S .J. Newman et al. J. Exp. Mar. Biol. Ecol. 255 2000 93 –110
99
Systat E 5.01 package for PC Systat. In addition, we performed two-way ANOVAs for
individual MAAs with time and treatment as factors, using StatView E 4.5 Abacus
Concepts. The effect of 36 days of starvation on concentration of individual MAAs in krill, i.e. the difference in mean MAA concentration between day 63 and day 98, was
tested using Student’s t-test Zar, 1996.
3. Results
The extraction efficiency from three serial extractions in 100 methanol was . 97 60.6. Extraction efficiency of different MAAs did not vary significantly one-way
ANOVA, df 5 5, F 5 1.04, P . 0.5. To adjust for this rate of efficiency, values for individual MAA concentrations were adjusted by a 3 increase.
Spectrophotometric analyses of methanol extracts obtained from P . antarctica grown
under PAR and PAR1UV light Table 1 showed marked differences in the UVA B region in a comparison of spectra normalised to chlorophyll a absorption at 663 nm
Fig. 1. UVA B-treated P . antarctica exhibited a 2.3–5-fold increase in MAA
absorbance as compared with cultures grown under PAR Table 1. The concentration of individual MAAs in P
. antarctica varied according to whether the algae were grown under UV light Fig. 2a,b. There were greater amounts of
porphyra-334 in algae grown under PAR-only-treated algae, whereas twice as much shinorine and significantly greater amounts of mycosporine-glycine:valine was measured
in P . antarctica grown under PAR1UVA B Fig. 2a. A two-way ANOVA without
replication showed that concentration of MAAs in culture did not vary over time Fig. 2a,b. Trace quantities of asterina-330 and palythine were observed in 22 and 15 of
HPLC analyses, respectively, but peak heights were too small to allow for peak area quantification.
A representative chromatogram from an extract of krill shows all identified MAAs Fig. 3, peaks 2 and 4–8 and a single unidentified peak Fig. 3, peak 3. This unknown
component has a wavelength of maximum absorbance ,280 nm and is not present in P .
antarctica. Examination using reverse-phase separation and weak ionic exchange separation on a bonded-phase amino column Helbling et al., 1996 confirmed this
component to be unrelated to MAAs. Concentration of individual MAAs in krill varied over time, according to the feeding
regime. For all starved animals, concentrations of most MAAs stayed at or about initial conditions, except asterina-330, which rose in concentration by the end of the
experiment. Krill fed algae grown under PAR1UVA B showed a strong increase in concentration of mycosporine-glycine:valine with time. In addition, concentrations of
both porphyra-334 and shinorine dropped initially, before recovering to concentrations similar to starved animals. Levels of mycosporine-glycine remained consistent through-
out the experiment. In contrast, krill fed algae grown under PAR-only appeared to accumulate porphyra-334 and, to a lesser extent, shinorine. This is analogous to the
MAA complement of the diet, in that algae grown under PAR-only were characterised by an abundance of porphyra-334 and a lack of mycosporine-glycine:valine. As was the
100 S
.J. Newman et al. J. Exp. Mar. Biol. Ecol. 255 2000 93 –110
Fig. 2. Phaeocystis antarctica. Concentration of MAAs in P . antarctica culture grown under either PAR-only,
or PAR1UVA B: a mycosporine-glycine:valine and porphyra-334, b mycosporine-glycine and shinorine. Two-way analysis of variance without replication showed that MAA concentration varied according to MAA
identity for P . antarctica grown under PAR-only F 5 14.98, df53, 0.05 . P . 0.025 and PAR1UVA UVB
F 5 12.87, df53, 0.05 . P . 0.025. The date of feeding did not have a significant effect on MAA concentration for P
. antarctica grown under PAR-only F 5 1.26, df57, P . 0.5 and PAR1UVA B F 5 1.087, df57, P . 0.5. A lack of replication prevented testing for between-factor interactions.
case with krill fed PAR1UVA B-treated algae, concentrations of mycosporine-glycine remained consistent throughout the experiment.
