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Onset of metamorphic competence in larvae of the gastropod Crepidula fomicata (L.), judged by a natural and an artificial cue
Exp. Mar. Biol. Ecol., 167 (1993) 59-72 0 1993 Elsevier Science Publishers BV. All rights reserved 0022-0981~93/$06.00
59
.I.
JEMBE 01913
Onset of metamorphic competence in larvae of the gastropod Crepidula fornicata (L.), judged by a natural and an artificial cue Jan A. Pechenik and Christopher C. Gee Biology department, Tufif ~~~ver~ity,Redford, ~assac~sett~,
USA
(Received 21 May 1992: revision received 23 October 1992; accepted 3 November 1992) Abstract: Exposing larvae of the marine prosobranch gastropod Crepidula fornicata L. to excess K+ triggers the metamorphosis of competent individuals. This paper considers whether larvae of C. fornicata become responsive to excess K + at the same time and size that they become responsive to adult-conditioned seawater and other apparently natural cues. Fourteen experiments were conducted at room temperature (ca. 23 “C), with 3-5 replicates per treatment and 6-10 larvae per replicate. Larvae became responsive to natural cues within 12-24 h after becoming responsive to excess K+, and responded sooner to natural cues under agitated (20-50 rpm) rather than static conditions (pt0.05). These results emphasize the importance of defining metamorphic competence with respect to specific external cues. Although it seems reasonable to estimate the onset of metamorphic competence using excess K + in this species, the data suggest that the excess K’ and the natural inducer act at different points in the met~orphi~ pathway.
Key words: Competence; Crepid~~u~rn~cutu; Met~orphosis
Free-living larvae are the major dispersal agents in the life cycles of many benthic marine invertebrates (Thorson, 1946, 1950). Frequently there is an obligate period before larvae become physiologically “competent” to metamorphose, followed by a period of time during which larvae defer, or “delay”, their met~orphosis in the absence of specific chemical or physical environmental cues (reviewed by Crisp, 1974; Gray, 1974; Scheltema, 1974, 1986; Burke, 1983, 1986; Woodin, 1986; Pechenik, 1990). We cannot determine how long larvae can delay their metamorphosis unless we first know when the precompetent period of development ends. Precompetent, obligate dispersal periods have been rigorously defined in the laboratory for only a few species, in part because the natural cues triggering metamorphosis are typically not known or are incompletely characterized; when larvae fail to metamorphose in a laboratory experiment, is it because they are not yet competent, or is it because the provided cues Correspondence USA
address: J.A. Pechenik, Biology Department, Tufts University, Medford, MA 02155,
60
J.A. PECHENIK AND C.C. GEE
are inadequate? Working with such natural cues as microbial films and adultconditioned seawater makes the question impossible to answer because larvae are exposed to unknown concentrations of unknown substances. In recent years (but see also Lynch, 1949, 1961) researchers have found that, for many marine invertebrate species, metamorphosis can be triggered by elevating seawater K’ concentrations or exposing larvae to various neuroactive substances and related compounds (Miiller & Buchal, 1973; Hadfield, 1984; Coon et al., 1985; Hirata & Hadfield, 1986; Trapido-Rosenthal & Morse, 1986a,b; Yool et al., 1986; Coon & Bonar, 1987; Pechenik & Heyman, 1987; Cameron et al., 1989; Coon et al., 199Oa,b; Pawlik, 1990; Chevolot et al., 1991; Pires & Hadfield, 1991; Todd et al., 1991). While the major goal of such work has been to determine the sequence of internal biochemical events leading to normal metamorphosis, some researchers have used specific chemical cues to explore the range of sizes at which larvae become competent to metamorphose (Pechenik & Heyman, 1987; Coon et al., 1990a) or to examine the influence of salinity and temperature on the time required for larvae to become metamo~hic~ly competent ~Zirnrne~~ & Pechenik, 1991). For species in which excess K’ induces metamorphosis, using it to judge metamorphic competence has the advantage of working with known concentrations of a fully defined and stable chemical. Moreover, for Crepidzda fomicata the response to excess K + is rapid; at 24 “C, all larvae competent to respond do so within about 6 h (Pechenik & Heyman, 1987). The sequence of steps through which the various chemical inducers of metamorphosis function, and where they act in or on the larva, is not clear (reviewed by Jensen et al., 1990; Pawlik, 1990; Todd et al., 1991), although a number of int~guing models have been proposed for two mollusc species (Trapido-Rosenthal & Morse, 1986a; Baxter & Morse, 1987; Coon et al., 1990a). It is generally believed that increasing external K+ concentration depolarizes artificially the external receptor cells that are normally involved in perceiving the natural chemical cue(s) (Yool et al., 1986; Jensen & Morse, 1990; Chevolot et al., 1991); this depolarization presumably then triggers a series of other, incompletely defined events inside the larva (Burke, 1983; Coon et al., 1985, 1990a,b; Hadfield, 1986; Trapido-Rosenthal & Morse, 1986a; Cameron et al., 1989; Todd et al., 1991), resulting in metamorphosis. However, Todd et al. (1991) point out that the excess K* could instead be acting later in the pathway, bypassing the external sensory cells that perceive the natural chemical stimulus. This hypothesis has not yet been tested. In the field, larvae of C. fomicata and C. plana probably metamorphose in response to cues produced by adults or microbial films associated with adults (Pechenik, 1980; Lima, 1983; Lima & Pechenik, 1985; McGee & Target, 1989). These chemical cues presumably interact with specific external receptors somewhere on the larva (e.g., Arkett et al., 1989). Excess Kt may operate in identical fashion as the natural cue for Crepiduh larvae, or it may bypass one or more steps in the pathway (Pechenik, 1990; Todd et al., 1991). Although it is not yet possible to determine directly how or where the excess K+ operates, we may address the following question: do larvae become
61
COMPETENCEFORLARVALMETAMORPHOSIS responsive
to elevated
K+ and to natural
the two cues may operate pathway. responsive
If, in contrast, to natural
through
cues at the same time in development?
the identical
larvae become
pathway
responsive
If so,
and at the same point in that
to excess K’
cues, it is more likely that the elevated
K’
before they become treatment
bypasses
one or more early steps in the normal pathway, or operates through a different pathway. Here we address this question using larvae of the slipper shell snail C.firnicata (L.). The results provide some information about the action of excess potassium on the larvae, and indicate whether the onset of metamo~hic competence in this species may be reliably judged by larval response to elevated external K’ concentration.
