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Projeto do CEBIMar

Dados do solicitante

Kátia Capel

Natureza do projeto

Projeto de docente ou pesquisador
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Projeto de Pós-Doutorado

Pesquisadores ou docentes associados

Marcelo Visentini Kitahara
Aurea Maria Ciotti
Samuel Coelho de Faria
Carla Zilberberg
Raquel Renó de Oliveira

Recursos

2020/15945-4
Fapesp

Descrição do projeto

Daily rhythms on zooxanthellate and azooxanthellate corals, an integrative approach of gene expression, microbiome, physiology, and environmental parameters
15-11-2021
30-09-2023
Environmental rhythms regulate the behavior and physiology of all living organisms. The most conspicuous rhythm is the light:dark cycle, regulating metabolic states, physiologic process, reproduction, and behavioral activities either by an endogenous circadian clock or as a simple response to light. The circadian clock mechanism as well as gene expression profiles are widely known for model organisms, but it has been explored for cnidarians only in the last few decades. Scleractinian corals are known to have the same core circadian genes observed in mouse and fly, suggesting that the ability to perceive, anticipate, and respond to constant changes in the environment arose early in animal evolution. Interestingly, some of the core circadian genes seem to behave differently in corals, losing rhythmicity under constant darkness. Furthermore, the symbiotic relationship with dinoflagellate algae from the family Symbiodiniaceae complicate studies, and most of the available results on circadian gene expression patterns are performed for the holobiont. Here we propose an unprecedented approach, in which the symbiont is isolated from the coral tissue after collection for an independent evaluation of the gene expression, microbiome, and physiological parameters of both parts in symbiosis, correlating them with environmental variables. The results will serve as a baseline about the functioning of healthy corals and how the host coral and its symbionts respond to the light:dark cycle. In this context, Madracis decactis, a zooxanthellate species, and Tubastraea coccinea, an azooxanthellate species will be studied and will compose the first study in the South Atlantic on the circadian clock in an integrative and transdisciplinary way.
cronobiologia
Scleractinia
Tubastraea coccinea
Madracis decactis
ritmos ambientais
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).
The first year will be dedicated to performing the experiments, extracting the RNA, preparing the libraries, and sequencing. All analyzes and manuscript preparation are predicted to happen from the second year of the project, during the Research Internship Abroad (BEPE – Bolsa Estágio de Pesquisa no Exterior) at the Dr. Oren Levy laboratory at the Bar-Ilan University. Successful achievement of each proposed activity can be used as a measurement of progress.

Solicitações

Laboratório molecular e estufa para a realização dos experimentos de laboratório.
Todos os equipamentos atuais do laboratório molecular e 2 tanques de ~1000 L para a realização dos experimentos de laboratório. Os demais equipamentos necessários para a realização do experimento de laboratório serão adquiridos.
Serão coletados pelo menos 24 fragmentos dos corais Madracis decactis e Tubastraea coccinea.
Arquipélago de Alcatrazes.
Não.
Não
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