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). Many species of holothurian forage during daylight hours in shallow water and thus may require protection from UV radiation. For this reason, holuthurians were included in ongoing surveys of coral reef organisms for the presence of UV radiation absorbing mycosporine-like amino acids (MAAs). A range of tissue types from 13 deposit feeding species of holothurian were analysed to examine differences in MAA content that may be related to differences in UV exposure.

海参类（Holothuroids）于1988年12月在希克斯礁（Hicks Reef）及1991年7月在虾礁（Shrimp Reef）的完全暴露、无遮蔽生境中，于日间采集。样本通过水肺潜水或浮潜于1-20米深度采集。所检测的物种包括刺参（Actinopyga echinites）、白底辐肛参（Actinopyga lecanora）、蛇目白尼参（Bohadschia argus）、黑海参（Holothuria (Halodeima) atra）、绿刺参（Holothuria (Halodeima) edulis）、棕环海参（Holothuria (Microthele) fuscopunctata）、糙海参（Holothuria (Microthele) nobilis）、格氏异手海参（Pearsonothuria graeffei）、绿刺参（Stichopus chloronotus）、花刺参（Stichopus variegatus）、梅花参（Thelenota ananas）、尖刺参（Thelenota anax）及斑锚参（Synapta maculata）。
从每个样本中切取小块组织（表皮、体壁、肠壁、呼吸树、性腺、纵肌、泄殖腔及完整触手）。切碎的组织在3 cm³ 100%甲醇中连续提取3次，每次20分钟。合并提取物经离心澄清后，在船上分析类菌孢素氨基酸（mycosporine-like amino acids，MAAs）。单个MAAs通过反相等度高效液相色谱（HPLC）分离。通过313 nm和340 nm处的紫外吸收检测峰。峰的鉴定通过波长法（313 nm与340 nm处检测的峰面积比）及与标准品共色谱法确认。峰面积在惠普（Hewlett-Packard）或光谱物理（SpectraPhysics）积分仪上积分，单个MAAs的定量通过已发表的摩尔消光系数校正提取效率。
经甲醇提取后的组织冷冻后送回澳大利亚海洋科学研究所（Australian Institute of Marine Science）测定蛋白质含量。复水组织在热1N NaOH中消化，冷却后中和并稀释，取三份等分试样，使用考马斯亮蓝（Coomassie Brilliant Blue）结合牛γ球蛋白标准品（Bio-Rad Laboratories）测定平均蛋白质含量。
许多海参物种在日间浅水区觅食，因此可能需要防护紫外线辐射。为此，海参被纳入珊瑚礁生物持续调查中，以检测其是否存在吸收紫外线的类菌孢素氨基酸（MAAs）。分析了13种食沉积物海参物种的多种组织类型，以探究MAAs含量差异是否与紫外线暴露差异相关。