GAMETE TRAITS AND MEASURES OF ADULT BODY CONDITION
Female egg size was assessed from three replicate counts using a Beckman
multisizer™ 3 Coulter counter (mean number of eggs per female = 8,371 ±
1140 SE). To estimate sperm size, digital photographs of sperm were
taken using a digital camera attached to a microscope (OLYMPUS IX53, 20X
objective). Sperm length was measured for each male using the software
ImageJ (http://rsb.info.nih.gov/ij). Sperm length was measured in μm as
the distance from the tip of the head (excluding the protruding
acrosomal cap or ‘beak’) to the end of the tail. Ten sperm were measured
for each male, and the average of the 10 measures was used in the
analysis.
Sperm motility was assessed using computer-assisted sperm analysis
(CEROS sperm tracker, Hamilton-Thorne, Beverley USA). Sperm motility was
assessed at two sperm concentrations (106 and
108). For each individual, two 4 μl aliquots of the
sperm solution were placed in two separate wells of a 12-well Multi-test
slide and then covered with a coverslip. Sperm motility was therefore
assessed in two subsamples for each male at both sperm concentrations.
Given the high reported repeatability of sperm motility measures in blue
mussels (Fitzpatrick et al. 2012), we recorded the average of the two
subsamples for the ensuing analysis, and caution was taken to analyse
sperm in a random order with respect to the sperm concentration used.
From the CASA analyses, we obtained the percentage of motile sperm and a
series of parameters describing velocity and trajectory of motile sperm.
These parameters include average path velocity (VAP), straight-line
velocity (VSL), curvilinear velocity (VCL), lateral head displacement
(ALH), beat cross frequency (BCF), straightness (STR) and linearity
(LIN). Cut offs for static cells were the same as those used in previous
experiments by Eads et al. (2016). An average of 276.8 ± 37.0 SE sperm
tracks were analysed for each male at each concentration.
As body condition may influence
fertilisation success (i.e. mussels in better condition may have higher
quality gametes) we controlled for this potential confounding effect in
our analysis. We recorded shell length (mm) and flesh mass (g) from each
brood parent and used these to calculate a condition index using the
residuals of flesh weight against shell length.