Field sampling of iDNA (leech) samples involves two people. One person serves as the ‘sampler’ and the other person serves as a ‘helper’. The helper can look up details in these instructions when needed, keep track of samples, handle objects that are contamination risks, serve as a second set of eyes for potential contamination, and ensure safety of the sampler in potentially hazardous field conditions.

Find two suitable sampling s within the water body representing different ecological systems, if possible.

At each sampling , leech traps should be placed in triplicate at least 10-20 meters apart from the water and sediment sample collection point, and each trap should be distant by 5 meters from each other.

After identifying sample sites in the water body, deploy the leech traps. Place a stake in the water body or flag a tree to indicate the sampling site .

Insert a piece of liver into the prepared traps, as instructed in Appendix 1. Leech Traps Construction (see Figures 1 and 2). Attach fishing line to trap and tie the remaining end around the flagged tree or stake to enable trap recovery.

Use nearby rocks to weigh the leech trap when placing it in water to ensure full submersion.

Repeat steps 4 - 6 to place traps at the second sampling in the water body.

Check traps after 2–3 hours (after completing tick/water and soil collections).

Recover traps and carefully open to expose the liver bait. Using forceps, check each side of the liver for leeches.

Leeches can gently be removed from the liver bait with tweezers and placed directly into the large (example size 500mL) pre-labeled field transport container with water collected from their original environment. Leeches can be aggregated from the three traps at the same sampling (1 OR 2) and stored in a single container with water collected from their original environment.

Place all labeled storage containers into a cooler with ice until arriving back to the lab to prevent active swimming during transport; which promotes the consumption of the blood-meal.

After collecting all leeches from the first sampling , advance to the second sampling and repeat steps 9-11.

Keep freshly collected leeches alive in glass jars which allow air passage (net-lidded or lids with multiple holes) and two-thirds filled with source water and tightly packed (so they will not spill) in a 4°C cold chain (cooler box), ensuring air movement until return to laboratory. Storage at 4°C is not recommended for long periods of time.

Once at the laboratory, if leech samples can not be processed on the same day of collection, remove from water and freeze individually at -80°C. Leeches can be stored at -80°C for up to 1 month.

Gently fold the pie pan in half until the edges meet (Figure 1A ).

Push the metal skewer through the edges to create 4 equally spaced holes to thread the ties through (Figure A1B, A1C).

Measure and cut a piece of mesh wire that fits around the folded pie pan.

Carefully sew the mesh wire around the pie pan ensuring to leave openings for liver placement and to allow leeches into the trap (Figure A2).

Cut a length of twine 3-4 m long. Tie the twine through the hole on one of the end holes. Thread through and twist tie two other holes shut, leaving one hole open (Figure A1D, A1E, and A1F).

Using rubber gloves, slip approximately 8 oz of liver (add the blood drippings too) into the cavity of the pre-prepared trap and seal the trap by threading and fixing the twist tie in the final hole.

Adjust the spaces between the tied parts of the open edge to ensure some space that allows the leeches to enter the trap (Figure A1G and A1H).

Move slightly away from the highest animal traffic area (so as not to damage the trap) and hold the trap in one hand and the end of twine in another, toss the leech trap into the water body, keeping hold of the cord.

Wait until it sinks and secure the cord of the leech trap to sturdy vegetation, or fashion ground stakes to secure.

Remove the soda bottle cap as this will not be needed for the trap (Figure A4).

Cut the bottle top just above the labeled line as shown in Figure A5.

Make holes on the bottom portion and around the bottle using an icepick or scissors as shown in Figure A6.

Place a piece of beef liver, approximately 8 oz, into the bottom of the bottle along with a medium-size rock. The rock will help sink the trap.

Invert the top of the soda bottle so it looks like a funnel and insert it into the bottom of the soda bottle as shown in Figure A7.

Using a stapler, staple the funnel into position.

Punch two holes, one at the top and one at the bottom portion of the bottle, to tie the fishing line for throwing and retrieving the trap.

Place a sewer grate onto both sides of the ‘T’ shaped drain.

Cut 6 in of the 10 ft x 4in PVC pipe using a hacksaw.

Place the 6 inch cut into the lower part of the T shaped drain. Then attach the 4 in sewer drain adapter to the other side.

Secure both the adapter and the Tee drain to the 6 in PVC section using nuts and bolts for both sides. (the image below only shows the top attached, the bottom segment will need to be attached with a bolt and nut as well).

Drill a hole using a 3/8 in drill bit through the top of the drain plug.

Push the 3/8 x 7 in eyebolt through the drain plug, then spin a 3/8 in hex bolt all the way to the top and add a second to the bottom.

Bait will be placed between these two hex bolts.

The 3/8 hex bolt might not go all the way to the top of the eyebolt and that is ok.

Attach rope to eyebolt loop.

Place a piece of beef liver, approximately 8 oz, into the bottom of the trap along with a medium-size rock. The rock will help sink the trap.

Close the trap using the latch.

Attach the rope to the trap for throwing and retrieving the trap.