Fatty acids as biological markers for bacterial symbionts in sponges from the Great Barrier Reef

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Samples were taken from three phototrophic, cyanobacteria containing sponge species (Dysidea herbacea, Phyllospongia papyracea and Pseudaxinyssa sp) and two nonphototrophic sponge species (Phakellia aruensis and Rhopaloeides odorabile) at Davies Reef.Lipids were extracted and a subsample saponified. The resultant fatty acids were converted to methyl esters, which were analysed on both apolar and polar capillary columns. Structures were confirmed by chromatographic comparisons with authentic standards, by comparison of equivalent chain lengths for the methyl esters with those of secondary standards and by capillary gas chromatography-mass spectometry (GC-MS).Pseudaxinyssa sp. was collected from Rib Reef and the sponge was separated into surface tissue, to a depth of 0.5 mm (including exopinacoderm and a distinct reddish-brown band of cyanobacteria) and interior tissues. Each portion was lyophilized prior to extraction and the isolated phospholipids were converted to methyl esters, which were analysed using gas chromatography. Electron microscopy was used to confirm the large volume of cyanobacteria within the surface tissue fraction and the almost complete absence of cyanobacteria within the interior tissue fraction.The contributions of symbiotic bacteria were determined on the basis that fatty acids extracted from sponge tissue originated in either the cell membranes of the sponge, or nonphototrophic bacteria or cyanobacteria. The minimum abundance of any specific acid in a suite of sponge samples was used as an estimate of the sponge contribution of that acid in other sponges.
This study was initiated to estimate the cyanobacterial and nonphototrophic bacterial abundance in sponges using a bacterial biological marker technique based on fatty acid content.

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Maintenance and Update Frequency: notPlanned

Statement: Statement: Lipid Extraction:Samples were immediately placed in solvent (chloroform/methanol, 1:1, v/v, plus 0.05% pyridine), with static lipid extraction (24 hr) proceeding in the dark at room temperature. Decantation was followed by reextraction of the sponge residue by grinding in two further changes of solvent.Fatty acids were converted to methyl esters by the procedures described in:Johns RB, Nichols PD and Perry GJ (1979) Phytochemistry 18, 799-802.Johns RB, Nichols PD and Perry GJ (1980) Comp. Biochem. Physiol. 65B, 207-214.Fatty acid analyses:Fatty acid methyl esters were analyzed on both apolar (50 m X 0.2 mm, SP2100, Hewlett Packard, Palo Alto, CA) and polar (50 m X 0.2 mm, Superox 0.1, S.G.E. Australia) capillary columns, and the raw data from electronic integration were interpreted using the computer programs described in:Gillan FT (1983) J. Chromatogr. Sci. 21, 293-297.Structures were confirmed by chromatographic comparison with authentic standards where available, by comparison of equivalent chain lengths (ECL) for the methyl esters with those of secondary standards and by capillary gas chromatography-mass spectrometry (GC-MS) (HP5970A ion selective detector coupled to an HP 5790B gas chromatograph).Bacterial community structure analysis:The respective contributions of symbiotic bacteria were determined according to the methods described in:Gillan FT and Hogg RW.(1984) J. Microbial. Methods 2, 275-293.Johns RB, Nichols PD, Gillan FT, Perry GJ and Volkman, JK (1981) Comp. Biochem. Physiol. 69B, 843-849.

Notes

Credit
Gillan, Francis T, Dr (Principal Investigator)