MATERIALSAND Adult
C. fornicara
were collected
METHODS
near Woods
Hole, MA and maintained
at room
temperature (ca. 23 ‘C) on a diet of the flagellated protist Dunalieh tertiolectu (Butcher). When larvae emerged from their egg capsules they were transferred to seawater filtered to 0.45 gum and fed with the unicellular protist Isochrysis g&ma Parke (clone T-ISO) at approximately 1.5 x lo5 cells*mll’. We maintained the larvae at room temperature and changed the seawater and food every two days. Each experiment involved larvae released by different females and cultured until at least 40% of subsampled larvae metamorphosed when external K’ concentration was raised by 20 mM (by adding KCl; Pechenik & Heyman, 1987). Larvae were typically 12-17 days old at the start of an experiment. The main goal in each experiment was to determine whether particular groups of larvae were equally responsive to excess K’ and the natural cue. In addition, when larvae were not comparably responsive to the two cues we sought to determine how much additional time was required for larvae to become comparably responsive. To begin an experiment, we divided larvae from a single culture among the various treatments, using 3-5 replicates of 6-10 larvae each. Larvae were tested in 9 cm glass dishes containing 45 ml of test solution. Usually twice a day, we determined the number of larvae that had metamorphosed in each dish, and the shell lengths of metamorphosed individuals. Juvenile shell lengths were measured at 50 x using a dissection microscope equipped with an ocular micrometer. Each experiment was continued for 3-4 days, or until the number of larvae metamorphosing in response to natural cues and excess K” became statistically comparable. The treatments for each experiment generally consisted of a filtered seawater control. seawater with the K ’ concentration (added as KCl) increased by 20 mM, and a “natural” treatment as described below. For the first set of experiments (Experiments 1-8, 14), the natural treatment consisted of adult-conditioned seawater plus one microbialty filmed shell fragment (Pechenik, 1980; Eyster & Pechenik, 1987; McGee & Target, 1989). Seawater was conditioned by allowing 5-8 adults to feed in it for 6- 16 h, and was then filtered to 0.45 nm before use. For the remaining experiments (Experi-
62 ments
J.A.PECHENIK AND CC. GEE 9-13),
to condition
two filmed shell fragments
were used per replicate,
adults were allowed
the seawater for about 16 h, and half the dishes containing
and adult-conditioned
seawater were agitated (at 20-108
shell fragments
rpm) on a rotary shaker table
(Model 3540, Lab-Line Instruments, Inc.). Agitation has been reported to increase percentage metamorphosis for larvae of the blue mussel, Mytilus edulis (Eyster & Pechenik, 1987), and the polychaetes Sabellaria alveolata (Wilson, 1968; but see Pawlik, 1988) and Phragmatopoma californica (Pawlik, 1988). Larvae exposed to excess potassium were never agitated. Larvae in all treatments (except those subjected to excess K+) were fed T-IS0 at an initial concentration of about at 1.5 x lo5 cells.mll’. All data were analyzed using one-way analysis of variance followed by Bonferonni pairwise comparisons where appropriate. More sophisticated analyses involving the pooling of data from different experiments were inappropriate for the following reasons: the concentration of natural cue may have varied among experiments, and larvae were not necessarily at identical stages of development at the start of each experiment. In this study, the comparisons of interest are within experiments, not among experiments. For at least two marine mollusc species (Phestilla sibogae - Hadfield & Scheuer, 1985; Haliotis rufescens - Trapido-Rosenthal & Morse, 1986a,b), maintaining precompetent larvae in low concentrations of natural inducer “habituates” the larvae, so that the larvae fail to metamorphose when their siblings become competent to do so. These habituated larvae must be transferred to control seawater for a time before they will metamorphose in the presence of the inducer. We therefore conducted two experiments (4-5 replicates per treatment, 6-8 larvae per replicate) to determine whether some larvae of C. fornicata might have habituated in our “natural” treatments. Such habituation could occur if some individuals were not competent to respond to natural cues at the start of an experiment; this would interfere with our ability to determine how much longer it took such individuals to become competent. To test for this possibility we kept some larvae in the natural treatment throughout an experiment but held other larvae in the natural treatment for about 8 h each day, transferring them to control seawater for the remaining portion of the day to allow time for dehabituation. As above, larvae in both treatments were fed T-IS0 at about 1.5 x 10’ cells.ml-‘. All larvae were transferred daily to fresh mixtures of the appropriate treatment, phosis was determined at frequent intervals for up to 4 days.
and percent metamor-
RESULTS We saw no indication that precompetent larvae of C. fornicata habituated to adultconditioned seawater. Figure 1 presents results of our most detailed study. Although few larvae metamorphosed in response to natural cues during the first 10 h of observation, the number of larvae metamorphosing increased considerably over the next 40-60 h, and increased comparably (p > 0.10) whether or not larvae were constantly bathed in adult-conditioned seawater. Comparable results were obtained in a pilot
COMPETENCE
FOR LARVAL
20
0
METAMORPHOSIS
40 60 Hours
80
63
100
Fig. 1. Influence of continuous exposure to natural inducer on metamorphosis of C.firnicata. A, Data for larvae continuously exposed to natural inducer (adult-conditioned seawater plus shell fragments coated with microbial films); A; data for larvae exposed to natural inducer about 8 h per day and then transferred to filtered seawater. Larvae were fed Zsochrysisgulbarza(clone T-ISO) at a concentration of 1 x 10’ cells.ml~’ except when exposed to excess K' Error bars represent 1 SD about the mean. N= five replicates per treatment, eight larvae per replicate.
experiment (four replicates per treatment, six larvae per replicate with metamorphosis determined at five intervals between 24 and 92 h): transferring larvae from adultconditioned seawater to filtered seawater had no statistically significant effect (p > 0.05) on the mean number of individuals metamorphosing. The relative responsiveness of C. fornicata larvae to excess potassium and natural cues depended on whether or not larvae were agitated during exposure to the natural cue. In all but two (Experiments 1 and 3) of the nine experiments conducted without
60
”
1
2’
3
4*
5*
6.
7’
a’
14%
Experiment Fig. 2. Effect of 20 mM increased external K + concentration and natural cues on the number of G. fornicata larvae metamorphosing within 24 h. No larvae were agitated in these experiments. Asterisks indicate experiments in which the mean percentage of larvae metamorphosing in response to excess K+ significantly exceeded the mean percentage metamorphosing in response to natural cues (p< 0.05). Error bars indicate 1 SD about the mean (n = 3-5 replicates per treatment, with 6-10 larvae per replicate).
64
J.A. PECHENIK
AND C.C. GEE
Experiment Fig. 3. Influence of agitation on the mean percentage of C. fomicatcr larvae metamorphosing in response to natural cues within 24 h. Agitation varied between 20 and 108 rpm in different experiments, as indicated. Error bars indicate 1 SD about the mean (n = 5 replicates per treatment, with 10 larvae per replicate).
agitation, significantly more (p < 0.05) larvae metamorphosed in response to elevated K+ than to natural cues over the first 24 h of incubation (Fig. 2). Similarly, excess potassium induced more larvae to metamorphose during the first 24 h than did static natural cues (p < 0.05) in three of the subsequent five experiments (Fig. 3, Experiments 9-l 1; compare first and second bars for each experiment). Moreover, the average size of individuals metamorphosing in response to excess K+ was often smaller than that of individuals metamorphosing in response to static natural cues (Fig. 4, Table I), even
1,600
g
1,200
P 2
800
z c
5
400
8 1
2
3’
4’
Experiment Fig. 4. Influence of elevated external K+ and natural cues (static treatments only) on the mean shell length of juvenile C. fornicafa measured within 24 h of exposure. Asterisks indicate significant differences in mean shell length for larvae induced to metamorphose by natural cues vs. excess K+ (p
ND
ND
10
11
1094+ 127 (12)
9X4* 121(X!)