A three-way ANOVA showed that all three factors day of analysis, MAA identity and treatment of algae had significant effects on the dry mass concentration of individual
MAAs in whole krill, and that there are statistically significant interactions between all combinations of factors. Possible explanations for the observed interactions appear in the
ANOVA table Table 3.
A series of two-way ANOVAs for individual MAAs examined the effect of time and diet on MAA concentration independent of MAA identity Table 4. Only con-
S .J. Newman et al. J. Exp. Mar. Biol. Ecol. 255 2000 93 –110
101
Fig. 3. Euphausia superba. Representative chromatogram of krill using a detection wavelength of 313 nm fed UV-treated algae. Identity of peaks: 1, salt; 2, mycosporine-glycine; 3, unknown substance not an MAA,
l , 280 nm; 4, shinorine; 5, porphyra-334; 6, mycosporine-glycine:valine; 7, palythine; 8, asterina-330.
max
Table 3 Euphausia superba. Three-way ANOVA examining the effect of time day, MAA identity and diet starved,
PAR-only, PAR1UVA B on MAA concentration of krill Factor
df F
P Comments
MAA identity 5
253.9 ,0.0001
Relative concentrations of different MAAs produced
by algae vary markedly Time day
2 37.7
,0.0001 Due to accumulation of
MAAs in krill tissues Diet
2 4.7
0.0105 Weakly significant due to
lower-than-expected difference between total MAAs in
PAR6UVA B treatments
Interactions probably due to: MAATime
10 7.9
,0.0001 Accumulation of different
MAAs in krill tissues MAADiet
10 14.2
,0.0001 Different MAAs induced by
different light treatments TimeDiet
4 9.8
,0.0001 Effect of UVA B on total
concentration of MAAs produced MAATimeDiet
20 6.7
,0.0001 Product of three highly significant
two-way interactions Residual
143
102 S
.J. Newman et al. J. Exp. Mar. Biol. Ecol. 255 2000 93 –110 Table 4
Euphausia superba. Two-way ANOVA of concentration of individual MAAs in krill with time and diet as a factor. Bold P-values denote statistical significance P ,0.05. Concentrations of mycosporine-glycine:valine,
porphyra-334 and shinorine in krill vary with time accumulation and diet effect of growth conditions. Palythine concentration varies only with time. Significant interactions between time and diet for mycosporine-
glycine:valine and porphyra-334 are most likely due to increasing availability of MAAs to krill as feeding progressed
MAA Factor
df F
P Mycosporine-
Time 2
21.94
,0.0001
glycine:valine Diet
2 23.56
,0.0001
TimeDiet 4
15.03
,0.0001
Residual 24
Porphyra-334 Time
2 13.62
0.0001
Diet 2
12.35 0.0002
TimeDiet 4
5.03 0.0043
Residual 24
Shinorine Time
2 5.42
0.0114
Diet 2
5.78 0.0089
TimeDiet 4
2.14 0.1065
Residual 24
Mycosporine- Time
2 2.43
0.1091 glycine
Diet 2
0.12 0.8876
TimeDiet 4
0.60 0.6663
Residual 24
Palythine Time
2 4.47
0.0229
Diet 2
2.39 0.1140
TimeDiet 4
0.95 0.4547
Residual 24
Asterina-330 Time
2 1.06
0.3596 Diet
2 1.09
0.3515 TimeDiet
4 1.64
0.1949 Residual
24
centrations of mycosporine-glycine:valine and porphyra-334 showed a statistically significant effect of time and diet, and an interaction between the two factors. Shinorine
showed a weaker effect of time and diet, and a non-significant interaction between the two. Asterina-330, palythine and mycosporine-glycine did not vary according to time
and diet, although palythine did vary significantly over time Table 4.
