An experimental test of shadow response function in ascidian tadpoles

J. Exp. Mar. Biol. Ecol., 1985, Vol. 85, pp. 165-175

165

Elsevier

JEM 409

AN EXPERIMENTAL

TEST OF SHADOW

RESPONSE FUNCTION IN

ASCIDIAN TADPOLES

CRAIG

M. YOUNG’ and Fu-SHIANG

CHIA

Department of Zoology, University of Alberta, Edmonton, Alberta T6G 2E9. Canada

(Received 12 March 1984; revision received 29 June 1984; accepted 17 October 1984) Abstract: Tadpole larvae of nearly all ascidians are induced to swim by a sudden decrease in light intensity. The function of this shadow response remains unknown; some workers have suggested that it may help larvae locate shaded settlement sites. In a small-scale laboratory experiment with eight species of solitary ascidians, tadpoles were offered shaded and unshaded surfaces facing up and facing down, while being maintained under conditions of continuous light, continuous dark, or alternating light and dark. Larvae induced to swim often by shadows were not distributed differently at settlement than larvae in the other treatments. Moreover, the total number of larvae undergoing metamorphosis successfully did not differ among treatments for most species. It was concluded that the mechanism by which larvae locate shaded habitats and overhangs is independent of the shadow response.

Key words: ascidian tadpoles; shadow response; habitat selection; ascidian settlement

INTRODUCTION

The behavior of ascidian tadpole larvae is highly variable, both within and among species (Grave, 1935; Crisp & Ghobashy, 197 1; Young Jz Braithwaite, 1980). However, the shadow response, in which an abrupt decrease in light intensity elicits active, generally upward, swimming, occurs almost universally. Only two species investigated to date, A4oZgulucitrina, which has no photoreceptor (Grave, 1926) and Metundrocarpa tuylori (Abbott, 1955) are reported not to exhibit this response. Although such a widespread phenomenon clearly demands a functional explanation, the few hypotheses that have been advanced have not been tested adequately with experiments. The shadow response may be considered a form of photokinesis (sensu Fraenkel & Gunn, 1940) in that the level of activity, and not the direction of movement, changes in response to the stimulus. Inactive larvae resting on the bottom, drifting passively, or sinking through the water column exhibit the response, while larvae that are already swimming do not. Ontogenetically, the response develops with the formation of the larval ocellus and continues until the time of metamorphosis. However, some species display the response less frequently with advancing age (Grave & Woodbridge, 1924; Grave, 1935). Larvae of most solitary ascidians respond to shadows at light intensities down to 0.1-0.5 pE.rnS2.s-’ (Young, 1982). ’ Present address: Department of Biological Science, Florida State University, Tallahassee, Fl 32306, U.S.A. 0022-0981/85/$03.30 0 1985 Elsevier Science Publishers B.V.

166

CRAIG M. YOUNG

AND FU-SHIANG

CHIA

Shadow responses of adult marine invertebrates, including serpulids, echinoids, and cirripedes, are generally considered defensive in function (reviewed by Steven, 1963). Larvae of the xanthid crab Rhithropanopeus hati.rii escape ctenophores and other pelagic predators by sinking or swimming downward when shaded (Forward, 1977). It seems unlikely that the ascidian shadow response has a similar escape function, since tadpoles would swim toward rather than away from a predator casting a shadow. Mast (192 1) and Abbott (1955) both noted that an ascidian larva swimming through the water in its characteristic spiral fashion regularly casts a shadow on its own ocellus. Mast (1921) suggested that this was a mechanism mediating phototactic orientation. In support of this idea, he noted that a tadpole of Styela plicata trapped under a cover slip would twitch its tail in different directions depending on the relationship of the light source to the ocellus. The hypothesis tested in the present paper was proposed initially by Woodbridge (1924). She suggested that the shadow response enables tadpoles of Botryllus schlosseri to locate the undersides of eelgrass blades, which are suitable sites for attachment and growth. Larvae drifting through the shadows cast by blades would presumably be stimulated to swim upward, thus contacting the plants. Woodbridge supported her hypothesis experimentally by demonstrating that more tadpoles settled on an eelgrass blade suspended diagonally across a culture dish than on the bottom or sides of the dish. Unfortunately, her experiment was not controlled for differences in substratum composition and texture, now known to be important cues used by tadpoles selecting habitats (Young & Braithwaite, 1980; Schmidt, 1982; Young, 1982). Juveniles of most subtidal ascidians seem to require cryptic sites for survival (Dybern, 1963; Goodbody, 1963; Young & Chia, 1984). Negative phototaxis is generally invoked as the mechanism whereby larvae select such habitats (Bert-ill, 1950; Crisp & Ghobashy, 197 1; Young, 1982). However, it is possible to envisage a hypothetical scenario in which the shadow response increases a larva’s chances of finding shaded sites. A larva drifting passively with the current would be stimulated to swim when passing under a ledge or near a surface of low reflectivity. If a larva increased its activity at this time, even by erratic swimming, the probability of encountering a surface would be greater than if the larva remained motionless. Furthermore, by swimming only when there is high probability of encountering a site, the larva would presumably conserve energy and thus be able to delay metamorphosis longer, again improving its chances for successful settlement. In the present study, we tested the modified Woodbridge (1924) hypothesis outlined above using tadpoles of eight species of solitary ascidians from the San Juan Islands, Washington. MATERIALS AND METHODS

