ALBERT

Description: The basic observed water characteristics (pH, temperature, salinity and total alkalinity (TA)) and calculated water carbonate chemistry in the field (22.44°N, 113.95°E, Pak Nai, Hong Kong) and laboratory (22.28°N, 114.14°E, the University of Hong Kong, Hong Kong) during a two-factor orthogonal experiment. To be specific, we designed a two-factor (low pH, as a proxy for OA, and Vibrio infection) orthogonal experiment to examine the immunological and molecular responses in the ecologically and economically important edible Hong Kong oyster, Crassostrea hongkongensis. Wild oyster population was collected from their native coastal area (22.44°N, 113.95°E, Pak Nai, Hong Kong). Vibrio parahaemolyticus, as a major global concern of foodborne human-related disease, was used in this study to investigate the antimicrobial characteristics of edible commercial oysters in the context of future OA. pH and temperature were monitored daily with pH meter (Mettler Toledo InLab®, Switzerland) calibrated with NBS standards. Salinity was monitored using a dual-scale refractometer (MingRui, China). The seawater was utterly changed every two days and 50 mL seawater was preserved with HgCl2 every week for total alkalinity (TA) measurement. The TA was measured using an alkalinity titrator (Mettler Toledo G20S Compact Titrator, Switzerland), and pCO2, CO32-, saturation states of calcite and aragonite were calculated by CO2SYS program with equilibrium constants K2 and KSO4 (Millero, F. J., Graham, T. B., Huang, F., Bustos-Serrano, H., & Pierrot, D. (2006). Dissociation constants of carbonic acid in seawater as a function of salinity and temperature. Marine Chemistry, 100(1), 80-94. https://doi.org/10.1016/j.marchem.2005.12.001).

Description: The basic observed water characteristics (pH, temperature, salinity) of Magallana angulata (also known as Crassostrea angulata) F2 generation from larval stage to adult in the hatchery located in Zhanjiang, China (21.07°N, 110.74°E) for around one year. The F2 larvae were acquired by artificial fertilization of matured F1 generation (F1_8.1-7.4 and F1_7.4-8.1). Embryos from F1_8.1-7.4 were separated into two pH levels: F2_8.1-7.4-8.1 and F2_8.1-7.4-7.4. The embryos from F1_7.4-8.1 were separated into two pH levels: F2_7.4-8.1-8.1 and F2_7.4-8.1-7.4. There were four treatments for F2 generation and reared till 2022 to test the transgenerational OA effects on oyster biological responses. Low pH was driven by bubbling of CO2-enriched air mixed with ambient air into filter seawater in the treatment tanks. pH and temperature were monitored daily with pH meter (Mettler Toledo InLab®, Switzerland) calibrated with NBS standards. Salinity was monitored using a dual-scale refractometer (MingRui, China). The seawater was utterly changed every two days during larval stage and every week for juvenile stage.

Description: The basic observed water characteristics (pH, temperature, salinity and total alkalinity (TA)) and calculated water carbonate chemistry of Magallana angulata (also known as Crassostrea angulata) F1 generation from larval stage to adult in the hatchery located in Zhanjiang, China (21.07°N, 110.74°E) for around one year. These F1 larvae were acquired by artificial fertilization of matured F0 generation (F0_8.1 and F0_7.4). Embryos from F0_8.1 were separated into two pH levels: F1_8.1-8.1 and F1_8.1-7.4. Likewise, embryos from F0_7.4 were separated into two pH levels: F1_7.4-8.1 and F1_7.4-7.4. Thus, there were four treatments for F1 generation, and each treatment had three independent 500L tank replicates (A, B, and C). F1 generation continued to be reared to the next year till most of them reached the first year of gonad maturation to test the transgenerational low pH effect on oyster biological responses. Low pH was driven by bubbling of CO2-enriched air mixed with ambient air into filter seawater in the treatment tanks. pH and temperature were monitored daily with pH meter (Mettler Toledo InLab®, Switzerland) calibrated with NBS standards. Salinity was monitored using a dual-scale refractometer (MingRui, China). The seawater was utterly changed every two days during larval stage and every week for juvenile stage. About 50 mL of seawater was preserved with HgCl2 for total alkalinity (TA) measurement. The TA was measured using an alkalinity titrator (Mettler Toledo G20S Compact Titrator, Switzerland), and pCO2, CO32-, saturation states of calcite and aragonite were calculated by CO2SYS program with equilibrium constants K2 and KSO4 (Millero, F. J., Graham, T. B., Huang, F., Bustos-Serrano, H., & Pierrot, D. (2006). Dissociation constants of carbonic acid in seawater as a function of salinity and temperature. Marine Chemistry, 100(1), 80-94. https://doi.org/10.1016/j.marchem.2005.12.001