9
13
1177+96(12)
5
1166186(12)
1056 2 107 (12)
4
12
9892 152(12)
3
6-18 h
7h St: 1214 ?: 92 (8) Ag: 12141 141 (8)
1080 + 143 (24)
1146 f 83 (38)
841 f 56 (47)
906 k 71139)
7h St: 965 It 71(3) Ag: 1020 + 182 (9) 6h St: 1055 i: 119(9) Ag: 953 i 94 ( 15) 6h St: 869+ 71(16) Ag: 914 f 89 (23) 13 h St: 1227 i: 96 (28) A8: 1235 ?: 107 (32)
18h 1295 _t 117 (25)
1208 + 108 (39)
982 f 95 (21)
8h 1127&106(14)
7h 1219+110(l) 10h 1068 f. 163 (23)
[Cl
Natural
Mean shell length
973 ?: 132 (39)
936 & 127 (32)
1202 & 97 (47)
PI
[Al
ND
K+ triggered
Initial
2
Expt.
treatment data.
24 h
St: 1219k 125 Ag: 1214~ 141 (8)
St: 1229+ 10 (38) Ag: 1217i: 119(39)
St: 889i 98 (24) Ag: 93 12 94 (34)
St: 1060 + 120 (14) Ag: 995 + 114 (23)
St: %2t65(1) Ag: 103 1f 140 (18)
1281+ 116 (35)
1113+130(20)
1082 _F155 (28)
1237+86(19)
PI
Natural
A A A B A n A l3 B 6.8 (5,184)
B vs Dst
B vs CAg B vs D
B vs Cst B vs I& 6.0 (6,209) p
3.8 (5,71) p = 0.004
B YS CAg B vs D,,
B vs C,, 9.4 (4,951 p<0.0001
vs B vs C YS D vs Cst vs B “S c vs D vs c w, DAg
B YS Cst B vs D
All
A YS C A YS D B vs C B YS D None
B vs C B vs D
B vs C B YS D A A A A A A A
YS B vs C YS D YS B vs C vs D YS I3
None
Significant (p
All
Not significant (p>O.O5)
Bonferroni comparisons
0.7 (5,68) p = 0.65
8.1 (3,107) pio.001
6.3 (4,89) p
0.7 (3.73) p = 0.53 6.0 (3.91) p < 0.0009
F value
ANOVA
C.fonricaru. Metamorphosed individuals in K+ treatment at 6-18 h and at 24 h. St, static conditions: Ag, agitated; ND, no
of metamorphosed
I
were measured
size QIN~ shell length)
in natural
on mean
individuals
and time of measurement
at 7-8 h. Metamorphosed
of treatment
were measured
Influcncc
TABLE
66
J.A. PECHENIK
AND C.C. GEE
TABLE
II
Time to competence: comparison between natural (but without agitation) “statistical equivalence”, mean percent of larvae that had metamorphosed between treatments. Expt.
Mean ( + SD) Pa metamorph. in exess K’ within 7 h
1 3
45~26 80 + 16.8 100 87.5 k 7.2 78.0 k 16.4 94 k 8.9 95 + 11.2 too 87.5 f 8.8
9 10 11 12 13 14
““‘t
Mean ( + SD) "" metamorph. in natural treatment within 24 h 32.5 65 67.5 45.8 28.0 48 92.7 70.8 17.5
Statistical equivalence by Xh
+ 28.8 + 20.5 f 16.8 + 26.7 + 19.0 * 22 k 25.2 5 18.3 f 14.3
Previous time examined (h)
6.5 9.5 40 8 24 47 0 7 24
24 24 48 24 30 54 24 24 32
q Stationary
@KC,
and excess K’ treatments. At was not significantly different
H
Agitated
0
Control
F t
1,200
_c +J cn 6 =
800
_F tn 5
400
8
0
9 (20
rpm)
10 (20
rpm)
11 (30
rpm)
12 (50
rpm)
13 (108
rpm)
Experiment Fig. 5. Mean she11 length ofjuvenile C. fornicatu induced by excess potassium and by natural cues with and without agitation. All measurements were made within 24 h of exposing larvae to the cues. There were no significant differences in mean shell length for larvae held in stationary or agitated conditions. Each mean typically represents measurements on 20-40 individuals, except as indicated by a number above a bar. Error bars indicate 1 SD about the mean (R = 5 replicates per treatment, with 10 larvae per replicate).
COMPETENCE FOR LARVAL METAMORPHOSIS
67
when juveniles were measured after only 6-8 h (Table I). These results suggest that larvae of C. fornicata became responsive to the elevated IS+ concentration somewhat before they became responsive to static natural cues, with many of the smaller individuals being competent to respond to excess K’ but not yet competent to respond to the natural cues. In such cases, larvae developed the ability to respond to natural cues later (Table II). In Experiment 11, for example (Table II), the response to adultconditioned seawater within 24 h was only about half the response to excess K’ , and a statistically significant difference in response (p < 0.05) was still evident at 47 h (last column). However, the response to the two cues was statistically equivalent by 54 h (column 4). On the other hand, natural cues were as effective (p> 0.10) as excess K’ in promoting metamorphosis when larvae in natural inducer were agitated up to 50 rpm (Experiments 9-12, Fig. 3, p
DISCUSSION
The data suggest that larvae of C. fornicata become responsive to natural cues (adult-conditioned water, microbial films) within 12 h (if larvae are agitated) to 24 h (in static culture) of becoming responsive to elevated K+ concentrations. This is short relative to the amount of time required for newly emerged larvae to become responsive to excess K’ ; Zimmerman & Pechenik (1991) found that the average C. fornicata Larva became competent to respond to excess K’ in 18-23 days at 20 “C, and in 12-19 days at 25 “C. Elevating external K + concentration thus seems a reasonable means of estimating when larvae of this species first become competent to metamo~hose. However, the data also suggest that C. fu$~~cata larvae did not become competent to metamorphose in response to all cues simultaneously. Larvae apparently became responsive to elevated potassium somewhat before they became responsive to natural cues under agitation, and responded to natural cues under agitation somewhat before they responded to the same cues under static conditions. This is indicated by differences in the percentage of larvae responding in each treatment within 24 h of being exposed to the cues provided. Moreover, the average size of individuals measured after they metamorphosed typically varied with treatment; the data suggest that larvae generally became responsive to the natural cue at a more advanced stage of development (assuming this correlates, in general, with a larger size), p~icul~ly if the larvae
68
J.A. PECHENIK AND C.C. GEE
were in static culture. Thus, the alternative expl~at~on that the process of metamorphosis simply requires less time to complete when triggered by some cues (e.g., elevated K ’ concentration), and more time when stimulated by others (e.g., natural cues), seems unlikely. However, the result could be an artifact of the differential effects of the treatments on larval growth. Larvae cease feeding while immersed in the KC1 solution (Pechenik & Heyman, 1987) and therefore were not given phytoplankton during the excess K+ treatment, but were allowed to feed in the other treatments. At 25 ‘C, C. fornicatu larvae grow up to about 60 pm-day - ’ under optimal conditions (one larva per bowl, unlimited food: Pechenik & Lima, 1984). Moreover, newly metamorphosed juveniles grow between 125 and ZOO~m.day-’ under optimal conditions (one juvenile per bowl, unlimited food) at this temperature (Pechenik & Eyster, 1989). Under such conditions, a combination of larval and juvenile growth could completely account for the average larger size of juveniles recovered from our “natural” treatments. Our data argue against this alternative hypothesis. First, the animals in our experiments were relatively crowded and probably food-limited, so that growth rates should have been well below the optima reported above. This suggestion is supported by Bonferroni analyses comparing the sizes of juveniles measured in the different treatments with average sizes of larvae at the start of an experiment; snails often showed negligible growth during the experiments (p> 0.1) (Table I, comparing column A with columns B-D). Moreover, in most cases for which we have data, mean sizes of juveniles resulting from natural cues were greater than the mean sizes of those metamorphosing in response to elevated K +, even when measurements were made as soon as 7-12 h after the start of an experiment (Table I). It appears, then, that juveniles were, on average, smaller when metamorphosing in response to elevated potassium not because larvae or juveniles were growing dramatically faster in other treatments, but rather because the smallest individuals in the natural treatments did not respond to the cue provided. The data emphasize the importance of defining metamorphic “competence” with respect to specific external cues. It is noteworthy that the average size of the few individuals metamorphosing within 24 h under control conditions was not subst~tially greater than that for individuals deliberately triggered to met~orphose in our experiments (Figs 4 and 5). So-called “spontaneous” metamorphosis is thought to reflect either increasing sensitivity to environmental cues, or an individual’s reaching the end of a genetically programmed larval lifespan (Pechenik, 1980, 1990; Coon et al., 1990a); in either case, the results confirm that larval size can be a poor predictor of physiological state (Coon et al., 1990a; Pechenik, 1990; Pechenik et al., 1990; Zimmerman & Pechenik, 1991). It is not yet clear what makes an individual larva competent to metamorphose. Likely possibilities include the differentiation or activation of external receptors, completion of specific neural pathways, differentiation of specific neurosecretory systems, or differentiation of specific receptors on target tissues (Hadfield, 1978; Highnam, 1981; Burke, 1983; Trapido-Rosenth~ & Morse, 1986a). Our data touch on this issue, al-
