Projeto de pesquisa

A colony of ​ Mussismilia hispida (zooxanthellate) was fragmented into 19 pieces and transplanted close to the original place in December 2018 (these fragments were followed since then and all of them recovered successfully; Figure 1). For Tubastraea coccinea (azooxanthellate), as the invasive population at the Alcatrazes Archipelago was shown to be clonal (one genotype, Capel et al., 2017; 2019), small colonies were sampled at the same location. By the time of sampling (late October/2020), each fragment of M. hispida and colony of T. coccinea was collected every 4 hours for 72 hours, in order to enable diel cycle replication. Abiotic data was collected in the meantime - also every 4 hours, using a CTD device with additional sensors to measure temperature, salinity, dissolved oxygen, PAR, turbidity and pH - and will later be integrated to the biological results in collaboration with Prof Áurea Maria Ciotti. Once sampled, fragments were flash frozen in liquid nitrogen and stored at -80 ºC upon return to the laboratory. A second in situ experiment involving the manipulation of abiotic factors (e.g. availability of light) will be performed using a similar experimental design. Analyses will take place in the Centre of Marine Biology of the São Paulo University or at the Department of Marine Science of the Federal University of São Paulo (DNA and RNA extraction and library preparation) as well as at the Genome Investigation and Analysis Laboratory (Genial - CEFAP; Next Generation Sequencing).
In the case of the zooxanthellate coral, Symbiodiniaceae will be separated from host tissue using consecutive centrifugations, as described by Krueger et al. (2015). If they are not successful, data may be separated computationally, by matching reads against datasets already available for cnidarians and Symbiodiniaceae (e.g. Sorek and collaborators (2018) using the SNAP software [http://snap.cs.berkeley.edu/]).
Gene expression in the host and Symbiodiniaceae should be assessed by RNA extraction followed by library preparation with a TruSeq Stranded mRNA Library Prep (Illumina) and sequencing on a NextSeq 550 platform as single-end (SE) reads Reads will be filtered for low quality, short and duplicated reads, as well as mapped to a reference genome.Relative proportions of mapped reads to each gene will be compared among different time points. The annotation will be performed by BLASTX (Altschul et al., 1990) against UniProt (The UniProt Consortium, 2019) obtaining Gene Ontology (Ashburner et al., 2000; The Gene Ontology Consortium, 2019) information for top hits.
In order to analyze changes in microbial community composition and abundance during the studied period, bacterial 16S rDNA will be amplified from total genomic DNA, purified, and prepared for sequencing following Illumina’s protocol for the 16S Metagenomic Sequencing Library Preparation (https://support.illumina.com/content/dam/illumina-support/
documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf). Libraries will be pooled and sequenced on an Illumina MiSeq platform. Following quality control, a binning analysis should be carried out against SILVA database (Quast et al., 2013). Relative proportions of taxa present from sample to sample will be analyzed on STAMP (Parks et al., 2014). Physiological parameters such as total antioxidant capacity (TAC), lipid peroxidation (LPO), and Ca-ATPase activity will be assessed in parallel to the present project, in collaboration with other researchers (e.g. Prof Samuel Fari​a, who is already associated with Cebimar-USP). Analyses will be performed at the Federal University of Rio Grande (FURG). Eventually, obtained information from holobiont components as well as different stages of their metabolism will be integrated in the form of networks using softwares such as Cytoscape (Shannon et al., 2003).