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
TLC showed no unconjugated FITC in the purified FD samples, either before or after uptake by the algae.
W .L. Zemke-White et al. J. Exp. Mar. Biol. Ecol. 245 2000 57 –68
61
3.1. Plasmolysis experiments All algal species were plasmolysed by treating them with mannitol 0.75 M in
seawater. The FD equilibrated between the plasmolysing solution and either the cell wall, or the aqueous volume between the plasma membrane and the cell wall. The 10K
FD was taken up within 10 min in all four of the algae tested, as indicated by the green fluorescence. This is shown in Fig. 1b, e, h and f, for Enteromorpha intestinalis, Ulva
rigida, Porphyra sp., and Polysiphonia strictissima, respectively. In P
. strictissima, the 10K FD was taken up into the spaces between the cell wall and the plasma membrane
created by the plasmolysis of the algal cells. Although a clear space between plasma membrane and cell wall was not apparent in E
. intestinalis, U. rigida or Porphyra sp, the 10K FD was evident throughout the cell wall of these algae. The 20K FD was taken up
by E . intestinalis, U. rigida and P. strictissima, although it took 30 min for significant
amounts to be found either within the space between the cell wall and the plasma membrane in P
. strictissima or within the cell walls of E. intestinalis and U. rigida. All larger FDs were excluded from these three algal species even after 2 h incubation. The
20K FD was not able to pass through the cell walls of the Porphyra sp. tested even after 2 h incubation in the plasmolysing solution.
3.2. pH treatments Every low pH treatment had the effect of increasing the cell-wall porosity in all the
algae in this study. After incubation in pH 2.0 for 60 min all four algal species took up the 250K FD within 5 min Fig. 1c, f, i and l. For each species of alga, there was a
gradient of effect on the cell-wall porosity depending on both the pH and treatment time Table 1. An examination of all of the low pH treatments showed that, among the algal
species, there was also a gradient of resistance to the effects on the cell-wall porosity due to low pH treatment. The rhodophytes were more resistant that the chlorophytes.
Within the rhodophytes, the subtidal Porphyra sp. was more resistant than the intertidal Polysiphonia strictissima. Within the chlorophytes, the subtidal Enteromorpha intes-
tinalis was more resistant than the inter-tidal Ulva rigida.
The higher resistance of Porphyra sp. was apparent in all pH treatments except for 60 min at pH 2.0. After 20 min at pH 2.0, Enteromorpha intestinalis, Ulva rigida and
Polysiphonia strictissima took up the 250K FD in under 5 min, but 60 min was required for Porphyra sp. to take up this FD. After 20 min at pH 2.5, a difference became
apparent between P . strictissima and the two chlorophytes: P. strictissima took up the
250K FD in under 60 min, but the two chlorophytes took this FD up in less than 30 min. After 20 min at pH 3.0, the difference between E
. intestinalis and U. rigida became apparent: 60 was min required for the former to take up significant amounts of the 250K
FD, but the latter required 30 min to take up this FD.
4. Discussion
