potential antifouling mechanisms using toxic chemicals in some british ascidians

Journal of Experimental Marine Biology and Ecology

188 (1995) 49-62

JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY

Potential antifouling mechanisms using toxic chemicals in some British ascidians

Serena L.-M. Tea*, John S. Ryland

School of Biological Sciences, University of Wales Swansea, Swansea SA2 8PP, UK

Received 24 November 1994; revision received 13 October 1994; accepted 2 November 1994

Abstract

Four ascidians - Clavelina lepadiformis, Aplidium prolgerum, Morchellium argus and Botryllus schlosseri - were examined for larvotoxic properties. In using aqueous extracts against invertebrate larvae, moderate levels of toxicity were detected. C. lepadiformis extract displayed strong toxicity to all larvae. Methanol extracts applied to a polystyrene surface reduced settlement of M. argus larvae, and produced toxic effects against larvae of the bryozoan Flustrellidra hispida and the tubeworm Spirorbis spirorbis. The observations suggest that the ascidians may contain potential anti-larval compounds and their possible role in preventing fouling is discussed.

Keywords: Allelochemical; Ascidian; Chemical defense; Larva; Larvotoxin

1. Introduction

In the marine environment, suitable habitats for growth of sessile fauna are often limited and competition between species within these may be intense (reviews by Jackson, 1977, 1979). Many sessile marine organisms have been demonstrated to contain offensive compounds with potential allelopathic functions, and chemical defence mechanisms are now known to play a major role in interspecific interactions (Co11 et al., 1985; Bakus et al., 1986; La Barre et al., 1986 a,b; Davis et al., 1989, 1991; Davis & Wright, 1990; Paul, 1992).

Antifouling mechanisms in ascidians may be broadly divided into: (1) physical/ structural and (2) chemical defence mechanisms; the two modes are not exclusive of each other.

* Corresponding author. Department of Zoology, The National University of Singapore, Singapore 05 11.

0022-0981/95/$9.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0022-0981(94)00185-5

50 S.L.M. Ten. J.S. Rylandl J. Exp. Mar. Biol. Ecol. 188 (1995) 49-62

I. I. Physical defence mechanisms

Among ascidians, exfoliation of epidermal tissues has been suggested for species such as Ascidia nigra and some polycitorids (Davis et al., 1989). This results in the regeneration of surfaces and is effective against both micro- and macro-fouling. Somewhat drastic, it would probably be most employed in long-lived solitary species. Production of copious mucus is not known for many ascidians (Dyrynda, 1986; Davis et al., 1989). The cyclic degeneration and regeneration of colonies may also result in lower levels of fouling where rates of epibiosis are moderate to low (Davis et al., 1989). The delicate texture of the tunic of species such as Diplosoma listerianum would discourage macro- foulants (Dyrynda, 1986). Many unitary ascidians, such as those in the families Asci- diidae, Pyuridae and Styelidae, are tolerant of heavy epibiosis; indeed the cover, often including hydroids, may well have a protective role (Kott, 1989). Such ascidians usu- ally have a thick leathery tunic immune to damage caused by the mechanical anchor- ing of epibionts. The spiny texture of the test of the Boltenia echinata may discourage larval settlement, though the spines of Halocynthia igaboja actually promote settlement by larvae of such epizoites as Boltenia villosa (Young, 1986 and pers. comm.). Many molgulid and other species occurring in sand/mud bottom habitats often incorporate solid particulate matter into their tunic, and may occur partially buried in the substra- tum. In general, most colonial ascidians lack hard structures that could contribute to structural defence, such as the spines and avicularia of bryozoans, though some didemnids are densely packed with calcareous granules and others incorporate inor- ganic particles into the test. Physical defence mechanisms are limited in function and lack versatility against fouling as potential settlers vary greatly in morphology and mode of colonization. As such, they are more likely to occur in concert with other antifouling mechanisms or in situations where the threat is specific.

I .2. Chemical defences

Stoecker (1978) proposed the acid-vanadium theory in ascidians and showed that low pH and high vanadium content of the tunic, as in Ascidia nigra, prevented settlement on it of larvae of the ascidian Ecteinascidia turbinata and the hydroid Pennaria tiarella. Subsequent workers have disputed the theory since high vanadium and low pH are not always correlated with low levels of fouling (Parry, 1984; Davis & Wright, 1989; Wahl, 1989). Moreover, as seawater acts as a powerful buffer, low pH levels would be difficult to maintain. Many secondary metabolites have been suggested as possible antifoulants. A wide range of bioactive extracts from ascidians have been shown to exhibit antibacterial (Carter & Rinehart, 1978; Stoecker, 1978; Ireland et al., 1982; Rinehart et al., 1984, 1987; Dyrynda, 1985; Davis & Wright, 1989; Biard et al., 1990) and anti-larval (Young & Chia, 1981; Dyrynda, 1985; Davis & Wright, 1989) properties.

