2.5. Net-seeding and nursery stageN

2.5. Net-seeding and nursery stage
Nori nets (20 cm2 mesh size, 1.7 m×1.7 m) were
obtained from Dr. P. He, at Shanghai Fisheries
University, Shanghai, China. The nets were made from
one line with three twisted components: a polyethylene
thread for spore attachment, and two polypropylene
threads to increase strength. In preparation for seeding,
the nets were washed three times in commercial washing
machines in warm water without detergent and air-dried.
This was done to remove any harmful residues left over
from the manufacturing process.
P. umbilicalis for net-seeding was collected from the
open shore at Dark Harbor, Grand Manan Island, New
Brunswick, Canada (44.75°N 66.85°W), on July 11,
2005 (voucher deposited at the University of Maine
Herbarium #DH071105 1–6) and kept at 4 °C in the
dark for 36 h before use. We used material from Grand
Manan because viable spores could not be obtained at
our Schoodic Point field sites in summer (see below).
Wild material with a well-defined deep red margin was
selected for seeding, because such a margin was
indicative of maturing neutral sporangia. We screened
1.0 kg of damp whole thalli to remove grazers by
rubbing the P. umbilicalis against a plastic screen with a
1 cm2 sized mesh and then air-dried the material in the
shade for 1 h, which killed remaining amphipods. These
“semi-dried” (∼3 times dry weight) P. umbilicalis
blades were placed in a plastic tub with 20 L of
20 μm-filtered seawater (SW) for 1 h at 16 °C. After 1 h,
the P. umbilicalis thalli were removed from the spore
suspension using plastic screen. The number of spores
was quantified using a special counting chamber for
spore enumeration (IOCAS, (1978). This chamber
consisted of a standard microscope slide with a piece of
2 mm thick white Plexiglas glued to its surface. A 0.4 mL
well was created by drilling a 16 mm diameter hole in the
plastic. The number of spores mL−1 was determined by
the average of 3 chambers, counting five randomly
selected fields (100×) per chamber and using a multiplier
for the chamber to determine the number of spores mL−1.
Following spore enumeration, thirty 1.7×1.7 m nets were
seeded with 1.8×109 neutral spores (see below). Spore
density and attachment were determined by observing
separate pieces of nori net that was included in the seeding
process.
The cleaned and dried nets were individually folded
like an accordion and placed side-by-side in a