Settlement of abalone larvae (Haliotis laevigata Donovan) in response to non-geniculate coralline red algae (Corallinales, Rhodophyta)

Journal of Experimental Marine Biology and Ecology, 234 (1999) 125–143

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Settlement of abalone larvae (Haliotis laevigata Donovan) in response to non-geniculate coralline red algae (Corallinales, Rhodophyta) Sabine Daume*, Sascha Brand-Gardner, Wm.J. Woelkerling Department of Botany, La Trobe University, Bundoora, Victoria 3083, Australia Received 30 June 1998; received in revised form 17 July 1998; accepted 31 July 1998

Abstract Settlement trials with larvae of the abalone Haliotis laevigata and three species of nongeniculate coralline red algae (NCA) revealed a species-specific response. The number of settled larvae was significantly greater on Sporolithon durum than on two other species of NCA (Mesophyllum engelhartii and Hydrolithon rupestre) co-occurring in the habitat. Settlement on Sporolithon durum commenced immediately after competent larvae were added. When offered a choice between two growth-forms of S. durum, larvae initially preferred the more complex lumpy growth-form to the encrusting growth-form, but this result was not significant after 48 h. This indicates that surface characteristics of the species influence settlement of Haliotis laevigata, but are not the main factors. When the algal thallus was damaged, the number of settled larvae was greater on damaged than on undamaged pieces of Sporolithon durum and Mesophyllum engelhartii, but less on damaged pieces than on undamaged pieces of Hydrolithon rupestre. Settlement on Sporolithon durum was also significantly greater when the photosynthetic pigments in the outermost cells of the thallus were present. These results indicate that inducers of settlement are highly variable and dependent on both the NCA species and species-specific characteristics. No larvae settled on the bottom or sides of the jars, suggesting that the inducers of settlement may not be soluble in water after their release from the algal thalli. Alternatively, inducers are not released in a large enough concentration, even when the algal thallus has been damaged. We conclude that the inducers are algal in origin. Larvae of the abalone Haliotis laevigata do not settle gregariously in response to recently-settled conspecific larvae. Gregarious settlement behaviour was, however, observed with 7-day-old conspecifics.  1999 Elsevier Science B.V. All rights reserved. Keywords: Abalone larvae; Bacteria; Coralline red algae; Diatoms; Gregarious settlement

*Corresponding author. Present address: Deakin University, Ecology and Environment, P.O. Box 423, Warrnambool, Victoria 3280, Australia. e-mail: [email protected]. 0022-0981 / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved. PII: S0022-0981( 98 )00143-9

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1. Introduction Patterns of larval settlement cause variation in population dynamics and community structure particularly in cases where the settlement rate is small (see review by Rodriguez et al., 1993). In some habitats, the supply and successful settlement of larvae may even be the dominant factor controlling local populations of invertebrates (Connell, 1985; Roughgarden et al., 1985; Menge and Sutherland, 1987) and promoted the idea of supply-side ecology (see Underwood and Fairweather, 1989). For many marine invertebrates, selection of an initial settlement site is a crucial and irreversible process (Morse, 1992). It is an active process and has to ensure correct selection of a specific benthic substratum (Crisp, 1974). Specificity to particular substrata by invertebrate larvae is well-known, but existing studies mostly concern sessile intertidal organisms such as barnacles (Gaines and Roughgarden, 1985; Wethey, 1986; Raimondi, 1988, 1990). These studies suggest that settlement of barnacles is not very specific and is induced by a variety of different natural substrata and co-occurring intertidal species. Rowley (1989); Pearce and Scheibling (1991), however, found that larvae of the sea urchin Strongylocentrotus droebachiensis settled on a variety of different red algal species. Morse and Morse (1984a) reported that larvae of the abalone Haliotis rufescens are induced to settle by a number of different coralline red algal species, but do not settle on intact foliose red algae. Furthermore, Morse et al. (1994) demonstrated that settlement of the coral Agaricia humilis was induced by the nongeniculate coralline red alga Hydrolithon boergesenii, whereas other species did not induce larvae to settle. Few studies have investigated the level of specificity or the effect of such specificity on natural populations (Rowley, 1989). Non-geniculate coralline red algae (NCA) (Corallinales, Rhodophyta) are known to induce settlement and metamorphosis of several species of abalone including Haliotis rufescens (Morse et al., 1979a,b, 1980; Morse and Morse, 1984a; Ebert and Houk, 1984; Morse, 1991), H. discus hannai (Takami et al., 1997), H. iris (Moss and Tong, 1992; Roberts and Nicholson, 1997), H. virginea (Roberts and Nicholson, 1997) and H. rubra (Garland et al., 1985; Daume et al., 1997). In addition, bacteria, diatoms and mucus on the surface of both NCA and on inert surfaces, such as plastic plates used in abalone hatcheries, all induce settlement (ZoBell and Allen, 1935; Seki and Kan-no, 1981; Crisp, 1984; Garland et al., 1985; Johnson et al., 1991a; Slattery, 1992). Species-specific relationships between NCA and Australian abalone have been proposed (Shepherd and Turner, 1985; Shepherd and Daume, 1996). In a boulder habitat at West Island, South Australia, juvenile abalone (Haliotis laevigata, H. scalaris) were found more frequently on certain species and growth-forms of NCA (eg. lumpy growth-form of Sporolithon durum). It is unknown, however, whether the preferences of juvenile abalone in the field are determined by settlement patterns or by post-settlement processes such as differential mortality or migration. Research projects on seeding of abalone larvae have been carried out worldwide in an effort to increase the size of natural populations (Tong et al., 1987; Schiel, 1992; Preece et al., 1997). Abalone habitats are rich in NCA species and thus, larvae experience some degree of choice in the natural environment. If larvae settle preferentially on some species of NCA and not on others, the selection of suitable habitat may be crucial for the success of larval seeding projects.