Description: The basic observed water characteristics (pH, temperature, salinity and total alkalinity (TA)) and calculated water carbonate chemistry in the hatchery located in Zhanjiang, China (21.07°N, 110.74°E). The two-year-old Portuguese oysters Magallana angulata (also known as Crassostrea angulata) were collected from Wuchuan, China (21.37°N, 110.74°E). To be specific, about 300 adult oysters were transferred into six black fibreglass tanks (500L each) randomly and each culture tank contained about 50 individuals. Six tanks were independent and completely randomized to be separated into two different pH levels: ambient pH 8.0~8.1 as control (named F0_8.1) and pH 7.4 as ocean acidification (OA) treatment (named F0_7.4) for about one month to produce next generation and test for transgenerational effect. Low pH was driven by bubbling of carbon dioxide (CO₂) enriched air mixed with ambient air into filter seawater in the treatment tanks. pH and temperature were monitored daily with pH meter (Mettler Toledo InLab®, Switzerland) calibrated with NBS standards. Salinity was monitored using a dual-scale refractometer (MingRui, China). The seawater was utterly changed every two days and 50 mL seawater was preserved with mercury dichloride (HgCl2) every week for total alkalinity (TA) measurement. The TA was measured using an alkalinity titrator (Mettler Toledo G20S Compact Titrator, Switzerland), and partial pressure of carbon dioxide (pCO₂), carbonate ion (CO₃²⁻), saturation states of calcite and aragonite were calculated by CO2SYS program with equilibrium constants K2 and KSO4 (Millero et al. 2006).

Description: The basic observed water characteristics (pH, temperature, salinity) of Magallana angulata (also known as Crassostrea angulata) F2 generation from larval stage to adult in the hatchery located in Zhanjiang, China (21.07°N, 110.74°E) for around one year. The F2 larvae were acquired by artificial fertilization of matured F1 generation (F1_8.1-8.1 and F1_7.4-7.4). Embryos from F1_8.1-8.1 were separated into two pH levels: F2_8.1-8.1-8.1 and F2_8.1-8.1-7.4. The embryos from F1_7.4-7.4 were separated into two pH levels: F2_7.4-7.4-8.1 and F2_7.4-7.4-7.4. There were four treatments for F2 generation and reared till 2022 to test the transgenerational OA effects on oyster biological and immunological responses. Low pH was driven by bubbling of CO2-enriched air mixed with ambient air into filter seawater in the treatment tanks. pH and temperature were monitored daily with pH meter (Mettler Toledo InLab®, Switzerland) calibrated with NBS standards. Salinity was monitored using a dual-scale refractometer (MingRui, China). The seawater was utterly changed every two days during larval stage and every week for juvenile stage.

Description: Unprecedented rate of increased CO2 level in the ocean and the subsequent changes in carbonate system including decreased pH, known as ocean acidification (OA), is predicted to disrupt not only the calcification process but also several other physiological and developmental processes in a variety of marine organisms, including edible oysters. Nonetheless, not all species are vulnerable to those OA threats, e.g. some species may be able to cope with OA stress using environmentally induced modifications on gene and protein expressions. For example, external environmental stressors including OA can influence the addition and removal of methyl groups through epigenetic modification (e.g. DNA methylation) process to turn gene expression “on or off” as part of a rapid adaptive mechanism to cope with OA. In this study, we tested the above hypothesis through testing the effect of OA, using decreased pH 7.4 as proxy, on DNA methylation pattern of an endemic and a commercially important estuary oyster species, Crassostrea hongkongensis at the time of larval habitat selection and metamorphosis. Larval growth rate did not differ between control pH 8.1 and treatment pH 7.4. The metamorphosis rate of the pediveliger larvae was higher at pH 7.4 than those in control pH 8.1, however over one-third of the larvae raised at pH 7.4 failed to attach on optimal substrate as defined by biofilm presence. During larval development, a total of 130 genes were differentially methylated across the two treatments. The differential methylation in the larval genes may have partially accounted for the higher metamorphosis success rate under decreased pH 7.4 but with poor substratum selection ability. Differentially methylated loci were concentrated in the exon regions and appear to be associated with cytoskeletal and signal transduction, oxidative stress, metabolic processes, and larval metamorphosis, which implies the high potential of C. hongkongensis larvae to acclimate and adapt through non-genetic ways to OA threats within a single generation.

