Using KCl to determine size at competence for larvae of the marine gastropod Crepidula fornicata (L.)

J. Exp. Mar. BioL EcoL, 1987, Vol. 112, pp. 27-38

27

Elsevier

JEM 00940

Using KCI to determine size at competence for larvae of the marine gastropod Crepidulafornicata (L.) Jan A. Pechenik and William D. Heyman Biology Department, Tufts University, Medford, Massachusetts, U.S. (Received 16 November 1986; revision received 3 June 1987; accepted 8 June

1987)

Abstract: Competent larvae ofthe marine gastropod Crepidulafomicata(L.) were induced to metamorphose (i.e., lose the velum) by elevating sea-water [KCI] by 5-50 mM. The response was optimal at 15-20-mM elevations, at which 5 0 ~ metamorphosis was obtained in < 4 h. Larvae that did not metamorphose during brief exposures ( i - 5 h) to elevated [KCI] generally maintained the larval form folloxsing transfer to control sea water, suggesting that competent larvae must be continuously immersed in the test solutions for metamorphosis to occur. The smallest larvae to respond to elevated [KCI] had shell lengths of 700-800/am, the range of shell lengths xsithin which larvae of this species become responsive to natural inducers. All larvae > ~ 1125 pm shell length metamorphosed in response to increased [KCI]. Rearing temperature may affect the size at which larvae of this species become responsive to K +. CaCI 2 (20-mM concentration elevations), GABA (4 x 10 - 7, 4 x I 0 - 6 M), and NaCI (I 0-20-ram concentration elevations) generally failed to trigger metamorphosis. Twenty-mM elevations of [RbCI] and [CsCI] induced 100~, metamorphosis but the juveniles were immobile and died after several days. Elevating IKCI] appears to be a reliable way to assess competence and trigger metamorphosis in larvae of C.fornicata. Key ~ords: Crepidulafornicata; Metamorphosis; Larva; Gastropod; Competence

INTRODUCTION

Precompetent larvae of marine benthic invertebrates are, by definition, incapable of metamorphosis; they eventually undergo a discrete, but little understood change rendering them capable (i.e., competent) to metamorphose to the benthic juvenile stage (Crisp, 1974; Chia, 1978; Hadfield, 1978). Once competent, larvae will metamorphose in response to specific environmental stimuli (Thorson, 1950; Crisp, 1974; Scheltema, 1974; Chia, 1978; Highnam, 1981; Burke, 1983a, Hadfield, 1986; Pawlik & Faulkner, 1986). In the absence of morphological or behavioral markers reliably indicating the onset of metamorphic competence in individual larvae, the only way to conclusively demonstrate that larvae have attained competence is to induce metamorphosis (Pechenik, 1984; Pechenik & Lima, 1984; Miller & Hadfield, 1986). Although natural inducers of metamorphosis are known for larvae of several molluscan species (Crisp, 1974; Hadfield, 1977, 1978; Morse et aL, 1979), the naturally active factors have rarely Correspondence address: J.A. Pechenik, Biology Department, Tufts University, Medford, MA 02155, U.S. 0022-0981/87/503.50 9 1987 Elsevier Science Publishers B.V. (Biomedical Division)

28

J.A. PECIIENIK AND W.D. IIEYMAN

been isolated in pure form; in consequence, inducing metamorphosis generally involves using unknown substances in unknown concentrations. Except for Hadfield (1977), working with a partially purified coral extract, it has therefore not been possible to determine a minimum concentration of natural inducer that will routinely trigger metamorphosis of newly competent larvae. As part of a study of juvenile fitness following metamorphosis in the prosobranch gastropod Crepidulafornicata (L.), we have tested the ability of several inexpensive and readily available chemicals to induce larval metamorphosis ofthis species, and then used one of these substances to ascertain the onset of competence in individual larvae. Competent larvae of the abalone Italiotis rufescens can be induced to metamorphose through exposure to increased extracellular [K § ] or [GABA] (Morse etal., 1979; Baloun & Morse, 1984; Yool etaL, 1986). Competent larvae of several other invertebrate species can be induced to metamorphose by elevating ambient [KCI] (Morse etaL, 1979; Baloun & Morse, 1984; Yool etal., 1986). In this study, we examined the potential usefulness of KCI, NaCI, CaCI2, RbCI, CsCI, and GABA as inducers of metamorphosis for veliger larvae of C.fornicata. We also consider: (1) the range of sizes over which larvae become responsive to elevated [KCI] (the most effective treatment tested); (2) the time course of the metamorphic response; (3) the minimum exposure period necessary to irreversibly initiate metamorphosis; and (4) the influence of rearing temperature on the sizes at which larvae of this species become responsive to elevated [KCI].

