First report of field sampling and identification of crown-of-thorns starfish larvae
First report of field sampling and identification of crown-of-thorns starfish larvae
复制标题
首次对棘冠海星幼虫进行现场采样和鉴定的报告
DOI：
10.3755/galaxea.17.15
复制
发表时间：
2015
期刊：
影响因子：
--
通讯作者：
K. Nadaoka
中科院分区：
文献类型：
--
作者：
N. Yasuda;K. Kajiwara;S. Nagai;Kota Ikehara;K. Nadaoka
到目前为止，还没有成功地对食珊瑚海星的幼虫进行实地取样和鉴定（Birkeland，1990年）。我们从5个不同深度（表面、3 m、7 m、11 m、15 m和底部附近）采集了水样（500 L），以找到A。2005年6月29日，在产卵期间，发现了一条扁尾轮虫幼虫（Yasuda等人，2010年）。样品取自日本冲绳宫古岛外的矢鼻矶礁（25°1′13.85 ″ N，125°14′49.20 ″ E，图1A），那里曾发生过尖吻金丝猴的种群爆发。观察到planci（图1B）。我们使用立体显微镜从保存在乙醇（>70%）中的样品中发现并分离出26只可能的海星幼虫（原肠胚和双羽状幼体阶段;分别见图1C和D）。我们使用DNeasy血液和组织试剂盒（Qiagen）提取DNA，并直接对使用之前发表的COI引物对（Vogler等人，2008）和推定的控制区引物对（Timmers等人，2011）扩增的部分线粒体DNA片段进行测序。后一对引物能区分A. planci来自一些常见的海星，例如Protoreaster nodosus、Linkia laevigata和Culcita novaeguineae。使用以下PCR条件：在94°C下变性步骤3分钟，然后是94°C下20秒、60°C下30秒和72°C下30秒的35个循环，最终延伸步骤在72°C下5分钟。PCR混合物含有3.84 μL ddH 2 O、5 μL KAPATaq Extra HotStart ReadyMix（Kapa Biosystems）、1 μL模板DNA和0.07 μL 50 mM引物。仅成功分析了10只幼虫，部分原因是DNA降解，但所有幼虫都被鉴定为A。通过BLAST搜索。在表层（2）、7 m（5）或近底层（2）样品中发现了9只原肠胚期幼虫，而在表层样品中发现了1只双羽幼虫期幼虫。盐度-温度-深度（STD）数据显示，在我们鉴定出的A. planci。由于成功发育的叶绿素a最低阈值密度为0.4微克/升（Fabricius等人，2010年），因此在研究期间，幼虫可能只能获得有限的食物。棘冠海星幼鱼野外采样鉴定初报
To date, field sampling and identification of the larvae of the coral-eating starfish Acanthaster planci have not been successfully performed (Birkeland 1990). We collected water samples (500 L) from 5 different depths (surface, 3 m, 7 m, 11 m, 15 m, and near the bottom) to find A. planci larvae on June 29, 2005, during the spawning period (Yasuda et al. 2010). The samples were obtained from Yabiji Reef, off Miyako Island, Okinawa, Japan (25°1′13.85′′N, 125°14′49.20′′E, Fig. 1A), where a population outbreak of A. planci was observed (Fig. 1B). We used a stereoscopic microscope to find and isolate 26 possible starfish larvae (gastrula and bipinnaria stages; Fig. 1C and D, respectively) from the samples, which were preserved in ethanol (>70%). We extracted DNA using the DNeasy blood & tissue kit (Qiagen), and directly sequenced partial mitochondrial DNA fragments amplified by using a previously published COI primer pair (Vogler et al. 2008) and a putative control region primer pair (Timmers et al. 2011). The latter primer pair can discriminate A. planci from some common sea stars such as Protoreaster nodosus, Linckia laevigata, and Culcita novaeguineae. The following PCR conditions were used: denaturing step at 94°C for 3 min, followed by 35 cycles of 94°C for 20 s, 60°C for 30 s, and 72°C for 30 s with a final extension step at 72°C for 5 min. The PCR cocktail contained 3.84 μL of ddH2O, 5 μL of KAPATaq Extra HotStart ReadyMix (Kapa Biosystems), 1 μL of template DNA, and 0.07 μL of 50 mM primers. Only 10 larvae were successfully analyzed, partly due to degradation of DNA, but all were identified as A. planci by BLAST search. Nine larvae at the gastrula stage were found in either the surface (2), 7 m (5), or near bottom (2) samples, while 1 larva at the bipinnaria stage was found in the surface sample. Salinity-Temperature-Depth (STD) data showed that the water temperature was 27.7-27.9°C and chlorophyll a concentration was 0.3-0.4 μg/L at the sampling points from where we identified larvae of A. planci. Since the minimum threshold density of chlorophyll a for successful development is 0.4 μg/L (Fabricius et al. 2010), the larvae might have had access to limited food during the study period. First report of field sampling and identification of crown-of-thorns starfish larvae