Proc. Nat. Acad. Sci. USAVol. 71, No. 9, pp. 3654-3658, September 1974

Metamorphosis of Aplysia californica in Laboratory Culture(development/neurobiology/behavior)

ARNOLD R. KRIEGSTEIN, VINCENT CASTELLUCCI, AND ERIC R. KANDEL

Department of Neurobiology and Behavior, The Public Health Research Institute of the City of New York, 455 First Avenue,New York, N.Y. 10016; and The Departments of Physiology and Psychiatry, New York University School of Medicine,550 First Avenue, New York, N.Y. 10016

Contributed by Eric R. Kdndel, July 8, 1974

ABSTRACT To utilize the advantages offered by thelarge identified nerve cells of the marine mollusc Aplysiacalifornica for cellular biological studies of development,we have devised simple techniques for growing this speciesin the laboratory in large number with a generation timeas short as nineteen weeks. We have used the culturedanimals to study the life cycle from fertilized egg to re-productive adult. The major developmental and behavioralchanges occur at metamorphosis, when the larvae settleon the seaweed Laurencia pacifica and the locomotor andfeeding behaviors are transformed into their adult forms.We have examined the timetable for the abandonment oflarval behaviors and the emergence of adult ones andfound that the transition from swimming to crawlingoccurs first and marks the onset of metamorpbosis. Thechange from ciliary feeding to radular feeding occurs laterand signals the end of metamorphosis. Other adult be-haviors, such as the reflex responses and fixed-action pat-terns of the mantle organs, appear after metamorphosis.

The large nerve cells in the marine gastropod mollusc Aplysiaare useful for studying the cellular biology of identified neu-rons and for relating individual cells to behavior (1-3). Thesefeatures could also be advantageous for studying the develop-ment of the nervous system and of the neural control of be-havior. It has not been possible to use Aplysia for develop-mental studies, however, because the animal could not besuccessfully cultured through metamorphosis. In this paperwe describe techniques for culturing the most extensivelyused species: Aplysia californica.* Since each animal laysmore than 105 eggs, and mortality is low with these tech-niques, an almost unlimited number of larvae can be culturedthrough metamorphosis and inbred strains can be established.With modifications, these techniques may be applicable toother Aplysia species and to related opisthobranchs used inneurobiological studies, such as Navanax, Hermissenda, andPleurobranchaea.We also describe the development of Aplysia californica

from fertilized egg to reproductive adult. The most dramaticchanges occur at metamorphosis. Particularly instructive foranalyzing the life cycle are the changes in locomotion whichcharacterize the onset of metamorphosis and the changes infeeding which mark its termination. We have examined thesetransformations and provide a timetable for the transition ofthis behavioral repertoire from larva to adult. This timetableprovides normative data for subsequent studies on the de-velopment of the nervous system.

* Four animals have previously been brought through meta-morphosis to reproductive maturity in the laboratories of thePacific Bio-Marine Supply Co., Venice, Calif. (James Vallee,personal communication). Although encouraging, the tech-niques have not yielded reproducible results.

RESULTSMethods of laboratory culture

We collected egg masses from four sexually mature adults(obtained from Dr. Rimmon C. Fay of the Pacific Bio-Marine Supply Co., Venice, Calif.), kept in a holding tank ofaerated sea water (from the Marine Biological Laboratory,Woods Hole, Mass.), and fed daily with sea lettuce (Ulvasp.). Animals were induced to lay eggs throughout the yearby keeping the temperature at 180 (4).Egg masses were transferred into 1-liter glass chambers

containing aerated, filtered sea water (Millipore pre-filter;Cat. no AP 2504700) which was changed daily and kept atroom temperature (220). The egg masses were screened with adissecting microscope at 8 days and discarded if embryosfailed to show normal rotatory movements (5). Only one outof 10 egg masses failed to develop. Hatched larvae (veligers,135 ,um) were siphoned into two stacked plastic filter tubeseach 10 cm long and 5 cm in diameter and each containing aNitex mesh (E. M. Mfg. and Instrument Co., Nanuet, N.Y.)at the bottom. The veligers passed through the upper 270-,ummesh Nitex filter, which retained debris, and were collected inthe lower 41-/Am Nitex filter tube. At all stages, caution wasexercised to prevent veligers from being trapped at the air-water interface. Veligers were, therefore, kept submerged andrinsed by adding sea water to the lower filter tube with awash bottle and then poured into a 4-liter jar of filtered seawater to a density of 100 veligers/liter. The jar was filled tothe top and covered with a 1/16 inch thick plastic lid to ex-clude air (D. Leibowitz, in preparation). The lid was coveredwith plastic wrap and secured with a rubber band to preventevaporation at the seal. The process of filtering the sea waterthrough the Millipore filter under partial vacuum de-aeratedthe sea water and eliminated bubble formation in the cham-bers. The water was changed every 2 days and the unicellularalga Isochrysis galbana was added as food at a concentrationof 104 cells per ml. Isochrysis was cultured in enriched mediaat -18° under continuous light (6, 7).To avoid exposing the veligers to air, they were transferred