COMPETENCE
FOR LARVAL METAMORPHOSIS
69
though indirectly. If excess K+ and natural cues act at identical points in the metamorphic pathway for C. for~~cata, we would expect larvae to become responsive to both cues at exactly the same time in development. In our experiments, however, the larvae of C. fornicata apparently became responsive to the elevated K’ before they could respond to natural cues. If so, the excess K’ must be acting at a later point in the pathway than the natural cues act, as suggested by Todd et al, (1991), and the last step to become functional in the pathway must precede the point affected by excess K’. Similarly, Trapido-Rosenth~ & Morse (1986b) found that exposing precompetent abalone larvae (~aliot~s r~~~cen~) to GABA made them unresponsive to GABA but not to excess K+ when exposed several days later, again implying that the excess K + acts downstream from the target of natural cues. This logic assumes that both cues operate through the same pathway. Since the morphological aspects of metamorphosis as triggered by natural cues and by elevating external K+ concentrations differ in C. fornicata (Pechenik & Heyman, 1987), this may not be a correct assumption for this species. Note also that even if excess potassium and natural cues acted at different points in the same pathway in our experiments, existing data cannot rule out the possibility that excess IV will act earlier in the pathway once larvae become competent to respond to natural cues. For example, the excess K’ may bypass epithelial receptor cells in larvae not yet competent to respond to natural cues, but might act on those very cells later in development. Larvae of C.fomicata did not habituate in our experiments: in each experiment, larvae met~orphosed at equiv~ent rates (number met~o~hosed within any given time period) and at equivalent sizes, whether continually immersed in inducer or not. In contrast, both the abalone Haliotis rufescens and the opisthobranch Phestilla sibogae appear to habituate to their natural (and related) chemical inducers (Hadfield & Scheuer, 1985; Hirata & Hadfield, 1986; Trapido-Rosenthal & Morse, 19&6a,b). That is, if those larvae are subjected to the inducer before they are competent to respond, they will not metamorphose at the appropriate age unless transferred for a time to control seawater and then re-exposed to inducer. We propose four explanations for the failure of C.~rn~eata to habituate in our experiments. One is that the “natural” chemical inducer active in our experiments is short-lived or volatile, so that larvae dehabituated between water changes. The longevity of adult-produced pheromone for C. fornicata has not been explored, and its identity is unknown (Pechenik, 1980; McGee & Target, 1989). Both Hadfield & Scheuer (1985) and Trapido-Rosenthal & Morse (1986a,b) used stable inducers (lyophilized coral extract and GABA, respectively). It is also possible that all or most C. fornicata were already competent when first placed in the adult-conditioned water, and so would have no opportunity to habituate. We began our experiments the day after at least 40% of tested larvae metamorphosed in response to excess K+, so that some (or many) individuals were probably also competent to respond to the natural cues. On the other hand, individuals from a
70
J.A.PECHENIK AND C.C.GEE
single larval culture become competent & Pechenik,
1991) and competent
to metamorphose
larvae exposed
at different ages (Zimmerman
to adult-conditioned
water or mi-
crobial films typically metamorphose within about 12 h (pers. obs.); C. fornicata larvae that metamorphosed only after 24 h or more in inducer thus were unlikely to have been competent to respond the possibility.
at the start of the experiment,
but we can’t completely
discredit
It is also possible that larvae of C.~~jc~~ff simply do not habituate to their natural cue while Iarvae of the other two species do. Additional experiments should be done in which C. for~zi~at~ larvae are exposed to inducer soon after they hatch, as in the studies of Trapido-Rosenthal & Morse (1986a,b), Hadfield & Scheuer (1985), and Hirata & Hadfield (1986). One final possibility, however, is that neither Haliotis rufescens or Phestilla sibogae truly habituate; suppose for example, that subjecting prccompetent larvae to inducer simply suppresses full differentiation of the metamorphic pathway. If so, such larvae would fail to metamorphose when exposed to inducer not because their receptors have been habituated or down-regulated, but because the receptors (or other key parts of the pathway) have not yet fully developed. Transferring larvae to control seawater, then, might simply allow differentiation to proceed, thus explaining the ability of larvae to metamorphose when later re-exposed to the appropriate cues. Seen in this light, the isotope binding data of Trapido-Rosenthal & Morse (1986b) could indicate not that habituation of abalone larvae reduced the number of external receptor sites, but that receptors failed to develop (or at least developed far more slowly) in the presence of the inducer. This might also explain why it took abalone larvae up to 2 weeks to fully “dehabituate” after they were transferred to control seawater following pre-exposure to GABA (Trapido-Rosenthal & Morse, 1986a). Such a pseudo-habituation seems a less likely possibility for Phe~t~~la sibogae, since those larvae became responsive to natural inducer within only l-5 h of being transferred to control seawater (Hadfield & Scheuer, 1985). Further studies are required to resolve this question. ACKNOWLEDGEMENTS This research was partially supported by a grant from the Hughes Tufts University. We thank D. Cochrane, M.G. Hadfield, A. Marsh, mous reviewer for commenting on the manuscript, statistical analyses, and V. Ri~ciardone for patiently script.
Foundation to and an anony-
D. Marshall for reviewing the preparing the tables and manu-
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mol-
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FOR LARVAL
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of marine invertebrate
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and the gregarious
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AND
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des Gibbs-Donna-
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durch monov-
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and metamorphosis
Pawlik, J. R., 1990. Natural
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Does it occur? Is
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individually
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in the nudibranch
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R. S., 1974. Biological
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Jagosl., Vol. 10, pp. 263-296. Scheltema,
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G., 1946. Reproduction
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Kommn Danm. Fish.-og Havundrs., Series Plankton, Vol. 4, pp. l-523. Thorson,
G.. 1950. Reproductive
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Bentley & J.N. Havenhand,
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Nudibranchia):
1991. Larval metamorphosis
of the opisthobranch
the effects of choline and elevated potassium
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ion concen-
.I. Mar. Biol. Assoc. UK., Vol. 71, pp. 53-72.