Low MW compounds detected in some ascidians delay and/or inhibit settlement of larvae without any marked toxic effects (Young & Chia, 1981; Davis & Wright, 1990; Davis et al., 1991). In most instances, the compounds have not been characterized and it is unclear if they have a true physiological effect on the larvae, act merely as nega-

S.L.M. Teo, J.S. Ryland/ J. Exp. Mar. Biol. Ecol. 188 (1995) 49-62 51

tive signal cues, or alter the character of the settlement surface to make it less preferred. Young & Chia (198 1) found that seawater previously containing Diplosoma macdonaldi

colonies delayed settlement of Bugulapaczfia larvae. Dyrynda (1985) found that methanol extracts of Didemnum candidum and Morchellium argus were moderately toxic to larvae of Bugula turbinata but not B. jabellata. Davis & Wright (1990) showed that eudistomins g and h (both soluble alkaloids) isolated from Eudistoma olivaceum are toxic to larvae of Bugula neritina at concentrations 0.5% of those naturally present. They also isolated, but could not purify, a fraction that inhibited settlement without any marked toxicity to the larvae. Cross comparisons must be treated cautiously as test animals and experimental techniques are often different. Species-specificity may be important (Dyrynda, 1985; Rittschof et al., 1988) especially if results are to be related to field observations.

In this study, four readily available, temperate water, colonial ascidians Clavelina

lepadiformis (Miiller), Aplidiumproliferum (Milne Edwards), M. orchellium argus (Milne Edwards), and Botryllus schlosseri (Pallas) were tested for anti-larval effects. Toxicity assays were conducted with crude aqueous extracts, against a standard test organism, Artemia sp. nauplii, and the easily obtained larvae of three common sessile organisms - the bryozoan Flustrellidra hispida (Fabricius), the tubeworm Spirorbis spirorbis (L.) and the hydroid Tubularia larynx (Ellis and Solander). A preliminary assay for anti- settlement effects using a crude methanol extract was also carried out, using the larvae of M. argus (Milne Edwards) in addition to those of the bryozoan and serpulid. These organisms are commonly found epiphytic on Fucus serratus L. and hard substrata on sheltered shores, occasionally occurring in the same community.

2. Materials and methods

2.1. Test animals

2.1 .I. Artemia sp. nauplii Brine shrimp eggs (San Francisco Bay Brand) were cultured in seawater. Three-day

old nauplii were used in the experiments.

2. I .2. Flustrellidra hispida larvae

Larvae were obtained from adult colonies collected from Bracelet Bay on Gower Peninsula, South Wales, during May-June. Colonies were rinsed with clean sea water and kept in the dark overnight. On return to seawater and light, larvae were released and collected using a wide-mouthed pipette.

2.1.3. Spirorbis spirorbis larvae Adults were collected from Bracelet Bay on Fucus serratus at low water of neap tides

during June-July. On return to seawater, larvae were released and collected using a wide-mouthed pipette.

52 S.L.M. Teo, J.S. Ryland / J. Exp. Mur. Biol. Ecol. 188 (1995) 49-62

2.1.4. Tubularia larynx larvae

The hydroid was collected from test panels at Texaco Jetty and other submerged structures at Angle, Milford Haven, southwest Wales in April-May. The colonies were maintained in seawater. Late stage actinulae were collected as released.

2.1.5. Morchellium argus larvae Brooding adult colonies of M. argus were collected from boulders at St. Catherine’s

Rock, Tenby, southwest Wales, in September-October. On return to the laboratory, they were placed in a large aquarium containing aerated sea water collected from Oxwich Bay, Gower Peninsula, with z lo-20 g wet weight of colonies to 1 1 of seawater. The colonies were maintained in total darkness at a constant 17 “C for 24- 36 h. Larvae were released about 1 h after exposure to light, and were very gently collected with a wide-mouthed glass pipette.

2.2. Preparation of extracts

The ascidians used for extraction were obtained from various localities in southwest Wales. Fresh material was blot-dried, deep frozen and stored at z -20 “C. A crude aqueous extract was prepared from the frozen ascidian material. 10 g wet weight of ascidian was macerated in 100 ml filtered sea water in a Waring blender for 5 min. It was then filtered through muslin and centrifuged at 2000 rpm for 20 min. The super- natant was used in experiments, diluted to the appropriate concentration with filtered sea water. Wet weight concentrations are given as weight of macerated frozen material per ml seawater (g wet wt:mll’).

Another 5 g freeze-dried ascidian material was macerated and extracted overnight in 250 ml methanol. Solid fragments were removed by filtering through Whatman’s No. 1 paper. The filtrate was concentrated by rotary evaporation to give 50 ml of extract. This methanol extract was insoluble in seawater. As no suitably non-toxic solvent was found, it was tested in a separate assay, by application on Petri dishes. Methanol extract concentrations are given as weight of freeze-dried material extracted per ml methanol solvent (g dry wt:ml -I).

2.3. Toxicity assay with aqueous extract

Only the four non-ascidian larvae were tested in this assay. Larvae were placed in cavity dishes in about lo-15 ml test solution. z lo-20 larvae were used in each replicate with five replicates per treatment. All dishes were incubated at 17 “C for 24 h. For Tubularia larynx, five actinulae were placed in each dish; any actinulae living after 24 h were transferred into fresh sea water and maintained for another 24 h.

