TITLE: Environmental DNA sampling protocols for the surveillance of marine non-indigenous species 
AUTHORS: Luca    Mirimin, Dulaney Miller, Sara Fernandez

[DESCRIPTION]
This document describes a series of protocols for the collection of environmental samples intended for the monitoring and surveillance of marine invasive species by means of eDNA metabarcoding analysis, as described in the associated publication (Fernandez et al. 2021: https://doi.org/10.1016/j.marpolbul.2021.112893).

[BEFORE_START]
MAKE SURE THAT BIOSECURITY MEASURES ARE PUT IN PLACE TO AVOID THE UNINTENTIONAL SPREAD OF HARMFUL OR UNWANTED ORGANISMS

[STEPS]
SECTION: HEALTH AND SAFETY
1. 
 

SECTION: Protocol for collection of water samples (low volume water)
2. SAMPLING

 

SECTION: Protocol for collection of water samples (low volume water)
2.1. Put on clean, single-use gloves.

Change gloves if a glove has contacted anything except the sampled water body or decontaminated equipment. For example, if your glove touches your clothes, skin, etc., change gloves. For ease of changing gloves regularly, it is advised to use double-gloves (e.g. only outer glove is replaced).
SECTION: Protocol for collection of water samples (low volume water)
2.2. For each site, pre-label the appropriate number of Whirl-Pak bags as follows:

1. "Site name/code", "Date", "LV Water field blank"
2. "Site name/code", "Date", "Rep 1"
3. "Site name/code", "Date", "Rep 2"
4. "Site name/code", "Date", "Rep 3"
...
n. "Site name/code", "Date", "Rep n"
SECTION: Protocol for collection of water samples (low volume water)
2.3. Take the pre-labelled field blank Whirl-Pak bag and fill with DNA-free water at the site location. 

This will be the field negative control sample. Keep sample blank in a cool, dark place (e.g. black bin bag or cooler box) until you are ready for filtration either in the filed or in the lab.
SECTION: Protocol for collection of water samples (low volume water)
2.4. Take the next pre-labelled Whirl-Pak bag (e.g. "Rep 1") and fill with site water either from the surface (using a DNA-free pole and bag adapter) or at depth by using a DNA-free niskin bottle.

If using a Niskin bottle, it is recommended to rinse 3 times with site water prior to sample collection.

It is also recommended to take at least one additional sample than intended, to account for potential loss of samples during transport/filtration.

Make sure to seal bags properly and keep them up right in a cool dark place during transport and prior to filtration. 
SECTION: Protocol for collection of water samples (low volume water)
2.5. Repeat Step 1.4 as many times as the number of samples required from a given site/location.

Record all metadata (e.g. "Site name/code", "GPS", "Date", "Rep #", "time of deployment", etc.).
SECTION: Protocol for collection of water samples (low volume water)
2.6. (OPTIONAL) If available, use a water parameter meter to record temperature, salinity, depth, pH, DO, etc.

Record all metadata.
SECTION: Protocol for collection of water samples (low volume water)
3. GEAR DECONTAMINATION

 

SECTION: Protocol for collection of water samples (low volume water)
3.1. While it is not necessary to decontaminate gear between replicate samples within a given site/location, it is necessary to decontaminate any reusable equipment or accessory prior to sampling the next location.

Wipe the Whirl-Pak pole and spray clamps and adapters with 20% bleach and leave to decontaminate for at least 15 minutes.

To reduce sampling time and risk of cross site contamination, it is recommended to have several pre-prepared sets of clamps and adapters (e.g. ideally as many as the intended number of sites to be visited).
SECTION: Protocol for collection of water samples (low volume water)
4. WATER FILTRATION

 

SECTION: Protocol for collection of water samples (low volume water)
4.1. Put on clean, single-use gloves and wipe clean the surface work area with 20% bleach. For ease of changing gloves regularly, it is advised to use double-gloves (e.g. only outer glove is replaced).
If filtering in the field, use a disposable or reusable plastic sheet (e.g. tarpaulin) as surface.

