COLLECTION OF GAMETES
Gametes were harvested by inducing spawning using standard thermal
stress protocols (Pettersen et al. 2010). This involved cycling seawater
temperature between 16-24 °C to induce spawning. Males and females were
identified at the time of gamete release, rinsed with filtered seawater,
and isolated into individual spawning chambers (120 × 175 × 70 mm). Eggs
were rinsed through a 125μm mesh, and sperm through a 30μm mesh, to
remove any debris released from the adult mussel during spawning. The
concentration of sperm for each male was determined from three replicate
counts using an improved Neubauer haemocytometer and sperm
concentrations were initially standardized to 2 × 108sperm ml-1. A serial sperm dilution was then carried
out to obtain the desired experimental concentrations of sperm for our
experiments: 2 × 102; 2 × 103; 2 ×
104; 2 × 105; 2 ×
106; and 2 × 108 sperm
ml-1. Egg concentrations were estimated from three
replicate counts using a Beckman multisizer™ 3 Coulter counter and
standardized to 2000 eggs ml-1 (stock egg solution).