Ultraviolet radiation absorbing mycosporine-like amino acids in holothurians from the Great Barrier Reef

Holothuroids were collected during daylight from fully exposed, unshaded habitats at Hicks Reef, in December 1988 and Shrimp Reef in July 1991. Specimens were collected by scuba diving or snorkeling at depths between 1 to 20m. The species examined were Actinopyga echinites, Actinopyga lecanora, Bohadschia argus, Holothuria (Halodeima) atra, Holothuria (Halodeima) edulis, Holothuria (Microthele) fuscopunctata, Holothuria (Microthele) nobilis, Pearsonothuria graeffei, Stichopus chloronotus, Stichopus variegatus, Thelenota ananas, Thelenota anax and Synapta maculata.

Small sections of tissues (epidermis, body wall, intestinal wall, respiratory trees, gonads, longitudinal muscle, cloaca and entire tentacles) were excised from each specimen. Minced tissues were sequentially extracted 3 times for 20 minutes in 3 cm³ of 100% methanol. The combined extract was clarified by centrifugation and analyzed aboard ship for mycosporine-like amino acids (MAAs).

Individual MAAs were separated by reverse-phase isocratic HPLC. Detection of peaks was by UV absorbance at 313 and 340 nm. Identities of peaks were confirmed by the wavelength method (ratio of peak areas detected at 313 nm/340 nm) and by co-chromatography with standards. Peaks were integrated on Hewlett-Packard or SpectraPhysics integrators, and quantification of individual MAAs was corrected for extraction efficiency using published molar extinction coefficients.

Methanol-extracted tissues were frozen and returned to the Australian Institute of Marine Science for determination of their protein content. Rehydrated tissues were digested in hot 1N NaOH, and after being cooled, neutralized and diluted, the mean protein content of triplicate aliquots was measured using Coomassie Brilliant Blue with bovine gamma globulin standards (Bio-Rad Laboratories).