DOI: 10.6084/m9.figshare.5177083.v1
title: Eads_meps11944_all data.xlsx

description: Gametes of marine broadcast spawners are highly susceptible to the threats of ocean<br>warming and acidification. Here, we explore the main and interacting effects of temperature and<br>pH changes on sperm motility and fertilization rates in the mussel Mytilus galloprovincialis. Additionally,<br>we determine how temperature and pH interact to influence the motility of aging sperm.<br>We show that the interactive effects of temperature (18°C or 24°C) and pH (ranging from 7.6 to 8.0)<br>on sperm motility depend on the time that sperm spend in these conditions. Specifically, sperm<br>linearity was influenced by a temperature × pH interaction when measured after a relatively short<br>exposure to the treatment conditions, while main effects of temperature and pH (but no inter -<br>action) on sperm motility became apparent only after prolonged exposure (2 h) to the treatments.<br>Despite the interactive effects of temperature and pH on sperm motility, these factors had independent<br>effects on fertilization rates, which were significantly higher at the ambient ocean pH<br>level and at the elevated temperature. This study highlights the importance of considering the<br>combined effects of predicted ocean changes on sperm motility and fertilization rates, and cautions<br>against using only sperm motility as a proxy for reproductive fitness. Detrimental effects of<br>pH and temperature may only be uncovered when these factors are examined together, or conversely,<br>negative impacts of one variable may be buffered by changes in another. Our results raise<br>the intriguing possibility that some species may cope better with ocean acidification if they simultaneously<br>experience ocean warming.<br><br>Mussels were collected by hand from a pontoon at<br>Woodman Point, 30 km south of Perth, Western Australia,<br>over multiple trips from July to September during<br>2012 to 2014 (permit no. 2141, Department of<br>Transport, Government of Western Australia). Experimental<br>replicates were taken over multiple seasons<br>due to poor spawning, and year was included in the<br>analyses to account for any seasonal variation (see<br>‘Statistical analyses’). Winter water conditions at the<br>collection site averaged 19.8°C and pH 8.1 over the<br>experimental seasons (Reynolds et al. 2007; https://<br>imos.aodn.org.au/imos123). Mussels were kept in<br>aerated aquaria of recirculating bio logically filtered<br>seawater (FSW) at the University of Western Australia<br>until required (within 3 wk of collection).<br><br>Seawater preparation<br>Experimental water temperatures were maintained<br>by placing containers in water baths in a temperature-<br>controlled room. A total of 4 water baths, compris -<br>ing 2 replicates of each experimental temperature,<br>were used in the experiment. Holding containers<br>with FSW were placed in each water bath and, after<br>water temperatures were stable, experimental pH<br>levels were set by bubbling CO2 through the FSW.<br>By using CO2 to alter seawater pH, the partial pressure<br>of CO2 (pCO2) is raised while simultaneously<br>lowering the pH and carbonate ion concentrations.<br>(Lowering the pH of water by adding an acid such as<br>HCl maintains the pCO2—unrealistic conditions in<br>which to investigate the impacts of anthropogenic<br>climate change—and prior research has shown<br>altered outcomes on fertilization rates using this<br>method; Sung et al. 2014).<br>Experimental water temperatures were 18°C and<br>24°C, while pH levels were set at 7.6, 7.8, and the<br>local ambient pH (~8.0). These values represent<br>current and predicted average winter conditions for<br>the local area in the coming century and are therefore<br>likely to be realistic for the focal population, particularly<br>considering increases in marine heatwave<br>occurrence and length along with more extreme<br>peaks in environmental fluctuations (IPCC 2013,<br>Pearce & Feng 2013). Water parameters (pH, tem -<br>perature, dissolved oxygen, and salinity) were measured<br>before and after conducting each experimental<br>‘block’ (= male) using a pH meter (TPS WP-81;<br>TPS Pty Ltd) calibrated with TPS buffers, and pCO2<br>ranges were calculated using CO2SYS software<br>(Pierrot et al. 2006; Table S1 in the Supplement at<br>www.int-res.com/ articles/ suppl/ m562 p101_ supp. pdf).