Adult ascidians were collected from the undersides of floating docks or from various rocky subtidal sites near Friday Harbor, Washington. Embryos were obtained from

ASCIDIAN

TADPOLE

SHADOW

RESPONSE

167

Corella inflata Huntsman and Corella willmeriana Herdman by exposing adults to light after holding them in the dark for at least 12 h. Oocytes and sperm were pipetted directly from the gonoducts of Ascidia callosa Stimpson and Ascidia paratropa (Huntsman) and mixed in vitro. Gonads were dissected from Pyura haustor (Stimpson), Boltenia villosa (Stimpson), Cnemidocalpa jhmarkiensis (Kiaer) and Styela gibbsii (Stimpson) and macerated through 253~pm nitex screen into filtered sea water, which was changed at least five times during the first 12 h to eliminate excess sperm and gonadal tissue. All cultures were maintained near ambient sea-water temperature (9-12 “C) in a shallow sea-water table. The experimental containers were the same as those used in previous work on ascidian phototaxis: 16-ml polystyrene Petri dishes with tight-fitting lids, half-shaded with black electrician’s tape and completely tilled with sea water (Young & Braithwaite, 1980; Young, 1982). Larvae chose between the dark side and the light side, and also between the top and bottom surfaces. To test the Woodbridge (1924) hypothesis, we incubated experimental dishes of larvae in continuous light, continuous darkness and alternating light and dark, then compared settlement patterns. Four replicate dishes, each containing between 49 and 325 tadpoles, were run for each treatment with each species. All treatments rested in a shallow aquarium of running sea water 30 cm under a 100-W incandescent light bulb. Dark treatment dishes were enclosed in small bags of opaque black plastic. Light intensity ranged from 50 PE. m _ 2 . s _ l at night to = 100 PE. m- 2. s _ ’ by day. Thus, background lighting conditions varied over the day, with the higher daytime light levels resulting from window light and overhead fluorescent lamps. The alternating light/dark condition was produced by a 12-cm diameter disk of Plexiglas, half-painted with matt black paint and rotated by a clock motor at 1 t-pmjust above the experimental dishes. Larvae responded to shadows cast by the rotating disk in the same way that they responded to other shadows. All experiments were terminated at least 1 day after tadpoles lost the capacity to undergo metamorphosis (as determined in separate experiments; Young, 1982). At this time, the number settled in each region of the dishes was counted under a Wild M-5 dissecting microscope, any unmetamorphosed larvae were noted, and a small sample (usually 20) of the latter were tested under a dissecting microscope illuminator to see if they still responded to shadows. RESULTS

Field experiments (Young & Chia, 1984) suggested that juvenile ascidians survive better in dark habitats than in light habitats and better on the undersides of overhangs than on upward-facing surfaces, because of the adverse effects of silt, tilamentous algae, and benthic herbivores. We thus predicted a priori that, if the shadow response helps larvae locate suitable settlement sites, one or both of the following should hold true: (1) larvae in alternating light/dark conditions should locate the top surface, the dark side, or the top/dark surface more readily than larvae in constant lighting conditions,

CRAIG M. YOUNG AND FU-SHIANG

168

StyHo gibbsii

Pyuro haustor

CHIA

Cnemidocortro

-I

finmorklensis

Bolteniovilloso

60

Core/lo wiiimeriona

Ascidio

poratropo

-I

Corelo

inffofo

Ascidio colloso

Fig. 1. Settlement distributions of ascidian larvae (eight species) under conditions of continuous light (open histogram bars) continuous darkness (black bars) and alternating light, in which dishes were shaded for half of each minute (shaded bars): each histogram bar represents the mean f 1 SE percentage of larvae settled in a particular region of the dish; the left group of bars shows percentage of larvae settled on top surface of dish, the middle group is for larvae settling on dark side of dish, and the right group of bars shows the percentage of larvae in each treatment that located the dark undersurface of the lid.

