To extract intracellular metabolites from lyophilized holobiont add 400 microliters of 100%cold methanol with internal standards to the lyophilized holobiont. Then add 10 milligrams of acid washed glass beads and place the tube in a pre chilled bead mill. Insert at 50 hertz for three minutes.
After lysis, add 600 microliters of cold methanol and vortex for one minute. Place the tube on a rotisserie shaker at four degrees celsius for 30 minutes. To extract metabolites from separated lyophilized symbiodiniacae cells, Add 200 microliters of cold methanol with internal standards to the dried symbiodiniacae cells.
Then add acid washed glass beads and place the tube in a pre chilled bead mill insert at 50 hertz. After three minutes, add 800 microliters of methanol and vortex for 30 seconds. For metabolite extraction from separated lyophilized host tissue, add one milliliter of cold methanol containing internal standards to the dried host material and vortex for 20 seconds.
Then place the tube in a floating tube holder for sonication at four degree Celsius for 30 minutes. Next for metabolite extract purification, centrifuge all samples at 3000G for 30 minutes at four degrees Celsius. Carefully transfer the supernatant into a new two milliliter micro centrifuge tube without disturbing the pellet.
Re-suspend the pellet in one milliliter of 50%cold methanol and vortex vigorously for one minute. Centrifuge again, then collect and pool the polar extracts supernatant with the semi-polar extracts from the same sample. Centrifuge the pooled extracts at 16, 100G for 15 minutes to remove precipitates.
For analysis, aliquot 50 microliters of each extract into a glass insert and concentrate using a vacuum concentrator for 30 minutes at 30 degrees celsius. GCMS analysis revealed 107 annotated metabolites across all the treatments, including a suite of amino acids, organic acids, carbohydrates, fatty acids, and sterile. K means clustering identified three distinct clusters of samples.
The holobiont samples were intermediate between the separated host and symbiont fractions. Although K means cluster distributions, parallel coordinates, and heat map visualization in the metabolite relative abundance indicated that the holobiont profile more closely matched the host fraction profile significantly differed from both the host and symbiont profiles. The host and symbiont profiles were significantly distinct from each other with 100 individual metabolites, significantly different between the host and symbiont fractions.
