construction of ventilated cultures for larvae of crepidula fornicata

Culturing Larvae of &lt;em&gt;Crepidula fornicata&lt;/em&gt; in Laboratory and Field Conditions

The slipper limpet, Crepidula fornicata (Gastropoda: Calyptraeidae), is well-represented in current and historical research literature because of its utility as a developmental model and because of its widespread impacts as an invasive species. It served as a foundational example of spiralian development in the classic age of experimental embryology1 and has experienced a rebirth of interest with the application of modern imaging and genomic tools to dissect mechanisms of lophotrochozoan early development2,3. At the other end of its life history, other investigations have focused on the impacts of adult populations of this ecosystem engineer in temperate coastal marine environments far removed from its original distribution in eastern North America4,5. In between embryo and adult, the veliger larvae of this species have been subjects of numerous studies of larval development and ecology, especially of factors influencing growth and acquisition of competence for metamorphosis, the internal and external cues mediating larval settlement, and the effects of larval experience on juvenile performance6,7,8,9,10,11. Recent studies have revealed the resilience of larvae and juveniles of C. fornicata to ocean acidification, yet another avenue for productive research use of this animal12,13,14,15,16.
An advantage of C. fornicata for studies of marine larval biology is that it is relatively easy to grow in the laboratory in natural or artificial seawater on a unialgal diet of the flagellate Isochrysis galbana. Culture methods have been detailed by the author in an earlier methods-focused print publication17. The reasons for the present contribution are twofold. First, the routine physical maneuvers involved in establishing and caring for cultures are conceptually very simple but difficult to perform correctly without hands-on or video demonstration. Second, two variations on previously described culture methods are described that are especially suited to laboratory and field studies of responses to environmental stressors such as ocean acidification, eutrophication, and oxygen depletion. The first of these is a low-volume (800 mL) culture system suited for manipulation of pH and dissolved oxygen in seawater via small volumes of bubbled gases, and the second is a larger volume (15 L) mesocosm system that can be placed in the field and that allows free exchange of ambient seawater.

Author Spotlight: Bridging the Gap Between Field Observations and Lab Manipulations in Larval Ecology Research

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 of 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 larvae deal with stressors that vary on short time scales in productive coastal environments that are especially susceptible to human impacts.

Construction of Ventilated Cultures for Larvae of Crepidula fornicata

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.

construction-of-ventilated-cultures-for-larvae-of-crepidula-fornicata

Research

JoVE Journal

Biology

The calyptraeid gastropod mollusk, Crepidula fornicata, has been widely used for studies of larval developmental biology, physiology, and ecology. Brooded veliger larvae of this species were collected by siphoning onto a sieve after natural release by adults, distributed into the culture at a density of 200/L, and fed with Isochrysis galbana (strain T-ISO) at 1 x 105 cells/mL. Shell growth and acquisition of competence for metamorphosis were documented for sibling larvae reared in ventilated 800 mL cultures designed for equilibration to ambient air or to defined atmospheric gas mixtures. Contrasting with these laboratory culture conditions; growth and competence data were also collected for larvae reared in a 15 L flow-through ambient seawater mesocosm located in a field population of reproductive adults. Growth rates and timing of metamorphic competence in the laboratory cultures were similar to those reported in previously published studies. Larvae reared in the field mesocosm grew much faster and metamorphosed sooner than reported for any laboratory studies. Together, these methods are suited for exploring larval development under predetermined controlled conditions in the laboratory as well as under naturally occurring conditions in the field.

The paper presents methods for larval culture of the gastropod Crepidula fornicata in a small-volume laboratory-scale system and in an ambient-seawater mesocosm system that can be deployed in the field.

The calyptraeid gastropod mollusk, Crepidula fornicata, has been widely used for studies of larval developmental biology, physiology, and ecology. Brooded ...

Collection of Larvae of Crepidula fornicata for Laboratory and Field Cultures

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 sea water on the bottom of the sieve to rinse the larvae into a glass bowl with filtered sea water.

Setup and Routine Maintenance of Laboratory Cultures of Crepidula fornicata

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 sea water. Feed the larvae with an appropriate volume of microalgae.Add filtered sea water 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 culture jar containing fresh, filtered sea water, then use a squirt bottle of filtered sea water to rinse the larvae into the jar.Add some microalgae as feed for the larvae and top off the jar with filtered sea water to within one centimeter of the jar's shoulder. The larvae of Crepidula fornicata appear to grow slower in the laboratory cultures relative to the mesocosm cultures. The larvae started to metamorphos by day eight, and were all competent for metamorphosis by day 12.

Field-Deployable Mesocosm Culture of the Larvae of Crepidula fornicata

To begin, use a handheld electric saber saw to cut four evenly spaced rectangular openings into the sides of a standard 7 gallon polyethylene bucket. With a table saw, slice the cutout pieces lengthwise into 2 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 top 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 metamorphose spontaneously between the fifth and sixth day.Almost all the larvae had metamorphosed by day seven.