MATERIALS AND METHODS

Adults of C. fomicata were collected from Nahant and Bamstable, Massachusetts, U.S., and maintained in the laboratory at g 25 ~ on a mixed diet of the phytoplankton Dunaliella tertiolecta (clone DUN), Isocho,sis galbana (clone ISO), and a Tahitian strain of Isochrysis sp. (TqSO). Following natural release, larvae were reared at 25 ~ ~ 75 ind.. 150 ml of algal suspension- i, on a diet of T-XSOat g 18 x 104 cells, ml- i; larval growth of C. fornicata is sustained with low mortality under these conditions (Pechenik, 1980, 1984). All sea water was collected at Nahant, Massachusetts, and Milliporefiltered to 0.45 Fm to prevent protozoan infestation of larval cultures. Water and algal medium were replaced daily, and the glass rearing dishes were cleaned at each change. Periodically, we tested the ability of KCI (concentration elevated by 5-50 raM), NaCI (concentration elevated by 10-20 raM), anhydrous CaCI 2 (20 mM), RbCl (20 raM), CsCl (20 mM), GABA (4 x 10 -7, 4 x 10 -6 M), or adult-conditioned sea water to trigger metamorphosis. Metamorphosis was judged as the complete loss of the larval swimming organ, the ciliated velum (Scheltema, 1961; Fretter, 1967; Hadfield, 1978; Pechenik, 1980, 1984). In one experiment, we examined the rate of the metamorphic response as a function of [KCI] (elevations of 5-20 mM), and in one especially detailed

KCI INDUCES CREPIDULA METAMORPHOSIS

29

study using an elevated [KC1] of 20 mM, we made hourly observations to determine the time course of the metamorphic response. To induce natural metamorphosis, sea water was conditioned by allowing adults to reside for 24-36 h in the water; the conditioned water was filtered to 0.45 pm before use in experiments. Biologically filmed glass slides were included in adult-conditioned sea-water treatments to further increase the likelihood of triggering natural metamorphosis. These treatments have proven effective inducers of metamorphosis for larvae of both C.fornicata and C.plana (Pechenik, 1980, unpubl, data; Lima & Pechenik, 1985). Unless otherwise specified, each experimental series used larvae that hatched on a single day. For each replicate, five larvae were transferred to 15-25 ml of the test solution without food. Three to five replicates were run for each treatment, including controls. Control larvae were maintained in unadulterated filtered sea water. Test solutions were prepared as 0.5-M stock solutions in deionized water and stored at 12 ~ until needed. Stock solutions were diluted with filtered sea water to yield desired test-solution concentrations. The osmotic concentration of each solution was determined using an advanced freezing-point depression osmometer. The osmolarity of control sea water was 932 mOsm. 1- ~. The osmolarity of test solutions was raised by < 2 ~ , the greatest increase being recorded in the elevated [CaC12] treatment, as expected (final osmotic concentration of ~ 950 mOsm.. 1-~). Since KCI was found to be the most effective of the solutions tested, we focused all subsequent studies on this substarice. In one study, we reared larvae at 18, 25, or 27.5 ~ to see if the size at which larvae become responsive to KC1 is influenced by rearing temperature. Larvae of C. fomicata grow at significantly different rates at these temperatures (Peehenik, 1984; Pechenik & Lima, 1984). Larval growth rates were determined by subsampling larvae at 3-4-day intervals, measuring individuals nondestructively, and returning them to the cultures (Pechenik, 1984; Pechenik & Lima, 1984). Shell lengths were measured at 32 or 50 x using a dissecting microscope equipped with an ocular micrometer. After some larvae in a culture reached 600 pm in shell length, experiments testing for competence were conducted daily, using filtered sea water or filtered sea water with [KCI] elevated by 20 mM. Each day, 30 previously untested larvae were randomly selected from the rearing dishes, pooled, and randomly distributed among the two treatments at each temperature in groups of five larvae, dish- ~. After 24 h, the shell lengths of all individuals, both metamorphosed and unmetamorphosed, were recorded. This experiment was repeated the following summer. To determine the minimum exposure time required for KCI to trigger metamorphosis, groups of larvae (15 per group, divided among three replicates) were immersed for either 1, 2, 3, 4, 5, or 24 h in sea water whose [KC1] was elevated by 20 mM, and then removed to filtered sea water for hourly observation over a 24-h period. Larvae from the parent culture were maintained in untreated filtered sea water as controls. This experiment was repeated with previously untested larvae several days later. Differences in the shell lengths of larvae in control and parallel test populations were evaluated using 1-way ANOVA.

