Animals

Gravid coral colonies (Acropora digitifera) were collected from a fringing reef near Oku fishery port in Okinawa Island, Japan. The sea cucumber, Holothuria spp. was collected at Sesoko Island, in the northern part of Okinawa, Japan. The animals were maintained in a running seawater tank under natural light conditions at Sesoko Station, Tropical Biosphere Research Center, University of the Ryukyus, Okinawa, Japan. The coral spawned around the full moon in May, June, and July, 2008. The coral gametes were collected after spawning according to Morita et al. (Reference Morita, Nishikawa, Nakajima, Iguchi, Sakai, Takemura and Okuno2006). The sea cucumber sperm were collected by squeezing the gonads according to Morita et al. (Reference Morita, Kitamura, Nakajima, Susilo, Takemura and Okuno2009).

Preparation of pH-adjusted seawater

To prepare pH-adjusted seawater, we used a pH-stat system similar to that described by Leclercq et al. (Reference Leclercq, Gattuso and Jaubert2002). Seawater was filtered by an inline filter system (0.45 μm). Filtered seawater was bubbled with pure CO₂ to specifically adjust the pH of the seawater using a controller connected to a pH electrode (Micro-pH, Aquabase). The pH conditions of the seawater were adjusted to pH 6.6, 7.3, 7.6, 7.7, 7.8, and 8.0 based on the total hydrogen ion concentration pH scale. The pH conditions of pH 7.3 and 7.6 were predicted for year 2100 and year 2200 by geochemical models (Caldiera & Wickett, Reference Caldiera and Wickett2003). In order to clarify whether the response of corals to low pH conditions is linear or non-linear, an extreme condition of pH 6.6 was included in our experiments. The pH controller was adjusted using a solenoid valve that opens when pH rises to 0.01 higher than the desired level, thereby injecting pure CO₂ from the compressed CO₂ tanks. The temperature was held at 26.3–27.3 °C, which is the typical water temperature during coral spawning season in Okinawa (Suwa et al., Reference Suwa, Hirose and Hidaka2008).

Aquaria (12 l) were filled with seawater adjusted to each pH value. Each aquarium was established as a flow-through system. The stability of the pH in each aquarium was confirmed daily using a pH meter connected to a combined glass/reference pH electrode (713 pH Meter, Metrohm), which were calibrated against the total hydrogen ion concentration pH scale buffers: TRIS and AMP (Dickson et al., Reference Dickson, Sabine and Christian2007). The chemical and physical conditions of each treatment are summarized in Table 1. The pH, temperature and mean salinity (34.0) were measured, and the aragonite saturation state was estimated from these parameter values and the mean total alkalinity of 2300 μmol/kg reported for the region (Fujimura et al., Reference Fujimura, Oomori, Maehira and Miyahira2001) by using the computer program CO2SYS (Lewis & Wallace, Reference Lewis and Wallace1998).

Table 1 Summary of physical and chemical conditions in each experimental aquarium (SWS) during the pH adjusted experiments.

The carbon parameters were calculated based on pH_SWS, temperature, salinity 34.0 and the assumed total alkalinity of 2300 μmol/kg/Ω_Arag, aragonite saturation state. ^aMeans ± SD.

Motility assessment

Sperm flagellar motility was assessed with a microscope equipped with a dark field condenser. We placed 2–3 eggs in 100 μl pH-adjusted seawater taken from aquaria prepared as described above on the slide glass and added 0.5 μl sperm suspension (about 10⁷ sperm/ml) there immediately. Flagellar motility was initiated in response to the eggs. Sperm movements were recorded in digital video by a CCD camera (63W1N, MINTRON) attached to the microscope. Sperm movements were captured with Premier 6.5 (Adobe systems) or I-movie (Apple), and sperm trajectories were traced with NIH image software (http://rsb.info.nih.gov/nih-image/). Flagellar motility was assessed from the captured movie using I-movie from five individuals of each species with 2 s of filming each.

Results and discussion

Sperm motility of a coral (A. digitifera) and a sea cucumber (Holothuria spp.) was assessed at pH 6.6 to pH 8.0 on a total hydrogen ion concentration pH scale, as described by Morita et al. (Reference Morita, Nishikawa, Nakajima, Iguchi, Sakai, Takemura and Okuno2006). In A. digitifera, even relatively small decreases in ambient pH (0.2 to pH 0.4 pH) resulted in significant decreases in sperm flagellar motility (69% of sperm were motile at pH 8.0, 46% at pH 7.8, less than 20% at pH ≤ 7.7) (Fig. 1A, C). Such effects should be viewed in the context of conservative estimates that decreases of 0.2 to 0.4 units in ocean pH are likely within 100 years (Orr et al., Reference Orr, Fabry, Aumont, Bopp, Doney, Feely, Gnanadesikan, Gruber, Ishida, Joos, Key, Lindsay, Maier-Reimer, Matear, Monfray, Mouchet, Najjar, Plattner, Rodgers, Sabine, Sarmiento, Schlitzer, Slater, Totterdell, Weirig, Yamanaka and Yool2005). In Holothuria spp., a significant decrease in flagellar motility was also detected with reductions in pH (73% of sperm were motile at pH 8.0, 72% at pH 7.8, <30% at pH ≤ 7.7) (Fig. 1B, C). Many sessile marine organisms release their gametes into the water column for fertilization, resulting in a rapid dilution of sperm concentration (Levitan & Petersen, Reference Levitan and Petersen1995). Therefore, a slight decrease in sperm motility could seriously threaten their life cycle, due to inefficiency with respect to fertilization. Whether corals can adapt to future ocean acidification is still under debate (Baird & Maynard, Reference Baird and Maynard2008), but relatively long generation times and a higher sensitivity of sperm to ocean acidification may restrict their potential for adaptation.

