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Title: Gene expression analysis during metamorphosis and the onset of calcification in the scleractinian coral M. faveolata      
Transcriptome or Gene expression
Similar to many marine invertebrates, scleractinian corals experience a dramatic morphological transformation, as well as a habitat switch, upon settlement and metamorphosis. At this time, planula larvae transform from non-calcifying, demersal, motile organisms into sessile, calcifying, benthic juvenile polyps. We performed a gene expression microarray analysis between planulae, aposymbiotic primary polyps, and symbiotic adult tissue to elucidate the molecular mechanisms underlying coral metamorphosis and early stages of calcification in the Robust/Short clade scleractinian coral Montastraea faveolata. Among the annotated genes, the most abundant upregulated transcripts in the planula stage are involved in protein synthesis, chromatin assembly and mitochondrial metabolism; the polyp stage, morphogenesis, protein catabolism and organic matrix synthesis; and the adult stage, sexual reproduction, stress response and symbiosis. Additionally, our results indicate that metamorphosis in M. faveolata planulae is likely regulated by: 1) a mechanism that resembles that described for hydrozoan cnidarians involving the neuropeptide LWamide; and 2) conserved cell adhesion and apoptosis mechanisms. Our results also suggest that calicoblast differentiation pathways may be regulated by transforming growth factors from the BMP family and Notch signalling pathway. We also present evidence showing that the planula and adult transcriptomes are more similar to each other than to the polyp trancriptome. Lastly, our results point to a large number of uncharacterized adult coral-specific genes likely involved in coral-specific functions such as symbiosis and calcification. Overall design: Collection of samples -- Montastraea faveolata gamete bundles and adult fragments were collected on 3 September 2007 during the annual spawning event in Puerto Morelos, Quintana Roo, Mexico. Gamete bundles were collected and fertilized as described in Miller and Szmant 2005. After 1 hour, excess sperm was rinsed away, and the eggs were examined for signs of cleavage. Embryos were maintained in 1 μm filtered seawater, with water changes several times per day, until they reached the planula stage. At this point, a subset of planula larvae was collected and preserved in RNAlater (Ambion) for posterior RNA extraction and microarray hybridizations. Remaining planulae were transferred to polycarbonate culture bins and allowed to metamorphose and settle. After a week, settled polyps were collected and preserved in RNAlater. Three adult fragments were obtained using a hammer and chisel from one colony of M. faveolata at 2.7 m depth near Puerto Morelos (20o52’28.77”N and 86o51’04.53”W) on 31 July 2007. Fragments were transported back to the station and acclimated to a flow-through (~0.6 L/min) 50 L aquarium for 27 days. Mean temperature of the aquarium was 28.4 ± 0.9oC (as measured by a HOBO Light/Temperature Data Logger – Onset Corp.). The fragments were frozen in liquid nitrogen prior to and during transport to Merced, CA. All samples were exported to the USA through a CITES permit (MX-HR-007-MEX). Microarray data analysis -- The experiment followed a loop design in which all life stages (i.e. planula, polyp, adult) were directly compared against each other. Hybridizations were performed in triplicate (n=3 for each developmental stage) and included dye swapping between technical replicates. Pre-processing of microarray intensity data was performed using TIGR TM4 software. Briefly, spot-finding was performed in SpotFinder 3.11 with background-subtracted median intensities being extracted. Using MIDAS 2.19, the data were LOWESS normalized, and the dye swap pairs were collapsed (thus leaving an output of 9 data files collapsed from 18 total arrays). Secondly, the in-slide duplicates were collapsed (also in MIDAS 2.19). Resulting intensities in both channels (for nine total microarrays) were compiled into a spreadsheet (AR_matrix1.xls). Genes were included in subsequent statistical analysis only if there were data in two out of three replicates for each developmental stage. Of 1310 possible genes, 1243 passed filtering criteria. The ratio between the fluorescence intensity of the two channels was used as input for BAGEL (Bayesian Analysis of Gene Expression Levels). Normalized, filtered data is provided as another matrix (AR_matrix2.xls). Note this file contains the ratio fo both channels NOT log2 transformed.
Orbicella faveolata
National Center for Biotechnology Information
NCBI BioProject