Laboratory and Field Culture of Larvae of The Slipper Limpet, Crepidula fornicata

I'm interested in how the development of marine larvae is influenced by multiple environmental factors and stressors, and how the larval nervous system integrates information from the environment to regulate settlement of larvae, metamorphosis to benthic juveniles, and recruitment into adult populations. Larvae is some marine species like the slipper limpet Crepidula fornicata are quite resilient to ocean acidification, but there are important interactions between acidification and other factors like nutrition and salinity. We don't know very much about how larvae grow, develop, and behave in nature.

Field mesocosm observations can fill in some of these gaps and suggest hypotheses to test with lab manipulations. We can begin to ask on neurobehavioral, developmental, and transcriptional levels how larva deal with stressors that vary on short time scales in productive coastal environments that are especially susceptible to human impacts. To begin, stop the aeration of the jar containing the adult Crepidula fornicata.

Let the jar stand for 15 to 20 minutes to allow for debris sedimentation. Next, fix a short length of five millimeter wide glass tubing to a soft plastic aquarium tubing. Once the larvae swim up, place a sieve over a 600 milliliter glass beaker.

Then siphon the larvae through the tubing into the sieve. Lift the sieve briefly, invert it and squirt filtered seawater on the bottom of the sieve to rinse the larvae into a glass bowl with filtered seawater. To begin, drill two five millimeter holes in lid of jar.

Install a tubing barb in each hole using a 10-32 nylon nut. Apply a dab of silicone rubber aquarium sealant on the aperture of the threaded inner portion of a tubing barb. Push a 20 centimeter long, two millimeter wide polyethylene tubing through the aperture.

Allow the sealant at the end of the tubing to cure. Then trim the protruding end so that it is flush with the end of the tubing barb. To begin, place a glass bowl containing the larvae of Crepidula fornicata on the stage of a dissecting microscope.

With a Pasteur pipette, manually transfer and count the desired number of larvae into a culture jar containing filtered seawater. Feed the larvae with an appropriate volume of microalgae. Add filtered seawater to bring the culture volume to within one centimeter of the shoulder of the jar.

Screw down the lid of culture jar and connect the ventilation air supply to the barb fitting on the tubing leading to the jar's bottom. Use an aquarium air pump with common aquarium gang valves to supply ventilation with ambient air. Then adjust the air flow to yield a steady, slow stream of bubbles.

To replace the culture water, pour the contents of the jar into a sieve over a beaker. Invert the sieve over the cultured jar containing fresh filtered seawater. Then use a squirt bottle of filtered seawater to rinse the larvae into the jar.

Add some microalgae as feed for the larvae and top off the jar with filtered seawater to within one centimeter of the jar shoulder. The larvae of Crepidula fornicata appear to grow slower in the laboratory cultures relative to the mesocosm cultures. The larvae started to metamorphose by day eight and were all competent for metamorphosis by day 12.

To begin, use a handheld electric saber saw to cut four evenly spaced rectangular openings into the sides of a standard seven gallon polyethylene bucket. With a table saw, slice the cutout pieces lengthwise into two centimeter wide strips. Cut out four panels of nylon mesh to overlap the cutout openings of the bucket.

Temporarily secure one edge of a mesh panel over the long edge of a cutout with small spring clamps. Now glue the edge of the mesh panel to the bucket opening with hot glue. Once the first edge is fastened, glue down the other edges while maintaining a taut tension on the panel.

Next, trim down the ends of the cutout pieces so that they fit over the glued area of the panel. Press each covering strip in place with a liberal amount of hot melt glue. Finally, secure the end of each strip with a nylon blind rivet installed from outside the bucket.

Use a utility razor to trim the excess glue and mesh from the edges of the reinforcing strips. The larvae of Crepidula fornicata appeared to grow faster in the mesocosm relative to the laboratory cultures. The larvae metamorphosed spontaneously between the fifth and sixth day.

Almost all the larvae had metamorphosed by day seven.