ASCIDIAN

TADPOLE

SHADOW

RESPONSE

169

and (2) more larvae overall should settle successfully in fluctuating light than in continuous light or darkness. Distributional data, which test the first hypothesis, are presented in Fig. 1. More tadpoles reached the top undersurface of the lid under alternating light/dark conditions in Styela gibbsii, Boltenia villosa, and Corella injlata, though the difference among treatments, as tested by one-way analysis of variance (Table I), was significant only in TABLE

I

Single factor analysis ofvariance on percentages of tadpoles selecting the “top” (undersurface oflid) position in continuous light, continuous dark, and alternating light/dark treatments: data were arcsine transformed for the analyses; ns, not significant. Source of variation

d.f.

ss

MS

F

P

Ascidia callosa

among within total

2 9 11

2323.569 4968.906 1292.415

1161.785 552.100

2.104

ns

Ascidia paratropa

among within total

2 9 11

74.568 3845.138 3919.706

37.284 427.231

0.087

ns

Corella injlata

among within total

2 9 11

373.461 613.514 986.975

186.730 68.168

2.739

ns

Corella willmeriana

among within total

2 9 11

707.32 1421.51 2134.89

353.66 158.62

2.23

ns

Boltenia villosa

among within total

2 9 11

391.105 1004.110 1395.215

195.552 111.569

1.753

ns

Pyura haustor

among within total

2 9 11

49.369 409.574 458.943

24.684 45.508

0.542

ns

Styela gibbsii

among within total

2 9 11

1253.395 1113.563 2366.958

626.697 123.729

5.065

< 0.05

Cnemidocarpa jnmarkiensis

among within total

2 9 11

1811.349 1166.129 2977.478

905.674 129.569

6.989

< 0.025

Species

Stylea gibbsii. Conversely, more Ascidia callosa, A. paratropa, Corella willmeriana, and Cnemidocarpafinmarkiensis reached the top in complete darkness. The difference was significant only in C. fmmarkiensis. The extremely low overall settlement on the undersurface of the lid in Pyura haustor was unusual for that species (Young, 1982) and may

have resulted from a culture of unhealthy tadpoles.

CRAIG

170

M. YOUNG

AND FU-SHIANG

CHIA

TABLE II Single factor analysis ofvariance on percentages of tadpoles selecting the dark sides of experimental dishes in continuous light and alternating light/dark treatments: data were arcsine transformed for the analysis; ns, not significant.

Source of variation

d.f.

ss

MS

F

P

Ascidia callosa

among within total

1 6 7

21.263 1845.726 1866.989

21.263 307.621

0.069

ns

Ascidia paratropa

among within total

1 6 7

208.794 2452.082 2660.876

208.794 408.680

0.511

ns

Corella injlata

among within total

1 6 7

209.405 374.386 583.791

209.405 62.397

3.356

ns

Corella willmeriana

among within total

1 6 7

17.110 1136.249 1153.359

17.110 189.375

0.090

ns

Bolienia villosa

among within total

1 6 7

41.359 274.054 315.413

41.359 45.675

0.905

ns

Pyura hausror

among within total

1 6 I

94.187 969.229 1063.416

94.187 161.538

0.583

ns

Styela gibbsii

among within total

1 6 7

332.433 774.619 1107.052

332.443 129.103

2.574

ns

Cnemidocarpa finmarkiensis

among within total

1 6 7

0.665 696.420 697.085

0.665 116.070

0.006

ns

Species

Larvae located neither the dark side of the dish (Table II) nor the dark/top surface (Table III) any more frequently in alternating light/dark than in continuous light (Fig. 1). The percentages of larvae selecting various regions of the dish were tested against expected random values (based on surface areas available) to determine whether larvae choose the “optimal” surfaces more frequently than random settlement would predict. Following F-max tests to establish homogeneity of variances, one-tailed t-tests were performed for each separate treatment (Table IV). Four of the eight species showed preferences (significantly higher than 50 % settlement) for the undersurface of the lid in continuous darkness, whereas none demonstrated the same preference in light treatments, and only one (Cnemidocarpafinmarkiensis) preferred the top surface in alternating light/dark. This finding supports the previous conclusion (Table I) that shadows do not increase the probability of locating the top surface. Five of the eight species demonstrated significant photonegative tendencies in light treatments (Table IV); five likewise