to fresh solution through a siphon composed of two funnelswith a Nitex mesh filter (41 MAm) clamped between them (D.Leibowitz, in preparation). The veligers were first collectedby siphoning the medium containing them through the doublefunnel. The larvae were retained by the Nitex filter and re-mained in the proximal funnel, where they could be washedby siphoning fresh sea water through the funnel in the same

direction. They were transferred to a new jar by siphoningfresh sea water through the filter in the opposite direction.When the veligers showed external signs of readiness to meta-morphose (e.g., the appearance of the propodium) they were

3654

Metamorphosis of Aplysia californica 3655

transferred in groups of 10 into dishes (diameter 50 mm Xheight 12 mm; Seal tight; Falcon) containing two to threetwigs (1 cm long) of the triggering substance, the red seaweedLaurencia pacifica (Pacific Bio-Marine Supply Co.). Thedishes were then sealed.

Laurencia pacifica was kept in filtered sea water at roomtemperature under constant illumination. New sprouts de-veloped in 4-5 days and these growing tips were fed to theyoung juveniles. The metamorphosing animals could bereadily observed inside the sealed dishes with a dissectingmicroscope. Animals were transferred each day by pasteurpipette to fresh medium. Pieces of Laurencia remaining ingood condition were also transferred; discolored or partlyeaten pieces were replaced. One to 2 weeks after metamorpho-sis, the animals were transferred into plastic containers (di-ameter 10 cm X height 7 cm) with 210-,gm mesh Nitex screen-ing covering the top and bottom, and were immersed in 20-liter aquaria where the water could be aerated without dis-turbing the small growing animals.When the juveniles grew to 1 cm in length, they were placed

in larger (20 cm X 10 cm X 10 cm) aerated, uncovered con-tainers and fed red seaweed: Laurencia sp., Polysyphonia sp.,or Daysia sp., depending on availability. Older animals couldbe fed on dried Porphyra (Laver, Vega Trading Co., N.Y.).

Delay of metamorphosis and survival rate

Metamorphosis can occur as early as 34 days after hatching,but can also be delayed. Approximately 90 veligers that hadbecome competent to metamorphose (34 days after hatchingand 4 days after the appearance of the propodium) were main-tained in culture for 4 additional weeks without exposure tothe triggering seaweed. The veligers did not develop further,their behavior did not change, and five to ten died. Theanimals were then presented with Laurencia; 85% settledand metamorphosed.These simple culturing techniques gave highly reproducible

results. Larvae were resistant to infection even in the absenceof antibiotics. We have now grown large numbers of indi-viduals from eight different egg masses with 70% survivalduring metamorphosis. Offspring of some of these animalshave also been reared through metamorphosis to reproductivematurity.

The life cycle of Aplysia californica

Pre-hatching Development-Aplysia is a hermaphroditicherbivore that normally grazes and lays egg masses on sea-weed and rocks in sublittoral and intertidal regions (8, 9).The cell lineage of the early stages and development up tohatching have been described at the beginning of this centuryby Carazzi (10) and Saunders and Poole (5). Movements ofthe embryos inside the egg capsules can be seen 4-5 days afterthe eggs are laid and spontaneous hatching occurs 5-6 dayslater (Fig. 1).

Post-hatching Development-On the basis of behavioral andmorphological criteria, the post-hatching development ofAplysia can be divided into three phases: planktonic, meta-morphic, and juvenile (Fig. 1). The growth rate of Aplysiais temperature dependent. At room temperature, the plank-tonic phase lasts a minimum of 34 days, during which themaximum diameter of the veliger shell increases from anaverage of 135 /M to an average of 400 MM. External changesinclude enlargement of the velar lobes, modification of theshell, and development of the eyes and the propodium (fordetails of staging, see Kriegstein, in preparation). Veliger

