First report of field sampling and identification of crown-of-thorns starfish larvae

Abstract

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℃ for 3 min, followed by 35 cycles of 94℃ for 20 s, 60℃ for 30 s, and 72℃ for 30 s with a final extension step at 72℃ for 5 min. The PCR cocktail contained 3.84 μL of ddH2O, 5 μL of KAPATaqTM 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℃ 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.