How does mesophotic fish diversity compare to shallow water fish diversity?

To target the Indonesian coelacanth with eDNA, I will collect water samples from several sites where coelacanths have been previously detected, particularly Manado, including Talise Island, Gangga Island and Waigeo Island in Raja Ampat, West Papua. Samples will be taken in triplicate and will consist of 5 liters of water per replicate collected from 100-200m depth using a Niskin bottle.

Following the methods of Miya et al. (2015), we will isolate eDNA from the water samples using Sterivex ™ filters (Millipore®, SIGMA MILLIPORE). eDNA extractions will be proceeded using DNeasy Blood & Tissue Kit (QIAGEN, Germany) following previous methods (Marwayana et al., 2021). We will amplify the extracted eDNA molecules using Multiplex PCR Kit (QIAGEN, Germany), and will target two mitochondrial genes, 12S and COI, to optimize the chances for successful implifications of eDNA from coelacanths. Table 2 shows the list of primer sets specifically designed for the Indonesian coelacanth (Sudarto et al., 2010).

     Tabel 2. eDNA Primers which will be used in Chapter I

PCR results will be visualized using gel electrophoresis and then purified using Sera-Mag ™ and Sera-Mag SpeedBeads Magnetic Particles (SIGMA-ALDRICH®). I will then pool the three PCR replicates and adjust final library concentration after quantification using the Qubit ™ 4 NGS Starter Kit (ThermoFisher). I will then indiex the PCR libraries using the Nextera DNA Library Preparation Kit (illumine®) using a specific combination of the Illumina Nextera i5 and i7 primer in the second PCR process. The final PCR products will be sequenced at UCLA using the Illumina MiSeq platform. For the bioinformatic analysis, I will use Anacapa pipeline (Curd et al., 2018) to analyze the eDNA signals.