Ž .
focus were grabbed with the aid of Microsoft’s Media Manager Video capture 32 and the resulting still images were saved as bitmap images on the computer’s hard disk.
Ž .
Then an image analysis software package Jandel Scientific Sigma Scan was used to measure the length of 60 larvae. This technique was compared with the photographic
technique by measuring the same slide of larvae. The mean values obtained by the two techniques did not differ by more than 5.
2.3. Statistical analysis Before applying any parametric test each data set was tested for normality and
homogeneity of variances, with a normal probability plot and with Barlett’s test, respectively. Significance was tested at the 95 level.
Logarithmic transformation of data resulted in more data sets having a normal distribution; however, in no instance were all the distributions of all data sets normal.
Even when one of the assumptions necessary for applying parametric tests was violated Ž
. in a small part of the data set typically one or two sets of larval lengths , parametric
tests were used. The experimental design was such that nested ANOVA was used in all experiments to assess whether significant differences existed between diets. This test
also reveals whether there is significant variation between the replicates of the same diet.
Ž In order to identify the groups that demonstrated significant differences 95 confidence
.
X
Ž limit , the multiple comparisons between means of T -method was used Sokal and
. Rohlf, 1987 . All tests were carried out with the aid of the statistical software package,
Minitab
w
, while preliminary calculations and graphs were made in Quattro
w
Pro. To identify any relationship between algal biochemical component and larval growth,
correlation analysis was used. This type of analysis was used instead of ordinary regression analysis because the assumptions of model I regression analysis do not hold
Ž .
true with this type of data Sokal and Rohlf, 1987 .
3. Results
The gross biochemical composition of the algal species grown under various nutrient Ž
. light and nutrient conditions is taken from Leonardos and Lucas 2000a and reproduced
here in Table 1 for their protein, carbohydrate and proportions of some of the major fatty acids. The percentage of change in per cell protein content in S. costatum, C. muelleri
Ž and P. lutheri as a result of the various culture conditions employed was smaller one-
. to twofold change than in R. reticulata where protein content varied from 4.17 pg
y1 y1
Ž .
cell to 18.27 pg cell
fourfold change . Protein levels of the two diatoms and of R. reticulata were increased under low light. S. costatum exhibited a peak of protein
content under phosphorus limitation in both light intensities, while R. reticulata for each corresponding nutrient condition should protein increase at high-light condition; at the
low-light nutrient conditions, protein content increased under nitrogen limitation. Protein levels of C. muelleri were not greatly affected under the culture conditions employed,
although they decreased to some extent with nutrient limitation and increased light intensity. The change in protein content of P. lutheri was not consistent.
Table 1 Responses of S. costatum, C. muelleri, R. reticulata and P. lutheri to various growth conditions
Ž .
HL s high light; LL s low light; fr2 s fr2 medium no nutrient limitation ; P s phosphorus-limited medium, Ns nitrogen-limited medium. Fatty acids are expressed as percentages of TIFA. SAFA ssaturated fatty acids;
PUFA s polyunsaturated fatty acids. BDs below detection levels. Reproduced from Leonardos and Lucas Ž
. 2000a . Three samples were analyzed for their protein and carbohydrate content while two samples underwent
fatty acid analysis. Numbers in parentheses indicate standard deviation. Treatment
Protein Carbohydrate 16:0
18:3ny3 20:5ny3 SAFA PUFA ny3 FAs
y1 y1
Ž . Ž
. pg cell
pg cell Ž
. Ž
. S. costatum HL fr2
3.8 1.03 1.22 0.07
8.47 1.26
20.07 23.62
56.81 25.98
Ž .
Ž .
HL P 5.8 0.87
2.71 0.57 8.16
0.79 19.9
26.1 53.9
25.1 Ž
. Ž
. HL N
6.3 0.30 1.79 0.22
9.10 0.54
19.8 29.1
51.4 26.5
Ž .
Ž .
LL fr2 6.4 0.08
2.07 0.09 11.57
2.42 15.66
18.91 46.42
24.09 Ž
. Ž
. LL P
6.3 0.23 2.56 0.01
12.98 1.26
12.52 29.91
34.42 17.60
Ž .
Ž .
LL N 3.9 0.88
1.06 0.16 15.91
1.12 12.65
30.00 37.79
18.31 Ž
. Ž
. C. muelleri HL fr2
9.2 1.80 3.19 0.81
11.10 0.86
17.79 24.33
50.99 22.44
Ž .
Ž .