2.4. Settlement assay with methanol extract

Methanol extracts were tested by application to new 90 mm disposable Sterilin@ polystyrene Petri dishes, allowing the solvent to evaporate away. To see if there were any non-toxic but repellent effects, only one side of each Petri dish was treated. A line

S.L.M. Teo. J.S. Ryland / J. Exp. Mar. Biol. Ecol. 188 (1995) 49-62 53

was drawn dividing each Petri dish into halves. In the first instance, the effect of the methanol solvent alone was tested by applying to one side a single coat (1 ml) of methanol using a 150 mm Bilbate @ disposable pipette. The Petri dish was tilted and excess solvent removed; E 0.2 ml remained. The treated dishes were air-dried in a fume cupboard for 1 h, and then oven-dried at 50 ‘C for 15 min to remove any remaining traces of solvent. 5-10 replicates were prepared for each treatment type depending on the availability of larvae. For S. spirorbis, Petri dishes were filmed in the aquarium and air-dried overnight before applying any treatments.

For the following treatments, a methanol coat was first applied to one side of the Petri dish and allowed to air-dry for 1 h. A coat of extract in solvent was then applied to the remaining side. The Petri dishes were then air-dried for l-2 h followed by 15 min in a 50 “C oven. Only the larvae of M. argus, Flustrellidra hispida and S. spirorbis were tested in this assay. For all larvae, the following treatment pairs were tested:

control (no treatment) vs control (methanol) control (methanol) vs C. Zepadiformis extract control (methanol) vs Aplidium pro&rum extract control (methanol) vs M. argus extract control (methanol) vs B. schlosseri extract

20-30 larvae were added to each Petri dish with z 20 ml filtered sea water. The Petri dishes were kept in total darkness at a constant temperature of 17 “C for 48 h. The numbers of larvae settled on each half of a dish were recorded. Also, the numbers dead, still swimming, and metamorphosed without attachment were recorded. As filming tended to increase variation between replicates, Petri dishes without any filming were used for M. argus and Flustrellidra hispida larvae, since satisfactory settlement could be obtained in 48 h. For S. spirorbis, filmed dishes were used as, without filming, the larvae may delay settlement for up to three days, by which time the extracts had begun to foul the seawater.

3. Results

3. I. Larvotoxicity

As it was sometimes difficult to separate effects arising from reduced water quality from actual toxic effects, the degree of toxicity was estimated more conservatively than by the standard LC,,. Dyrynda (1985) used >75% mortality as an indication of toxicity. In this study, the following scale was used:

Non-toxic: < 50% mortality. Light-moderately toxic: > 50% mortality; some settlement. Toxic: > 75 y0 mortality; no settlement.

The results from the assays using aqueous extracts are summarized in Tables l-4. C. lepadiformis extract was the toxic to all the larvae. The polyclinid ascidians, Aplidium

54

Table 1

S.L.M. Tea. J.S. Rylundl J. Exp. Mar. Biol. Ecol. 188 (19951 49-62

Toxicity of aqueous ascidian extracts to Artemia sp. nauplii (five replicates each of 10 larvae)

Species extract Concentration

(g wet wt.ml- ‘)*

Mortality

Mean (*SD)

Control (filtered sea water)

Clavelina lepadiformis

Apiidium prolijbum

Morchellium urgus

Botryllus schlosseri

0.2 (0.4)

0.01 4.8 (1.5)

0.05 9.8 (0.4)

0.01 0.2 (0.4)

0.05 0.8 (0.8)

0.01 1.0 (1.0)

0.05 4.0 (0.7)

0.01 0.6 (0.5)

0.05 1.0 (1.0)

The condition of the nauplii was scored after 24 h as either live or dead. * Wet weight concentrations are given as weight of macerated frozen material per ml seawater

pro&rum and M. argus, gave moderate effects and B. schlosseri extracts produced only slightly toxic effects. This order of toxicity obtained for the marine larvae corresponds with the results obtained for Artemia sp. nauplii, although these are obviously less sensitive.

Table 2

Toxicity of aqueous ascidian extracts to Flustrellidrtr hispida larvae

Species extract Concentration

(g wet wt.ml- ‘)

Larval condition

Settled Swimming Dead

Mean ( + SD) Mean (SD) Mean (k SD)

Control 1 (filtered sea water) 76.05 (6.28) 18.41 (2.96) 5.54 (8.74)

Control 2 (filmed dish) 92.44 (7.21) 7.56 (7.21)

Cluvelincr lepadiformis 0.02 100.00 (0.00)

0.01 100.00 (0.00)

Aplidium prolferum 0.02 23.74 (8.73) 76.48 (8.44)

0.01 29.37 (6.42) 70.63 (6.42)

Morchellium argus 0.02 13.31 (5.46) 86.69 (5.46)

0.01 44.25 (10.37) 1.14 (2.55) 54.60 (8.33)

Botryllus schlosseri 0.02 40.89 (13.34) 59. I1 (13.34)

0.01 45.45 (7.59) 18.66 (11.22) 35.89 (16.49)

The condition of larvae was scored after 24 h. There were five replicates of zz 20 larvae; figures are percent-

ages.