T~dpido-Rosenthal,
H. G. & D. E. Morse,
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1986a. Regulation
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Wilson, D. P., 1968. Some aspects of the development
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Yool, A. .I., S. M. Grau, sium induces
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M. G. Hadfield,
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R.A. Jensen,
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D.A.
Markell & D. E. Morse,
invertebrate
1991. How do femperature
and time to metamorphic
1986. Excess potas-
species. Biol. Bull. ~~~oodsHole, Mass.), and salinity affect relative rates of growth,
competence
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Crepidula plana? Biol. BUN. (Wood.7 Hole, Mass.), Vol. 180, pp. 372-386.
gastropod
59
.I.
JEMBE 01913
Onset of metamorphic competence in larvae of the gastropod Crepidula fornicata (L.), judged by a natural and an artificial cue Jan A. Pechenik and Christopher C. Gee Biology department, Tufif ~~~ver~ity,Redford, ~assac~sett~,
USA
(Received 21 May 1992: revision received 23 October 1992; accepted 3 November 1992) Abstract: Exposing larvae of the marine prosobranch gastropod Crepidula fornicata L. to excess K+ triggers the metamorphosis of competent individuals. This paper considers whether larvae of C. fornicata become responsive to excess K + at the same time and size that they become responsive to adult-conditioned seawater and other apparently natural cues. Fourteen experiments were conducted at room temperature (ca. 23 “C), with 3-5 replicates per treatment and 6-10 larvae per replicate. Larvae became responsive to natural cues within 12-24 h after becoming responsive to excess K+, and responded sooner to natural cues under agitated (20-50 rpm) rather than static conditions (pt0.05). These results emphasize the importance of defining metamorphic competence with respect to specific external cues. Although it seems reasonable to estimate the onset of metamorphic competence using excess K + in this species, the data suggest that the excess K’ and the natural inducer act at different points in the met~orphi~ pathway.
Key words: Competence; Crepid~~u~rn~cutu; Met~orphosis
Free-living larvae are the major dispersal agents in the life cycles of many benthic marine invertebrates (Thorson, 1946, 1950). Frequently there is an obligate period before larvae become physiologically “competent” to metamorphose, followed by a period of time during which larvae defer, or “delay”, their met~orphosis in the absence of specific chemical or physical environmental cues (reviewed by Crisp, 1974; Gray, 1974; Scheltema, 1974, 1986; Burke, 1983, 1986; Woodin, 1986; Pechenik, 1990). We cannot determine how long larvae can delay their metamorphosis unless we first know when the precompetent period of development ends. Precompetent, obligate dispersal periods have been rigorously defined in the laboratory for only a few species, in part because the natural cues triggering metamorphosis are typically not known or are incompletely characterized; when larvae fail to metamorphose in a laboratory experiment, is it because they are not yet competent, or is it because the provided cues Correspondence USA
address: J.A. Pechenik, Biology Department, Tufts University, Medford, MA 02155,
60
J.A. PECHENIK AND C.C. GEE
are inadequate? Working with such natural cues as microbial films and adultconditioned seawater makes the question impossible to answer because larvae are exposed to unknown concentrations of unknown substances. In recent years (but see also Lynch, 1949, 1961) researchers have found that, for many marine invertebrate species, metamorphosis can be triggered by elevating seawater K’ concentrations or exposing larvae to various neuroactive substances and related compounds (Miiller & Buchal, 1973; Hadfield, 1984; Coon et al., 1985; Hirata & Hadfield, 1986; Trapido-Rosenthal & Morse, 1986a,b; Yool et al., 1986; Coon & Bonar, 1987; Pechenik & Heyman, 1987; Cameron et al., 1989; Coon et al., 199Oa,b; Pawlik, 1990; Chevolot et al., 1991; Pires & Hadfield, 1991; Todd et al., 1991). While the major goal of such work has been to determine the sequence of internal biochemical events leading to normal metamorphosis, some researchers have used specific chemical cues to explore the range of sizes at which larvae become competent to metamorphose (Pechenik & Heyman, 1987; Coon et al., 1990a) or to examine the influence of salinity and temperature on the time required for larvae to become metamo~hic~ly competent ~Zirnrne~~ & Pechenik, 1991). For species in which excess K’ induces metamorphosis, using it to judge metamorphic competence has the advantage of working with known concentrations of a fully defined and stable chemical. Moreover, for Crepidzda fomicata the response to excess K + is rapid; at 24 “C, all larvae competent to respond do so within about 6 h (Pechenik & Heyman, 1987). The sequence of steps through which the various chemical inducers of metamorphosis function, and where they act in or on the larva, is not clear (reviewed by Jensen et al., 1990; Pawlik, 1990; Todd et al., 1991), although a number of int~guing models have been proposed for two mollusc species (Trapido-Rosenthal & Morse, 1986a; Baxter & Morse, 1987; Coon et al., 1990a). It is generally believed that increasing external K+ concentration depolarizes artificially the external receptor cells that are normally involved in perceiving the natural chemical cue(s) (Yool et al., 1986; Jensen & Morse, 1990; Chevolot et al., 1991); this depolarization presumably then triggers a series of other, incompletely defined events inside the larva (Burke, 1983; Coon et al., 1985, 1990a,b; Hadfield, 1986; Trapido-Rosenthal & Morse, 1986a; Cameron et al., 1989; Todd et al., 1991), resulting in metamorphosis. However, Todd et al. (1991) point out that the excess K* could instead be acting later in the pathway, bypassing the external sensory cells that perceive the natural chemical stimulus. This hypothesis has not yet been tested. In the field, larvae of C. fomicata and C. plana probably metamorphose in response to cues produced by adults or microbial films associated with adults (Pechenik, 1980; Lima, 1983; Lima & Pechenik, 1985; McGee & Target, 1989). These chemical cues presumably interact with specific external receptors somewhere on the larva (e.g., Arkett et al., 1989). Excess Kt may operate in identical fashion as the natural cue for Crepiduh larvae, or it may bypass one or more steps in the pathway (Pechenik, 1990; Todd et al., 1991). Although it is not yet possible to determine directly how or where the excess K+ operates, we may address the following question: do larvae become
61
COMPETENCEFORLARVALMETAMORPHOSIS responsive
to elevated
K+ and to natural
the two cues may operate pathway. responsive
If, in contrast, to natural
through
cues at the same time in development?
the identical
larvae become
pathway
responsive
If so,
and at the same point in that
to excess K’
cues, it is more likely that the elevated
K’
before they become treatment
bypasses
one or more early steps in the normal pathway, or operates through a different pathway. Here we address this question using larvae of the slipper shell snail C.firnicata (L.). The results provide some information about the action of excess potassium on the larvae, and indicate whether the onset of metamo~hic competence in this species may be reliably judged by larval response to elevated external K’ concentration.
