This protocol describes how mussels were sourced, how they were induced to attach to standardized mica substrates, and how their byssal threads were collected and stored for mechanical testing in the PSMFC byssus pilot. The same platform design can be reused for any experiment that needs threads on a smooth, uniform surface.

1. Specimen collection

About 500 mussels (Mytilus trossulus and M. galloprovincialis) were obtained from Penn Cove Shellfish (Coupeville, WA). Mussels were taken directly from the aquaculture lines rather than from packed product, to avoid the stress of commercial harvesting and packaging. According to the shipping tags, the two lots were:

Lot (as labeled) Species Harvest location Harvested Shipped Quantity
Penn Cove mussels M. trossulus Penn Cove rafts, Whidbey Island, WA 21 Oct 2021 21 Oct 2021 5 lb
Mediterranean mussels M. galloprovincialis Totten Inlet, WA (harvest area WA-0046-SP) 20 Oct 2021 21 Oct 2021 5 lb
Rows of mussel rafts floating in Penn Cove under an overcast sky
Mussel rafts in Penn Cove, Whidbey Island.
A work boat moored beside mussel rafts
Work boat alongside the rafts.
Culture lines hanging in rows from a mussel raft
Culture lines hanging from a raft.
A culture line densely covered with mussels lifted from the water
Mussels growing on a culture line.
Penn Cove Shellfish tag: Penn Cove mussels, harvested 10/21/21 at Penn Cove Rafts, WA, 5 lb
Shipping tag, M. trossulus lot.
Penn Cove Shellfish tag: Mediterranean mussels, harvested 10/20/21 at Totten Inlet, WA, 5 lb
Shipping tag, M. galloprovincialis lot.

2. Thread production

2.1 Choosing a substrate

Mussels were induced to attach to mica. Muscovite mica is hydrophilic and cleaves into smooth, crystalline sheets, giving a reproducible surface on which differences in adhesion are not confounded by surface roughness or chemical heterogeneity. Cellulose acetate is a reasonable alternative. Metals and hydrophobic surfaces should be avoided unless they are part of the study design.

The main challenge is finding enough natural mica sheets of the right size and thickness. Search for natural “muscovite” mica sheets or tiles sold for crafts or electrical insulation, and check that they are natural rather than reconstituted. The smallest sheets I have used successfully were 2 in × 2.3 in, about 0.1 in thick.

Mica option Suitable? Notes Example listing (2022-23)
Pressed (reconstituted) mica, e.g. microwave waveguide covers No Ground mica mixed with a heat-resistant binder and pressed into shape; the surface is not crystalline. Amazon B09ZB5R4F1
Natural mica slabs sold for art and stenciling Rarely Natural mica, but usually too thick, irregular in shape, and expensive. Amazon B00161O5UI
Natural mica sheets for microwave repair No Inexpensive and natural, but cut too small. Amazon B07P4CG33S
Clear mica tiles, 2×3, 3×3, and 3×5 in, packs of 10 Yes Works; buy several sizes and trim to a uniform size. Relatively expensive. Etsy listing 820460402
Bulk natural muscovite sheets from a manufacturer Yes Cuts to size with bulk pricing; worth contacting for large or repeat orders. Alibaba listing 1600553787122

2.2 Building the attachment platforms

Each mica sheet is held on a small acrylic block that snaps onto a LEGO baseplate, so platforms can be arranged, moved, and replaced individually.