Description: The majority of common edible oysters are projected to grow more slowly and have smaller impaired shells because of anthropogenic CO2-induced reductions in seawater carbonate ion concentration and pH, a process called ocean acidification (OA). Recent evidence has shown that OA has carryover effects, for example, larvae exposed to OA will also exhibit either positive or negative effects after metamorphosis. This study examined the hidden carryover effects of OA exposure during parental and larval stages on post-metamorphic traits of the commercially important oyster species Crassostrea hongkongensis. Adults of C. hongkongensis were exposed to control pH (pHNBS 8.0) and OA-induced low pH (pHNBS 7.4) conditions. Their larval offspring were then exposed to the same aquarium conditions before being out-planted as post-metamorphic juveniles at a mariculture site for 10 months. Initially, larval offspring were resilient to low pH with or without parental exposure. The larvae exposed to low pH had significantly faster development and higher percentage of settlement success compared to control groups. The out-planted juveniles with parental exposure had improved survival and growth compared to juveniles without parental exposure, regardless of the larval exposure history. This implies that transgenerational effects due to parental exposure not only persists but also have a greater influence than the within-generational effects of larval exposure. Our results shed light on the importance of linking the various life history stages when assessing the OA-induced carryover capacity of C. hongkongensis in the natural environment. Understanding these linked relationships helps us better predict the species rapid adaptation responses in the face of changing coastal conditions due to OA.

Description: Biomineralization is one of the key biochemical processes in calcifying bivalve species such as oysters that is affected by ocean acidification (OA). Larval life stages of oysters are made of aragonite crystals whereas the adults are made of calcite and/or aragonite. Though both calcite and aragonite are crystal polymorphs of calcium carbonate, they have different mechanical properties and hence it is important to study the micro and nano structure of different life stages of oyster shells under OA to understand the mechanisms by which OA affects biomineralization ontogeny. Here, we have studied the larval and juvenile life stages of an economically and ecologically important estuarine oyster species, Crassostrea hongkongensis, under OA with focus over shell fabrication under OA (pHNBS 7.4). We also look at the effect of parental exposure to OA on larvae and juvenile microstructure. The micro and nanostructure characterization reveals directional fabrication of oyster shells, with more organized structure as biomineralization progresses. Under OA, both the larval and juvenile stages show directional dissolution, i.e. the earlier formed shell layers undergo dissolution at first, owing to longer exposure time. Despite dissolution, the micro and nanostructure of the shell remains unaffected under OA, irrespective of parental exposure history.

Description: Ocean acidification (OA) has important effects on the intrinsic phenotypic characteristics of many marine organisms. Concomitantly, OA can alter the extended phenotypes of these organisms by perturbing the structure and function of their associated microbiomes. It is unclear, however, the extent to which interactions between these levels of phenotypic change can modulate the capacity for resilience to OA. Here, we explored this theoretical framework assessing the influence of OA on intrinsic (immunological responses and energy reserve) and extrinsic (gut microbiome) phenotypic characteristics and the survival of important calcifiers, the edible oysters Crassostrea angulata and C. hongkongensis. After one-month exposure to experimental OA (pH 7.4) and control (pH 8.0) conditions, we found species-specific responses characterised by elevated stress (hemocyte apoptosis) and decreased survival in the coastal species (C. angulata) compared with the estuarine species (C. hongkongensis). Phagocytosis of hemocytes was not affected by OA but in vitro bacterial clearance capability decreased in both species. Gut microbial diversity decreased in C. angulata but not in C. hongkongensis. Overall, C. hongkongensis was capable of maintaining the homeostasis of the immune system and energy supply under OA. In contrast, C. angulata's immune function was suppressed, and the energy reserve was imbalanced, which might be attributed to the declined microbial diversity and the functional loss of essential bacteria in the guts. This study highlights a species-specific response to OA determined by genetic background and local adaptation, shedding light on the understanding of host-microbiota-environment interactions in future coastal acidification.

Description: 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.