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 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 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.
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 not break up adult stacks. Seasonality of reproduction and methods for conditioning adults for spawning out of season have been previously described17. Larvae are best collected within 2-3 h after release when they are strongly geonegative and will concentrate near the surface of the jar.
2. Construction of ventilated cultures for larvae of C. fornicata
NOTE: The recommended glass jar (Table of Materials) has a polypropylene lid, which is inert to seawater and has the right thickness for fastening tubing barb inlets for a ventilating gas stream.
3. Construction of a field-deployable mesocosm culture for larvae of C. fornicata
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 15 L mesocosm containing 600 larvae, deployed in Buzzards Bay, MA, USA, under similar conditions of pH, temperature, and salinity as the lab cultures, but fed by natural phytoplankton in the ambient seawater that flowed through the mesh panels of the mesocosm. The pH measured was 7.8-8.3, temperature was 20-24 °C, and salinity was 26-28 ppt. Growth was determined as a change in shell length (Figure 3). Larvae grew faster in the mesocosm (71 µm/day) than in the laboratory cultures (54 µm/day) during the first 4-5 days after hatching (Figure 4). Between the 5th and 6th days, the larvae in the mesocosm began to metamorphose spontaneously, and the larvae that remained in the mesocosm on day 6 were, on average, 181 µm bigger than on day 5. There were no further larval measurements from the mesocosm because most individuals had metamorphosed by day 7. Larvae in the laboratory cultures grew at a much slower rate from day 4 to day 8 (41 µm/day) and from day 8 to day 13 (31 µm/day). These larvae began to become competent for metamorphosis on day 8 and were nearly all competent by day 12, as determined by stimulation of subsamples with elevated [K+], described in8. There was little spontaneous metamorphosis in the laboratory cultures (<10%) through day 13, when the experiment was terminated. Survivorship was 91%-100% in the lab cultures and was not determined in the mesocosm because of the difficulty in inspecting and recovering all individuals from the mesocosm volume and surfaces.
Figure 1: Ventilated culture setup. The figure shows 800 mL ventilated cultures containing larvae of Crepidula fornicata, stocked at a density of 1 larva/5 mL. Note a thin stream of bubbles, especially visible in jars 2 and 4. Please click here to view a larger version of this figure.
Figure 2: Field-deployable mesocosm culture setup. The figure shows 15 L mesocosm, (A) lateral view, (B) top view, and (C) setup deployed in floating rack. Please click here to view a larger version of this figure.
Figure 3: Veliger larva of Crepidula fornicata at 4 days post-hatching. Dashed line indicates axis of shell length measurement. Abbreviations: s = shell; v = velum; f = foot; o = operculum. Scale bar = 100 µm. Please click here to view a larger version of this figure.
Figure 4: Growth of larvae of Crepidula fornicata in lab and field cultures. Growth of larvae in 800 mL laboratory cultures (dashed line, open circles) and 15 L field mesocosm (solid line, filled squares). Each point represents the mean of 15 larvae from each of 4 replicate laboratory cultures or the mean of 25 larvae from a single mesocosm. Error bars are ± 1 SD. Please click here to view a larger version of this figure.
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, bright, and free of visible fouling by stalked ciliate protists or sessile pennate diatoms, which spread rapidly in crowded cultures. Shell fouling inhibits swimming and feeding, causes mortality, and is usually an indication that the density of larvae in the culture is too high.
The larval growth rates and timing of competence documented here in the ventilated laboratory cultures are similar to published results from laboratory experiments using an earlier version of this method under similar conditions of larval density, pH, temperature, salinity, and food ration13,14,16. The only substantive difference between the method described here and the one used in the latter studies is that in the present experiment, the ventilating gas stream was introduced through a 2 mm (outer diameter) tube that created a stream of bubbles from the bottom of the culture jar, rather than being passed across the headspace at the surface of the jar. The water circulation and mixing created by the bubble stream is thus not deleterious to larval growth and acquisition of competence and may have advantages for experiments requiring rapid equilibration of culture seawater to changing gas mixes, e.g., for experiments investigating effects of diel cycles in the partial pressures of CO2 and dissolved O2 such as occur in productive nearshore marine environments20,21.
Mesocosm results yielded larval growth rates in excess of any that have been published from laboratory studies, as well as the shortest time to acquisition of competence for metamorphosis22,23. Although the present results were obtained under field conditions that were clearly very favorable for larval growth and development, the method should be most informative for exploring larval performance in field sites that exhibit naturally-occurring combinations of environmental stressors, including at-risk and degraded habitats that are of interest for purposes of management and remediation24.
The authors have nothing to disclose.
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.
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 |