MATERIALSAND Adult
C. fornicara
were collected
METHODS
near Woods
Hole, MA and maintained
at room
temperature (ca. 23 ‘C) on a diet of the flagellated protist Dunalieh tertiolectu (Butcher). When larvae emerged from their egg capsules they were transferred to seawater filtered to 0.45 gum and fed with the unicellular protist Isochrysis g&ma Parke (clone T-ISO) at approximately 1.5 x lo5 cells*mll’. We maintained the larvae at room temperature and changed the seawater and food every two days. Each experiment involved larvae released by different females and cultured until at least 40% of subsampled larvae metamorphosed when external K’ concentration was raised by 20 mM (by adding KCl; Pechenik & Heyman, 1987). Larvae were typically 12-17 days old at the start of an experiment. The main goal in each experiment was to determine whether particular groups of larvae were equally responsive to excess K’ and the natural cue. In addition, when larvae were not comparably responsive to the two cues we sought to determine how much additional time was required for larvae to become comparably responsive. To begin an experiment, we divided larvae from a single culture among the various treatments, using 3-5 replicates of 6-10 larvae each. Larvae were tested in 9 cm glass dishes containing 45 ml of test solution. Usually twice a day, we determined the number of larvae that had metamorphosed in each dish, and the shell lengths of metamorphosed individuals. Juvenile shell lengths were measured at 50 x using a dissection microscope equipped with an ocular micrometer. Each experiment was continued for 3-4 days, or until the number of larvae metamorphosing in response to natural cues and excess K” became statistically comparable. The treatments for each experiment generally consisted of a filtered seawater control. seawater with the K ’ concentration (added as KCl) increased by 20 mM, and a “natural” treatment as described below. For the first set of experiments (Experiments 1-8, 14), the natural treatment consisted of adult-conditioned seawater plus one microbialty filmed shell fragment (Pechenik, 1980; Eyster & Pechenik, 1987; McGee & Target, 1989). Seawater was conditioned by allowing 5-8 adults to feed in it for 6- 16 h, and was then filtered to 0.45 nm before use. For the remaining experiments (Experi-
62 ments
J.A.PECHENIK AND CC. GEE 9-13),
to condition
two filmed shell fragments
were used per replicate,
adults were allowed
the seawater for about 16 h, and half the dishes containing
and adult-conditioned
seawater were agitated (at 20-108
shell fragments
rpm) on a rotary shaker table
(Model 3540, Lab-Line Instruments, Inc.). Agitation has been reported to increase percentage metamorphosis for larvae of the blue mussel, Mytilus edulis (Eyster & Pechenik, 1987), and the polychaetes Sabellaria alveolata (Wilson, 1968; but see Pawlik, 1988) and Phragmatopoma californica (Pawlik, 1988). Larvae exposed to excess potassium were never agitated. Larvae in all treatments (except those subjected to excess K+) were fed T-IS0 at an initial concentration of about at 1.5 x lo5 cells.mll’. All data were analyzed using one-way analysis of variance followed by Bonferonni pairwise comparisons where appropriate. More sophisticated analyses involving the pooling of data from different experiments were inappropriate for the following reasons: the concentration of natural cue may have varied among experiments, and larvae were not necessarily at identical stages of development at the start of each experiment. In this study, the comparisons of interest are within experiments, not among experiments. For at least two marine mollusc species (Phestilla sibogae - Hadfield & Scheuer, 1985; Haliotis rufescens - Trapido-Rosenthal & Morse, 1986a,b), maintaining precompetent larvae in low concentrations of natural inducer “habituates” the larvae, so that the larvae fail to metamorphose when their siblings become competent to do so. These habituated larvae must be transferred to control seawater for a time before they will metamorphose in the presence of the inducer. We therefore conducted two experiments (4-5 replicates per treatment, 6-8 larvae per replicate) to determine whether some larvae of C. fornicata might have habituated in our “natural” treatments. Such habituation could occur if some individuals were not competent to respond to natural cues at the start of an experiment; this would interfere with our ability to determine how much longer it took such individuals to become competent. To test for this possibility we kept some larvae in the natural treatment throughout an experiment but held other larvae in the natural treatment for about 8 h each day, transferring them to control seawater for the remaining portion of the day to allow time for dehabituation. As above, larvae in both treatments were fed T-IS0 at about 1.5 x 10’ cells.ml-‘. All larvae were transferred daily to fresh mixtures of the appropriate treatment, phosis was determined at frequent intervals for up to 4 days.
and percent metamor-
RESULTS We saw no indication that precompetent larvae of C. fornicata habituated to adultconditioned seawater. Figure 1 presents results of our most detailed study. Although few larvae metamorphosed in response to natural cues during the first 10 h of observation, the number of larvae metamorphosing increased considerably over the next 40-60 h, and increased comparably (p > 0.10) whether or not larvae were constantly bathed in adult-conditioned seawater. Comparable results were obtained in a pilot
COMPETENCE
FOR LARVAL
20
0
METAMORPHOSIS
40 60 Hours
80
63
100
Fig. 1. Influence of continuous exposure to natural inducer on metamorphosis of C.firnicata. A, Data for larvae continuously exposed to natural inducer (adult-conditioned seawater plus shell fragments coated with microbial films); A; data for larvae exposed to natural inducer about 8 h per day and then transferred to filtered seawater. Larvae were fed Zsochrysisgulbarza(clone T-ISO) at a concentration of 1 x 10’ cells.ml~’ except when exposed to excess K' Error bars represent 1 SD about the mean. N= five replicates per treatment, eight larvae per replicate.
experiment (four replicates per treatment, six larvae per replicate with metamorphosis determined at five intervals between 24 and 92 h): transferring larvae from adultconditioned seawater to filtered seawater had no statistically significant effect (p > 0.05) on the mean number of individuals metamorphosing. The relative responsiveness of C. fornicata larvae to excess potassium and natural cues depended on whether or not larvae were agitated during exposure to the natural cue. In all but two (Experiments 1 and 3) of the nine experiments conducted without
60
”
1
2’
3
4*
5*
6.
7’
a’
14%
Experiment Fig. 2. Effect of 20 mM increased external K + concentration and natural cues on the number of G. fornicata larvae metamorphosing within 24 h. No larvae were agitated in these experiments. Asterisks indicate experiments in which the mean percentage of larvae metamorphosing in response to excess K+ significantly exceeded the mean percentage metamorphosing in response to natural cues (p< 0.05). Error bars indicate 1 SD about the mean (n = 3-5 replicates per treatment, with 6-10 larvae per replicate).
64
J.A. PECHENIK
AND C.C. GEE
Experiment Fig. 3. Influence of agitation on the mean percentage of C. fomicatcr larvae metamorphosing in response to natural cues within 24 h. Agitation varied between 20 and 108 rpm in different experiments, as indicated. Error bars indicate 1 SD about the mean (n = 5 replicates per treatment, with 10 larvae per replicate).
agitation, significantly more (p < 0.05) larvae metamorphosed in response to elevated K+ than to natural cues over the first 24 h of incubation (Fig. 2). Similarly, excess potassium induced more larvae to metamorphose during the first 24 h than did static natural cues (p < 0.05) in three of the subsequent five experiments (Fig. 3, Experiments 9-l 1; compare first and second bars for each experiment). Moreover, the average size of individuals metamorphosing in response to excess K+ was often smaller than that of individuals metamorphosing in response to static natural cues (Fig. 4, Table I), even
1,600
g
1,200
P 2
800
z c
5
400
8 1
2
3’
4’
Experiment Fig. 4. Influence of elevated external K+ and natural cues (static treatments only) on the mean shell length of juvenile C. fornicafa measured within 24 h of exposure. Asterisks indicate significant differences in mean shell length for larvae induced to metamorphose by natural cues vs. excess K+ (p
ND
ND
10
11
1094+ 127 (12)
9X4* 121(X!)
