Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton

The overall goal of this procedure is to dissect and flat-mount the Stickleback Branchial Skeleton. This method can help answer key questions in the fields of development, genetics and evolution. Such as how animals adapt to new environments and how variation in adult morphology arises during development.

The main advantage of this technique is that by dissecting out and flat-mounting the branchial skeleton, on can visualize a variety of trophic traits that can't be studied in whole-mount. In preparation, review the relevant head skeletal morphology of the three spine stickleback. After preparing the fish for the dissection, begin by inserting sharp number five forceps into the side of the eye, at a 45 degree angle to puncture the membrane covering the eye.

Then, peel away the eye membrane, like the lid of a yogurt container. Next, insert open forceps behind the eye, grab a hold of the optic nerve, and remove the eye. Be careful to avoid puncturing the eye, or it will leak melanin.

Repeat this process with the eye on the opposite side. Then, place the blade of a small scissors under the posterior side of the opercle flap, and cut through the soft tissue dorsally above the opercle, towards the eye socket. Next cut through the frontal bone, which is dorsal to the eye socket.

Then, cut through the mid line parasphenoid bone, around the center of the eye socket. Repeat the opercle cut on the opposing side. For the next steps, the pelvic spines can be used as handles.

They fold out, by gently pulling them directly away from the body. Now, insert the forceps under the opercle, and slowly peel the face away from the body. Trimming away any soft tissue that is still attached in the process.

Be careful around the gill rakers, and take care not to disrupt them. It's easy to accidentally grab the first ceratobranchial bone, along with the percoll when removing the facial skeleton. Look under their percoll as you peel it forward to be sure you don't have a hold of anything extra.

Now, with forceps, detach the ceratohyals from both sides of the midline basihyal, while peeling away and removing the anterior cranial facial skeleton. Repeat on the opposing side. The anterior cranial facial skeleton includes the entire jaw, the entire hyoid skeleton, the underlying dorsal and endochondrial elements and the anterior part of the skull.

Next, insert closed forceps just below the gut tube, and drag them anteriorally. Teasing apart the remaining muscles and ligaments attached to the branchial skeleton in the process. Then, use the tip of the closed forceps to scrape away the muscles that attach the dorsal branchial skeleton to the ventral brain case.

Scrape with a posterior to anterior motion. Repeat the muscle removal process on the opposite side of the stickleback. Next, remove the branchial skeleton, by grasping the base of the gut tube and pulling anteriorally.

Separate the gut tube using a perpendicular cut, posterior to the fifth ceratobranchial. A common mistake is to grasp the gut tube too anterior which leaves the ventral frontal tube way behind. Be sure to grasp deep in posterior in the thoracic cavity and you can always remove the excess gut tube later.

After removing any remaining bone fragments from the brain case on the dorsal side of the branchial skeleton. Insert scissors into the branchial basket, and make a dorsal cut between the bi-lateral sets of dorsal tooth plates. Keep this cut centered to avoid causing damage to the dorsal tooth plates.

Next, make two shallow lateral cuts in the rubbery gut lumen at the posterior end of the branchial skeleton. At the antieror end of the gut tube. To assist with opening the branchial skeleton.

Now, transfer the skeleton into a microsentrifuge tube loaded with 50 percent glycerol, zero point two five percent potassium hydroxide for gentle clearing. Or instead, transfer it to pure glycerol if no further clearing is required. If re staining is needed, details are given in the text protocol.

To prepare to mount, change the solution to pure glycerol, and shake the tube for at least five minutes. Then, remove the branchial skeleton and place it near the bottom of a 22 by 60 millimeter glass cover slip with the dorsal side up. Now, transfer a few drops of glycerol onto the skeleton.

Next, roll out two small balls of modeling clay, and play one on either end of the cover slip, they function as spacers. Then, loosely place a second cover slip over the preparation, with enough pressure to flatten the anterior branchial skeleton. Now, peel open the left dorsal flap, including the dorsal tooth plates, and slide the flap between the cover slips.

Then do the same with the right dorsal flap. And push the entire branchial skeleton away from the edge of the cover slip. To complete the procedure, lightly press on the top cover slip to flatten the clay balls.

Just enough so they keep the branchial skeleton mounted flat. But, not so much that it is crushed. If during the process, a row of rakers is obscured, slide forceps between the coverslips and reorient the ceratobranchial or the entire preparation.

When flat mounted in pure glycerol, the preparation can be stored flat at room temperature for at least a decade. This protocol results in a dissected and flat-mounted branchial skeleton. Where a variety of important trophic traits can be chronified.

From a dorsal view, all rows of gill rakers, all pharyngeal tooth plates, and nearly all branchial bones can be easily visualized and chronified. Alizarin red S, also floureses on a rhodamine or similar red filter, allowing for double labeling with other markers and alternative methods for visualization. In this image, gill rakers are marked with carats.

Teeth are marked with arrow heads, and bones are marked with asterisks. Once mastered, the dissection can be done in a few minutes and be performed on hundreds of animals relatively quickly. And while we've demonstrated a stickleback branchial skeleton dissection, we hope this procedure can be used as a guide for a variety of fish species.

By dissecting out the branchial skeleton, we can quickly and easily visualize a variety of important trophic traits, not visible in whole mounts. Allowing for a better understanding of the development and evolution of the cranial facial skeleton.