Table 3 Toxicity of aqueous ascidian extracts to Spirorbis spirorbis larvae

Species extract Larval condition

Swimming

Mean (k SD)

Lethargic Dead

Mean ( f SD) Mean ( f SD)

Control (filtered sea water) 100.00 (0.00) Aplidium prolijkm 35.25 (9.03) 64.75 (9.03)

Morchellium argus 100.00 (0.00) Botryllus schlosseri 36.30 (9.24) 63.70 (9.24)

There were five replicates of z 20 larvae; figures are percentages. Results are tabulated for a concentration

of 0.002 g wet wt.ml- I* (at 0.01 g wet wt.ml- ’ all larvae were dead after 24 h). Condition of larvae was

scored as: actively swimming; on bottom, lethargic; dead.

* Wet weight concentrations are given as weight of macerated frozen material per ml seawater.

Table 4

Toxicity of aqueous ascidian extracts to Tubularia larynx actinulae

Species

extract

Concentration Dura- Larval condition Mor-

(g wet wt.ml- ‘) tion tality

(h) Growing Attached Alive Dead (%)

Mean Mean Mean Mean

(+s”) (*SD) (*SD) (?sD)

Control 24

(filtered sea water) 48

Clavelina lepadiformis 0.1 24

Aplidium proliferum

Morchellium argus


0.05 24

0.01 24

48

0.1 24

0.5 24

48

0.01 24

48

0.10 24

0.05 24

48

0.01 24

48

0.1 24

48

0.05 24

48

5.00 (0.00) 5.00 (0.00)

0.80 (0.84)

0.60 (0.89)

2.00 (1.41)

0.40 (0.55)

2.00 (1.00) 3.00 (1.00)

2.20 (1.30)

1.20 (1.10)

4.20 (0.84) 0.80 (0.84) 3.00 (1.00) 2.00 (1.00)

0.20 (0.45) 3.40 (1.14)

3.40 (1.52) 2.00 (1.87) 3.00 (1.87)

2.60 (0.89) 2.40 (0.89)

0

0

5.00 (0.00) 100

5.00 (0.00) 100

4.20 (0.84)

4.40 (0.89) 88

5.00 (0.00) 100 3.00 (1.41)

4.60 (0.55) 92

2.80 (1.30) 56

5.00 (0.00) 100

3.80 (1.10) 100

5.00 (0.00)

0

1.10 (1.34)

1.60 (1.52) 32

0

There were five replicates of five actinulae. The condition of the T. larynx was scored as: polyp formed and

with stolonal outgrowths; polyp newly attached; live actinula; dead.

56 S.L.M. Tea. J.S. Ryland 1 J. Exp. Mar. Biol. Ecol. 188 (1995) 49-62

Table 5 Settlement of A4orchellium argus larvae in paired experiments using (A, C) 0.01 and (B) 0.1 g dry wt.ml ’ methanol extract? (five replicates except where indicated)

(A) 0.01 g dry wt.ml- ’ Mean u, Total s;,

Settled ( + SD) Settled ( + SD)

Ctrl (not treated)

Ctrl (methanol)

Ctrl (methanol)

Clavelina lepadiformis

Ctrl (methanol)

C. lepadiformis (three coatings)

Ctrl (methanol)

Aplidium prol~erum

Ctrl (methanol)

Morcheilium argus

23.92 (9.52) 40.85 (9.30)

16.94 (8.42) (N= 10)

28.20 (8.77) 52.37 (10.58)

24.16 (9.81) (N= 10)

23.75 (9.82) 28.21 (12.85)*

4.46 (4.69) 48.43 (7.10) 49.43 (7.38)**

1.00 (1.67) (N = 10)

48.46 (14.51) 53.95 (17.60)**

5.49(7.11) (N= 10)

(B) 0.1 g dry wt.ml-’

Ctrl (methanol)

Botryllus schlosseri Ctrl (methanol)

A. prol[ferum Ctrl (methanol)

M. argus Ctrl (methanol)

C. lepadiformis

26.30 (2.21) 26.30 (2.21)*

0.00 (0.00) 33.78 (18.34) 35.61 (16.02)*

1.83 (2.53) 42.19 (8.95) 59.78 (11.31)*

17.59 (7.59) 87.82 (9.28) 91.62 (7.01)*

3.79 (6.66)

(C) 0.01 g wet wt,ml~ ’ (N= 5)

Response

Mean ( f SD)

Settled Swimming Unattached Dead

24 h Ctrl (not treated)

Ctrl (methanol)

Ctrl (methanol)

A. proliferum

Ctrl (methanol) M. argus

48 h Ctrl (not treated)

Ctrl (methanol)

Ctrl (methanol)

A. proliferum

Ctrl (methanol)

M. argus

32.89 (11.78) 65.68 (14.50) 1.43 (3.19) 0.00 (0.00)

30.94 (9.68)* 41.91 (8.95) 27.14(12.07) 0.00 (0.00)

35.67 (10.36)* 54.55 (12.08) 10.70 (7.80) 0.00 (0.00)

45.91 (4.42) 50.23 (7.53) 3.86 (5.34) 0.00 (0.00)

69.03 (18.06)* 30.97 (18.60) 0.00 (0.00) 0.00 (0.00)

74.58 (17.80)* 20.88 (9.44) 0.00 (0.00) 4.55 (10.16)

Experiments of 48 h duration except (in C) as noted. Significance testing between treated and control sur-

faces was done using the Sign test (Siegel, 1956). ’ Dry weight concentrations are given as weight of freeze dried material extracted per ml methanol solvent.

