Name: Author Spotlight: Bridging the Gap Between Field Observations and Lab Manipulations in Larval Ecology Research
Uploaded: 2024-01-05T14:40:00
Description: Read this Scientific Article Protocol about Laboratory and Field Culture of Larvae of The Slipper Limpet, Crepidula fornicata at JoVE.com

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.

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 ...

Culturing Larvae of Crepidula fornicata in Laboratory and Field Conditions

<ol>
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K., Pires, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+correlates+of+settlement+in+veliger+larvae+of+the+gastropod,+Crepidula+fornicata.">Neural correlates of settlement in veliger larvae of the gastropod, Crepidula fornicata.</a> Invertebrate Biol. 132 (1), 14-26 (2013).</li><li>Meyer-Kaiser, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carryover+effects+of+brooding+conditions+on+larvae+in+the+slipper+limpet+Crepidula+fornicata.">Carryover effects of brooding conditions on larvae in the slipper limpet Crepidula fornicata.</a> Mar Ecol Prog Ser. 643, 87-97 (2020).</li><li>Noisette, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+encapsulation+protect+embryos+from+the+effects+of+ocean+acidification?+The+example+of+Crepidula+fornicata.">Does encapsulation protect embryos from the effects of ocean acidification? The example of Crepidula fornicata.</a> PLoS One. 9 (3), e93021 (2014).</li><li>Kriefall, N. G., Pechenik, J. A., Pires, A., Davies, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resilience+of+Atlantic+slippersnail+Crepidula+fornicata+larvae+in+the+face+of+severe+coastal+acidification.">Resilience of Atlantic slippersnail Crepidula fornicata larvae in the face of severe coastal acidification.</a> Front in Mar Sci. 5, 312 (2018).</li><li>Bogan, S. N., McMahon, J. B., Pechenik, J. A., Pires, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Legacy+of+multiple+stressors:+Responses+of+gastropod+larvae+and+juveniles+to+ocean+acidification+and+nutrition.">Legacy of multiple stressors: Responses of gastropod larvae and juveniles to ocean acidification and nutrition.</a> Biol Bull. 236 (3), 159-173 (2019).</li><li>Pechenik, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+ocean+acidification+on+growth,+onset+of+competence,+and+perception+of+cues+for+metamorphosis+in+larvae+of+the+slippershell+snail,+Crepidula+fornicata.">Impact of ocean acidification on growth, onset of competence, and perception of cues for metamorphosis in larvae of the slippershell snail, Crepidula fornicata.</a> Mar Biol. 166, 128 (2019).</li><li>Reyes, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+marine+gastropod+Crepidula+fornicata+remains+resilient+to+ocean+acidification+across+two+life+history+stages.">The marine gastropod Crepidula fornicata remains resilient to ocean acidification across two life history stages.</a> Front Physiol. 12, 702864 (2021).</li><li>Pires, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+seawater+culture+of+the+gastropod+Crepidula+fornicata+for+studies+of+larval+settlement+and+metamorphosis.+In:+Carroll,+D.,+Stricker,+S.+(eds)+Developmental+biology+of+the+sea+urchin+and+other+marine+invertebrates.">Artificial seawater culture of the gastropod Crepidula fornicata for studies of larval settlement and metamorphosis. In: Carroll, D., Stricker, S. (eds) Developmental biology of the sea urchin and other marine invertebrates.</a> Methods Mol Biol. 1128, 35-44 (2014).</li><li>Bashevkin, S. M., Pechenik, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interactive+influence+of+temperature+and+salinity+on+larval+and+juvenile+growth+in+the+gastropod+Crepidula+fornicata+(L.).">The interactive influence of temperature and salinity on larval and juvenile growth in the gastropod Crepidula fornicata (L.).</a> J Exp Mar Biol Ecol. 470, 78-91 (2015).</li><li>Strathmann, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Reproduction and Development of Marine Invertebrates of the Northern Pacific Coast: Data and Methods for the Study of Eggs, Embryos, and Larvae. , (1987).</li><li>Clark, H. R., Gobler, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diurnal+fluctuations+in+CO2+and+dissolved+oxygen+concentrations+do+not+provide+a+refuge+from+hypoxia+and+acidification+for+early-life-stage+bivalves.">Diurnal fluctuations in CO2 and dissolved oxygen concentrations do not provide a refuge from hypoxia and acidification for early-life-stage bivalves.</a> Mar Ecol Prog Ser. 558, 43114 (2016).</li><li>Gobler, C. J., Baumann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+and+acidification+in+ocean+ecosystems:+coupled+dynamics+and+effects+on+marine+life.">Hypoxia and acidification in ocean ecosystems: coupled dynamics and effects on marine life.</a> Biol Lett. 12 (5), 20150976 (2016).</li><li>Pechenik, J. A., Hilbish, T. J., Eyster, L. 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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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bucket, Polyethylene, 7 gallon</td>
			<td>US Plastic</td>
			<td>16916</td>
			<td>for mesocosm</td>
		</tr>
		<tr>
			<td>Crepidula fornicata</td>
			<td>Marine Biological Laboratory, Marine Resources Center</td>
			<td>760</td>
			<td>adult broodstock</td>
		</tr>
		<tr>
			<td>Hotmelt glue, Infinity Supertac 500</td>
			<td>Hotmelt.com</td>
			<td>INFINITY IM-SUPERTAC-500-12-1LB</td>
			<td>good for bonding polyethylene</td>
		</tr>
		<tr>
			<td>Jar, glass, 32 oz, with polypropylene lid</td>
			<td>Uline</td>
			<td>S-19316P-W</td>
			<td>for 800 mL ventilated cultures</td>
		</tr>
		<tr>
			<td>Nitex mesh, 236 &#181;m</td>
			<td>Dynamic Aqua Supply Ltd.</td>
			<td>NTX236-136</td>
			<td>for mesocosm</td>
		</tr>
		<tr>
			<td>Nut, hex, nylon, 10-32 thread</td>
			<td>Home Depot</td>
			<td>1004554441</td>
			<td>for fastening tubing barbs</td>
		</tr>
		<tr>
			<td>Rivets, nylon, blind, 15/64&#34; diameter, 5/32&#34;-5/16&#34; grip range, pack of 8</td>
			<td>NAPA auto parts</td>
			<td>BK 6652844</td>
			<td>4 packs needed per mesocosm</td>
		</tr>
		<tr>
			<td>Tubing barb 1/8&#34; x 10-32 thread</td>
			<td>US Plastic</td>
			<td>65593</td>
			<td>2 needed per culture jar</td>
		</tr>
		<tr>
			<td>Tubing, polyethylene, 2.08 mm OD</td>
			<td>Fisher Scientific</td>
			<td>14-170-11G</td>
			<td>for ventilating gas stream inside culture jar</td>
		</tr>
		<tr>
			<td>Tubing, Tygon, 1/8&#34;x3/16&#34;x1/32&#34;</td>
			<td>US Plastic</td>
			<td>57810</td>
			<td>fits barbs for ventilating cultures</td>
		</tr>
	</tbody>
</table>

laboratory and field culture of larvae of the slipper limpet, crepidula fornicata

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.

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.

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 &#176;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...

Read this Scientific Article Protocol about Laboratory and Field Culture of Larvae of The Slipper Limpet, Crepidula fornicata at JoVE.com

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 ...