Eads_meps11944_all data.xlsx

Name: Eads_meps11944_all data.xlsx
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Published: 2020-09-02 06:28:57
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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 and pH 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 below). 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.

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