* Significant at 5 “/0, ** at 1 “:,,


3.2. Assays with methanol extract

The results are summarized in Tables 5-7. Most of the extract-treated plates produced some degree of mortality. The effect was more marked than that obtained in the larvotoxicity trials with aqueous extracts, despite the much lower concentrations. This suggests that methanol extracted greater amounts or obtained more toxic components.

The effect obtained from the treatments also differed between the types of larvae tested. Against M. argus, mortality was relatively low, although the larvae tended either to settle away from the treated half of the dishes or metamorphosed without proper attachment to any surface. FZustreZZidra hispida larvae sometimes attached but did not metamorphose; most were either still swimming or dead. Any settlement was on the untreated half of the dishes. Most S. spirorbis larvae were killed.

Comparing treatments, the extracts of the colonial ascidians, Aplidium pro&-urn, M. argus and B. schlosseri significantly reduced settlement of all target larvae. C. lepadiformis extract was less effective against the ascidian but produced marked mortality against Flustrellidra hispida and S. spirorbis. These differences could be a result of the much greater solubilities of the C. lepadzfirmis extract; films of which tended to dissolve after being left for 48 h whereas the extracts of the remaining ascidians produced vis- ibly oily films on the surface of the dish. It is likely that the nature and type of compounds are different.

3.2.1. Experiments with Morchellium argus larvae (Table 5) In the choice experiments, the Sign test (Siegel, 1956) was used to test differences

in settlement between the two sides of the dishes. There was no significant difference between the control (no treatment) and methanol-treated sides. Two concentrations of extract were tested. At 0.01 g.ml -l, settlement was prevented except for C. lepadzfirmis (Table 5A). With coatings of 0.1 gemll’ extracts, all four extracts significantly prevented settlement on the treated side (Table 5B). The presence of the organic extracts appeared to speed up metamorphosis in most cases. Although total settlement was not always much greater than in the methanol control dishes, free-swimming time was reduced and, even when the larvae did not successfully attach, they nonetheless metamorphosed and resorbed their tails. After 48 h, 50% of larvae in the control dishes were still swimming compared to 20-30% for extract-treated dishes (Table 5C). In general, mortality of larvae was not significant.

3.2.2. Experiments with Flustrellidra hispida larvae (Table 6) The presence of methanol reduced total settlement but there was no difference be-

tween the methanol treated and untreated sides. For all four extracts at 0.1 g.ml-‘, significant numbers of larvae were killed. Only in the case of B. schlosseri extracts, were nearly half (47%) of the larvae still swimming after 48 h; they were able to settle successfully when placed in a separate dish with fresh filtered sea water.

3.2.3. Experiments with Spirorbis spirorbis larvae (Table 7) Poor settlement was obtained with new, unfilmed Petri dishes. With biofilming, 75 y0

settlement was obtained. No significant effect was observed from methanol treatment

58

Table 6

S.L.M. Tea, J.S. Ryland / J. Exp. Mar. Biol. Ecol. 188 119951 49-62

Settlement of Flustrellidra hispida larvae using methanol extracts (0.1 g wet wt.ml~ ‘) of the named ascid-

ians

Treatment Mean ( + SD) 9;

Settled Total settled Dead Swimming

No filming

Filmed

Ctrl (methanol)

Ctrl (no treatment)

Ctrl (methanol)

Clavelina lepadijknis

Ctrl (methanol)

Aplidium prol~erwn

Ctrl (methanol)

Morchellium argus

Ctrl (methanol)


80.04 (4.84)

92.44 (7.21)

25.54 (5.01) 69.76 (8.19) 4.80 (5.37) 19.64 (5.73)

34.52 (9.94)

6.87 (5.49) 11.64(?) (6.80) 87.49 (6.31) 0.87 (2.75)

4.77 (3.22)

17.37 (7.52) 28.36(?) (11.76) 68.72 (11.60) 1.05 (2.54)

10.99 (8.45)

28.45 (7.08) 39.94(?) (11.76) 59.71 (12.30) 0.34 (1.03) 11.91 (1.90)

8.71 (4.64) 10.44(?) (5.51) 41.78 (9.13) 46.61* (18.80)

1.55 (1.90)

Experiments of 48 h duration. In experiments indicated (?) the larvae had attached but did not metamor-

phose and subsequently died. N= 10.

* Swimming larvae in the Bofryllus schlosseri extract treatment were able to settle successfully in a separate

dish with fresh filtered sea water.

of the filmed surface. However, all four extracts led to > 85 yO mortality and < 1 yO settlement. The methanol ascidian extracts are thus very toxic to S. spirorbis larvae, corresponding well with the larvotoxicity trials.

