Eads_meps11944_all data.xlsx



Gametes of marine broadcast spawners are highly susceptible to the threats of ocean warming and acidification. Here, we explore the main and interacting effects of temperature andpH changes on sperm motility and fertilization rates in the mussel Mytilus galloprovincialis. Additionally,we determine how temperature and pH interact to influence the motility of aging sperm.
We show that the interactive effects of temperature (18°C or 24°C) and pH (ranging from 7.6 to 8.0) on sperm motility depend on the time that sperm spend in these conditions. Specifically, sperm linearity was influenced by a temperature × pH interaction when measured after a relatively short exposure to the treatment conditions, while main effects of temperature and pH (but no inter -action) on sperm motility became apparent only after prolonged exposure (2 h) to the treatments.Despite the interactive effects of temperature and pH on sperm motility, these factors had independent effects on fertilization rates, which were significantly higher at the ambient ocean pH level and at the elevated temperature. This study highlights the importance of considering the combined effects of predicted ocean changes on sperm motility and fertilization rates, and cautions against using only sperm motility as a proxy for reproductive fitness. Detrimental effects of pH and temperature may only be uncovered when these factors are examined together, or conversely, negative impacts of one variable may be buffered by changes in another. Our results raise the intriguing possibility that some species may cope better with ocean acidification if they simultaneously experience ocean warming.

Mussels were collected by hand from a pontoon at
Woodman Point, 30 km south of Perth, Western Australia,
over multiple trips from July to September during
2012 to 2014 (permit no. 2141, Department of
Transport, Government of Western Australia). Experimental
replicates were taken over multiple seasons
due to poor spawning, and year was included in the
analyses to account for any seasonal variation (see
‘Statistical analyses’). Winter water conditions at the
collection site averaged 19.8°C and pH 8.1 over the
experimental seasons (Reynolds et al. 2007; https://
imos.aodn.org.au/imos123). Mussels were kept in
aerated aquaria of recirculating bio logically filtered
seawater (FSW) at the University of Western Australia
until required (within 3 wk of collection).

