Methods
Summary
Before BRUVs deploy, about 2.5 L water sample collected using Van Dorn water sampler. Those 2.5 L will be subsampled as 500 mL each and were filtered on the same day as collection using 50 ml single-use syringes (Terumo) and 0.45 μm Sterivex filters (Merck Millipore, Darmstadt, Germany). Make sure to remove the remaining liquid from the Sterivex unit by pushing air. Cap both ends of the Sterivex. If the units have a male tip outlet, Parafilm can be used to cap them. Be aware of potential leakage, and make sure the Sterivex is dried and well-sealed. The filters were stored at 4 °C for 2 days prior to transportation to the laboratory, transported in cool boxes with ice packs and then stored at −20 °C until DNA extraction.
After eDNA sample collected, BRUVs will be deployed. BRUVs consist of a GoPro mounted on a metal frame that has a bait- source mounted on a pole extending ~ 1 m into the camera’s field of view. Baits consist of oily fish that is chopped into small chunks, crushed up, and then weighed out to 1 kg and placed in a bait bag. A minimum of 150 BRUVs will be deployed around three Gili Islands. The BRUVs are left for at least 70 minutes, allowing it to film continuously for ~ 60 min after settling to the bottom. Soak times of 60 minutes are necessary to ensure sampling in FinPrint 2 is comparable to the first FinPrint.
BRUVs works
Video footage will analyse using EventMeasure software (11). The maximum number of any one species seen at once during the recording period will use as a measure of relative abundance(MaxN), and is generally acknowledged to provide a conservative estimate of abundance (12). The relative abundance will standardize to MaxN per hour, which is consistent with previous studies of shark abundance from BRUVs (13, 14). Where possible, sharks will record to species level and sexed. Other taxa of interest will also count from BRUVs footage. This included species of importance to dive tourism (turtles, moray eels and sea-snakes).
Habitat features, for each BRUV replicate will estimate, including the percent cover of 9 habitat types (hard coral, soft coral, encrusting coral, sand, coral rubble, algae, rock, mud and other), habitat coverage, visibility (in meters) and current (low, medium and high) will were visually estimate from the video imagery.
eDNA works
DNA will extract from the filters in a dedicated eDNA clean room following the Mu-DNA protocol for water sample (15) with a final elution volume of 100 μl. Field controls will be extracted first, followed by the eDNA samples. Five DNA extraction negative controls (one for each day of extractions) containing only extraction buffers will also include. All surfaces will sterilise with 50% bleach and then washed with 70% ethanol. Tweezers and scissors will place in a UV Stratalinker® before, in-between and after extracting each sample to reduce the risk of cross-contamination.
DNA extracts will store at −20 °C until PCR amplification. The Elas02 primer pair (Elas02-F, 5′-GTTGGTHAATCTCGTGCCAGC-3′; Elas02-R, 5′-CATAGTAGGGTATCTAATCCTAGTTTG-3′) will use to target an ∼180 bp amplicon from a variable region of the 12S rRNA mitochondrial gene (16, 17). The PCR mix recipe is as follows: A total volume of 24 μl consist of 12.5 μl Qiagen™ Multiplex PCR kit, 1 μl of the 5 μM pre-mixed forward and reverse primers (Macrogen™), 3 μl of a standardised amount (10-15 ng/μl) of DNA, and 7.5 μl sterile water. The PCR profile include a 15-minute initial denaturing step at 95 °C, 40 cycles at 94 °C for 1 minute, 59 °C for 30 seconds, 72 °C for 1 minute and a 5-minute final extension step at 72 °C. Samples will amplify in triplicate to minimize amplification stochasticity, and replicates will later pool into a single representative sample. Amplification will validate using 1.5% agarose gel electrophoresis stained with ViSafe Red Gel Stain (Vivantis).
Amplicon sequence library preparation will be conducted using the SQK-LSK109 kit and PCR barcoding kit 96 (PCB-096) (Oxford Nanopore Technologies Ltd., UK), according to the manufacturer’s instructions, with the following adaptations: barcoding PCR will be performed in a total volume of 15µL containing 0.3µL PCR barcode primer and 10-50ng amplicon. The applied barcode PCR program is as follows: initial denaturation at 95°C for 180s, 15 cycles of 95°C for 15s, 62°C for 15s, 65°C for 90s, followed by a final extension at 65°C for 180s.
After the barcoding PCR, sample concentration will be estimated using the Qubit HS kit on the non-purified barcoded PCR products, and samples are pool in equimolar ratios. The pooled amplicon sequence library will be cleaned using magnetic beads, washed once with 70% ethanol and once with a mixture of Long Fragment Buffer (LFB) and Short Fragment Buffer (SFB) (2:1) to enrich for the 2kb target size fragments. During final clean-up, the library again washes in a mixture of LFB and SFB in a ratio of 2:1. A maximum of 100ng DNA will load on a primed flow cell to prevent overloading of the flow cell. If necessary, the flow cell will be refuelled using a mixture of Sequencing Buffer (SQB) and nuclease free water (1:1). Sequencing will run until an estimated average sequencing depth of 30k reads per barcode was achieved. Base-calling will be performed using Guppy (Version 4.2.2, Oxford Nanopore Technologies Ltd., UK) in high accuracy (HAC) mode. The sequencing will conduct in Centre Laboratory of Sequencing, National Research and Innovation Agency, Indonesia.
Challenges
Bioinformatics analyses of previous study (18) found the existing 12S marker sequence database had significant gaps and limited resolution to identify several species, such as hammerhead sharks (Sphyrna spp.) and wedgefishes (Rhynchobatus spp.). Moreover, to avoid contamination, two dedicated people will work on BRUVs and eDNA sampling separately, with routine sterilisation conducted regularly.
Pre Analysis Plan
To overcome this hindrance in taxonomic resolution, a custom reference from (18) will use for further analysis. This database consisted of 94 samples representing 45 species.
Protocols
This project has not yet shared any protocols.