Table 7 Settlement of Spirorbis spirorbis larvae using methanol extracts (0.1 g wet wt,ml- ‘) of the ascidian indicated

Treatment Mean ( f SD) 9;

Settled Dead Swimming

Filmed

Ctrl (no treatment) Ctrl (methanol)

Ctrl (methanol) Clavelina lepadiformis

Ctrl (methanol) Aplidium pro&rum

Ctrl (methanol) Morchellium

argus

Ctrl (methanol) Botryllus schlosseri

74.7 1 (16.79) 5.36 (4.39) 19.91 (13.97)

71.37 (16.30) 8.16 (8.99) 20.48 (13.19)

> 0.00 (0.00) 100.00 (0.00) 0.00 (0.00)

1 0.0 (0.00) 92.00(11.51) 8.00(11.51)

> 1.00 93.62 (2.24) (9.60) 5.38 (10.03)

> 0.00 (0.00) 93.43 (8.89) 6.57 (8.89)

Experiments of 48 h duration. Five replicates.

S.L.M. Teo, J.S. Ryland 1 J. Exp. Mar. Biol. Ecol. 188 (1995) 49-62 59

4. Discussion

Anti-larval activity was observed in all the assays above. Only C. lepadiformis was found to be markedly toxic but all four ascidians exhibited moderate to high toxicity against invertebrate larvae. None of the larvae tested have been observed to occur epizoically on the ascidians. Under field conditions, it is plausible that they may settle close to (but not on) the ascidians as a result of dilution of bioactive substances under naturally turbulent conditions. No feeding deterrent effects were found in assays against fishes but C. lepadiformis and Aplidiumproliferum displayed antibacterial activity against Gram-positive bacteria (Teo & Ryland, 1995). No necrosis has been observed in near neighbours in overgrowth interactions, and competitive dominance appears to be a result of high growth rate and colony morphology (pers. obs.).

The four ascidians are not known to contain high vanadium levels or acidity (Webb, 1939; Ciereszko et al., 1963; Parry, 1984). An alkaloid, lepadin A, has been isolated from the ascidian C. Zepadiformis (Steffan, 1991). Cytotoxic and antibacterial agents have been isolated from two other species of Clavelina (Copp et al., 1991; Raub et al., 1991). Sebens (1986) observed that Aplidium pallidurn was overall competitive dominant in overgrowth interactions but may be overgrown by other species when it occurs in sheet form instead of the thick lobed form; predation limited its dominance in the assemblage. In the literature, most studies on polyclinid ascidians report anti-cancer activity (e.g. Carter & Rinehart, 1978; Howard & Clarkson, 1979; Copp et al., 1989). Dyrynda (1983) reported some antibacterial activity in extracts of M. argus, but no toxicity to fish. However, the extracts displayed larvotoxicity to Bugula turbinata but not to B. jlabellata. No toxicity was reported for B. otryllus schlosseri. Grosberg (1981) observed that invertebrate larvae of competitively inferior species tended to avoid settlement near B. schlosseri colonies, but allelopathy was unlikely. Consolidation of these observations requires in situ experiments with live animals, and controlled experiments with purified extracts. Assuming that chemical antifoulants are present, species distribution should depart from random. This remains to be tested.

The physical effects arising from the application of extracts to test surfaces poses another problem. In the experiments of Rittschof et al. (1985, 1988), antibryozoan fractions of gorgonian extracts were ineffective against barnacle larvae and vice versa; experiments in vitro suggest that this may have been a consequence of the way in which each fraction altered the physical properties of polystyrene. In most studies, artificial hard substrata are used. Although these make a poor comparison with the dynamic surfaces of living animals, the results may be relevant for commercial antifouling use. The use of crude extracts should not be discounted as it provides the widest possible screen of substances present and synergism between components of an extract may be important. Current research in larval settlement emphasises the importance of chemical mediation, either via a surface dependent interaction or diffusible “signal” molecules (Rittschof & Bonaventura, 1986; Rittschof et al., 1991).

The presumption remains that each of the ascidians investigated may employ different strategies against hazards such as predation, epibiosis and overgrowth competition; and the occurrence of chemical defences within the class may not be universal but integrated with specific ecological situations. Such appears to be the case for soft

60 S.L.M. Tea, J.S. Ryland / J. Exp. Mar. Bioi. Ecol. 188 ll995J49-62

corals (Sammarco & Coil, 1990). The presence of a toxic substance does not, of course, necessarily imply an allelopathic agent but suggest the likelihood of a chemical defense system that may be elucidated with detailed experiments after isolation of the bioactive component. From our observations, it appears that, for the four species examined, any overt toxicity is lacking. It is unlikely that chemicals are important in defense against predation or overgrowth, but potential chemical antifouling mechanisms may be present.

Acknowledgements

This paper represents part of a thesis submitted by S. Teo to the University of Wales. Financial support is acknowledged from an Overseas Research Scholarship awarded by the CVCP of the Universities of the UK. The authors wish to thank Bob Woollacott and Craig Young for constructive comments on a draft of this paper.

