U . Karsten, J.A. West J. Exp. Mar. Biol. Ecol. 254 2000 221 –234
223
include damage to molecular targets such as DNA, RNA and proteins and inhibition of physiological processes such as photosynthesis and growth Britt, 1995; Buma et al.,
¨ 1995; Franklin and Forster, 1997; Hader and Figueroa, 1997; Aguilera et al., 1999. An
important protective mechanism that allows macroalgae living in high-light habitats to survive and reproduce involves the biosynthesis and accumulation of UV sunscreens.
The most common substances with a potential role as UV-sunscreens in marine organisms are the mycosporine-like amino acids MAAs, a suite of chemically closely
related, water-soluble compounds. MAAs typically absorb between 310 and 360 nm Karentz et al., 1991, their function as intracellular screening agents has been inferred,
for example, from the partial prevention of UV-induced inhibition of photosynthesis Neale et al., 1998.
Because there is a lack of detailed ecophysiological studies of seasonally changing environmental factors on the quantitative concentrations of heterosides and MAAs in
Bangia atropurpurea the present investigation was undertaken. Samples of this species were collected over the course of the growth season from the same population in the
field at Williamstown, Melbourne Australia.
2. Materials and methods
2.1. Plant material The marine red alga Bangia atropurpurea Roth C. Agardh was collected from the
upper eulittoral zone of a rock platform at Williamstown Port Philips Bay, Melbourne, Australia Fig. 1. This species was regularly collected over the growth season of the
gametophytic-bladed stage between November 1995 and May 1996. Samples were taken at low tide from the intertidal zone at intervals of 2–4 weeks. In the field algal samples
were stored on ice, air-dried or oven-dried at 408C overnight in the laboratory, air-mailed to the Alfred-Wegener-Institute and chemically analyzed. This treatment did
not affect the heteroside or MAA concentrations, and such samples could be stored for many months without any degradation when kept under dry, cool and dark conditions.
2.2. Environmental data At all sampling dates salinity was determined using a hand refractometer. Data on air-
and surface water temperature and rainfall were collected from the bureau of meteorolo- gy, Melbourne. Data on UV radiation total UV [290–400 nm], UVB [290–315 nm]
were kindly provided by Dr. H.P. Gies Australian Radiation Laboratory, Melbourne, Australia, and sun rise and sun set times for the calculation of daylengths were received
from Melbourne Observatory.
13
2.3. C-NMR measurements
13
For C-NMR nuclear magnetic resonance measurements 100–250 mg of algal dry
weight DW was usually extracted in 5 ml of 70 v v ethanol for 3 h in a waterbath
224 U
. Karsten, J.A. West J. Exp. Mar. Biol. Ecol. 254 2000 221 –234
Fig. 1. Map showing location of Williamstown, Port Phillip Bay, Victoria. Inset with arrow shows location in Australia.
at 708C. After centrifugation at 5000 g, the supernatant was evaporated to dryness under
13
vacuum and redissolved in 0.5 ml of D O 99.98 for NMR spectroscopy. C NMR
2
spectra were recorded on a Bruker AM-500 spectrometer at 125.77 MHz. Typically, a sweep width of 30 000 Hz, 16 000 time domain points and a 608 pulse of 3.5 ms were
used for acquisition, with composite pulse decoupling. The free induction decay was zero-filled to 32 000 data points and processed with a line broadening of 1.5 Hz.
Samples were contained in spinning 5 mm tubes and spectra were run at 278C and referenced from added dioxane 67.4 ppm. Floridoside,
D
-isofloridoside and
L
-iso- floridoside were verified by comparing with standards isolated from red algae Karsten
et al., 1993. 2.4. Heteroside quantification
Floridoside,
D
-isofloridoside and
L
-isofloridoside were quantified by GLC. For these analyses, the algal samples 10–15 mg DW were extracted in 1 ml of 70 v v
ethanol for about 3 h in a waterbath at 708C. These were then centrifuged for 5 min at
21
5000 g. An internal standard 10 ml of myo-inositol 1 mg ml was added to 50–100
U . Karsten, J.A. West J. Exp. Mar. Biol. Ecol. 254 2000 221 –234
225
ml of the sample supernatant, or to a standard of known concentration, and evaporated to dryness under a stream of dry air. The residue was redissolved in 150 ml of pyridine and
75 ml of acetic anhydride and acetylated by heating for at least 1 h at 708C prior to GLC analysis. This derivatization protocol did not, however, separate floridoside and
L
- isofloridoside. In addition, therefore, 50–100 ml aliquots of the extracts and standards
were silylated after evaporation to dryness. The residue was redissolved in 180 ml pyridine and 20 ml of N-trimethylsilylimidazole TMSI; Pierce Chemical Co., shaken
for 30 s and left overnight prior to GLC analysis.