Seawater preparation
Experimental water temperatures were maintained
by placing containers in water baths in a temperature-
controlled room. A total of 4 water baths, compris -
ing 2 replicates of each experimental temperature,
were used in the experiment. Holding containers
with FSW were placed in each water bath and, after
water temperatures were stable, experimental pH
levels were set by bubbling CO2 through the FSW.
By using CO2 to alter seawater pH, the partial pressure
of CO2 (pCO2) is raised while simultaneously
lowering the pH and carbonate ion concentrations.
(Lowering the pH of water by adding an acid such as
HCl maintains the pCO2—unrealistic conditions in
which to investigate the impacts of anthropogenic
climate change—and prior research has shown
altered outcomes on fertilization rates using this
method; Sung et al. 2014).
Experimental water temperatures were 18°C and
24°C, while pH levels were set at 7.6, 7.8, and the
local ambient pH (~8.0). These values represent
current and predicted average winter conditions for
the local area in the coming century and are therefore
likely to be realistic for the focal population, particularly
considering increases in marine heatwave
occurrence and length along with more extreme
peaks in environmental fluctuations (IPCC 2013,
Pearce & Feng 2013). Water parameters (pH, tem -
perature, dissolved oxygen, and salinity) were measured
before and after conducting each experimental
‘block’ (= male) using a pH meter (TPS WP-81;
TPS Pty Ltd) calibrated with TPS buffers, and pCO2
ranges were calculated using CO2SYS software
(Pierrot et al. 2006; Table S1 in the Supplement at
www.int-res.com/ articles/ suppl/ m562 p101_ supp. pdf).
For each ‘block’, we placed 3 × 150 ml glass jars, each
containing 30 ml of water set at one of the 3 pH levels
(~8.0, 7.8, or 7.6), in each water bath (18°C or 24°C).
Spawning and gamete collection
Mussels were induced to spawn using a temperature
shock by moving them from their holding tanks
(at ~17°C) to a large tray preheated to ~26°C using an
aquarium heater (SONPAR automatic, 200W) (Galley
et al. 2010). Females that began spawning were
rinsed in FSW, placed in a glass jar containing 30 ml
of ambient FSW, and left for approximately 1 h to
spawn. When a male commenced spawning, it was
removed from the tray, rinsed in FSW, and wrapped
in a wet paper towel to halt spawning until enough
eggs were collected (see below). When required,
each male was placed in a glass spawning jar containing
30 ml of ambient FSW and left to spawn for
approximately 10 min until sperm were sufficiently
concentrated (as judged initially by eye) for the
sperm motility and fertilization assays (see below).
Sperm density in the spawning jar was quantified
using an improved Neubauer haemocytometer (Hirsch -
mann Laborgeräte). We then extracted a known
quantity of sperm from the spawning jars to make up
concentrations of 5 × 106 sperm per ml in each treatment
jar, a concentration appropriate for the sperm
motility analysis and fertilization trials below (see
Characterizing sperm motility
Sperm motility of a subset of males (n = 14) was
assessed using computer-assisted sperm analysis
(CASA; CEROS sperm tracker, Hamilton-Thorne
Research), 20 min after sperm were added to the
treatment water, and then again 2 h post-addition to
treatment conditions (hereafter referred to as Time 1
and Time 2, respectively). For each sample, 1.5 μl of
sperm was pipetted into 2 separate wells of a 12-well
multitest slide (MP Biomedicals) and covered with a
coverslip. We used a phase-contrast Olympus CX41
microscope (×10 objective) and captured 30 frames at
50 f s−1. We defined static cells below the threshold
values of 19.9 μm s−1 for smoothed average path
velocity (VAP) and 4 μm s−1 for straight-line velocity
(VSL) (see ‘Statistical analyses’ for details about the
CASA parameters), minimum cell size as 2 pixels,
and measured an average of 193 ± 8 SE sperm tracks
per sample. The slides were coated with 1% poly -
vinyl alcohol (Sigma-Aldrich) to avoid sperm sticking
to the glass (Wilson-Leedy & Ingermann 2007). We
randomized the order in which sperm motility was
analyzed by treatment among males.
Fertilization trials
In each water bath (18°C or 24°C), 3 × 50 ml plastic
tubes were floated in a polystyrene frame, each containing
10 ml of water set at one of the 3 pH levels
(~8.0, 7.8, or 7.6). Eggs from all females spawned on
a given day were pooled (range: 2−6 females) to provide
a ‘homogenous’ genetic background for the
fertilization trials (Fitzpatrick et al. 2012), thus reducing
variance in fertilization rates attributable to specific
male-by-female interactions (i.e. compatibility),
which are known to occur in this system (Evans et al.
2012, Oliver & Evans 2014). We estimated egg density
from a 5 μl sub-sample of pooled eggs, then
added eggs to each aforementioned treatment tube
at a density of 15 000 per ml.
After sperm and eggs had been separately exposed
to the treatments for 10 min, an aliquot of sperm from
each treatment jar was added to the eggs in the treatment
tubes equilibrated under the same conditions,
at a ratio of 20:1 (sperm density: 300 000 per ml) to
give moderate fertilization rates while avoiding ceiling
or basement effects (Fitzpatrick et al. 2012), and
gently swirled to homogenize the samples. Fertilization
was halted after 1 h by adding 1% formalin to
each tube. Fertilization rates were assessed under a
microscope as the percentage of eggs showing signs
of cleavage and/or with polar body formation among
approximately 100 haphazardly chosen eggs per rep -
licate (Longo & Anderson 1969). For logistical reasons,
fertilization rates were estimated at one time
point only (n = 32 males), and sperm motility assays
were only undertaken on a subset of the same individual
males in the fertilization assays.
Date made available6 Jul 2017

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