Projeto de pesquisa

Corals samples will be obtained in three nested spatial scales: regions, sites and habitats. Two regions (i.e. latitudinal bands) will be surveyed along the Brazilian coast: the Abrolhos Bank (Bahia) and the coast of São Paulo. Two sites across the shelf will be sampled within each region: Parcel das Paredes and Parcel dos Abrolhos, in Abrolhos, and Alcatrazes Archipelago and south of Ilhabela, in São Paulo. Finally, two habitats (horizontal reef tops, 2-4 m depth, and vertical walls, 5-15 m depth) will be sampled within each site.
Five coral colonies will be collected per habitat per site using SCUBA, totaling 40 colonies for the entire study. Colonies will be collected at least 10 m apart from each other to avoid collecting clones (cf. Schoepf et al. 2022). Collection, handling, and preservation of samples will follow the guidelines provided by Thurber et al. (2022), while experimental procedures will follow the guidelines from Grottoli et al. (2021). The first measurements of tissue/bleaching necrosis and maximum photochemical efficiency (Fv/Fm) will be obtained in situ using Pulse Amplitude Modulated Fluorometry and photographic records (see below). In addition, one fragment from each colony (~2 cm2) will be frozen (-20°C) immediately after collection for baseline measurements (see Table 1). The live colonies will be shipped to the facilities of the Centre for Marine Biology (CEBIMar-USP) in the São Paulo coast. In the laboratory, each colony will be fragmented into 12 pieces (~2 cm2) that will be acclimated for 15 days under their native temperature and light conditions, as measured by the HOBO sensors. After this acclimation period, one coral fragments from each colony will be subjected to twelve treatments considering all possible combinations of native/acute/chronic temperature, low/high light levels and presence/absence of zooplankton in a typical aquarium-based ramp and hold experiment (Nielsen et al. 2022). Temperature stress will be set considering two scenarios: one in which emissions will continue to increase rapidly (SSP5-8.5), with a mean SST increase of about 3.5°C predicted for the end of the century (IPCC 2021) and another mimicking the conditions faced by Brazilian corals during episodes of marine heatwaves in the last decade, with a mean SST increase equivalent to about 6°C for the 15-day period of our experiment (i.e. 12 Degree Heating Weeks, see Banha et al. 2020) (Figure 3). Light regimes will be set at 12:12h dark:light cycle and light stress will be determined based on values of light intensity for the most lit habitats (possibly the reef tops of the Abrolhos Bank, see Figure 1), as estimated by the HOBO sensors. Temperature increases will be done with glass heaters with a ramping rate of +0.5 °C every 12 h (cf. McRae et al. 2022). Standardized amounts of zooplankton will be provided every 5 days along the entire experiment (cf. Aichelman et al. 2016). Temperature and light stress will be maintained for 15 days (stress phase). After this period, coral fragments will be frozen and used for physiological analyses. In total, 520 coral fragments will be analyzed for the entire study (40 fragments for baseline measurements and 480 fragments for the laboratory experiments).
To estimate the area of each fragment with bleaching or necrosis, digital photographs will be taken with a reference color card (cf. Nielsen et al. 2020, 2022). After removing the coral tissue using a water pik (Johannes & Wiebe 1970), Symbiodiniaceae density will be measured using a hemocytometer (cf. Stimson et al. 2002). Chlorophyll-a will be extracted using acetone and the supernatant will be read with a spectrophotometer after centrifugation (cf. Marangoni et al. 2019). Both Symbiodiniaceae density and chlorophyll-a content will be standardized to the area of coral tissue.
A respirometry chamber will be used for the oxygen consumption estimates (cf. Gravinese et al. 2021). Photosynthetic efficiency will be measured with a Pulse Amplitude Modulated Fluorometry (Diving-PAM) for dark-adapted samples (cf. Voolstra et al. 2020). Lipid peroxidation (LPO) and total antioxidant capacity (TAC) will be used as proxies for the oxidative stress experienced by the corals and their susceptibility to oxidative stress, respectively (Marangoni et al. 2017, 2019). Lipid peroxidation will be estimated through the fluorimetric method described by Oakes and van der Kraak (2003), while TAC will be estimated using the OxiSelect™ Total Antioxidant Capacity (TAC) Assay Kit (Cell Biolabs Inc., San Diego, CA, USA), which detect all classes of antioxidants (cf. Marangoni et al. 2017).
The relative contribution of the coral autotrophic and heterotrophic feeding modes will be evaluated using fatty acid analyses. The relative abundance of fatty acids exclusively derived from photosymbionts (stearidonic acid, 18:4ω3, and docosapentaenoic acid, 22:5ω3) and from heterotrophic feeding (cis-gondoic acid, 20:1ω9) will be measured with gas chromatography and used as biomarkers following the protocols in Mies et al. (2018).
Screening for metabolite biomarkers will be performed using a liquid (LC) and gas (GC) chromatography coupled to a mass spectrometer (MS). Coral extracts will be prepared with methanol and hexane solvents. For LC-MS samples from methanol portion will be analyzed using a Shimadzu LC-20A model coupled to a UV-DAD detector (CBM20A) and to an UltrOTof Bruker Daltonics mass spectrometer with electrospray ionization mode (ESI). The “untargeted” method will be employed whereas ions up to 1x103 of intensity will be selected for fragmentation spectrum acquisition (MS/MS). In GC-MS analysis, hexane coral extracts will be injected in a Shimadzu GC-MS QP2010 coupled to an MSD mass spectrometer with impact ionization mode electrons (IE) and a quadrupole type analyzer will be employed. A DB5-MS capillary column (30 m x 0.25 mm x 0.25 mm + 10m Duraguard pre-column) will be used. Helium will be used as a gas of drag. The metabolic identification by GC-MS will be performed through the independent parameters: Kovats index and fragmentation profile. The fragmentation profiles and high-resolution masses will be used for in situ detection of molecules through comparation in chemical data bases and a molecular networking for each coral extract will be constructed using GNPS platform (Wang et al., 2016).
Changes on Symbiodiniaceae composition will be evaluated through Next Generation Sequencing (NGS), by the amplification of the Internal Transcribed Spacer 2 rDNA (ITS2) using primers from Hume et al. (2013) linked to Illumina adapters. Reads will be processed in the FastQC on-line platform and paired/filtered/grouped using the Geneious software. Sequencies will be then classified using the GeneBank (NCBI) and SymPortal (Hume et al. 2019).