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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 focus....

Protocol

1. Routine maneuvers for establishing and maintaining larval cultures of C. fornicata

NOTE: This method starts with a gallon (3.8 L) jar of seawater containing adult C. fornicata that have just released brooded veliger larvae. Adults may be field-collected or obtained from a supplier given in the Table of Materials. The adults are protandrous hermaphrodites that live in mating stacks with sessile brooding females at the bottom of the stack. Do .......

Representative Results

Larval growth and acquisition of competence for metamorphosis were measured in 4 simultaneous replicates of the 800 mL ventilated cultures, each containing 160 larvae, derived from a sibling batch of larvae that hatched from a single egg mass and that were fed Isochrysis galbana at a density of 1 x 105 cells/mL. The pH was 7.9-8.0, temperature was 20-21 °C, and salinity was 30-31 ppt. Growth and metamorphosis were also determined with a different sibling batch of larvae in a single trial of the 1.......

Discussion

Although larvae of C. fornicata are relatively easy to culture compared to other planktotrophic marine larvae, attention to fundamentals of good culture practice is still essential17,19. Healthy larvae should begin to feed immediately after hatching. This is easily verified on the day after hatching by observing their full guts, packed with algal cells, using a dissecting microscope with transillumination. Shells of healthy larvae should remain clean, br.......

Acknowledgements

Initial development of the low-volume ventilated culture system was supported in part by the National Science Foundation (CRI-OA-1416690 to Dickinson College). Dr. Lauren Mullineaux kindly provided laboratory facilities at the Woods Hole Oceanographic Institution, where the data presented for this system (Figure 4) was collected.

....

Materials

NameCompanyCatalog NumberComments
Bucket, Polyethylene, 7 gallonUS Plastic16916for mesocosm
Crepidula fornicataMarine Biological Laboratory, Marine Resources Center760adult broodstock
Hotmelt glue, Infinity Supertac 500Hotmelt.comINFINITY IM-SUPERTAC-500-12-1LBgood for bonding polyethylene
Jar, glass, 32 oz, with polypropylene lidUlineS-19316P-Wfor 800 mL ventilated cultures
Nitex mesh, 236 µmDynamic Aqua Supply Ltd.NTX236-136for mesocosm
Nut, hex, nylon, 10-32 threadHome Depot1004554441for fastening tubing barbs
Rivets, nylon, blind, 15/64" diameter, 5/32"-5/16" grip range, pack of 8NAPA auto partsBK 66528444 packs needed per mesocosm
Tubing barb 1/8" x 10-32 threadUS Plastic655932 needed per culture jar
Tubing, polyethylene, 2.08 mm ODFisher Scientific14-170-11Gfor ventilating gas stream inside culture jar
Tubing, Tygon, 1/8"x3/16"x1/32"US Plastic57810fits barbs for ventilating cultures

References

  1. Conklin, E. G. The embryology of Crepidula: a contribution to the cell lineage and early development of some marine gastropods. Gasteropods. , (1897).
  2. Henry, J. J., Collin, R., Perry, K. J.

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Crepidula FornicataSlipper LimpetLarval DevelopmentVeliger LarvaeLaboratory CultureField CultureGrowth RateMetamorphosisIsochrysis GalbanaMesocosm

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