Study Site and Design
Sampling was conducted in the main tank at The Aquarium of Western Australia (AQWA; aqwa.com.au), which offered a relatively controlled system containing 50 known fish species in three million litres of seawater. This system draws incoming seawater from 0.5 m below the seabed (natural sand filter) of the nearshore ocean waters. It is then filtered (pressure glass media filter) before entering the AQWA facility where the water supplies several display tanks before entering the main tank of the mesocosm. The main tank has its own gravity filter system (volume of filter tank is 2 million litres) that uses a 50 cm sand bed with 2 mm (± 0.5 mm) size particles, over 50 cm of 6 mm (± 3 mm) gravel. The turnover rate between the gravity filter and main tank is 5 million litres every 2 hours. Passive eDNA sampling was conducted between 8am and 4pm on January 21 and 22, 2021, by submerging nine different membrane materials just below the surface in the mesh pockets of a pearl oyster aquaculture frame (Fig.1, see Bessey et al. 2021). Each of the nine membrane materials were deployed in quadruplicate for specified time intervals (5, 10, 30, 60 minutes and overnight for 18 hours) to examine whether increased submersion time led to increased eDNA collection. Of the quadruplicate samples, three were used for eDNA extractions while the other was used for scanning electron microscopy to visualise how eDNA collected on the different membrane surfaces.
Membrane Materials
We trialled nine different membrane materials (Table 1). The first was a cellulose ester membrane (0.45 µm Pall GN-6 Metricel®) commonly used in eDNA studies (Tsuji et al. 2019). To investigate whether chitosan coating would increase eDNA capture, the cellulose membranes were impregnated with either 1% or 3% chitosan (w/w) which was then crosslinked under glutaraldehyde vapour to confer stability. Loadings of chitosan on the membranes were confirmed by Fourier-transform infrared spectroscopy (FT-IR) as well as by staining with the anionic dye Eosin Y. Chitosan is a polycation polymer that efficiently binds anionic DNA under acidic conditions and has been used for DNA enrichment and purification (Pandit et al. 2015). Chitosan is derived from chitin in crustacean shells and is readily available, inexpensive and biocompatible. To investigate if eDNA would become entrapped in highly complex materials, we trialled overlaying the cellulose esters with electrospun nanofibres, while also trialling a combination of electrospun nanofibers that were subsequently covered in a 1% chitosan (w/w). Electrospinning is a technique for producing fibres from submicron down to nanometer in diameter with high surface area (Bhardwaj and Kundu 2010). We used solution electrospinning, where the polymer(s) and other additive materials are firstly dissolved in a suitable solvent at an optimized concentration before electrospinning. A high Voltage electric field is applied to the droplet of fluid coming out of the tip of a die or spinneret, which acts as one of the electrodes. When the electric field supply is strong enough, it will lead to the droplet formation and finally to the ejection of a charged jet from the tip of the cone accelerating toward the counter collector electrode leading to the formation of a nanofibrous membrane. These nanofibrous membranes have found applications in many areas, including biomedical areas (e.g., scaffolds for tissue engineering, drug delivery, wound dressing, and medical implants), filtration, protective textiles, and battery cells (Gao et al. 2014). Our electrospinning was carried out using polyether based thermoplastic polyurethane (TPU) grade (RE-FLEX® 585A, Townsend Chemicals) with a 10% w/v solution in dimethyl formamide solvent (DMF) using a 23 G needle spinneret, with an applied voltage of 20kV at 15 cm from the collecting drum. To ensure sufficient physical robustness for use in the marine environment, a composite was prepared using a thermal bonding [Protechnic 114P (13 gsm)] net material to bond the electrospun membrane attached through thermal adhesive. This backing plate was needed to prevent the nanofibre cellulose membranes from curling, and therefore, we also trialled these backing plates separately in the downstream processing to determine their effect on eDNA capture. We also trialled natural fibres, cotton and hemp, which were contained in a nylon bag for practical deployment purposes so they would remain anchored within the mesh of the pearl frame. A subset of nylon bags was retained for downstream processing in the same fashion as the trialled materials. These cotton and hemp fibres were 5 mm in diameter and cut into 40 mm lengths so they could fit in a 2 mL Eppendorf tube for DNA extraction. Finally, we trialled two sponge materials that would be highly robust in aquatic settings: one was a tightly woven filter pad with 100% active carbon (Aqua One®), while the other was a tightly woven filter pad with zeolite (Aqua One®). The sponge was cut into 40 mm rectangular lengths and had a 5 mm width and depth. All materials were placed under ultra-violet sterilizing light for a minimum of 30 min, except for the cellulose membranes which were certified sterile upon purchase.