171

ASCIDIAN TADPOLE SHADOW RESPONSE TABLE III

Single factor analyses of variance on arcsine-transformed percentages of tadpoles selecting the “top/dark” position in continuous light and alternating light/d~k treatments: ns, not si~ificant. Source of variation

d.f.

ss

Ascidiu callosa

among within total

1 6 7

445.062 3322.059 3767.121

Ascidia paratropa

among within total

1

6 I

Coreila injluta

among within total

Corella wilheriana

Boltenia villosa

Species -.

pvUra haustor

Styela gibbsii

-

F

P

445.062 553.676

0.804

ns

527.961 5745.239 6273.200

527.961 957.539

0.551

ns

1 6 7

0.120 1065.131 1065.251

0.120 177.540

0.001

ns

among within total

1 6 1

160.202 1168.202 1328.404

160.202 194.700

0.823

ns

among within total

1

34.150 845.950 880.1~

34.150 140.990

0.242

liS

6 I

5.070 59.576

0.085

ns

MS

-.--

1

among within total

6 1

5.070 357.456 362.526

among within total

1 6 7

1089.206 1393.866 2483.072

1089.206 232.311

4.688

ns

among within total

1

60.332 469.983 530.315

60.332 78.330

0.770

ns

6 1

selected the dark side dispropo~ionately in alternating light/dark, though two of the species significant in alternating light were not significant in the light treatment. Only three species showed a significant preference for the top/dark surface: two in the light treatment and one in the alternating light/dark treatment. In most experiments with aseidian larvae, a small percentage delay met~o~hosis until they die. Table V gives the percentage of larvae successfully locating a settlement site and undergoing metamorphosis by the time the experiment was terminated. The difference among treatments was only significant in BoZteniu villosa, tadpoles of which demonstrated the predicted response. In four of the other species the same pattern (higher settlement in alternating light than in other treatments) was seen, though the differences among treatments were non-si~i~c~t. We expected that larvae induced to swim frequently might ultimately become exhausted or unresponsive to shadows. This was not the case. In each species, between

Ascidia callosa Ascidia paratropa Corella infata CoreNa willmeriana Boltenia villosa Pyura hatutor Styela gibbsii Cnemidoca~a ~~markiens~

0.74

1.64

nS ns nS

ns ns ns

ns

IlS

P

1.62

t

light

6.96

3.24

3.21 5.46

t

P

<;:05

nS

<0l”o25 ns

< 0.025
dark

Percent top

2.12 2.77

1.95 1.09 0.73 2.31

t

<:05

2.02 2.36 2.45

4.00

IlS

ns

1.61 1.38 4.21 8.97

1

ns ns

P

< 0.05 < 0.05

ns

< 0.025 < 0.025

< 0.025

light ~____

ns ns ns ns

P

alternating t

ns ns ns co.01 < 0.025 < 0.05 < 0.05


P

alternating

5.39 1.08 1.86 2.31 5.21 3.50 3.17 2.35

-~

Percent dark

1.24 2.88

0.85 1.21 0.46 3.29

t

IlS

ns

XX3

P

ns ns ns < 0.05

< 0.025

light

2.29 2.26

13.71 0.37 0.37 0.54

t

tIS ns

ns ns ns ns ns

< 0.00 1

P

alternating

Percent top/dark

One-tailed t-tests of the hypotheses H,: percent top > 50%; percent dark > 50%; percent top/dark > 25%; where no r-value is given, the hypothesis was rejected automatically because the mean was less than the “random settlement” value; ns, not significant.

TABLE IV

ASCIDIAN TADPOLE SHADOW RESPONSE

173

TABLE V

Percentage of animals metamorphosed at termination of experiment in continuous light, continuous dark, and alternating light/dark: significance tested with Kruskal-Wallace test.

