Exposure of Nassarius reticulatus veligers in 24-well plates to assess effects of ocean acidification and warming projections on larval survival and behavior

• ¹ Centre for Environmental and Marine Studies CESAM, University of Aveiro, Biology Department, Portugal

The increase in the atmospheric CO2 has a straight relation with the augment of the oceanic pCO2 affecting the seawater chemistry, particularly by reducing the pH and the calcium carbonate saturation. In turn, it has also an important role on the rise of the atmospheric temperature and, therefore, on the oceanic warming. Although the combined effects of ocean acidification (OA) and warming (W) are not fully understood, it will certainly wield a negative impact on marine organisms, especially those producing biogenic carbonates such as gastropod molluscs. The present study aims to assess the combined effect of the temperature rise and the pH decrease on early life stages of the gastropod Nassarius reticulatus, a species widely distributed in the Northeast Atlantic coast, in the Mediterranean, Black and Baltic Seas. A factorial experimental design of two pH levels at four different temperatures was applied, allowing exposure to eight OA-W simulated scenarios. Concerning pH, an undisturbed condition corresponding to the present-day pH level (≈ 8.1 at the specimens’ collection site: Ria de Aveiro, NW Portugal at 40°38’34.65’’N - 8°44’06.80’’W) was used as control, plus a long-term projection by the Intergovernmental Panel on Climate Change (IPCC) of pH ≈ 7.5 (IPCC, 2013). Relatively to temperature, two control values were considered (the mean seawater surface temperature at the Ria de Aveiro mouth – 16 °C – and the mean at the same site during the species spawning season – 18 °C; Barroso and Moreira, 1998) plus two other scenarios corresponding to the IPCC projection of +0.2 °C/decade (20 and 22 °C occurring by the end of the century, depending on the control considered, 16 or 18 °C, respectively; IPCC, 2013). Exposure media consisted in a control of artificial seawater at 30 ± 1 psu or the same seawater acidified by CO2 bubbling (previously equilibrated), kept under defined temperatures by continuously controlled water baths (±1 °C). Nearly-hatched veligers were obtained by caesarean of mature egg capsules collected at Ria de Aveiro. After a 24h-period of acclimation, being fed and appropriate microalgae blend (Chatzinikolaou and Richardson, 2017), veligers were individually exposed in 24-well plates for 14 days. Exposure media was changed every 2-3 days (in order to maintain the chemical and microbiological quality of the media and add fresh microalgae). The following conditions were obtained throughout the exposure period (mean ± standard deviation of probe measurements performed right before and after media exchange in 5 randomly selected wells per plate): for pH, 8.11 ± 0.07 and 7.40 ± 0.07; for temperature, 16.4 ± 0.19, 18.3 ± 0.29, 20.3 ± 0.14 and 22.1 ± 0.15 °C. Mortality, geotaxis, development stage and shell loss were inspected after 2, 4, 7, 10 and 14 days of exposure (T2d to T14d, respectively). Our results indicate that the mortality is significantly increased by the acidic condition tested from T10d onwards. Geotaxis is also significantly affected by pH alone until T7d, being positive for a higher percentage of larvae exposed to acidity, while at T14d the same behaviour seems to be determined by the interaction between temperature and pH. However, as the development stage is significantly driven by temperature and N. reticulatus veligers approach the bottom to crawl preparing settlement (Zupo and Patti, 2009), the effect of temperature on inducing an increased percentage of larvae with positive geotaxis must be considered cautiously, as it can, in fact, indicate an expected behaviour. Still, regarding shell loss, which was only observed under the acidic condition from T7d onwards, it seems to be intensified by the temperature increase. Exception made, in this case, to more developed and resistant survivors in the 22 ºC treatment at T14d, in which shell loss was not observed. This work indicates that, in case the tested scenarios become effective, an increased mortality associated with altered geotaxis and larval shell damage and loss under acidification and warming may reduce larval fitness and jeopardize this species survival.