Surgical Removal of a Complex Sensory Organ in Highly Regenerative Ctenophores

Our lab is interested in understanding the origins and evolution of animal life history strategies. Meaning reproduction, growth and development, including regeneration. Ctenophores are excellent phylogenetically informative models for these phenomena.

We're still learning a lot about ctenophores regeneration and life history, even though people have been studying both for over a hundred years. A paper from last year showed that when two ctenophores are fused during regeneration, some of their behaviors synchronize. I showed that the dramatic changes in body shape that take place as our study species grows happen after the onset of reproduction, which is quite different from our usual ideas about metamorphosis.

To begin, adjust the bunsen burner to produce a sharp blue cone of flame. Hold the center of a glass capillary micro pipette in the flame until it softens under gentle pressure. Remove the pipette from the flame and immediately pull both ends straight apart to create a short, strong needle.

Alternatively, 26 gauge half inch needles can also be used effectively, particularly for the larger end of the focal size range. To prepare measuring pipettes, cut plastic transfer pipettes using sharp scissors or a razor blade to form openings with the preferred internal diameter. Prepare at least one pipette for the top and one for the bottom of the desired size range.

Select animals that appear morphologically normal and are of the desired size and stage. Verify that the animals do not pass through the bottom of the range sizing pipette, but fit comfortably into the top of the range sizing pipette. Use a transfer pipette wider than the animal's body to move it into a 35 millimeter polystyrene dish filled with sterile seawater.

Under a dissecting microscope, roll one animal onto its side to access the aboral organ easily. Focus the microscope on the aboral organ. Using the non-dominant hand, gently hold the animal in place with one glass needle and with the dominant hand insert the tip of a needle just below the aboral organ.

Make an oblique cut from one edge to below the base. Withdraw the needle and make a second cut to remove a wedge-shaped piece containing the aboral organ structures, examine both the animal and excise tissue to verify successful removal. Use the same transfer pipette to move the dissected animal into a new dish filled with sterile seawater.

Next, treat a standard microscope slide with a silanizing agent such as a water repellent glass treatment, and allow it to dry completely. Use a transfer pipette to place one post-surgical animal onto the prepared slide, and confirm that the water beads up with the animal inside. Place approximately 1.5 milligrams of modeling clay on each corner of the cover glass to form feet that will serve as a spacer to lift the cover glass away from the animal.

Gently place the cover glass, clay side down over the animal and roll it into position. Press gently to slightly compress and hold the animal in place. Adjusting is needed for optimal imaging.

For imaging longer than 30 minutes, apply a thin layer of petroleum jelly around the edges of the cover glass to prevent the specimen from drying. Wound closure was visibly completed within 20 minutes after aboral organ removal in cydippid stage mnemiopsis leidyi with tissue spanning the wound site that was initially open and exposing the gut. Regeneration progressed gradually with early tissue contraction evident by 60 minutes post-surgery, and full regeneration of the aboral organ observed by 72 hours.

In lobate stage cydippid stage mnemiopsis leidyi wound closure progressed steadily and was complete by 240 minutes post-surgery, with full regeneration of the aboral organ, clearly visible at 120 hours.