growth reaches a plateau after approximately 25 days, withno further increase in size until after metamorphosis. At theend of the planktonic phase (day 34), the larvae settle on theseaweed Laurencia pacifica and metamorphose. The meta-morphic phase lasts about 3 days; during which the overallsize of the animals does not change. At the end of the meta-morphic phase, animals begin eating seaweed and becomejuveniles. Gradually the mantle shelf grows over the shell, andparapodia, siphon, gill, rhinophores, and anterior tentaclesappear. Young juveniles acquire a red coloring which laterdevelops into a mottled brown. At first, juvenile growth islogarithmic, animals doubling their weight every 10 days, but90 days after hatching, growth tapers off (Fig. 1). Reproduc-tive maturity is reached at approximately 120 days afterhatching.Behavioral transformations at metamorphosisThe life cycle of Aplysia is punctuated by a number of be-havioral changes, the most striking of which occur at meta-morphosis. The onset of metamorphosis results in a transitionin locomotor behavior from swimming to crawling, and thecompletion of metamorphosis involves the assumption ofadult feeding behavior. We have examined the invariant as-pects of the transformations of these two behaviors quali-tatively, emphasizing the timetable for their emergence inadult forms (Fig. 2). These observations are based on cul-tures from eight different egg masses reared through meta-morphosis, each containing about 100 animals. The timetabledescribes the minimum time required for these transforma-tions.

Transformation of the Locomotor Pattern-Prior to meta-morphosis, Aplysia veligers swim by means of the coordinatedbeating of long velar cilia. Swimming is occasionally inter-rupted by a withdrawal response whereby a veliger retractsits velar lobes and foot into its shell. Deprived of the actionof locomotor cilia, the veliger sinks before resuming ciliaryswimming (Fig. 3Alb). The first sign of a transformation inthe swimming pattern becomes evident about 30 days afterhatching when an extension of the foot, the propodium, de-velops. When the propodium develops, a veliger has the abilityto crawl but continues to spend most of its time swimming(Fig. 1, 34 days and Fig. 3A1).Aplysia, like many molluscs, initiates metamorphosis by

settling on a particular triggering substance (11-13). When aveliger competent to metamorphose (34 days after hatching),is exposed to a small piece of seaweed, Laurencia pacifica,it usually adheres to it and begins crawling. By the end of 1hr the veliger generally stops crawling and attaches itself tothe seaweed by its metapodium. The velum and propodiumare both retracted and the operculum is partly closed. Unlessdisturbed, the veliger remains in this position during the earlystages of metamorphosis. Veligers were found to select aspecific alga for metamorphosis. When exposed to six dif-ferent algae (Plocamium sp., Laurencia sp., Polysiphonia sp.,Daysia sp., Chondrus sp., or Ulva sp.), veligers crawled uponLaurencia preferentially and only rarely upon any of theothers. The veligers only metamorphosed on Laurencia.tWithin 3 hr after settlement, the veligers shed their long

velar cilia, signalling the beginning of metamorphosis. After a

t Because very small Aplysia californica congregate on Laurenciapacifica in the wild (14), this seaweed is almost certainly a trig-gering substance in nature. However, we cannot exclude thepossibility that seaweeds we have not tested can serve a similarfunction.

Proc. Nat. Acad. Sci. USA 71 (1974)

3656 Physiology: Kriegstein et al.

SIPHON PARAPODIA

20

0-.-2-

mz -r)I--

0341 DAYS

.2 -

34 DAYS 37 DAYS

PLANKTONIC .02=SEA WATER - 22°

0 20 40 60 80 DO 120 140

500

15

C)-4

.50

.015

I mm AGE(DAYS)FIG. 1. Life cycle and growth of Aplysia californica. Adult depositing an egg mass consisting of a long string of egg capsules, each

containing 15 to 20 eggs. Graph of animals' weight (0) and length (-) as a function of age (time after hatching). Each point of the graphis the average of five measurements. Maximum shell diameter was used to measure the length of veligers and extended body length tomeasure juveniles. Post-hatching development can be divided into three major phases: the planktonic phase (days 1-34), the meta-morphic phase (days 34-37), and the juvenile phase (day 37 to sexual maturity at approximately day 120). External features of animalsfrom each of these phases are illustrated in the drawings on the left. Drawings are to the same scale, with the exception of the 60-dayjuvenile.

few more hours, the velar lobes begin to shrink. Once the velarcilia are shed, the larvae can only crawl; they can no longerswim. The disappearance of the velum results in obligatorycrawling and indicates the end of the planktonic phase andthe beginning of the metamorphic phase (Fig. 3A2).The adult locomotor pattern develops gradually. At first,

crawling depends on the action of pedal cilia as well as onmuscular contraction. The contribution of integrated pedalwaves, characteristic of the adult, increases progressivelyduring the early juvenile phase.