30

J.A. P E C H E N I K A N D W.D. I I E Y M A N RESULTS

In most experiments, control and test populations of larvae did not differ significantly in mean shell length ( P > 0.05, 1-way ANOVA), so that responses to the various treatments do not reflect larval size differences between control and test populations. In the few cases in which treatment populations did differ in mean shell length, the mean shell length of the control population exceeded that of the test population. Of the various substances tested, only KCI, RbCI, and CsCI caused substantial metamorphosis of C.fornicata (Table I). Larvae induced by RbCI and CsCI did not fight themselves after metamorphosis; they remained immobile and died several days after exposure. In contrast, all larvae metamorphosing in response to KCI survived and grew over a 6-day observation period. TABLE I

Effect of various chemicals on metamorphosis of larval Treatment

Expt. A Control 4 x 10 - 6 4 x 10 - 7 20 m M 10 m M 20 m M 5 mM 10 m M 15 m M 20 m M Expt. B Control 20 m M 25 m M 30 m M 40 m M 50 m M Expt. C Control 20 m M 20 m M 20 m M

Number of larvae tested

Mean shell length of larvae tested 4- sD 0~m)

C.fornicata. Percent metamorphosis after 24 h

1~I G A B A 1~I G A B A CaCI2 NaCI NaCI KCI KCI KCI KCI

40 10 10 30 15 30 25 15 15 55

1037 899 982 1054 1074 1061 973 1050 1035 1029

4- 100 + 82 4- 93 4- 75 + 63 4- 109 + 103 +_ 107 +_ 58 + 78

0.0 0.0 0.0 0.0 13.3 10.0 48.0 93.3 100.0 100.0

KCI KCI KCI KCI KCI

15 15 15 15 15 15

989 4- 85 1012 4- 65 1050_+ 66 965 _+ 73 985 + 72 1037 _+ 112

0.0 100.0 73.3 46.7 53.3 93.3

KCI RbCl CsCI

15 15 15 15

1084 1095 1048 1109

0.0 100.0 100.0 100.0

+ 44+

85 67 83 67

Elevating [KCI] by 20 mM had the most dramatic effect on metamorphosis, with 100~o of the tested larvae (mean shell length 1029pm) metamorphosing in <5 h (Fig. I). This treatment was far more effective than exposing larvae to adult-conditioned sea water (Treatment AC, Fig. 1); adult-conditioned sea water triggered metamorphosis

KCI I N D U C E S CREPIDULA M E T A M O R P t l O S I S

31

in ~ 2 5 ~ of tested larvae in < 5 h (Fig. 1), but was thereafter less effective in comparison with even the lowest [KCI] elevation tested (5 mM). While metamorphosis could be induced with higher [KCI] (Table I), elevating [KCI] by > 30 mM was toxic POmM o

I00

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Fig. I. Influence of elevated [KCI] on rates of metamorphosis of larval C.fornicata at 2 4 " C ; l a r v a e - t r e a t m e n t - z . AC, adult-conditioned sea water.

15

within 48 h at'ter exposure. Added [KCI] <20 mM induced metamorphosis, but more time was required to trigger metamorphosis in a given percentage of larvae (Fig. 1). In a more detailed study of responses in the In:st 6 h of exposure to 20 mM-added KCI, 50~ metamorphosis was attained in <3 h after immersion and 100% metamorphosis was recorded in < 6 h (Fig. 2). When placed into a solution of sea water with [KCI] elevated by 5-50 mM, the larvae of C.fornicata responded with small but continuous velar convulsions and intermittent swimming for 5-10 min. Eventually, the larvae withdrew partially into the shell and exhibited no substratum searching behavior or swimming activity. In response to KCI, competent larvae shed the velum abruptly; velum disintegration and ciliary detachment followed. The size at which the larvae of C.fornicata became responsive to the added KCI (20-mM elevations) varied greatly among individuals. The smallest larva was induced to metamorphose at 729 lzm in shell length, while some larvae as large as 1123/tm did not respond to a 24-h immersion (Fig. 3; Table lI). All individuals > ~ l1251tm

32

J.A. PECIIENIK AND W.D. HEYMAN

metamorphosed under these conditions of elevated [KCI]. Most larvae with conspicuous shell brims (Pechenik, 1980), which typically form at = 800-900 pm shell length (Pechcnik, 1980; Pcchenik & Lima, 1984), metamorphosed in the KCi treatments. The temperature at which the larvae were reared may have affected the size at which they became competent; larvae generally began responding to elevated [KCI] at a larger size at higher temperatures (Table II). % M~to~or 3hosis tO0 90 80 70 60 50 40 30 20

l0 (9

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Fig. 2. Effect of 20mM-elevated [KCI] on the rate ofmetamorphosis of C.fornicata at24 ~ made hourly on 15 larvae. Open circles represent controls.

