Influence on zoospore attachment
A slide glass with various sub-micro particles was deposited in a container (outer diameter 61.8 mm, height 125.2 mm) filled with seawater. Zoospores were poured from the surface of the water, and the number of zoospores that had attached to the slide glass was counted. The effect of the particles on attachment was investigated. Here, particles A, B and C were used (silicon carbide–SiC–particles with different size distributions) as the sediment particles. Particles A and B had one peak in the size distribution, and average particle sizes of 1.1 µm and 3.9 µm, respectively. Particle C had two peaks at 0.090 µm and 4.6 µm, and the average particle size was 1.5 µm (Supplementary Fig. S1 online).
When about 5 × 104 of E. bicyclis zoospores were placed in the container, after 12 h an average attachment of 13.5 ind./mm2 was observed on the slide glass without sediment particles. The relationship between the attachment percentage (%) of zoospores and amount of sediment particles of SiC is shown in Fig. 1a. The attachment percentage, expressed as the number of attached zoospores without sediments, was 100%.
Negative influences of sediment on zoospore attachment and gametophyte survival; (a,b) zoospore attachment percentage and gametophyte survival percentage, respectively.
In the case of particle A (mean diameter 1.1 µm), which had one peak in the size distribution, the zoospore attachment percentage (mean ± SD) at 0.05 mg/cm2 and 0.1 mg/cm2 of sediments were 25.9 ± 14.2% and 10.2 ± 6.17%, respectively (Fig. 1a, Supplementary Table S1 online). In the case of particle B (mean diameter 3.9 µm), the attachment percentage was 53.9 ± 24.8% at 0.05 mg/cm2 and 41.1 ± 23.1% at 0.1 mg/cm2. In the case of particle A, few attachments were found at sediment levels of 0.3 mg/cm2.
The attachment percentage decreased exponentially as the amount of sediment on the substrate increased at any particle size. A significant negative correlation (Spearman’s rank correlation, p < 0.05, rs = − 1 and − 1 for particles A and B, respectively) was observed between the amount of sediment and the attachment percentage for each particle size. For each particle, the approximate expression by the nonlinear least squares method and the pseudo-determination coefficient were Eqs. (1) and (2):
$$ {text{Particle A }}left( {{text{mean diameter 1.1}},upmu {text{m}}} right);A_{r} { = 100};{exp}left( – 23.7,Q right),quad left( {r^{2} = 0.{988}} right) $$
(1)
$$ {text{Particle B }}left( {{text{mean diameter 3}}.{9},upmu {text{m}}} right);A_{r} { = 100};{exp}left( { – {10}{text{.6}},Q} right),quad left( {r^{2} = 0.{988}} right) $$
(2)
where Ar is the zoospore attachment percentage (%) and Q is the amount of sediment (mg/cm2).
In the case of particle C, with two peaks in particle size distribution, the attachment percentage was 31.8 ± 25.8% at 0.05 mg/cm2 and 8.55 ± 10.9% at 0.1 mg/cm2, and it became less than 1% at 0.2 mg/cm2. The attachment percentage for particle C decreased exponentially as the amount of sediment on the substrate increased. The change in particle C was similar to that of particle A. We found a significant negative correlation (Spearman’s rank correlation, p < 0.05, rs = − 0.964) between the amount of sediment and the attachment percentage. The approximate expression of the nonlinear least squares method and pseudo-determination coefficient for the relationship between the zoospore attachment percentage and the amount of sediment in particle C is shown in Eq. (3):
$$ {text{Particle C }}left( {text{two peaks}} right);;A_{r} = 100{text{ exp}}left( { – {20}{text{.2 }}Q} right),quad left( {r^{2} = 0.962} right) $$
(3)
Influence on survival and growth of gametophytes
The slide glass on which the zoospores were attached was set in a Petri dish (diameter: 150 mm, height: 90 mm) filled with seawater. Various amounts of fine particles were deposited on the slide glass, and the influence of the sediment particles on the growth and survival of gametophytes was examined. Here, particles A, B and C, which are the same as those used in the attachment experiment, were used as the sediment particles.
The relationships between the survival percentage of the gametophytes and the amount of sediment are shown in Fig. 1b. The survival of gametophytes placed in Petri dishes with sediments is expressed as a percentage of the gametophytes surviving 12 days in Petri dishes without sediments.
When particle A (mean diameter 1.1 µm) with one peak in particle size distribution was deposited, the survival percentages (mean ± SD) of the gametophyte were 83.2 ± 12.9% at 0.1 mg/cm2 and 11.4 ± 4.04% at 1.0 mg/cm2 sediments. The survival percentage decreased remarkably as sediment amounts increased. The survival percentages for particle B (mean diameter 3.9 µm) were 73.8 ± 10.3% at 0.1 mg/cm2, and 26.4 ± 7.04% at 1.0 mg/cm2. The survival percentage decreased exponentially as the amount of sediment on the gametophyte increased. In the cases of particles A and B, there was a significant negative correlation (Spearman’s rank correlation, p < 0.05, rs = − 1 and − 1 for particles A and B, respectively) between the amount of sediment and the survival percentage. The relational expression approximated by the nonlinear least squares method and the pseudo-determination coefficient is shown in Eqs. (4) and (5):
$$ {text{Particle}};{text{A}};left( {{text{mean}};{text{diameter}}, 1.1,upmu {text{m}}} right) ;;S_{r} = 100;exp left( { – 2.06,Q} right),quad left( {r^{2} = 0.999} right) $$
(4)
$$ {text{Particle}};{text{B}};left( {{text{mean}};{text{diameter}};3.9,upmu {text{m}}} right) ;;S_{r} = 100 exp left( { – 1.49 Q} right),quad left( {r^{2} = 0.970} right) $$
(5)
where Sr is the survival percentage of gametophytes (%) and Q is the amount of sediment (mg/cm2).
