to September, 1997, at HMSC. Dietary treatments consisted of dulse cultured under one of six different combinations of seawater exchange rate and light exposure
Ž .
Ž .
Table 1 , while the seventh treatment consisted of fronds cut into 1 in. squares Ž
. removed from freshly collected N. luetkeana bull kelp . Treatments were replicated
fourfold. High-density polyethylene containers with a 370-ml seawater capacity and 221 cm
2
of submerged surface area served as experimental chambers, each of which housed 10 abalone. The containers were covered with a black plastic film to inhibit diatom growth
Ž as well as to promote maximum feeding by juvenile abalone Ebert and Houk, 1984;
. Tutschulte and Connell, 1988 . Filtered seawater entered the containers at a rate of
Ž .
Ž Fig. 1. Morphologies of dulse
P. mollis after 1 year of culture under different light 0 or 24 h light
y1
. Ž
y1
. supplementation d
and seawater exchange rate 1 or 35 vols. d combinations. Broad thalli were typical
Ž . Ž .
of dulse in the 24 hr35= treatment A , narrow thalli were typical of dulse in the 0 hr1= treatment D , and Ž
. Ž .
intermediate thalli between A and D were typical of the 0 hr35= treatment C . Very fine thalli were typical Ž .
of slow-growing dulse in the 24 hr1= treatment B .
approximately 100 ml min
y1
, or 389 volume exchanges d
y1
. The containers were randomly distributed in a flow-through water bath. The containers were checked daily to
ensure food availability and to monitor water flows. Seawater temperature was recorded Ž
. at 2-h intervals with a temperature logger StowAway, Onset, MA, USA . Average
Ž .
seawater temperature was 13.6 1.58C 1 standard deviation, range s 11.8–18.58C during the experimental period. Salinity of incoming water was automatically measured
every 6 min by an OMEGA non-contact conductivity sensor, model CDCN-108.
y1
Ž Average salinity was 31.3 1.2 g l
1 standard deviation, range s 25.1–33.1 g
y1
. l
during the experimental period. Abalone were fed ad libitum throughout the experiment. Each week, uneaten food
was removed from the container with forceps, blotted dry between paper towels for 30 s and weighed. Containers were subsequently cleaned to remove any waste accumulation
or growth of microalgae. Fresh food was then added to the containers. Algae samples were collected from culture containers at the beginning and end of the study and stored
at y808C for biochemical analysis. Control containers with additions of each diet, but without abalone, were included in the study, and the percentage change in algal weight
was used to adjust food consumption measurements.
Table 1 Experimental dietary treatments tested with juvenile red abalone
Treatment Mean initial Diet
Dulse culture method Frond
Presence
a b
abalone size morphology of epiphytes
Supplemental light Exchange rate
y1 y1
Ž .
mm1 SD Ž
. Ž
. h d
vols. d 1
11.300.16 P. mollis
1 fine
low 2
11.250.12 P. mollis
6 medium
low 3
11.310.16 P. mollis
35 wide
low 4
10.720.21 P. mollis
24 1
fine high
5 10.760.12
P. mollis 24
6 medium
moderate 6
10.960.12 P. mollis
24 35
wide moderate
c
7 11.490.14
N. luetkeana –
– wide
low
a
Ž Supplemental light refers to number of hours of exposure to artificial lighting 21.1–24.2 mol photons
y2 y1
. Ž
y2 y1
. m
d in addition to ambient light 7.0 mol m
d .
b
Exchange rate expressed as the number of total volume seawater exchanges of 110-l culture vessels over a 24-h period.
c
N. luetkeana was collected at the north jetty of the Yaquina estuary, Newport, Oregon.
2.3. Measurements SL was recorded on day 1 and on day 56 of the study by measuring the longest axis
Ž .
of the shell to the nearest 0.1 mm with calipers. Mean SL increase for the experimental
y1
Ž .
period was reported in mm d for each treatment. Specific growth rate SGR , feed
Ž .
Ž .
conversion efficiency FCE , and daily feed consumption DFC were measured and reported using ash-free dry weights of both abalone tissue and algae, unless otherwise
stated. SGR, expressed as percent change in natural log of the ash-free dry wt. d
y1
, was calculated using the formula:
SGR s 100
U
ln W y ln W rd 1
Ž .
Ž .
f i
where W was the final mean ash-free dry weight of tissue, W was the initial mean
f i
Ž .
ash-free dry weight of tissue, and d was the experimental period days . Ž
. Initial wet weights were determined after blotting-dry the abalone see above . Four
groups of 10 abalone, randomly sampled from the same population used for the growth experiment, were weighed at the beginning of the experiment for determination of initial
wet tissue weight, dry tissue weight, and ash-free dry tissue weight. Tissues were dried at 608C for 24 h to determine dry weight. Dried abalone tissues were subsequently
placed in a muffle furnace for 24 h at 4208C for ash weight determination. Ash-free dry weights of tissue were calculated by subtracting ash weights from dry tissue weights.