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MEAN S H E L L

LENGTH

(um)

Fig. 3. Percent metamorphosis of C.fornicata larvae of different shell lengths (675-1250 ttm), in response to 20-mM elevations of IKCI] at 27.5 ~ Shaded areas represent metamorphosed individuals while unshaded areas represent those larvae not responding within 24 h. Numbers at bar tops represent the number of individuals within the 50-ltm range that each bar represents. Data obtained for larvae reared and tested at 18 and 25 ~ are summarized in Table II.

CREPIDULA

KCI INDUCES

METAMORPHOSIS

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34

J.A. PECIIENIK AND W.D. IIEYMAN

Brief exposure (1, 2, 3, 4, or 5 h) to clevated [KCI] (20 mM) in sea water did not trigger all competent individuals to metamorphose; larvae metamorphosed only while in the elevated [KCI] solution or in the hour following their removal to filtered sea water (Fig. 4). For example, only three individuals (of the 15 tested) metamorphosed during

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Fig. 4. Effect of brief exposures to solutions of 20 mM-added [KCI] on percent metamorphosis of larval C.fornicata. After indicated exposure times, larvae were removed to 0.45-tim filtered sea water. 9 control (no [KCI] elevation); O, continuous exposure to KCI; 17, l-h exposure; x , 2-h exposure; A, 3-h exposure; II, 4-h exposure. This experiment was repeated several days later with comparable results (not shown).

a 2-h exposure period, and only two more larvae metamorphosed in the hour following transfer to control conditions (filtered sea water, normal ionic concentrations). None of the 10 remaining larvae metamorphosed over the next 20 h in sea water, even though continuous exposure to elevated [KCI] in the parallel treatment induced all 15 larvae to metamorphose in < 7 h (Fig. 4). Comparable results were obtained in a replicate experiment conducted several days later (not shown).

DISCUSSION

Although ineffective in triggering cyprid metamorphosis in the barnacle Balanus amphitrite (Rittschof etaL, 1986), excess K + does induce metamorphosis of H. rufescens, the opisthobranch Phestilla sibogae, the prosobranch Astraea undosa, and the polychaete Phragmatopoma californica (Baloun & Morse, 1984; Yool et al., 1986). To this list of species responding to elevated [K § ], we now add C.fornicata. As with other responsive species, the K § ion is the effective agent rather than increased osmolarity or elevated concentrations of associated anions; 20-mM increases of [NaCI], [CaCI2], and [KCI] all raised sea-water osmolarity but only the KCI treatment

KCI INDUCES CREPIDULA METAMORPtlOSIS

35

induced significant metamorphosis of C.fornicata. Similarly, additions of NaCI and CaCI 2 did not induce metamorphosis of H. rufescens larvae, but comparable additions of both KC! and K 2 S O 4 w e r e effective (Baloun & Morse, 1984). In our study, Rb § and Cs § also induced metamorphosis of competent larvae; these ions are similar to K + in hydrated size (Cotton & Wilkinson, 1972) and have previously been shown to induce metamorphosis in planulae ofItydractinia echinata (Muller & Buchal, 1973; as cited by Yool et ak, 1986). Future studies with C.fornicata might include experiments using lower [RbCl] and [CsC1], to see if these substances can induce metamorphosis in C.fornicata without the associated toxicity recorded in the present study. The response of C.fornicata to elevated [K i ] is more rapid than that previously reported for larvae of other species treated similarly. For Phestilla sibogae, H. n4fescens, and Astraea undosa, > 24 h was required to exceed 50~ larval response at the optimal [K § ] (48 h required for P. sibogae and H. rufescens) (Yool et al., 1986), whereas this level of response was exhibited in <3 h for C.fomicata. Similarly, Coon et al. (1985) report rapid metamorphic responses of oyster larvae Crassostrea gigas to low [epinephrine] and [norepinephrine]. Larvae of C.fonzicata responded similarly to adult-conditioned sea water and the lowest elevations of [KCI] for the first 5 h of exposure (Fig. 1), but a relatively small proportion of tested larvae ultimately metamorphosed in the adult-conditioned treatment; in some other studies (unpubl. data), adult-conditioned sea water triggered metamorphosis in 9 0 - I 0 0 ~ of tested larvae. The present result may reflect differing individual sensitivities to natural inducer (Hadfield, 1977), and illustrates the difficulties of testing for competence using unknown substances in unknown concentrations. Brief exposures of larval C.fornicata to 20-raM [KCI] elevations did not irreversibly initiate metamorphosis; those individuals not metamorphosing during exposure generally failed to metamorphose following their return to control sea water. Larvae of C. gigas responded to various neuroactive compounds similarly, continuous immersion in test solutions being prerequisite to metamorphosis (Coon et aL, 1985). In contrast, larvae of P. sibogae continued to metamorphose over the 24 h following their transfer from natural inducer to control sea water (Hadfield, 1978). Concentrations of 4 x 10 -7- and 4 x 10-6-M GABA induce metamorphosis in competent abalone larvae (Morse etal., 1979; Baloun & Morse, 1984; TrapidoRosenthal & Morse, 1986), but do not induce metamorphosis of C.fornicata (this study), Phestilla sibogae (Hadfield, 1984), or the chiton Katherina tunicata (Rumrill & Cameron, 1983). Response to GABA is apparently less widespread than is the response to K + . Some authors suggest that elevating [K § ] in sea water depolarizes an excitable membrane in the larval sensory system, triggering an uncertain chain of events that eventually culminates in metamorphosis (Hadfield, 1978; Burke, 1983a; Rumrill & Cameron, 1983; Baloun & Morse, 1984; Hirata & Hadfield, 1986; Yool etal., 1986). Cameron & Hinegardner (1974) and Burke (1983b) successfully triggered metamorphosis of the sea urchins Lytechim~s pictus and Dendraster excentricus electrically,