Transformation of the Feeding Pattern-Unlike locomotion,the adult feeding behavior does not emerge until metamorpho-sis is complete. Prior to metamorphosis, veligers feed by

DAYS 0

sweeping algal cells into their mouths, using thle water cur-rents generated by velar and subvelar cilia (Fig. 3B1; for othergastropod veligers, see refs. 15 and 16). After settlementon Laurencia pacifica, which is destined to serve as their dietafter metamorphosis, veligers stop eating unicellular algaeand do not feed until metamorphosis is complete. Three daysafter settlement (as early as 37 days post-hatching), the meta-morphosed animals resume eating by feeding on the growingtips of Laurencia (Fig. 2).Adult feeding behavior consists of two components: an

appetitive component where the animal rears up its head andwaves it from side to side orienting toward the seaweed;and a consummatory component involving an all-or-none

JUVENILE

50 60

LOCOMOTION

FEEDING _SlllllllllCIUARYFIG. 2. Timetable for the transition of locomotor and feeding behaviors from larval to adult form. This table describes the minimal

time necessary for the behavioral changes at 220. Whereas larval and adult locomotor behaviors overlap at the onset of metamorphosis,the termination of ciliary feeding is separated from the onset of radular (adult) feeding by 3 days. During these 3 days of metamorphosis,the animal does not feed. The time scale begins at hatching and extends to adulthood.

ANTERIORTENTACLE

DAY 34 DAYS

PLANKTONIC METAMORPHIC

10 2) 30 40

Proc. Nat. Acad. Sci. USA 71 (1974)

L\\\\ Ell RADULAR

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Metamorphosis of Aplysic californica 3657

PLANKTONIC30 DAYS

VJLUM M

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METAMORPHICLOCOMOTION

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34 DAYS

ANTERIOR TENTACLE

C H U M~~~~u

FEEDING APPETMVE 35 DAYS

30 DAYSo o a

bi PRECURSOR CONSUMMATORY 35-36 DAYS0

0

To

b2 DEFINMVE CONSUMMATORY 37 DAYS

200 jlm

FIG. 3. Changes in locomotion and feeding at metamorphosis. (A) Locomotion. (Al.) Veliger swimming by means of velar cilia. (Aib)Veliger withdrawal response. The veliger stops swimming and retracts its velar lobes and foot into the shell. Deprived of the action oflocomotor cilia, the veliger first sinks before resuming ciliary swimming. (A2) Metamorphic animal (34 days) crawling. The velar ciliahave been shed, the velar lobes have shrunk, and the larval propodium and metapodium have developed into a crawling foot. (B) Feeding.(B1) Veliger feeding on unicellular algae and diatoms; these are swept into the mouth by velar cilia. (B2.) The appetitive response-Afterthe first day of metamorphosis (day 35) an animal will exhibit the essential features of the appetitive feeding behavior. It anchors itsmetapodium on a substrate and waves its head and body from side to side in an orienting fashion. (B2bt) Precursor consummatory response

-During the first and second days of metamorphosis (days 35-36) the animal only partially protracts and retracts the buccal mass

but does not ingest food. (B2b2) Definitive consummatory response-On day 37 the metamorphosed animal exhibits full consummatorybehavior and ingests pieces of seaweed. All animals are drawn to the same scale.

ingestive movement of the buccal mass, radula, and esoph-agus (for discussion see ref. 17). The two components ofthe feeding behavior develop sequentially. The appetitiveresponse emerges first. Even before metamorphosis, on day 32,a veliger will wave its propodium from side to side, apparentlysearching while crawling. Once the anterior tentacles takeshape, halfway through the metamorphosis, an animal willexhibit all the essential elements of the appetitive response.

If removed from the seaweed, it will anchor the posteriorsegment of its foot and rear up its propodium and head,swaying its body from side to side (Fig. 3B25). At this same

stage, an animal will exhibit only incomplete movements ofthe consummatory response. When anchored to the seaweed,

it will rotate its radula and buccal mass in excursions thatresemble a consummatory response, but the response is partialand the animal will never bite or swallow seaweed (Fig. 3B2b1).The full consummatory response-biting and swallowing-

does not appear until 1 day later (day 37) when metamorphosisis complete (Fig. 3B2b2). A metamorphosed animal exhibitsfully integrated adult feeding behavior: the appetitive com-

ponent in which it locates food, followed by the full consum-

matory response.