Materials

Item Specification Example listing (2022-23)
Acrylic sheet Clear acrylic (plexiglass), cut into blocks the same size as the mica sheets Amazon B0899QVSY1
LEGO-compatible bricks 2×4 bricks work best; 2×3 also work Amazon B07YN6K5M4
Tubing 1/4 in OD polyethylene tubing, cut into short pieces and slit lengthwise with a razor blade Amazon B071D9G94B
Baseplate Green LEGO baseplate (LEGO 6384599); weight it down so it does not float Amazon B09JKVKC47
Superglue Gel cyanoacrylate (Loctite Super Glue Ultra Gel Control) for bricks and tags Amazon B01EZTPXEO

Steps

  1. Cut the acrylic sheet into blocks the same size as the mica sheets.
  2. Superglue a brick to the underside of each acrylic block.
  3. Lay a mica sheet on the block and hold it in place with two pieces of slit tubing slid over opposite edges, pinching the mica against the acrylic.
  4. Snap the blocks onto the baseplate in an array. Weight the baseplate so it does not float; rubber-banding it to a tile works.
  5. Place two mussels on each platform, valve opening down, and wrap a rubber band around the platform to hold both mussels in place.
An empty green LEGO baseplate
1. Baseplate.
A red LEGO brick glued to the underside of a clear acrylic block
2. Brick glued to an acrylic block.
The acrylic block being snapped onto the baseplate
3. Block snapped onto the baseplate.
A mica sheet on the acrylic block next to two pieces of slit tubing
4. Mica sheet and two pieces of slit tubing.
Hand placing the mica sheet onto the block
5. Mica placed on the block.
Hand sliding slit tubing over the edge of the mica and acrylic
6. Tubing slid over the edge to clamp the mica.
A finished platform with tubing on two opposite edges
7. Finished platform.
Six finished platforms arranged on the baseplate
8. Platforms arrayed on the baseplate.
Pairs of mussels held on platforms with rubber bands on a baseplate
Mussels secured to platforms with rubber bands (photo from an earlier experiment using the same design).
Many baseplates with platforms arranged in a seawater table
Baseplates arrayed in a seawater table (earlier experiment).

3. Exposure and thread collection

  1. Tag each mussel. Glue a numbered wire marker (e.g. Klein Tools 56250 wire-marker book, Amazon B072SVZKQ5) to one valve with gel superglue.
  2. Expose. Hold mussels on their platforms under the treatment conditions for three days. Record the number of threads each mussel produced, and compare with the number it produced before the exposure.
  3. Harvest threads. Cut each thread at the shell. When more than one mussel attached to the same mica sheet, cut the sheet apart and label each piece with the ID of the mussel that made the threads.
  4. Store dry. Lay the mica sheets between two paper towels, press them between two pieces of cardboard, and clamp with binder clips (Amazon B074XTRX7G).
Laboratory bench with controllers, header buckets, tubing, and treatment tanks
Laboratory exposure system, October 2021.
Close-up of a mussel attached to a mica sheet by several byssal threads
A mussel attached to mica by its byssal threads.
Tagged mussels above labeled mica sheets with threads on blue paper towel
Tagged mussels and their labeled mica sheets after thread harvest.

Close-ups of adhesive plaques on mica:

Microscope view of a byssal thread and its adhesive plaque on mica
Adhesive plaque, dissecting microscope.
Microscope view of a byssal adhesive plaque spreading on mica
Adhesive plaque.
Microscope view of a single plaque with threads crossing the field
Adhesive plaque.

4. Next step: thread testing

The stored threads were pull-tested to failure by Monica Klopp between April and July 2022. Her procedure, recorded in her thread-testing notebook entries, was:

  1. Rewet each sample with seawater and photograph the plaque (AmScope) to measure its area.
  2. Grip the thread with a hemostat hung from a force gauge (Omega DFG51-2, logged in LabVIEW).
  3. Run the motor until the sheet lies flat with slack in the thread, hold the sheet down with forceps, and zero the gauge.
  4. Start logging and run the motor in the other direction to pull the thread until it fails.
  5. Record the failure mode: tearing (the thread pulls out of the plaque, or part of the plaque stays on the mica), peeling (the plaque fails asymmetrically), or adhesive (the whole plaque detaches as one unit), as defined in George and Carrington (2018).

Gene expression in the same mussels is analyzed in PSMFC byssus pilot analysis Part 1 (Tag-seq processing) and Part 2 (transcriptome assembly).