Final weights of abalone used in the study were calculated using the same procedures. Dry weights and ash contents of macroalgal diets were also determined in this manner.
FCE, used as a measure of how efficiently the diet was used for growth, was calculated using the formula:
FCE s 100
U
W y W r F y F 2
Ž .
Ž .
Ž .
f i
g u
Ž . where W was the final ash-free dry weight g of abalone tissue, W was the initial
f i
Ž . Ž .
mean ash-free dry weight g of tissue, F was the ash-free dry weight g of food given,
g
Ž . and F was the ash-free dry weight g of food uneaten for the whole experimental
u
period. The DFC rate was estimated as the ash-free dry weight of algae consumed per day,
Ž
y1
. expressed as a percentage of the ash-free dry tissue weight of abalone BW d
, Ž
. according to a formula modified from Britz et al. 1997 :
DFC s F y R r W
U
T 3
Ž . Ž
. Ž .
Ž
y1 y1
. where DFC was the daily consumption g algae g
abalone d , F was the ash-free
Ž . dry weight g of feed offered over the experimental period, R was the ash-free dry
Ž . weight g of remaining feed collected over the experimental period, W was the average
Ž .
weight of abalone ash-free dry weight of tissue over the experimental period, and T Ž
. was the experimental period days .
2.4. Biochemical composition Protein, carbohydrate, and lipid content of the seaweed diets were analyzed using
Ž .
methods modified from Mann and Gallager 1985 . At the beginning and end of the experiment, one sample was collected for each diet by randomly sampling dulse from
each of the culture conditions. The samples were stored at y808C until analysis. Frozen
y3
Ž samples were freeze-dried for 24 h at less than 133 = 10
mbar LABCONCO
. freeze-dryer . Freeze-dried samples were subsequently pulverized in a Fritsch planetary
micromill and stored in glass scintillation vials at 48C until analysis. Protein was precipitated from 25 to 30 mg sub-samples of each diet with 5 wrv
Ž .
trichloroacetic acid TCA . Protein was then extracted from the precipitate by heating at 608C for 30 min in 5 ml of a solution of 2 wrv Na CO dissolved in 0.1 M NaOH,
2 3
followed by a second extraction with 5 ml 1 M NaOH under the same temperature conditions. Supernatants from the two extractions were combined. The concentration of
Ž .
dissolved protein was determined using the BCA protein assay method Pierce , in which 100 ml of the protein solution was mixed with 2 ml of BCA reagent mixture, and
the absorbance of the resulting solution was determined at 562 nm after incubation at Ž
. 378C for 30 min. Bovine serum albumin BSA was used for construction of a standard
curve. Soluble carbohydrate was determined in 2–3 mg freeze-dried, powdered macroalgae
Ž .
samples by extraction with 10 ml 0.1N H SO for 1 h Morgan and Simpson, 1981a .
2 4
Ž .
The phenol–sulfuric acid method described by Dubois et al. 1956 was subsequently used to determine soluble carbohydrate by reading absorbance at 490 nm, and compar-
ing absorbances with glucose standards. Lipids were extracted from 25 to 30 mg sub-samples of the prepared samples with
Ž .
chloroform and methanol as described by Mann and Gallager 1985 and weights Ž
. gravimetrically determined. Fish oil controls Zapata Haynie, feed grade menhaden oil
were used to correct for weight changes during lipid extraction and weighing. Residual weights were calculated by subtracting measured ash, protein, soluble
carbohydrate, and lipid dry weights from original total dry algal weights. Carbon, nitrogen, and hydrogen content of macroalgal samples were determined by CHN
analysis at the Marine Science Institute, University of California, Santa Barbara, California, USA.
2.5. Statistical analyses Ž
. Single-factor ANOVA P - 0.05 was used to determine whether there was a dietary
treatment effect on SL, SGR, FCE, and DFC. When null hypotheses were rejected, Ž
. Tukey’s HSD multiple range tests
P - 0.05 were used to determine significant
differences among treatments. Transformation of data was deemed unnecessary after Ž
testing for homogeneity of variances by box plot analysis. Two-factor ANOVA P - .
0.05 was used to determine overall effects of light and water volume exchange rate on algal food quality for abalone. Simple linear regressions were used to determine if
significant relationships existed between specific variables.
3. Results