DISCUSSION

The Laboratory Culture of Aplysia californica. Culture of-Aplysia in the laboratory can supply an unlimited stock of a

A

B

Proc. Nat. Acad. Sci. USA 71 (1974)

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To To

3658 Physiology: Kriegstein et al.

species that is in great demand and in danger of depletion at.collection sites. Although we have used sea water and freshseaweed, the culturing techniques are simple and we haveused them effectively in an inland laboratory. In the future itshould be possible to culture the necessary algae and to sub-stitute artificial for natural sea water.The culturing of Aplysia allows the study of neural and

behavioral development in terms of well-studied identifiedcells with known interconnections and behavioral functions(1, 3). It may now be possible to examine the differentiationof the electrical and biochemical properties of specific identi-fied cells of known behavioral function, and to study themorphological and physiological correlates of synapse for-mation between them. This type of program is particularlysuitable for Aplysia because its egg is mosaic; defined regionsdevelop into specific parts of the embryo (10, 5). The egg isalso determinate. The cells of the blastula assume charac-teristic shapes or positions and are, therefore, identifiable.As a result, one might be able to trace the lineage of the vari-ous identified nerve cells from specific cells in the blastula totheir fully differentiated form.

Although its generation time-formerly thought to be 1year (8)-is still long (19 weeks), Aplysia nonetheless could beuseful for certain genetic studies of behavior. Aplysia is herma-phroditic and may be amenable to autofertilization (18), whichwould allow one to generate inbred strains within five genera-tions (24 months). The animals that have short generationtimes and are more useful for behavioral genetics (e.g., nema-tode worms, fruit flies, crickets) are not as favorable for cellu-lar neurobiological studies because of the small size of theirnerve cells. Aplysia could, therefore, be a good compromise forselected problems.

Sequence in the Development of an Adult Behavioral Reper-toire. Since Aplysia larvae are free-swimming and transparentup to the early juvenile stage, it is possible to observe internalmovements and to study the sequence in which different adultbehaviors appear. We have found that the adult locomotorpattern begins to develop before the adult feeding pattern.Other adult behaviors (3), such as the reflex responses andfixed-action patterns of the mantle organs, siphon, gill, andmantle shelf, appear only later. This order of developmentmay provide clues to the sequence with which the criticalneuronal controls develop. With feeding, we have also exam-ined the sequence in which the appetitive and consummatorycomponents appear. We found that the appetitive behaviorappears first and is complete before the consummatory be-havior-develops. We also explored whether the behaviors ofthe adult repertoire appear in their definitive forms or developthrough a series of preliminary steps before culminating in thefully developed behavior. Our results suggest that some be-haviors develop gradually. For example, the consummatoryfeeding response undergoes a well-defined precursor stagebefore developing into the stereotyped adult response. Itwould be interesting to see whether the precursor movementsare a necessary pre-requisite for proper development of theadult response.

Interaction Between Environmental and Inborn Processes inthe Development of Aplysia. Perhaps the most remarkablefeature of the development of Aplysia and other invertebrates

having an obligatory planktonic larval phase is how meta-morphosis and development depend on behavior. In lowervertebrates and some invertebrates, metamorphosis involvesthe response of the organism to internal hormonal eventsthat do not require an external triggering factor (19). InAplysia, a behavioral step is interposed in the developmentalprocess. Once competent to metamorphose, Aplysia larvaemust find and settle upon an external triggering substance, aspecific species of seaweed. This external triggering factor inturn sets off a series of internal metamorphic events, includingchanges that permit the animal to assume adult behaviors.Thus, the life cycles of Aplysia and related opisthobranchs(11, 12) provide an opportunity for studying how externaltriggering factors and internal processes interact in deter-mining the development of the nervous system and of be-havior. It would, therefore, be interesting to identify thecritical ingredient in Laurencia pacifica that triggers meta-morphosis and to learn how it is recognized by the animal andhow it initiates the changes characteristic of metamorphosis.

We thank Lise Castellucci for participating in the early phasesof this work; David Leibowitz for assistance in developing tech-niques for rearing the larvae; Elizabeth Taylor and especiallyDoris Bull for technical assistance; Kathrin Hilten for help withthe illustrations; and Drs. L. Provasoli, R. Guillard, and J. L.Culliney for their advice on culture techniques. We also bene-fitted from critical comments by Drs. T. J. Carew, I. Kupfermann,J. H. Schwartz, and W. A. Spencer on an earlier draft of thismanuscript. This research was supported by an NIH TrainingGrant from the National Institute of General Medical Sciences5-TO5-GMO 1668 to A.R.K., Research Career DevelopmentAward 5KO4 NS 70346-02 to V.C., Research Scientist AwardMH 18558 to E.R.K., and Research Grants NS-09361 andMH-19795.

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Proc. Nat. Acad. Sci. USA 71 (1974)