9
13
1177+96(12)
5
1166186(12)
1056 2 107 (12)
4
12
9892 152(12)
3
6-18 h
7h St: 1214 ?: 92 (8) Ag: 12141 141 (8)
1080 + 143 (24)
1146 f 83 (38)
841 f 56 (47)
906 k 71139)
7h St: 965 It 71(3) Ag: 1020 + 182 (9) 6h St: 1055 i: 119(9) Ag: 953 i 94 ( 15) 6h St: 869+ 71(16) Ag: 914 f 89 (23) 13 h St: 1227 i: 96 (28) A8: 1235 ?: 107 (32)
18h 1295 _t 117 (25)
1208 + 108 (39)
982 f 95 (21)
8h 1127&106(14)
7h 1219+110(l) 10h 1068 f. 163 (23)
[Cl
Natural
Mean shell length
973 ?: 132 (39)
936 & 127 (32)
1202 & 97 (47)
PI
[Al
ND
K+ triggered
Initial
2
Expt.
treatment data.
24 h
St: 1219k 125 Ag: 1214~ 141 (8)
St: 1229+ 10 (38) Ag: 1217i: 119(39)
St: 889i 98 (24) Ag: 93 12 94 (34)
St: 1060 + 120 (14) Ag: 995 + 114 (23)
St: %2t65(1) Ag: 103 1f 140 (18)
1281+ 116 (35)
1113+130(20)
1082 _F155 (28)
1237+86(19)
PI
Natural
A A A B A n A l3 B 6.8 (5,184)
B vs Dst
B vs CAg B vs D
B vs Cst B vs I& 6.0 (6,209) p
3.8 (5,71) p = 0.004
B YS CAg B vs D,,
B vs C,, 9.4 (4,951 p<0.0001
vs B vs C YS D vs Cst vs B “S c vs D vs c w, DAg
B YS Cst B vs D
All
A YS C A YS D B vs C B YS D None
B vs C B vs D
B vs C B YS D A A A A A A A
YS B vs C YS D YS B vs C vs D YS I3
None
Significant (p
All
Not significant (p>O.O5)
Bonferroni comparisons
0.7 (5,68) p = 0.65
8.1 (3,107) pio.001
6.3 (4,89) p
0.7 (3.73) p = 0.53 6.0 (3.91) p < 0.0009
F value
ANOVA
C.fonricaru. Metamorphosed individuals in K+ treatment at 6-18 h and at 24 h. St, static conditions: Ag, agitated; ND, no
of metamorphosed
I
were measured
size QIN~ shell length)
in natural
on mean
individuals
and time of measurement
at 7-8 h. Metamorphosed
of treatment
were measured
Influcncc
TABLE
66
J.A. PECHENIK
AND C.C. GEE
TABLE
II
Time to competence: comparison between natural (but without agitation) “statistical equivalence”, mean percent of larvae that had metamorphosed between treatments. Expt.
Mean ( + SD) Pa metamorph. in exess K’ within 7 h
1 3
45~26 80 + 16.8 100 87.5 k 7.2 78.0 k 16.4 94 k 8.9 95 + 11.2 too 87.5 f 8.8
9 10 11 12 13 14
““‘t
Mean ( + SD) "" metamorph. in natural treatment within 24 h 32.5 65 67.5 45.8 28.0 48 92.7 70.8 17.5
Statistical equivalence by Xh
+ 28.8 + 20.5 f 16.8 + 26.7 + 19.0 * 22 k 25.2 5 18.3 f 14.3
Previous time examined (h)
6.5 9.5 40 8 24 47 0 7 24
24 24 48 24 30 54 24 24 32
q Stationary
@KC,
and excess K’ treatments. At was not significantly different
H
Agitated
0
Control
F t
1,200
_c +J cn 6 =
800
_F tn 5
400
8
0
9 (20
rpm)
10 (20
rpm)
11 (30
rpm)
12 (50
rpm)
13 (108
rpm)
Experiment Fig. 5. Mean she11 length ofjuvenile C. fornicatu induced by excess potassium and by natural cues with and without agitation. All measurements were made within 24 h of exposing larvae to the cues. There were no significant differences in mean shell length for larvae held in stationary or agitated conditions. Each mean typically represents measurements on 20-40 individuals, except as indicated by a number above a bar. Error bars indicate 1 SD about the mean (R = 5 replicates per treatment, with 10 larvae per replicate).
COMPETENCE FOR LARVAL METAMORPHOSIS
67
when juveniles were measured after only 6-8 h (Table I). These results suggest that larvae of C. fornicata became responsive to the elevated IS+ concentration somewhat before they became responsive to static natural cues, with many of the smaller individuals being competent to respond to excess K’ but not yet competent to respond to the natural cues. In such cases, larvae developed the ability to respond to natural cues later (Table II). In Experiment 11, for example (Table II), the response to adultconditioned seawater within 24 h was only about half the response to excess K’ , and a statistically significant difference in response (p < 0.05) was still evident at 47 h (last column). However, the response to the two cues was statistically equivalent by 54 h (column 4). On the other hand, natural cues were as effective (p> 0.10) as excess K’ in promoting metamorphosis when larvae in natural inducer were agitated up to 50 rpm (Experiments 9-12, Fig. 3, p
DISCUSSION
The data suggest that larvae of C. fornicata become responsive to natural cues (adult-conditioned water, microbial films) within 12 h (if larvae are agitated) to 24 h (in static culture) of becoming responsive to elevated K+ concentrations. This is short relative to the amount of time required for newly emerged larvae to become responsive to excess K’ ; Zimmerman & Pechenik (1991) found that the average C. fornicata Larva became competent to respond to excess K’ in 18-23 days at 20 “C, and in 12-19 days at 25 “C. Elevating external K + concentration thus seems a reasonable means of estimating when larvae of this species first become competent to metamo~hose. However, the data also suggest that C. fu$~~cata larvae did not become competent to metamorphose in response to all cues simultaneously. Larvae apparently became responsive to elevated potassium somewhat before they became responsive to natural cues under agitation, and responded to natural cues under agitation somewhat before they responded to the same cues under static conditions. This is indicated by differences in the percentage of larvae responding in each treatment within 24 h of being exposed to the cues provided. Moreover, the average size of individuals measured after they metamorphosed typically varied with treatment; the data suggest that larvae generally became responsive to the natural cue at a more advanced stage of development (assuming this correlates, in general, with a larger size), p~icul~ly if the larvae
68
J.A. PECHENIK AND C.C. GEE
were in static culture. Thus, the alternative expl~at~on that the process of metamorphosis simply requires less time to complete when triggered by some cues (e.g., elevated K ’ concentration), and more time when stimulated by others (e.g., natural cues), seems unlikely. However, the result could be an artifact of the differential effects of the treatments on larval growth. Larvae cease feeding while immersed in the KC1 solution (Pechenik & Heyman, 1987) and therefore were not given phytoplankton during the excess K+ treatment, but were allowed to feed in the other treatments. At 25 ‘C, C. fornicatu larvae grow up to about 60 pm-day - ’ under optimal conditions (one larva per bowl, unlimited food: Pechenik & Lima, 1984). Moreover, newly metamorphosed juveniles grow between 125 and ZOO~m.day-’ under optimal conditions (one juvenile per bowl, unlimited food) at this temperature (Pechenik & Eyster, 1989). Under such conditions, a combination of larval and juvenile growth could completely account for the average larger size of juveniles recovered from our “natural” treatments. Our data argue against this alternative hypothesis. First, the animals in our experiments were relatively crowded and probably food-limited, so that growth rates should have been well below the optima reported above. This suggestion is supported by Bonferroni analyses comparing the sizes of juveniles measured in the different treatments with average sizes of larvae at the start of an experiment; snails often showed negligible growth during the experiments (p> 0.1) (Table I, comparing column A with columns B-D). Moreover, in most cases for which we have data, mean sizes of juveniles resulting from natural cues were greater than the mean sizes of those metamorphosing in response to elevated K +, even when measurements were made as soon as 7-12 h after the start of an experiment (Table I). It appears, then, that juveniles