HL P 8.6 0.74
5.60 0.22 17.22
0.24 14.38
29.93 42.08
17.57 Ž
. Ž
. HL N
7.2 0.45 4.85 0.47
23.41 B.D.
16.51 32.20
37.11 19.85
Ž .
Ž .
LL fr2 12.5 1.5 3.20 0.30
10.75 0.49
21.45 23.22
52.02 25.11
Ž .
Ž .
LL P 10.5 2.0
2.61 0.17 22.69
B.D. 9.35
42.71 29.53
13.11 Ž
. Ž
. LL N
10.3 1.0 2.88 0.39
14.44 0.95
13.59 27.99
37.87 17.28
Ž .
Ž .
R. reticulata HL fr2 25.9 3.49 4.2 0.78
7.08 15.92 11.67
17.04 69.71
59.77 Ž
. Ž
. HL P
29.0 3.94 7.6 1.43
11.04 15.07 3.87
24.12 53.36
34.33 Ž
. Ž
. HL N
36.2 2.12 18.3 0.8
25.09 13.43 2.08
49.64 33.21
27.32 Ž
. Ž
. LL fr2 25.4 1.68
8.0 2.31 6.10 12.31
9.95 12.93
78.09 52.44
Ž .
Ž .
LL P 28.2 2.89
4.7 0.04 8.37 17.44
8.63 12.33
75.39 59.01
Ž .
Ž .
LL N 32.8 3.66
6.3 0.25 13.03 12.36
8.10 23.16
66.24 42.64
Ž .
Ž .
P. lutheri HL fr2
3.7 1.25 0.51 0.06
18.50 0.99
27.88 27.71
55.71 43.61
Ž .
Ž .
HL P 6.6 0.51
0.94 0.09 25.20
1.48 16.51
34.55 40.84
28.18 Ž
. Ž
. HL N
4.1 0.2 0.45 0.17
17.62 1.88
17.66 27.11
57.40 39.69
Ž .
Ž .
LL fr2 4.5 0.54
0.55 0.02 14.15
1.31 22.18
22.97 64.07
49.00 Ž
. Ž
. LL P
5.7 1.93 0.54 0.15
17.75 2.44
25.63 26.01
59.84 42.81
Ž .
Ž .
LL N 7.1 0.74
0.74 0.08 16.29
1.53 27.45
24.07 62.53
47.24
In all four algal species used, carbohydrate content per cell was not greatly affected by the culture conditions showing one- to twofold changes. Carbohydrate content of S.
costatum was generally increased with decreased irradiance except for the nitrogen- limited cultures where carbohydrate content remained practically unchanged at both
irradiance levels. R. reticulata, on the other hand, showed a different response with conservative changes in carbohydrate content; at the higher-light intensity, carbohydrate
content was increased only under phosphorus limitation and at the lower-light under nitrogen limitation. The carbohydrate content of C. muelleri was affected more by
nutrient limitation at the higher-light intensity while at low-light, there was little change. In contrast, P. lutheri showed little change in its carbohydrate content under the
conditions employed.
With regard to the fatty acid content of the algal species used, it appeared that Ž
. saturated fatty acids SAFA were increased as a proportion of the total identifiable fatty
Ž .
acids TIFA under the higher-light intensity in both diatom species. A more complex
Fig. 1. Length of D-stage larvae as well as final length of M. edulis larvae after the 14-day feeding trial using the Control, the S. costatum and P. lutheri as food cultured under various light and nutrient conditions.
Results shown are average of three replicate cultures and vertical lines indicate standard deviation. Ž
. fr2 s fr2 medium no nutrient limitation ; P s phosphorus-limited medium, Ns nitrogen-limited medium.
Fig. 2. Length of D-stage larvae as well as final length of M. edulis larvae after the 14-day feeding trial using the Control, the C. muelleri and the R. reticulata diets as food cultured under various light and nutrient
conditions. Results shown are average of three replicate cultures and vertical lines indicate standard Ž
. deviation. fr2 s fr2 medium no nutrient limitation ; P s phosphorus-limited medium, Ns nitrogen-limited
medium.
Ž .
picture emerged for the PUFA and the n y 3 series of fatty acids n y 3 . In S.
costatum, PUFA and n y 3 decreased with nutrient limitation under the higher-light intensity, but under low-light, PUFA proportions increased in the phosphorus-limited
cells and decreased in the nitrogen-limited cells. C. muelleri increased its SAFA proportion under nutrient stress at both light intensities, while n y 3 FA decreased under
the same conditions. A similar decrease occurred with PUFA, except with phosphorus limitation at low light, where its proportions were approximately the same as with the
Ž .
non-limiting nutrient fr2
conditions. The proportion of PUFA and n y 3 in R.