References

Bakus, G.J., N.M. Targett & B. Schulte, 1986. Chemical ecology of marine organisms: an overview. J. Chem.

Ecol., Vol. 12, pp. 951-987.

Biard, J.F., C. Grivois, J.F. Verbist, C. Debitus & J.B. Carrc, 1990. Origin of bistramidc A identified in

Lissoclinum bistratum (Urochordata): possible involvement of symbiotic Prochlorophyta. J. Mar. Biol.

Assoc. U.K., Vol. 70, pp. 741-746.

Carter, G.T. & K.L. Rinehart, Jr., 1978. Aplidiasphingosine, an antimicrobial and antitumour terpenoid from

an Aplidium sp. (marine tunicate). J. Am. Chem. Sot., Vol. 100, pp. 7441-7442.

Ciereszko, L.S., E.M. Ciereszko, E.R. Harris & CA. Lane, 1963. Vanadium content of some tunicates.

Comp. Biochem. Physiol. Vol. 8, pp. 137-140.

Coil, J.C., B.F. Bowden, D.M. Tapiolas, R.H. Willis, P. Dyura, M. Streamer, L. Trott & L. Webb., 1985.

Studies of Australian soft corals. XXXV. The tcrpcnoid chemistry of soft corals and its implications.

Tetrahed., Vol. 41, pp. 1085-1091. Copp, B.R., J.W. Blunt & M.H.G. Munro, 1989. A biologically active 1,2,3-trithiane derivative from the

New Zealand ascidian Aplidium sp. Tetrahed. Let?.. Vol. 30, pp. 3703-3706.

Copp, B.R., C.M. Ireland & L.R. Barrows, 1991. Wakayin: a novel cytotoxic pyrroloiminoquinone alkaloid

from the ascidian Cluvelina species. J. Org. Chem., Vol. 56, pp. 4596-4597. Davis, A.R. & A.E. Wright, 1989. Interspecific differences in fouling of two congcncric ascidians (Eudi.@omu

olivaceum and E. caps&turn): is surface acidity an effective dcfcnce? Mar. Biol., Vol. 102, pp. 491-497.

Davis, A.R. & A.E. Wright, 1990. Inhibition of larval settlement by natural products from the ascidian

Eudistomcr olivaceum (Van Name). J. Chem. Ecol.. Vol. 16, pp. 1349-1358. Davis, A.R., N.M. Targett, O.J. McConnell & CM. Young, 1989. Epibiosis of marine algae and benthic

invertebrates: natural products chemistry and other mechanisms inhibiting settlement and overgrowth. In,

Bioorganic marine chemisrry, edited by P.J. Scheucr, Springer-Verlag, Berlin and Heidelberg, Vol 3, pp. 85- 114.

Davis, A.R., A.J. Butler & I. van Altena, 1991. Settlement behaviour of ascidian larvae: preliminary evidence for inhibition by sponge allelochemicals. Mar. Ecol. Prog. Ser.. Vol. 72, pp. 117-123.

Dyrynda, P.E.J., 1983. Modular sessile invertebrates contain larvotoxic allelochemicals. Del’. Con?p. Immu-

nol., Vol. 7, pp. 621-624.

Dyrynda, P.E.J., 1985. Chemical defences and the structure of subtidal cpibcnthic communities. In, Proc.

19th Europ. Mar. Biol. Symp., edited by P.E. Gibbs, CUP, Cambridge, pp. 411-421. Dyrynda, P.E.J., 1986. Defensive strategies of modular organisms. Philos. Truns. R. Sot. Land.. Vol. B313.

pp. 227-243.

S.L.M. Tea, J.S. Ryland 1 J. Exp. Mar. Biol. Ecol. 188 (1995) 49-62 61

Grosberg, R.K., 1981. Competitive ability influences habitat choice in marine invertebrates. Nature (London),

Vol. 290, pp. 700-702. Howard, B. & K. Clarkson, 1979. Simple prenylated hydroquinone derivatives from the marine urochordate

Aplidium californicum. Natural anticancer and antimutagenic agents. Tetrahed. Lett., Vol. 46, pp. 4449-

4452. Ireland, C.M., A.R. Durso, R.A. Newman & M.P. Hacker, 1982. Antineoplastic cyclic peptides from the

marine tunicate Lissoclinum patella. J. Org. Chem., Vol. 47, pp. 1807-1811.

Jackson, J.B.C., 1977. Competition on marine hard substrata: adaptive significance of solitary and colonial

strategies. Am. Nat., Vol. 111, pp. 743-767.

Jackson, J.B.C., 1979. Overgrowth competition between encrusting cheilostome ectoprocts in a Jamaican

cryptic reef environment. J. Anim. Ecol., Vol. 48, pp. 805-823.

Kott, P., 1989. Form and function in the Ascidiacea. Bull. Mar. Sci., Vol. 45, pp. 253- 276. La Barre, S.C., J.C. Co11 & P.W. Sammarco, 1986a. Competitive strategies of soft corals (Coelenterata:

Octocorallia): III. Spacing and aggressive interactions between alcyonaceans. Mar. Ecol. Prog. Ser., Vol.