Figure 1 Trajectories of swimming sperm under different pH conditions. Sperm movements of (A) Acropora digitifera and (B) Holothuria spp. were recorded during 2-s periods. (C) Percentage of sperm motility in A. digitifera and Holothuria spp. in several pH conditions. Values are mean ± SE, n = five of each species with 2 s of filming each. Letters indicate statistical significance (a:b, p < 0.0001; ab:b, p < 0.01; c:d, p < 0.001 ANOVA/Bonferroni test).

In general, sperm of broadcast spawners including the coral A. digitifera and the sea cucumber Holothuria spp. do not become motile just after they are released into the sea, but are motile in the vicinity of eggs (Miller, Reference Miller1985; Morita et al., Reference Morita, Nishikawa, Nakajima, Iguchi, Sakai, Takemura and Okuno2006, Reference Morita, Kitamura, Nakajima, Susilo, Takemura and Okuno2009). Egg-derived compound(s) attach to the sperm and induce several signal cascades including motility activation and chemotaxis (Yanagimachi, Reference Yanagimachi1957; Miller, Reference Miller1985; Yoshida et al., Reference Yoshida, Inaba and Morisawa1993; Coll et al., Reference Coll, Bowden, Meehan, Konig, Carroll, Tapiolas, Aliño, Heaton, De'Nys, Leone, Leone, Maida, Aceret, Willis, Babcock, Willis, Florian, Clayton and Miller1994; Eisenbach & Tur-Kaspa, Reference Eisenbach and Tur-Kaspa1994; Morita et al., Reference Morita, Nishikawa, Nakajima, Iguchi, Sakai, Takemura and Okuno2006). During the course of motility activation, a difference between the extracellular and intracellular region is utilized as a signal to activate sperm flagellar motility in response to the egg-derived compound(s). A pHi causes activation of flagellar motility in many marine invertebrates, including sea urchin, starfish, sea cucumber, and coral (e.g. Christen et al., Reference Christen, Schackmann and Shapiro1982; Nakajima et al., Reference Nakajima, Morita, Takemura, Kamimura and Okuno2005; Morita et al., Reference Morita, Kitamura, Nakajima, Susilo, Takemura and Okuno2009). If sperm from many marine invertebrates require a pHi to activate their flagellar motility, then it might be reasonable to assume that sperm could lose their ability to move as a result of a decline of ambient pH (pHo), such as occurs with acidification of seawater.

Nevertheless, sea urchin sperm motility is suppressed in acidic seawater (Havenhand et al., Reference Havenhand, Buttler, Thorndyke and Williamson2008) because sea urchin sperm flagellar motility is activated after release into seawater. In the gonad, the pH is maintained below pH 7.3 by stable CO₂ tension (Johnson et al., Reference Johnson, Clapper, Winkler, Lee and Epel1983), and sperm flagellar motility is activated when sperm are released into seawater accompanied by a reduction of intracellular proton concentration, causing a pHi up to 7.5 to 7.6. As a consequence, pHi induces the activation of dynein ATPase and mitochondrial respiration resulting in the initiation of motility (Christen et al., Reference Christen, Schackmann and Shapiro1982). In contrast, coral and sea cucumber sperm do not become motile after being released into seawater (Miller et al., Reference Miller1985; Morita et al., Reference Morita, Nishikawa, Nakajima, Iguchi, Sakai, Takemura and Okuno2006, Reference Morita, Kitamura, Nakajima, Susilo, Takemura and Okuno2009). In these species, egg-derived substance(s) stimulate protein phosphorylation of flagellar proteins leading to the activation of motility (e.g. Morita et al., Reference Morita, Kitamura, Nakajima, Susilo, Takemura and Okuno2009). Therefore, there is a diversity of motility activation reactions among marine invertebrates that would be exposed to acidified seawater. Analysis of flagellar motility in several marine organisms in response to acidified seawater is required to describe a specific effect of ocean acidification on sperm motility. These surveys will provide us with an estimate of which marine organisms are likely to be endangered by ocean acidification.