The increase of CO2 by anthropogenic activities leads to a decrease of pH in the ocean surface due to ocean acidification (OA) process. Generally, OA not only reduces the rate of calcification in marine environments but also affects various physiological activities, especially in calcifiers, including edible oysters. Quantitative real-time PCR (qRT-PCR) is often used to detect gene expression in response to OA, which relies on the stability of internal control. However, the appropriate internal controls for OA experiments remain scarce especially in the marine calcifiers. Hence, this study developed internal controls for qRT-PCR assays using the Hong Kong oyster (Crassostrea hongkongensis) as a model to reveal gene expression profile during development under OA. In this study, 17 housekeeping genes were selected as the possible candidate of the internal controls. After a comprehensive interpretation from the multiple algorithms and software, GAPDH paired with RL23 is recommended for the normalization for planktonic larvae and benthic juveniles, but beyond that, TUBB and EF2 are recommended for post-metamorphic stage. Moreover, GAPDH and EF2 were suitable for various pH treatments, and TUBB, RL35 and RL23 could be the alternatives for OA experiments. These results are instrumental for the selection of internal control in Crassostrea hongkongensis during the development, and shed light on other molecular OA experiments in marine invertebrates for reference.

In order to allow full comparability with other ocean acidification data sets, the R package seacarb (Gattuso et al, 2021) was used to compute a complete and consistent set of carbonate system variables, as described by Nisumaa et al. (2010). In this dataset the original values were archived in addition with the recalculated parameters (see related PI). The date of carbonate chemistry calculation by seacarb is 2022-06-20.The species name used in the paper is Crassostrea hongkongensis.