Methods and materials Directory UMM :Data Elmu:jurnal:J-a:Journal of Experimental Marine Biology and Ecology:Vol245.Issue1.MAr2000:

W .L. Zemke-White et al. J. Exp. Mar. Biol. Ecol. 245 2000 57 –68 59 on the porosity of algal cell walls. We examined four marine macroalgae: the subtidal Ulva rigida Adams Chlorophyta: Ulvaceae and Polysiphonia strictissima C. Agardh Rhodophyta: Rhodomelaceae, and the intertidal Enteromorpha intestinalis Chloro- phyta: Ulvaceae and Porphyra sp. Tag no. PYGRosP13 5 98, Museum of New Zealand, Te Papa this genus is currently under taxomonic revision, pers. com., Glenys Knight, Museum of New Zealand, Te Papa, New Zealand. We determined the ability of the algal cells to take up fluorescein isothiocyanate FITC conjugated to dextrans of different molecular size to: 1 determine the porosity of algal cell walls in their native state, and 2 determine any changes in that porosity after immersion in seawater with a lowered pH.

2. Methods and materials

2.1. FITC dextran FD preparations 3 FDs with the molecular sizes 10K K 5 10 M , 20K, 40K, 70K, 150K and 250K r were obtained from Sigma St. Louis, MO, USA. As some commercial preparations of FD have been shown to contain unconjugated FITC Cole et al., 1990, all FDs used in this study were purified by gel chromatography, using the method of Cole et al. 1990. 21 FD 50 mg ml in water was loaded onto Sephadex G-25-150 Sigma packed in a prewashed 25 3 1.5 cm glass column. The FD was allowed to settle onto the column and 21 was then eluted using water at a flow-rate of 100 ml min . The FD was collected as the first fluorescent band and freeze–dried. All of these procedures were carried out at 58C in low light. For the uptake experiments, each purified FD was dissolved 10 mg 21 ml in seawater FD solution that had been filtered through a 0.2-mm pore size filter. We used thin-layer chromatography TLC to test for unconjugated FITC in both the FD preparations and in the algae from the uptake experiments. The purified FD and the supernatant from homogenised algae was chromatographed on Silica gel TLC plates F-1500, Schleicher and Schuell, Germany using chloroform–ethanol 3:1, v v as the solvent. Pure FITC Sigma was used as a standard in all experiments. The plates were allowed to dry and viewed in UV radiation. 2.2. Algal collection The algae were collected from South Piha Beach 368589S, 1748289E, near Auckland, New Zealand. They were kept in a 20-l aquarium under natural light conditions, and the seawater changed daily for the duration of the experiments six days maximum. All four algae used in this study are commonly found in the diet of New Zealand’s marine herbivorous fishes. This was determined by examining the gut contents of four species of herbivorous fish from New Zealand: Girella tricuspidata Quoy and Gaimard, 1824 Girellidae n 5 24; Parma alboscapularis Allen and Hoese, 1975 Pomacen- 60 W .L. Zemke-White et al. J. Exp. Mar. Biol. Ecol. 245 2000 57 –68 tridae n 5 19; Aplodactylus arctidens Richardson, 1839 Aplodactylidae n 5 28; ¨ and Kyphosus sydneyanus Gunther, 1886 Kyphosidae n 5 30. These fish were caught between August 1996 and August 1999 from various locations in the Hauraki Gulf, North-eastern New Zealand. These results were supplemented by data from a seasonal diet analysis of P . alboscapularis McMurtry, 1999, and from Vial 1997, who examined the gut contents of 43 K . sydneyanus. 2.3. Uptake experiments To determine the porosity of the cell walls of live algae, samples of the algae were placed in a plasmolysing solution seawater containing mannitol, 0.75 M final con- centration containing the FD. Mannitol has been used in similar experiments on angiosperms Baron-Epel et al., 1988, and preliminary experiments indicated that a 0.75 M solution in seawater was required to plasmolyse the algae in this study. The algae were left in these solutions for 10–120 min. At the end of the allotted time the algal samples were removed and washed three times in filtered seawater containing mannitol 0.75 M. The samples were then mounted on slides and the coverslips sealed to the slide with nail varnish. To test the effects of pH on the cell-wall porosity, algal samples were placed for 20 or 60 min in seawater filtered through a 0.2-mm pore size filter and adjusted to pH 2.0, 2.5 or 3.0 with HCl. These times of incubation were chosen as we wanted our findings to be comparable to previous work on acid lysis which used these incubation times Lobel, 1981; Zemke-White et al., 1999. The algae were then removed and washed three times before being placed into the FD solution. After incubation for 5–120 min the algae were removed, rinsed, and mounted on slides as above. Controls were run for all of the FDs, where the algae were placed in each of the FD solutions without pH treatment. All treatments in the uptake experiments were replicated twice with different algal specimens. 2.4. Microscopy The mounted samples were viewed with a Leica TCS 4D confocal laser scanning microscope system CLSM. The fluorescence of the FD was collected using a FITC filter set excitation 488 nm, emission approx. 525–545 nm. The chloroplasts within the algae autofluoresced strongly and this fluorescence was collected with a rhodamine filter set excitation 568 nm, emission . 590 nm. The FITC and rhodomine images from each optical section were then false coloured and combined using Adobe Photoshop 3.0 Microsoft.

3. Results