GLC was performed using a Hewlett-Packard 5890A GLC equipped with a flame- ionization detector. Data were analysed and processed using a Hewlett-Packard GC
ChemStation. For the acetylated samples a fused-silica column BPX70, 12 m, 0.32 mm I.D. was chosen, with hydrogen as the carrier gas at a head pressure of 0.068 kPa using
the following temperature program: the oven temperature was kept for 0.1 min at 1008C
21
following manual injection 1 ml and then raised at 358C min to 1708C, where it was
21
left for 1 min, then again raised at 208C min to 2508C, and left for another minute at
this temperature. The injection port and the detector were heated to 2608C. A fused- silica capillary column BP5, 25 m, 0.32 mm I.D. was used for the silylated samples,
with hydrogen as the carrier gas at a head pressure of 0.034 kPa. Floridoside was separated from
L
-isofloridoside with a temperature program: the oven temperature was
21
kept for 0.1 min at 1008C following injection 1 ml and then raised at 408C min to
2608C, where it was left for 2 min. The injection port and the detector were heated to 3008C.
2.5. MAA quantification Thalli of about 10–20 mg dry weight DW were extracted for 1.5–2 h in screw-
capped centrifuge vials filled with 1 ml 25 aqueous methanol v v and incubated in a waterbath at 458C. After centrifugation at 5000 g for 5 min, 800 ml of the supernatants
were evaporated to dryness under vacuum Speed Vac Concentrator SVC 100 H. Dried extracts were re-dissolved in 800 ml 100 methanol and vortexed for 30 s. After passing
through a 0.2 mm membrane filter, samples were analysed with a Waters HPLC system according to the method of Karsten and Garcia-Pichel 1996, modified as follows.
MAAs were separated on a stainless-steel Knauer Spherisorb RP-8 column 5 mm, 250 3 4 mm I.D. protected with a RP-8 guard cartridge 20 3 4 mm I.D.. The mobile
phase was 5 aqueous methanol v v plus 0.1 acetic acid v v in water, run
21
isocratically at a flow-rate of 0.7 ml min . MAAs were detected at 330 nm and
absorption spectra 290–400 nm were recorded each second directly on the HPLC- separated peaks. Identification was done by spectra, retention time and by co-chromatog-
raphy with standards extracted from the marine red macroalgae Chondrus crispus ¨
Stackhouse Karsten et al., 1998b and Porphyra umbilicalis Linnaeus Kutzing, which were kindly provided by Dr. L.A. Franklin, Biologische Anstalt Helgoland, Germany, as
well as from ocular lenses of the coral trout Plectropomus leopardus, kindly sent by Dr. David Bellwood, James Cook University, Townsville, Australia. Quantification was
made using the molar extinction coefficients given by Karsten et al. 1998c.
226 U
. Karsten, J.A. West J. Exp. Mar. Biol. Ecol. 254 2000 221 –234
2.6. Statistical treatment
21
The concentrations of the different heterosides are expressed as mmol kg dry
21
weight, the MAA contents are given as mg g dry weight. All values represent the
mean6S.D. of 4 replicate measurements, i.e. of 4 separate thalli collected at the same field site from the same tidal height. Seasonal variation in mean values of total
heteroside and total MAA concentrations were statistically tested using one-way analysis of variance ANOVA; Sokal and Rohlfs, 1981 followed by Tukey-Kramer multiple
comparison test InStat, GraphPad, San Diego, CA, USA.
3. Results