were, on average, smaller when metamorphosing in response to elevated potassium not because larvae or juveniles were growing dramatically faster in other treatments, but rather because the smallest individuals in the natural treatments did not respond to the cue provided. The data emphasize the importance of defining metamorphic “competence” with respect to specific external cues. It is noteworthy that the average size of the few individuals metamorphosing within 24 h under control conditions was not subst~tially greater than that for individuals deliberately triggered to met~orphose in our experiments (Figs 4 and 5). So-called “spontaneous” metamorphosis is thought to reflect either increasing sensitivity to environmental cues, or an individual’s reaching the end of a genetically programmed larval lifespan (Pechenik, 1980, 1990; Coon et al., 1990a); in either case, the results confirm that larval size can be a poor predictor of physiological state (Coon et al., 1990a; Pechenik, 1990; Pechenik et al., 1990; Zimmerman & Pechenik, 1991). It is not yet clear what makes an individual larva competent to metamorphose. Likely possibilities include the differentiation or activation of external receptors, completion of specific neural pathways, differentiation of specific neurosecretory systems, or differentiation of specific receptors on target tissues (Hadfield, 1978; Highnam, 1981; Burke, 1983; Trapido-Rosenth~ & Morse, 1986a). Our data touch on this issue, al-
COMPETENCE
FOR LARVAL METAMORPHOSIS
69
though indirectly. If excess K+ and natural cues act at identical points in the metamorphic pathway for C. for~~cata, we would expect larvae to become responsive to both cues at exactly the same time in development. In our experiments, however, the larvae of C. fornicata apparently became responsive to the elevated K’ before they could respond to natural cues. If so, the excess K’ must be acting at a later point in the pathway than the natural cues act, as suggested by Todd et al, (1991), and the last step to become functional in the pathway must precede the point affected by excess K’. Similarly, Trapido-Rosenth~ & Morse (1986b) found that exposing precompetent abalone larvae (~aliot~s r~~~cen~) to GABA made them unresponsive to GABA but not to excess K+ when exposed several days later, again implying that the excess K + acts downstream from the target of natural cues. This logic assumes that both cues operate through the same pathway. Since the morphological aspects of metamorphosis as triggered by natural cues and by elevating external K+ concentrations differ in C. fornicata (Pechenik & Heyman, 1987), this may not be a correct assumption for this species. Note also that even if excess potassium and natural cues acted at different points in the same pathway in our experiments, existing data cannot rule out the possibility that excess IV will act earlier in the pathway once larvae become competent to respond to natural cues. For example, the excess K’ may bypass epithelial receptor cells in larvae not yet competent to respond to natural cues, but might act on those very cells later in development. Larvae of C.fomicata did not habituate in our experiments: in each experiment, larvae met~orphosed at equiv~ent rates (number met~o~hosed within any given time period) and at equivalent sizes, whether continually immersed in inducer or not. In contrast, both the abalone Haliotis rufescens and the opisthobranch Phestilla sibogae appear to habituate to their natural (and related) chemical inducers (Hadfield & Scheuer, 1985; Hirata & Hadfield, 1986; Trapido-Rosenthal & Morse, 19&6a,b). That is, if those larvae are subjected to the inducer before they are competent to respond, they will not metamorphose at the appropriate age unless transferred for a time to control seawater and then re-exposed to inducer. We propose four explanations for the failure of C.~rn~eata to habituate in our experiments. One is that the “natural” chemical inducer active in our experiments is short-lived or volatile, so that larvae dehabituated between water changes. The longevity of adult-produced pheromone for C. fornicata has not been explored, and its identity is unknown (Pechenik, 1980; McGee & Target, 1989). Both Hadfield & Scheuer (1985) and Trapido-Rosenthal & Morse (1986a,b) used stable inducers (lyophilized coral extract and GABA, respectively). It is also possible that all or most C. fornicata were already competent when first placed in the adult-conditioned water, and so would have no opportunity to habituate. We began our experiments the day after at least 40% of tested larvae metamorphosed in response to excess K+, so that some (or many) individuals were probably also competent to respond to the natural cues. On the other hand, individuals from a
70
J.A.PECHENIK AND C.C.GEE
single larval culture become competent & Pechenik,
1991) and competent
to metamorphose
larvae exposed
at different ages (Zimmerman
to adult-conditioned
water or mi-
crobial films typically metamorphose within about 12 h (pers. obs.); C. fornicata larvae that metamorphosed only after 24 h or more in inducer thus were unlikely to have been competent to respond the possibility.
at the start of the experiment,
but we can’t completely
discredit
It is also possible that larvae of C.~~jc~~ff simply do not habituate to their natural cue while Iarvae of the other two species do. Additional experiments should be done in which C. for~zi~at~ larvae are exposed to inducer soon after they hatch, as in the studies of Trapido-Rosenthal & Morse (1986a,b), Hadfield & Scheuer (1985), and Hirata & Hadfield (1986). One final possibility, however, is that neither Haliotis rufescens or Phestilla sibogae truly habituate; suppose for example, that subjecting prccompetent larvae to inducer simply suppresses full differentiation of the metamorphic pathway. If so, such larvae would fail to metamorphose when exposed to inducer not because their receptors have been habituated or down-regulated, but because the receptors (or other key parts of the pathway) have not yet fully developed. Transferring larvae to control seawater, then, might simply allow differentiation to proceed, thus explaining the ability of larvae to metamorphose when later re-exposed to the appropriate cues. Seen in this light, the isotope binding data of Trapido-Rosenthal & Morse (1986b) could indicate not that habituation of abalone larvae reduced the number of external receptor sites, but that receptors failed to develop (or at least developed far more slowly) in the presence of the inducer. This might also explain why it took abalone larvae up to 2 weeks to fully “dehabituate” after they were transferred to control seawater following pre-exposure to GABA (Trapido-Rosenthal & Morse, 1986a). Such a pseudo-habituation seems a less likely possibility for Phe~t~~la sibogae, since those larvae became responsive to natural inducer within only l-5 h of being transferred to control seawater (Hadfield & Scheuer, 1985). Further studies are required to resolve this question. ACKNOWLEDGEMENTS This research was partially supported by a grant from the Hughes Tufts University. We thank D. Cochrane, M.G. Hadfield, A. Marsh, mous reviewer for commenting on the manuscript, statistical analyses, and V. Ri~ciardone for patiently script.
Foundation to and an anony-
D. Marshall for reviewing the preparing the tables and manu-
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