<br>For each ‘block’, we placed 3 × 150 ml glass jars, each<br>containing 30 ml of water set at one of the 3 pH levels<br>(~8.0, 7.8, or 7.6), in each water bath (18°C or 24°C).<br>Spawning and gamete collection<br>Mussels were induced to spawn using a temperature<br>shock by moving them from their holding tanks<br>(at ~17°C) to a large tray preheated to ~26°C using an<br>aquarium heater (SONPAR automatic, 200W) (Galley<br>et al. 2010). Females that began spawning were<br>rinsed in FSW, placed in a glass jar containing 30 ml<br>of ambient FSW, and left for approximately 1 h to<br>spawn. When a male commenced spawning, it was<br>removed from the tray, rinsed in FSW, and wrapped<br>in a wet paper towel to halt spawning until enough<br>eggs were collected (see below). When required,<br>each male was placed in a glass spawning jar containing<br>30 ml of ambient FSW and left to spawn for<br>approximately 10 min until sperm were sufficiently<br>concentrated (as judged initially by eye) for the<br>sperm motility and fertilization assays (see below).<br>Sperm density in the spawning jar was quantified<br>using an improved Neubauer haemocytometer (Hirsch -<br>mann Laborgeräte). We then extracted a known<br>quantity of sperm from the spawning jars to make up<br>concentrations of 5 × 106 sperm per ml in each treatment<br>jar, a concentration appropriate for the sperm<br>motility analysis and fertilization trials below (see<br>below).<br>Characterizing sperm motility<br>Sperm motility of a subset of males (n = 14) was<br>assessed using computer-assisted sperm analysis<br>(CASA; CEROS sperm tracker, Hamilton-Thorne<br>Research), 20 min after sperm were added to the<br>treatment water, and then again 2 h post-addition to<br>treatment conditions (hereafter referred to as Time 1<br>and Time 2, respectively). For each sample, 1.5 μl of<br>sperm was pipetted into 2 separate wells of a 12-well<br>multitest slide (MP Biomedicals) and covered with a<br>coverslip. We used a phase-contrast Olympus CX41<br>microscope (×10 objective) and captured 30 frames at<br>50 f s−1. We defined static cells below the threshold<br>values of 19.9 μm s−1 for smoothed average path<br>velocity (VAP) and 4 μm s−1 for straight-line velocity<br>(VSL) (see ‘Statistical analyses’ for details about the<br>CASA parameters), minimum cell size as 2 pixels,<br>and measured an average of 193 ± 8 SE sperm tracks<br>per sample. The slides were coated with 1% poly -<br>vinyl alcohol (Sigma-Aldrich) to avoid sperm sticking<br>to the glass (Wilson-Leedy & Ingermann 2007). We<br>randomized the order in which sperm motility was<br>analyzed by treatment among males.<br>Fertilization trials<br>In each water bath (18°C or 24°C), 3 × 50 ml plastic<br>tubes were floated in a polystyrene frame, each containing<br>10 ml of water set at one of the 3 pH levels<br>(~8.0, 7.8, or 7.6). Eggs from all females spawned on<br>a given day were pooled (range: 2−6 females) to provide<br>a ‘homogenous’ genetic background for the<br>fertilization trials (Fitzpatrick et al. 2012), thus reducing<br>variance in fertilization rates attributable to specific<br>male-by-female interactions (i.e. compatibility),<br>which are known to occur in this system (Evans et al.<br>2012, Oliver & Evans 2014). We estimated egg density<br>from a 5 μl sub-sample of pooled eggs, then<br>added eggs to each aforementioned treatment tube<br>at a density of 15 000 per ml.<br>After sperm and eggs had been separately exposed<br>to the treatments for 10 min, an aliquot of sperm from<br>each treatment jar was added to the eggs in the treatment<br>tubes equilibrated under the same conditions,<br>at a ratio of 20:1 (sperm density: 300 000 per ml) to<br>give moderate fertilization rates while avoiding ceiling<br>or basement effects (Fitzpatrick et al. 2012), and<br>gently swirled to homogenize the samples. Fertilization<br>was halted after 1 h by adding 1% formalin to<br>each tube. Fertilization rates were assessed under a<br>microscope as the percentage of eggs showing signs<br>of cleavage and/or with polar body formation among<br>approximately 100 haphazardly chosen eggs per rep -<br>licate (Longo & Anderson 1969). For logistical reasons,<br>fertilization rates were estimated at one time<br>point only (n = 32 males), and sperm motility assays<br>were only undertaken on a subset of the same individual<br>males in the fertilization assays.<br>