36

J.A. PECIIENIK A N D W.D. HEYMAN

suggesting that membrane depolarizations are indeed involved, but the morphological or physiological changes that render individual larvae competent to metamorphose remain to be determined. Present data are consistent with onset of competence coinciding with (I) the development or activation of sensory receptors for specific environmental cues, (2) the completion ofspecific neural pathways, (3) the development of particular neurosecretory cells, or (4) the development or activation of receptor sites on target tissues (Hadfield, 1978; Highnam, 1981; Burke, 1983a; Trapido-Rosenthal & Morse, 1986). Further work will be required to distinguish among these possibilities. Normal molluscan metamorphosis typically involves a period of substratum searching and selection followed by anirreversible loss ofthe larval velum (Bayne, 1965; Fretter, 1967; Crisp, 1974; Coon et al., 1985). When larvae of C.fomicata are induced naturally, using adult-conditioned sea water or biologically filmed glass slides or shells (Pechenik, 1980, unpubl, data), the velum is resorbed following a period of substratum searching. Metamorphosis induced by elevated [KCI] differed from normal metamorphosis in two respects: when induced with KC1, the larvae of C.fonzicata showed no period of substratum examination; and the velum was shed abruptly rather than resorbed gradually. Similarly, metamorphosis of C. gigas is not preceded by a crawling exploratory phase when triggered with epinephrine or norepinephrine; L-DOPA, however, stimulates both characteristic crawling behavior and metamorphosis in C. gigas (Coon et al., 1985). Despite the abnormality of the metamorphic process when triggered by elevated [K § ], all C.fornicata larvae triggered to metamorphose using [KCI] elevations of 5-20 mM survived and grew as juveniles for a subsequent 6-day observation period. Further studies on the growth and metabolism of juveniles induced naturally and by means of elevated [KCI] are in progress. Pechenik (1984), Pechenik & Lima (1984), and Lima & Pechenik (1985) have shown that over a temperature range of 12-29 ~ larval growth rate is inversely related to maximum length of larval life for both C.fornicata and C.plana, and that rearing temperature may influence size at spontaneous metamorphosis in these two species. Presumably, temperature, through its influence on rates of differentiation, will also influence the time required to become competent; in our study, temperature may also have altered the size at which larvae became competent to metamorphose, suggesting a differential influence of temperature on rates of differentiation and growth (Pechenik, 1984, in press). However, larvae showed great variation in the sizes at which they became competent even though there is little variation in larval shell length upon release from the parent (Pechenik, 1980, 1984). In addition, although most larvae with shell brims (Pechenik, 1980) were competent to metamorphose, some larvae were competent before brimming, as is also the case for larvae of C. plana (Lima & Pechenik, 1985). The onset of competence is therefore not neatly predictable, for individual larvae, on the basis of either growth rate, shell length, or shell geometry.

KCI INDUCES CREPIDULA METAMORPttOSIS

37

ACKNOWLEDGEMENTS This research was supported by Grant OCE 8500857 from the National Science Foundation.

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