G. caespitosa is used as a model species in studies on the evolution of polyandry and sperm competition (e.g., Evans and Marshall, 2005, Styan et al., 2008 and McLeod and Marshall, 2009) as well as in ecotoxicological assessments ( Moran and Grant, 1993 and Ross and Bidwell, 2001). Here, we focused on among-male variation in sperm swimming responses to future ocean acidification. Following the A1FI scenario (IPCC, 2007), R428 clinical trial we exposed sperm to seawater conditions predicted for near- (pCO2 = 970 μatm, year 2100) and far-future CO2 scenarios future (pCO2 = 1600 μatm, year 2300), and recorded impacts on the proportion

of motile sperm and their swimming speeds. Based on a previous study on individual variation in sperm swimming in sea urchins ( Schlegel et al., 2012), we hypothesized that there will be substantial variation in the responses of swimming capabilities in individual GSI-IX order sperm. Filtered seawater (FSW; 0.22 μm filtered) was aerated with a CO2/air mixture to achieve CO2 treatments. Seawater temperature and salinity (Table 1) were measured for each replicate (n = 23) using an IQ Sensor net (MIQ/T2020, WTW). Microprocessor CO2 injection units were set to maintain stable

pHNBS levels of 8.1 (controls, no CO2 added; pCO2 = 427 μatm), 7.8 (pCO2 = 971 μatm) and 7.6 (pCO2 = 1597 μatm), following the A1FI scenario ( IPCC, 2007). Total alkalinity was determined for every third replicate (n = 7) by titration (HI 3811 Alkalinity kit, Hanna Instruments), all other parameters of the CO2 system were calculated using CO2-SYS ( Lewis and Wallace, 1998) and the dissociation constants of Dickson and Millero, (1987) ( Table 1). Clumps Glycogen branching enzyme of large G. caespitosa (tube openings of 2+mm diameter) were collected from intertidal rock platforms in Fairlight, Sydney, Australia (33°48′1′′S,

151°16′3′′E) in November and December 2011, and held in a recirculating seawater system at Macquarie University. Individuals were used in experiments within 48 h of collection. Collection of gametes followed the protocol by Kupriyanova and Havenhand, (2002). Individual G. caespitosa were carefully removed from their calcareous tubes and inspected for ripeness. Individual males, characterized by creamy white lower abdomens, were placed into separate petri dishes. Removal of the males from their tubes caused instantaneous spawning in mature individuals. Males that did not immediately release gametes were discarded. Sperm from spawning individuals were collected with Pasteur pipettes from each male, and held “dry” on ice in Eppendorf tubes (one for each individual) until immediately prior to use (within 15 min of release). A total of 23 mature males were tested. Sperm motility experiments were conducted in a temperature-controlled room at 20 ± 0.5 °C and followed established protocols (Havenhand et al., 2008, Havenhand and Schlegel, 2009 and Schlegel et al., 2012). “Dry” sperm (∼0.