28, pp. 147-156.

La Barre, S.C., J.C. Co11 & P.W. Sammarco, 1986b. Defensive strategies of soft corals (Coelenterata:

Octocorallia) of the Great Barrier Reef: II. Relationship between toxicity and palatability. Biol. Bull., Vol.

171, pp. 565-576.

Parry, D.L., 1984. Chemical properties of the test of ascidians in relation to predation. Mar. Ecol. Prog. Ser.,

Vol. 17, pp. 279-282.

Paul, V.J., 1992. Chemical defenses of benthic marine invertebrates. In, Ecological roles of marine natural

products, edited by V.J. Paul, Comstock Publications, Ithaca, pp. 164-188.

Raub, M.F., J.H. Cardellina II, M.I. Choudhary, C-Z. Ni, J. Clardy & M.C. Alley, 1991. Clavepictines A

& B: cytotoxic quinolizidines from the tunicate Clavelina picta. J. Am. Chem. Sot., Vol. 113, pp. 3178-

3 180.

Rinehart K.L., Jr., J. Kobayashi, G.C. Harbour, R.G. Hughes, Jr., S.A. Mizsak & T.A. Scahill, 1984.

Eudistomins C, E, K and L. Potent antiviral compounds containing a novel oxathiazepine ring from the Caribbean tunicate Eudistoma olivaceum. J. Am. Chem. Sot., Vol. 106, pp. 1524-1526.

Rinehart K.L., Jr., J. Kobayashi, G.C. Harbour, J. Gilmore, M. Mascal, T.G. Holt, L.S. Shield & F.

Lafargue, 1987. Eudistomins A-Q, beta-carbolines from the antiviral Caribbean tunicate Eudistoma oli-

vaceum. J. Am. Chem. Sot., Vol. 109, pp. 3378-3387.

Rittschof, D. & J. Bonaventura, 1986. Macromolecular cues in marine systems. J. Chem. Ecol., Vol. 12,

pp. 1013-1023.

Rittschof, D., I.R. Hooper, E.S. Branscomb & J.D. Costlow, 1985. Inhibition of barnacle settlement and

behaviour by natural products from whip corals Leptogorgia virgulata. J. Chem. Ecol., Vol. 11, pp. 551-564.

Rittschof, D., I.R. Hooper & J.D. Costlow, 1988. Settlement inhibition of marine invertebrate larvae:

comparison of sensitivities of bryozoan and barnacle larvae. In, Marine biodeterioration advanced techniques

applicable to the Indian Ocean, edited by M.F. Thompson, R. Sarojini & R. Nagabhushanam, Oxford &

IBH Publ., New Delhi, pp. 599-608.

Rittschof, D., A.R. Schmidt, I.R. Hooper, D.J. Gerhart, D. Gunster & J. Bonaventura, 1991. Molecular

mediation of settlement of selected invertebrate larvae. In, Bioactive compounds from marine organisms,

edited by M.F. Thompson, R. Sarojini & R. Nagabhushanam, Oxford & IBH Publ., New Delhi, pp. 3 17- 330.

Sammarco, P.W. & J.C. Coll, 1990. Lack of predictability in terpenoid function. Multiple roles and inte-

gration with related adaptations in soft corals. J. Chem. Ecol., Vol. 16, pp. 273-289.

Sebens, K.P., 1986. Spatial relationships among encrusting marine organisms in the New England subtidal

zone. Ecol. Monogr., Vol. 56, pp. 73-96.

Siegel, S., 1956. Nonparametric statistics for the behavorial sciences. McGraw-Hill Book Co., New York,

312 pp. Steffan, B., 1991. Lepadin A, a decahydroquinoline alkaloid from the tunicate Clavelina & lepadiformis.

Tetrahedon, Vol. 47, pp. 8729-8732.

Stoecker, D., 1978. Resistance of a tunicate to fouling. Biol. Bull., Vol. 155, pp. 615-626.

Teo, S.L. M. & J.S. Ryland, 1994. Toxicity and palatability of some British ascidians. Mar. Biol., Vol. 120, pp. 297-303.

62 S.L.M. Tea. J.S. Ryland 1 J. Exp. Mar. Biol. Ecol. 188 il995J 49-62

Wahl, M., 1989. Marine epibiosis. I. Fouling and antifouling: some basic aspects. Mar. Ecol. Prog. Ser., Vol.

58, pp. 175-189. Webb, D.A., 1939. Observations on the blood of certain ascidians, with special reference to the biochem-

istry of vanadium. J. Exp. Biol., Vol. 16, pp. 499-523. Young, C.M., 1986. Defenses and refuges: alternative mechanisms of coexistence between a predatory

gastropod and its ascidian prey. Mar. Bid., Vol. 91, pp. 513-522.

Young, C.M. & F.S. Chia, 1981. Laboratory evidence for the delay of larval settlement in response to a

dominant competitor. Int. J. Invert. Reprod., Vol. 3, pp. 221-226.

potential antifouling mechanisms using toxic chemicals in some british ascidians

Documents