Table 2 Percent survival of larvae after the S. costatum, R. reticulata, C. muelleri and P. lutheri feeding trials, for
each of the triplicate cultures Ž
. Numbers in parentheses indicate standard deviation. fr2 s fr2 medium
no nutrient limitation ; P s phosphorus-limited medium; Ns nitrogen-limited medium.
Diet High-light trial
Low-light trial High-light trial
Low-light trial S. costatum
R. reticulata Ž
. Ž
. Ž
. Ž
. Control 1
74.4 5.67 47.2 13.2
39.8 5.79 39.8 5.79
Ž .
Ž .
Ž .
Ž .
Control 2 92.8 16.9
44.4 11.0 38.0 4.61
38.0 4.61 Ž
. Ž
. Ž
. Ž
. Control 3
88.9 15.1 35.6 2.08
39.2 7.89 39.2 7.89
Ž .
Ž .
Ž .
Ž .
Unfed 1 87.2 3.42
30.0 9.81 7.0 1.43
7.0 1.43 Ž
. Ž
. Ž
. Ž
. Unfed 2
52.8 2.08 55.0 4.91
9.4 0.83 9.4 0.83
Ž .
Ž .
Ž .
Ž .
Unfed 3 90.0 10.6
38.9 4.37 14.6 5.03
14.6 5.03 Ž
. Ž
. Ž
. Ž
. fr2 1
98.9 14.1 28.9 3.14
31.0 5.79 34.5 4.14
Ž .
Ž .
Ž .
Ž .
fr2 2 91.1 10.9
38.9 0.79 35.1 4.30
32.8 5.96 Ž
. Ž
. Ž
. Ž
. fr2 3
81.1 6.98 30.6 2.08
32.2 5.79 25.1 0.83
Ž .
Ž .
Ž .
Ž .
P 1 91.7 1.36
18.3 2.72 40.9 2.98
37.4 3.61 Ž
. Ž
. Ž
. Ž
. P 2
93.3 10.6 18.9 1.57
28.7 5.79 32.8 2.19
Ž .
Ž .
Ž .
Ž .
P 3 77.2 8.31
17.2 2.08 26.9 0.83
39.2 7.21 Ž
. Ž
. Ž
. Ž
. N 1
87.2 4.16 28.9 3.42
19.3 1.43 29.8 4.96
Ž .
Ž .
Ž .
Ž .
N 2 86.1 5.15
15.0 3.60 19.9 2.98
30.4 2.19 Ž
. Ž
. Ž
. Ž
. N 3
84.4 12.6 12.2 5.67
28.7 5.96 35.7 3.31
C. muelleri P. lutheri
Ž .
Ž .
Ž .
Ž .
Control 1 74.4 5.67
47.2 13.2 35.0 2.36
35.0 2.36 Ž
. Ž
. Ž
. Ž
. Control 2
92.8 16.9 44.4 11.0
40.0 7.20 40.0 7.20
Ž .
Ž .
Ž .
Ž .
Control 3 88.9 15.1
35.6 2.1 43.3 3.60
43.3 3.60 Ž
. Ž
. Ž
. Ž
. Unfed 1
87.2 3.42 30.0 9.8
36.7 1.36 36.7 1.36
Ž .
Ž .
Ž .
Ž .
Unfed 2 52.8 2.08
55.0 4.9 29.4 1.57
29.4 1.57 Ž
. Ž
. Ž
. Ž
. Unfed 3
90.0 10.6 38.9 4.4
35.6 4.16 35.6 4.16
Ž .
Ž .
Ž .
Ž .
fr2 1 82.8 8.20
20.6 5.5 43.3 8.28
37.2 6.71 Ž
. Ž
. Ž
. Ž
. fr2 2
84.4 8.20 25.0 6.2
38.9 8.75 44.4 3.14
Ž .
Ž .
Ž .
Ž .
fr2 3 93.3 16.0
32.8 5.2 35.6 3.42
32.8 4.37 Ž
. Ž
. Ž
. Ž
. P 1
73.3 13.0 23.9 3.9
36.1 5.50 39.4 3.42
Ž .
Ž .
Ž .
Ž .
P 2 87.8 4.37
42.2 6.3 27.2 4.37
37.8 13.2 Ž
. Ž
. Ž
. Ž
. P 3
74.4 9.26 19.4 4.2
27.2 0.79 37.8 2.83
Ž .
