More Like this

1. Quantification of Total Biomass in Ground Coral Samples

   https://doi.org/10.17504/protocols.io.bdyai7se  

   Mclachlan, R ; Dobson, K ; Grottoli, AG ( April 2020 , Protocolsio)

   null (Ed.)

   This protocol outlines a method for quantifying the total biomass of Scleractinian coral samples which have been ground into a homogenous paste consisting of aragonite skeleton, coral host tissue, and endosymbiotic Symbiodiniaceae cells. There are four parts to quantifying total biomass: 1) grind coral fragments into a homogenous paste, 2) partition the biomass subsample, 3) quantify the ash-free dry weight [AFDW], and 4) standardize AFDW to the colony surface area. This method has been reported in several publications by Grottoli's team (e.g., Rodrigues & Grottoli 2007). This protocol was written by Rowan McLachlan (03-19-20) and was reviewed by all co-authors. dx.doi.org/10.17504/protocols.io.bdyai7se 

   more » « less
2. Microplate Assay for Quantification of Soluble Protein in Ground Coral Samples

   https://doi.org/10.17504/protocols.io.bdc8i2zw  

   Mclachlan, R ; Price, H ; Dobson, K ; Weisleder, N ; Grottoli, AG ( April 2020 , Protocolsio)

   null (Ed.)

   This method for extracting protein from ground coral samples is based on the Bradford assay for the colorimetric detection and quantification of total protein (Bradford, 1976) and is compared to a known standard dilution of bovine serum albumin (BSA). Pierce Inc. and Bio-Rad have developed the reagents and standards necessary for completing the extraction. There are five parts to quantifying total soluble protein in ground corals: 1) grind and sub-sample the coral and store at -80 °C until ready to extract, 2) solubilize protein via cell disruption [detergent lysis and freeze-thaw lysis], 3) separate the dissolved protein from tissue and skeletal particles, 4) quantify the protein concentration via Bradford microassay procedure, and 5) standardize the protein concentration to ashfree dry weight (AFDW). This method was originally developed by Rowan McLachlan with the assistance of Jamie Price and Kerri Dobson and with the guidance of Dr. Noah Weisleder and Andréa Grottoli at The Ohio State University. This protocol was written by Rowan McLachlan and reviewed by all co-authors. dx.doi.org/10.17504/protocols.io.bdc8i2zw 

   more » « less
3. Extraction of Total Soluble Lipid from Ground Coral Samples

   https://doi.org/10.17504/protocols.io.bc4qiyvw  

   Mclachlan, R ; Munoz-Garcia, A ; Grottoli, A ( March 2020 , Protocolsio)

   null (Ed.)

   This method is adapted and updated from methods originally published in Grottoli et al. (2004) and is based on the original methods of Folch & Stanley (1957), and Bligh & Dyer (1959). There are five parts to extracting lipids from ground corals: 1) grind and sub-sample the coral and store at -80 °C until ready to extract, 2) freeze-dry the sample, 3) extract the lipids from the freeze-dried samples, 4) standardize the lipid concentration to ash-free dry weight (AFDW), and 5) resuspend the extracted lipid for long-term storage and possible later analysis of lipid classes or isotopes. The lipid extraction procedure must be conducted in a fume hood with the sash as low as possible with the researcher wearing protective eyewear, gloves, and lab coat at all times. Important considerations regarding lipid analysis were gained from reading Chapter 1.3 “Lipid extraction, storage, and sample handling” from the textbook Lipid Analysis by Christie (2003). This method was originally developed by Andréa Grottoli and refined by Rowan McLachlan (06-11-18) with the guidance of Dr. Agus Muñoz-Garcia at The Ohio State University. This protocol was written by Rowan McLachlan (03-12-2020). dx.doi.org/10.17504/protocols.io.bc4qiyvw 

   more » « less
4. Airbrushed coral sample preparation for organic stable carbon and nitrogen isotope analysis

   https://doi.org/10.17504/protocols.io.bgi7juhn  

   Price, J ; Smith, A ; Dobson, K ; Grottoli, AG ( July 2020 , Protocolsio)

   null (Ed.)

   This method for separating coral tissues from algal endosymbiont (Symbiodiniaceae) for stable isotope analysis is modified from previously published methods (Hughes et al. 2010). There are three parts to preparing coral samples for stable carbon and nitrogen isotope analysis: 1) airbrush to remove coral tissue and algal cells from skeleton and store at -80 °C until ready to separate, 2) separate the coral tissue from the algal cells through centrifugation and filtering, and 3) dry and pack separated tissues into tin capsules for analysis in a stable isotope ratio mass spectrometer. This method was modified from Hughes et al. (2010) by James Price with the assistance of Alex Smith and Kerri Dobson and with the guidance of Andréa Grottoli at The Ohio State University. dx.doi.org/10.17504/protocols.io.bgi7juhn 

   more » « less
5. Direct evidence of sex and a hypothesis about meiosis in Symbiodiniaceae

   https://doi.org/10.1038/s41598-021-98148-9  

   Figueroa, R. I. ; Howe-Kerr, L. I. ; Correa, A. M. ( December 2021 , Scientific Reports)

   null (Ed.)

   Abstract Dinoflagellates in the family Symbiodiniaceae are obligate endosymbionts of diverse marine invertebrates, including corals, and impact the capacity of their hosts to respond to climate change-driven ocean warming. Understanding the conditions under which increased genetic variation in Symbiodiniaceae arises via sexual recombination can support efforts to evolve thermal tolerance in these symbionts and ultimately mitigate coral bleaching, the breakdown of the coral-Symbiodiniaceae partnership under stress. However, direct observations of meiosis in Symbiodiniaceae have not been reported, despite various lines of indirect evidence that it occurs. We present the first cytological evidence of sex in Symbiodiniaceae based on nuclear DNA content and morphology using Image Flow Cytometry, Cell Sorting and Confocal Microscopy. We show the Symbiodiniaceae species, Cladocopium latusorum , undergoes gamete conjugation, zygote formation, and meiosis within a dominant reef-building coral in situ. On average, sex was detected in 1.5% of the cells analyzed (N = 10,000–40,000 cells observed per sample in a total of 20 samples obtained from 3 Pocillopora colonies). We hypothesize that meiosis follows a two-step process described in other dinoflagellates, in which diploid zygotes form dyads during meiosis I, and triads and tetrads as final products of meiosis II. This study sets the stage for investigating environmental triggers of Symbiodiniaceae sexuality and can accelerate the assisted evolution of a key coral symbiont in order to combat reef degradation. 

   more » « less