In contrast, the survival percentage of gametophytes in the case of particle C sediment (mean diameter 1.5 µm) with two peaks in the particle size distribution was 41.6 ± 10.8% at 0.1 mg/cm2 and 10.2 ± 5.38% at 1.0 mg/cm2. The negative effect of particle C on survival was greater than the effect of the same amount of particles A and B. In particle C, a significant negative correlation (Spearman’s rank correlation, p < 0.05, rs = − 1) was observed between the amount of sediment and the survival percentage. The relational expression approximated between the survival percentage of the gametophyte and the amount of sediment by the nonlinear least squares method and the pseudo-determination coefficient is shown in Eq. (6):
$$ {text{Particle C }}left( {text{two peaks}} right);;S_{r} = 100 exp left( { – 6.31 Q} right),quad left( {r^{2} = 0.919} right) $$
(6)
where Sr is the survival percentage of gametophytes (%) and Q is the amount of sediment (mg/cm2).
The culture of E. bicyclis gametophytes was performed with sediment of each particle size, and the total length of the gametophytes was measured on the 6th and 12th days. The total length of gametophytes without sediments was 37.7 ± 10.5 µm on day 6, and was 94.1 ± 26.0 µm for female and 137 ± 27.4 µm for male on day 12. The total length of the gametophytes on the 12th day, when particles of all sizes had been deposited, is shown in Table 1.
If the total length on the 12th day with no sediments was 100%, the relative total length for particles A (mean diameter 1.1 µm) in 0.1 and 1.0 mg/cm2 sediment amounts were 91.7 and 66.7% for female gametophytes, and 91.6 and 69.5% for male gametophytes, respectively. When the sediment amount was 5 mg/cm2, the gametophytes did not grow to a size that enabled discrimination between male and female. With respect to particle B (mean diameter 3.9 µm), the total length at sediment volumes of 0.1 and 1.0 mg/cm2 were 82.5 and 71.7% for female gametophytes, and 88.9 and 80.7% for male gametophytes, respectively. Gametophyte growth was worse as the amount of sediment increased, and the negative effect was greater when the particle size of the sediment was small. In particle C with two peaks, the total length at sediment volumes of 0.1 and 1.0 mg/cm2 were 92.3 and 74.4% for female gametophytes, and 91.1 and 77.0% for male gametophytes, respectively. When the sediment volume was 3.0 mg/cm2, the total lengths of female and male gametophytes were 71.8% and 63.0%, respectively.
Covariance analysis (ANCOVA) was performed on the total length as a response variable. Among the explanatory variables, particle size (mean diameter) and sediment volume were treated as continuous variables, and the sex of the gametophyte was treated as a categorical variable. It was shown that the growth of gametophytes was inhibited for each volume of sediment and at each particle size (p < 0.001; Table 2). The total length of female gametophytes was significantly smaller than that of males (p < 0.001). There was an interaction between particle size and sex (p < 0.001) (Fig. S2a), and the negative effect was found to be strong in female gametophytes. On the other hand, there was no interaction between sediment amount and sex (p = 0.215) (Fig. S2b).
Effects of gaps on substrate on zoospore attachment and gametophyte survival
We examined the relationship between the space on the substrate not covered by particles (the ‘percentage gap’) and zoospore attachment percentage, and the relationship between the percentage gap and gametophyte survival percentage. The percentage gap (%) is the ratio of the gap area to the substrate area (the ratio of voids between particles).
In the case of 1.1 and 3.9 µm particles, the attachment percentage decreased exponentially with decreasing percentage gap (Fig. 2a). In both particle size ranges, when the percentage gap was 0%, the zoospore attachment percentage was almost 0%. However, in the 48.2–599 µm particle range26, the attachment percentage decreased linearly as the percentage gap decreased (Fig. 2c). This result suggested that the attachment percentage of zoospores was governed by the percentage gap on the substrate.
Relationship between percentage gap on substrate and attachment percentage of zoospore (a) or survival percentage of gametophyte (b). Data of particle size 15.0–599 µm are shown from the relational expression of Watanabe et al.26 in (c,d).
The survival percentage of gametophytes deposited on 1.1 and 3.9 µm particles tended to decrease linearly as the percentage gap decreased (Fig. 2b). In the case of the 48.2–599 µm particle range, the survival percentage indicated the same trend in decrease (Fig. 2d). This change was different from the case of the attachment percentage, and showed the same change regardless of the particle diameter. However, even when the percentage gap was 0%, the survival percentage indicated a range of 0% to ca 70%, and the survival percentage decreased as the sediment thickness increased.
Sediment amount and size distribution in kelp communities deforested by sediment particles
The large kelp communities off Mio, Wakayama Prefecture, Japan, have been deforested owing to turbidity caused by Hidakagawa River inflow in the 1990s34. We measured the amount and size distribution of seabed sediment particles off Mio, and also in nearby off Noshima where the kelp communities are maintained (Supplementary Fig. S3 online).
The amount of sediment particles in Mio and Noshima was 2.03 ± 0.643 mg/cm2 and 2.07 ± 1.95 mg/cm2, respectively, and no significant difference was observed (Student-t test; P > 0.05). Figure 3 shows the particle size distribution of sediment particles in both seas. The mode and the average particle size of distribution in Mio were 79.3 µm and 39.1 µm, respectively. In contrast, the mode and average particle size in Noshima were 420 µm and 185 µm, respectively. Assuming that the sub-micro size is less than 10 µm, particles of that size were found at a level of 10.1% in Mio compared to 3.7% in Noshima.
Particle size distribution of seabed sediments in deforested sea area.
Source: Ecology - nature.com
