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

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 development^2,3. 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 .......

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 essential^17,19. 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.......

Materials

Name	Company	Catalog Number	Comments
Bucket, Polyethylene, 7 gallon	US Plastic	16916	for mesocosm
Crepidula fornicata	Marine Biological Laboratory, Marine Resources Center	760	adult broodstock
Hotmelt glue, Infinity Supertac 500	Hotmelt.com	INFINITY IM-SUPERTAC-500-12-1LB	good for bonding polyethylene
Jar, glass, 32 oz, with polypropylene lid	Uline	S-19316P-W	for 800 mL ventilated cultures
Nitex mesh, 236 µm	Dynamic Aqua Supply Ltd.	NTX236-136	for mesocosm
Nut, hex, nylon, 10-32 thread	Home Depot	1004554441	for fastening tubing barbs
Rivets, nylon, blind, 15/64" diameter, 5/32"-5/16" grip range, pack of 8	NAPA auto parts	BK 6652844	4 packs needed per mesocosm
Tubing barb 1/8" x 10-32 thread	US Plastic	65593	2 needed per culture jar
Tubing, polyethylene, 2.08 mm OD	Fisher Scientific	14-170-11G	for ventilating gas stream inside culture jar
Tubing, Tygon, 1/8"x3/16"x1/32"	US Plastic	57810	fits barbs for ventilating cultures