Set up the filter pump, power source (mains, battery, car) and (if available) the multi-filer manifold, ensuring that all connections are tight and that the flow of water/air will follow the intended direction (e.g. away from the filters).
SECTION: Protocol for collection of water samples (low volume water)
4.2. Pre-label 15 mL falcon tubes (pre-filled with 4mL silica beads) following the same labeling as that suggested in Step 1.2. It is recommended to secure the label by covering it with a strip of sellotape to avoid risk of mislabeling.
SECTION: Protocol for collection of water samples (low volume water)
4.3. Place DNA-free adapter-funnel-filter assemblies in the tubing (or manifold), ensuring that the funnel is kept vertical and is secure (to avoid any spillage). 

Filter each sample gradually, ensuring that any suspended particle is also poured into the funnel by swirling the liquid in the Whirl-Pak bag. Using DNA-free forceps, remove the filter from the funnel (top membrane, not the lower thick support pad), fold onto itself to create a semi-circle shape, and place in its respective pre-labelled falcon tube. Store tubes in a cool dark place (for short-term storage) and place in a -20°C as soon as possible (for medium-long term storage).

Record metadata (e.g. time of filtration of each sample).


SECTION: Protocol for collection of water samples (high volume water)
5. DEPLOYMENT AND RETRIEVAL

 

SECTION: Protocol for collection of water samples (high volume water)
5.1. Put on clean, single-use gloves.

Change gloves if a glove has contacted anything except the sampled water body or decontaminated equipment. For example, if your glove touches your clothes, skin, etc., change gloves. For ease of changing gloves regularly, it is advised to use double-gloves (e.g. only outer glove is replaced).
SECTION: Protocol for collection of water samples (high volume water)
5.2. 
Ensure that all equipment has been decontaminated with 20% bleach prior to deployment.

Check that the sampler's battery has sufficient charge.

Deploy the sampler by inserting and securing the starter plug prior to releasing in the water. Note that the sampler inflow valve should be facing upward.

Record all metadata (e.g. "Site name/code", "GPS", "Date", "Rep #", "time of deployment", etc.)
SECTION: Protocol for collection of water samples (high volume water)
5.3. Retrieve the sampler after the filtration cycle has completed (e.g. 25 hours).

Remove the filter capsule and remove any excess water through the outflow (not inflow). This step can be done either using a pump or large syringe or by gravity, but making sure to use DNA-free equipment/materials to avoid potential contamination of the sample.

Fill the capsule with fixative, inject 1 mL of Internal Positive Control (OPTIONAL), and seal both ends using caps and/or Parafilm. Label each sample and store the sample in a cool dark place for short-medium term until extraction.
SECTION: Protocol for collection of water samples (high volume water)
5.4. If a field negative is required, fill a filter capsule with fixative and IPC while on site and store/process as per other samples.
SECTION: Protocol for collection of water samples (high volume tow net)
6. SAMPLE COLLECTION

 

SECTION: Protocol for collection of water samples (high volume tow net)
6.1. Put on clean, single-use gloves.

Change gloves if a glove has contacted anything except the sampled water body or decontaminated equipment. For example, if your glove touches your clothes, skin, etc., change gloves. For ease of changing gloves regularly, it is advised to use double-gloves (e.g. only outer glove is replaced).
SECTION: Protocol for collection of water samples (high volume tow net)
6.2. Prior to deployment, each net and other reusable equipment must be decontaminated in order to remove any DNA  trace form previous use. While most materials and small equipment can be easily decontaminated by soaking in 20% bleach for at least 20 minutes (with occasional mixing/turning), it is recommended that nets must be fully submerged in bleach in a large deep tray and rinsed with DNA-free water at least 3 times while rubbing and "massaging" the net and seams by hand to remove any excess bleach. 
SECTION: Protocol for collection of water samples (high volume tow net)
6.3. For each site, pre-label the appropriate number of 50 mL falcon tubes as follows:

1. "Site name/code", "Date", "Net field blank"
2. "Site name/code", "Date", "Rep 1"
3. "Site name/code", "Date", "Rep 2"
4. "Site name/code", "Date", "Rep 3"
...
n. "Site name/code", "Date", "Rep n"

It is recommended to secure the label by covering it with a strip of sellotape to avoid risk of mislabeling.
SECTION: Protocol for collection of water samples (high volume tow net)
6.4. Using the 100% ethanol squeeze bottle, rinse the pre-decontaminated net and collect 40-50 mL of liquid in the falcon tube labeled as "Net field blank".
SECTION: Protocol for collection of water samples (high volume tow net)
6.5. For each subsequent sample, tow the net for the intended distance/time (e.g. 50 meters transects) and making sure that the net is fully submerged. Prior to retrieval, make sure to concentrate most of the suspended material trapped in the net into the dolphin bucket by performing several vertical “dips”.

Remove as much excess water as possible by holding the net at an angle and letting the water sip through the mesh of the dolphin bucket. If this is taking a long time, close off a portion of the net just above the dolphin bucket, and use the additional surface area to drain water. Draining the water is essential to have proper preservation of the eDNA.

Using the ethanol squeeze bottle, rinse down the bottom of the net into the dolphin bucket and detach the dolphin bucket. Then, rinse the dolphin bucket screens and transfer material to the 50 mL tube. Keep the net high in the air while rinsing down to keep ethanol in the dolphin bucket. If necessary, split the sample into an additional falcon tube. Aim for at least 80% ethanol in each sample for adequate preservation.
SECTION: Protocol for collection of water samples (high volume tow net)
6.6. Store samples out of direct sunlight, preferably on ice and in a rack. Keep the samples upright as much as possible to prevent leakage and contamination.
SECTION: Protocol for collection of sediment samples
7. SAMPLE COLLECTION

 

SECTION: Protocol for collection of sediment samples
7.1. Put on clean, single-use gloves.

Change gloves if a glove has contacted anything except the sampled water body or decontaminated equipment. For example, if your glove touches your clothes, skin, etc., change gloves. For ease of changing gloves regularly, it is advised to use double-gloves (e.g. only outer glove is replaced).
SECTION: Protocol for collection of sediment samples
7.2. Ensure that all equipment has been decontaminated with 20% bleach prior to deployment.

For each site, pre-label the appropriate number of 50 mL falcon tubes as follows:

1. "Site name/code", "Date", "Sediment field blank"
2. "Site name/code", "Date", "Rep 1"
3. "Site name/code", "Date", "Rep 2"
4. "Site name/code", "Date", "Rep 3"
...
n. "Site name/code", "Date", "Rep n"

It is recommended to secure the label by covering it with a strip of sellotape to avoid risk of mislabeling.
SECTION: Protocol for collection of sediment samples
7.3. Using the 100% ethanol squeeze bottle, rinse the inside of the decontaminated box corer and capture 40-50 mL of liquid in a falcon tube labeled as "Sediment field blank".

Store the sample blank out of direct sunlight, preferably on ice and in a rack. Keep the sample upright as much as possible to prevent leakage and contamination. Note that only the blank sample will be stored using ethanol, and will have to be extracted using a separate protocol from the actual sediment samples.
SECTION: Protocol for collection of sediment samples
7.4. Deploy and retrieve the box corer. Let any seawater on top of the sediment drain off and then use a falcon tube to scoop off approximately 10 mL of sediment from the top layer of sediment (approximately 1-2 cm).
SECTION: Protocol for collection of sediment samples
7.5. Rinse with site water and redeploy for the next replicate sample.

Repeat the above steps until the intended number of replicate samples has been collected.
SECTION: Protocol for collection of sediment samples
7.6. Fill sediment tubes with 30 mL of Silica beads on top of the sediment or more if the sediment sample exceeds 10 mL (3:1).

Store tubes in a cool dark place (for short-term storage) and place in a -20°C as soon as possible (for medium-long term storage).

Record metadata (e.g. "Site name/code", "GPS", "Date", "Rep #", "time of deployment", etc.).