Ž .
Ž .
Ž .
N 1 85.0 8.28
19.4 4.4 26.7 3.60
42.2 12.9 Ž
. Ž
. Ž
. Ž
. N 2
81.7 10.8 23.3 3.6
33.3 4.91 31.1 9.06
Ž .
Ž .
Ž .
Ž .
N 3 85.6 8.31
22.2 2.1 33.3 4.71
40.6 6.43
reticulata showed smaller changes under low irradiance, whereas at the high light, there was a peak of PUFA when nutrients were not limited. It was also noticeable that there
was a marked increase in the SAFA proportion in R. reticulata grown under nitrogen limitation at the higher-light conditions. For P. lutheri, there was little change in overall
SAFA components with the various treatments. The exception occurred under high-light phosphorus limitation where the SAFA proportion was considerably increased and there
was a corresponding reduction in the PUFA and n y 3 proportions.
The final length of M. edulis larvae achieved after the 14-day feeding trial, as well as the corresponding length of the control unfed and at the initial D-stage, is illustrated in
Fig. 1, when the larvae were fed either on S. costatum or P. lutheri and in Fig. 2, when fed on C. muelleri or R. reticulata. With the aid of nested ANOVA and analysis of
means, the S. costatum diets were ranked, in decreasing order of exhibited larval
Ž .
growth, with ‘‘ ’’ indicating significant difference 95 , as follows: LL N s LL fr2 Control HL fr2 s HL P s HL N LL P. The C. muelleri diets were ranked,
in decreasing quality, in the high-light experiment as: HL N Control HL fr2 s HL P, and for the low-light experiment, LL fr2 s LL N Control s LL P. The R.
reticulata diets were ranked as: HL N s LL fr2 s Control LL N HL fr2 s HL P s LL P. Finally, the P. lutheri diets were ranked, again in decreasing quality order as:
HL P HL fr2 s HL N LL N s LL P LL fr2 s Control.
Ž .
It becomes clear that survival varied between experiments Table 2 , which were carried out using different batches of larvae, while in the same experiment, survival
could not be correlated with the algal diet. This argument is supported by the variation Ž
in survival between triplicates of the same diet showing significant differences P -
. 0.05 .
Fig. 3. Relationship between proportion of dietary fatty acids of P. lutheri and length of the M. edulis larvae achieved after feeding on the corresponding diets. SAFA s total saturated fatty acids. PUFA s total poly-
unsaturated fatty acids.
Ž .
Fig. 4. Relationship between 16:0 proportion as percentage of total identifiable fatty acids, TIFA , protein and carbohydrate content of R. reticulata and larval length achieved after feeding for 2 weeks on this species.
Protein content is shown colour-coded.
There were few occasions that a coherent relationship between algal biochemical components and larval size could be established. In P. lutheri, there was very clear
Ž .
Fig. 5. Relationship between 18:3ny3 proportion as percentage of total identifiable fatty acids, TIFA , protein and carbohydrate content of R. reticulata and larval length achieved after feeding for 2 weeks on this
species. Protein content is shown colour-coded.
Ž .
Fig. 6. Relationship between total ny3 FA proportion as percentage of total identifiable fatty acids, TIFA , protein and carbohydrate content of R. reticulata and larval length achieved after feeding for 2 weeks on this
species. Protein content is shown colour-coded.
positive correlation between the 16:0 and SAFA, with growth and negative correlations Ž
. existing between PUFA and n y 3 FA with larval size Fig. 3 , with correlation values
of 0.97, 0.98, y0.98 and y0.97, respectively. Protein and carbohydrate content were not significantly correlated with larval growth, with correlation values of 0.19 and 0.57,
respectively. However, protein, carbohydrate and some fatty acids of R. reticulata were correlated with larval growth. Generally, lower correlation values were calculated with
0.73 and 0.48 for protein and carbohydrate content and 0.55, y0.91, 0.56, y0.6 for 16:0, 18:3n y 3, SAFA and n y 3 FA, respectively. Four-dimensional plots of larval
length, algal protein, carbohydrate and one of these algal fatty acids components are shown in Figs. 4–6 for the 16:0, 18:3n y 3 and n y 3 FA, respectively. Collectively,
these graphs, together with the correlation analysis, indicate that there is a positive correlation between algal 16:0 and SAFA with larval growth while algal 18:3n y 3 and
n y 3 FA were negatively correlated with larval growth. Despite careful examination of the data for C. muelleri and S. costatum, no consistent relationship could be established.
4. Discussion