The two- and three-way between-factor interactions found in the three-way ANOVA of MAA concentration are listed in Table 3. Most can be explained in terms of
differential MAA availability and MAA accumulation into tissues. Due to the observed strong interaction between factors, post-hoc tests are unlikely to be meaningful Zar,
1996. However, the data show a clear accumulation of mycosporine-glycine:valine over time in krill fed PAR1UVA B algae Fig. 4. Tukey honestly significant difference
HSD tests showed significant differences in mean mycosporine-glycine:valine con- centration between krill fed ‘PAR1UV B’ algae and other diets for days 35 and 63
S .J. Newman et al. J. Exp. Mar. Biol. Ecol. 255 2000 93 –110
103
Fig. 4. Euphausia superba. Effect of 63 days of feeding on concentration of major MAAs shinorine, porphyra-334, mycosporine-glycine:valine, mycosporine-glycine in krill fed on a diet of P
. antarctica grown under varying light conditions. Bars represent one standard error n 5 4. See Tables 3 and 4 for results of two-
and three-way ANOVA.
P , 0.001. Accumulation of porphyra-334 in krill fed PAR-only algae is also evident, although it is accompanied by an unexpected drop in concentration of porphyra in krill
fed PAR1UVA B algae Fig. 4. Tukey HSD tests showed a significant difference in mean porphyra-334 concentration between krill fed PAR-only algae and other treatments
on day 63 P , 0.001.
Although starvation appears to lead to a drop in MAA concentrations of fed krill for most MAAs except palythine, Table 5, some of these trends are not statistically
significant, according to Student’s t-test Table 5. Previously starved krill retained their background concentrations of all MAAs except asterina-330, which increased in
concentration Table 5. Krill fed PAR1UVA B algae lost significant amounts of mycosporine-glycine:valine, porphyra-334 and shinorine, and a weakly significant
amount of mycosporine-glycine P 5 0.048, Table 5. Additionally, krill fed PAR-only algae lost a weakly significant amount of porphyra-334 P 5 0.0380, Table 5
104 S
.J. Newman et al. J. Exp. Mar. Biol. Ecol. 255 2000 93 –110 Table 5
Euphausia superba. MAA concentrations in krill tissue after the final feeding 63 days and following 35 days
21
of starvation 98 days. Data in nmol g approx. dry wt. Figures in parentheses are S.D. Student’s t-test
performed on individual MAA concentrations from days 63 and 98. Bold P-values indicate statistical significance P ,0.05
MAA Treatment
Starved Fed PAR-only algae
Fed PAR1UVA B
algae 63
98 63
98 63
98 Mycosporine-
2.25 1.68
46.50 32.80
379.54 113.13
glycine:valine 1.9
0.68 7.63
4.25 76.54
16.00 df510, t 5 0.448
df58, t 5 1.568 df56, t 5 3.406
P , 0.332 P , 0.078
P , 0.007
Porphyra-334 273.12
268.91 446.77
356.42 295.69
174.92 28.84
25.07 22.53
38.16 27.80
14.21 df510, t 5 0.109
df58, t 5 2.038 df56, t 5 3.868
P , 0.457 P , 0.038
P , 0.004
Shinorine 135.25
129.05 187.09
156.60 129.20
87.75 16.50
15.61 17.89
13.73 11.13
8.27 df510, t 5 0.268
df58, t 5 1.352 df56, t 5 2.989
P , 0.397 P , 0.107
P , 0.012
Mycosporine- 143.07
128.24 133.91
129.44 164.54
106.18 glycine
24.34 27.78
34.06 27.26
20.44 21.27
df510, t 5 0.381 df58, t 5 0.102
df56, t 5 1.978 P , 0.356
P , 0.460 P , 0.048
Palythine 0.78
1.78 0.93
1.74 1.26
2.30 0.37
0.91 0.92
0.25 1.26
0.81 df510, t 5 0.636
df58, t 5 1.887 df56, t 5 1.579
P , 0.270 P , 0.048
P , 0.082 Asterina-330
2.69 0.93
1.98 0.68
2.11 1.19
0.49 0.26
0.55 0.43
0.54 0.44
df510, t 5 3.434 df58, t 5 1.842
df56, t 5 1.302
P , 0.003 P , 0.051
P , 0.120
4. Discussion