Species Corella inJlata Corella willmeriana Ascidia callosa Ascidia paratropa Styela gibbsii Cnemidocarpa finmarkiensis Boltenia villosa

Duration of experiment (days)

SE)

Percent metamorphosed

(Sz+

Light

Dark

Alternating

57.8 & 7.1

49.5 f 5.6

61.3 f

2 7 12 8 10

31.3 31.0 5.9 94.3 85.8

53.1 41.3 10.6 87.6 88.3

31.7 43.5 19.5 85.4 78.9

10

57.1 + 6.8

2

+ * f. f +

2.7 7.7 0.2

1.0 4.7

+ * f f k

6.3 9.0 4.4 5.8 4.6

62.8 + 3.5

6.1

P 0.329

+ 6.9 k 6.4 f 15.3 f 1.7 k 4.5

0.059 0.472 0.925 0.208 0.412

84.0 & 6.8

0.037

70 and 100% of the unmetamorphosed larvae still responded to shadows at the conclusion of the experiment. The among-treatment differences were all non-significant by the Kruskal-Wallace test.

DISCUSSION

Our data do not support the hypothesis that the ascidian shadow response helps larvae locate shaded or overhanging settlement sites. In comparing the distributions of metamorphosed ascidians under three experimental light regimes (continuous light, continuous dark, alternating light/dark) we saw no consistent pattern. Moreover, the outcome predicted by the habitat selection hypothesis (more larvae selecting “optimal” habitats in alternating light/dark than in other treatments) occurred infrequently. In both continuous light and alternating light treatments, larvae of most species located dark regions more often than expected by chance. This result is consistent with the frequently cited idea that phototaxis rather than photokinesis mediates the selection of cryptic settlement sites (Berrill, 1950, 1979; Crisp & Ghobashy, 1971; Young, 1982). Laboratory experiments with larvae give only a rough approximation of phenomena that might occur in the field. It is entirely possible that we saw no significant effects in our experiment simply because it was run on a very small scale and under artificial conditions. One problem derives from the fact that larvae in continuous light treatments were offered a choice between light and dark. Larvae swimming horizontally in such dishes might be expected to turn upward when passing from the light to the dark (Mast, 192 l), thus encountering the underside of the lid. This confounding behavior is not the same as the shadow response; in the terminology of Fraenkel 8c Gunn (1940) it is “photo-klino-taxis”. One could probably minimize the confounding effects of taxes by running the experiments in larger dishes or by reducing the relative proportion of shaded substratum available. Previous workers have reported that larvae induced to swim frequently by regularly occurring shadows begin metamorphosis sooner than larvae maintained in constant

174

CRAIG M. YOUNG

AND FU-SHIANG

CHIA

light (Grave, 1935; Crisp & Ghobashy, 1971). Although we have no data on the time course of metamorphosis, our data on total number settling in the various treatments support the observation of these previous workers only for Bolteniu villosa. In three other species, the treatment means ranked in the predicted order, but differences were not significant. Grave (1935) attempted to show that frequently swimming larvae produced a larger quantity of some metabolic product that induced metamorphosis. It seems equally probable that swimming larvae are forced to settle sooner than quiescent ones because the former exhaust their yolk supplies. Pechenik (1980) has studied the energetics of gastropod veligers delaying metamorphosis. A similar study is needed for ascidian tadpoles in order to determine whether swimming reduces energy supplies enough to shorten the period of metamorphic competency. In the few species induced to settle by shadows, the shadow response could be involved in habitat selection indirectly by increasing the probability that a larva will settle before drifting out of an area with many shaded sites. However, since most larvae also display phototaxis, an indirect mechanism of this sort would be of little value. The universality of the shadow response in ascidian tadpoles suggests that either a more fundamental function remains undiscovered or the response is an incidental and non-adaptive consequence of the tadpole’s method of orientating to light with a single photoreceptor.

REFERENCES

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H., 1924. Botryllus schlosseri (Pallas): The behavior of the larva with special reference to the habitat. Biol. Bull. (Woods Hole, Mass.), Vol. 47, pp. 223-230. YOUNG, C.M., 1982. Larval behavior, predation and early post-settling mortality as determinants of spatial distribution in subtidal solitary ascidians of the San Juan Islands, Washington. Ph.D. dissertation, University of Alberta, 260 pp. YOUNG,C.M. & L. F. BRAITHWAITE,1980. Larval behavior and post-settling morphology in the ascidian, Chelysoma productum Stimpson. J. Exp. Mar. Biol. Ecol., Vol. 42, pp. 157-169. YOUNG,C. M. & F. S. CHIA, 1984. Microhabitat-associated variability in survival and growth of subtidal solitary ascidians during the first 21 days after settlement. Mar. Biol., Vol. 81, pp. 61-68.

WOODBRIDGE,