Projeto de pesquisa

Field experiment
The project includes in situ and laboratory experiments. The 72 hours in situ experiment was performed at the Alcatrazes Archipelago in October 2020. Samples of Madracis decactis and Tubastraea coccinea were taken every four hours, totaling 18 samples per species. Each sample were divided into three sub-samples for physiological measurements, circadian molecular analyzes, and microbiome description. Madracis decactis endosymbionts from both, field and laboratory experiments, will be isolated from the host tissue by consecutive centrifugations, as described by Krueger et al. (2015). Abiotic measures were taken every two hours during sampling using a multi-parameter equipment. Additionally, a second multi-parameter equipment was anchored near the experiment, measuring several parameters every 10 minutes during the 72 hours of sampling.
Laboratory experiment
For the laboratory experiment, at least 24 fragments of M. decactis (from the same colony sampled for the in-situ experiment), and at least 24 colonies of T. coccinea (also from the same sampling area), will be collected and maintained in two separated semi-open systems with 20oC filtered seawater under a 12-hour light and 12-hour dark regime (12:12 LD) (12 samples per species in each system). Light conditions will simulate the luminosity from the collection site at 11 meters deep (where the experiment was set), with a maximum of 326 μmol quanta m-2 s-1 (maximum measurement obtained during in situ experiments), using LED lamps with wavelengths from 400 to 700 nanometers. Water temperature also followed the observed at the collection site at 11 meters deep (in situ temperature ranged between 17oC and 23oC) and will be kept constant by chillers and monitored during the experiment with a digital aquarium thermometer, a 55 dissolved oxygen instrument with temperature sensor, and Tidbit data loggers, to record temperature every minute. The pH will be measured twice a day. Corals will be fed during acclimatization at early night every day with zooplankton filtered through a 180- micron net. Feeding will be suspended two days before the experiment to avoid sequencing the zooplankton. After an acclimation period of six days, samples will be divided into two treatments: 1) a control treatment, with no changes in LD conditions (12:12 LD); and 2) a constant-dark treatment (12:12 DD). After two hours from the start of the treatments, one fragment or colony of each specimen/species/treatment will be sampled every four hours for 48 hours, totaling 12 samples per species/treatment. Samples will be divided into three sub- samples and immediately preserved in liquid nitrogen, ethanol 100%, and CHAOS solution (Fukami et al., 2004), as described for the field experiment. The 48-hour experiment was based on previous studies showing that after 48 hours in constant darkness most of the genes with different expressions in periods of 24 hours in anemones lose their rhythmicity (Leach and Reitzel 2019).