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Settlement induced by conspecific adults has been described for several benthic marine invertebrates, including abalone (Seki and Kan-no, 1981; Connor and Quinn, 1984; Hahn, 1989; Slattery, 1992), and could account for the patchy distribution of populations (Rodriguez et al., 1993). In abalone hatcheries, plastic plates covered with diatom films previously grazed by juveniles or adults are used as substrata for larval settlement (Crisp and Ghobashy, 1971; Seki and Kan-no, 1981; Cloney and Torrence, 1984; Slattery, 1992; Takami et al., 1997). It is unknown, however, whether abalone settle close to conspecific larvae or post-larvae. In the natural environment, multiple spawning events could be beneficial for larval settlement if larvae settle in response to conspecific larvae and post-larvae. Gregarious settlement behaviour could also be important for abalone hatcheries or for larval seeding in the field. In abalone hatcheries, settlement plates could be resettled during a single spawning season. In the present study, we tested the hypothesis that settlement of the abalone Haliotis laevigata Donovan occurs preferentially on particular species of NCA. We examined (1) whether the frequency of occurrence of bacteria and diatoms differed on the surfaces of those species, (2) whether the species-specific response can be explained by the differences in morphology of the tested species of NCA (3) whether the released cellular compounds from damaged cells of the NCA thalli and the presence of photosynthetic pigments affects larval settlement and (4) whether conspecific larvae and post-larvae induce gregarious settlement. We discuss the results in the light of possible implications for the abalone fishery in terms of selecting suitable localities for seeding.

2. Materials and methods

2.1. Study site and field collection Small rocks encrusted with NCA were collected from a boulder habitat on the western side of Taylor Island, South Australia, (34852’48‘S, 135859’54‘E) at 11 m depth. Two commercial species of abalone are present here, the greenlip abalone, Haliotis laevigata Donovan, and the blacklip abalone, Haliotis rubra Leach. This site was used for seeding trials of abalone larvae by Preece et al. (1997). At least ten species of NCA representing six genera in two families were recorded in the habitat. Three species of NCA, Hydrolithon rupestre (Foslie) Penrose, Mesophyllum engelhartii (Foslie) Adey and Sporolithon durum (Foslie) Townsend et Woelkerling were chosen for the settlement experiments because each has a distinctive and easily recognisable growth-form (encrusting, warty and lumpy, respectively: see Woelkerling et al., 1993) at this locality. H. rupestre is the most common species in this habitat (Daume, unpublished data) and has a 49% frequency of occurrence. M. engelhartii and S. durum occurred less frequently (both 19%), but both species are known to recruit abalone juveniles in another boulder habitat in South Australia (Shepherd and Daume, 1996). Pieces of each species were removed from the rocks with a razor blade or chisel. Each replicate for each experiment was obtained from a different plant to ensure independence, and each NCA piece was ¯ 1 cm 2 in size.

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2.2. Settlement experiments Three different batches of larvae were obtained from two hatcheries in Port Lincoln, South Australia. The larvae were derived from brood stock consisting of five males and five females collected around Taylor Island. Gametes from all animals were mixed for fertilization and formed one batch of larvae. Only larvae from the same batch were used for a single settlement experiment except for the gregarious experiment which contained 7-day-old post-larvae from a previous batch. Larvae were judged to be competent for settlement when the third tubule of the cephalic tentacle appeared and larvae started to explore the surface (Hahn, 1989). The density of abalone larvae was estimated by counting the larvae in ten 1-ml subsamples drawn from the whole population. The experiments were done in the laboratories at the Lincoln Marine Science Centre in Port Lincoln, South Australia, during December 1996 and December 1997. Jars from all experiments were kept at 20618C, with a 12 h L:D photo cycle. All jars contained 200 ml UV sterilised seawater and approximately 100 competent larvae were added to each jar. Three jars without pieces of NCA were used as controls for each experiment. Larval behaviour was observed under a dissecting microscope. The term ‘settlement’ describes the permanent attachment of the larvae to the NCA after shedding of the velum. Both initial shell-growth (visible after 24 h) and ciliary processes in the mantle cavity indicate that metamorphosis was completed (Hahn, 1989).

2.3. Sporolithon durum versus seawater experiment Three jars with sterilised seawater only (i.e. without any possible inducer) and three jars each with one piece of Sporolithon durum were tested at the same time; settled larvae were counted after 24 and 48 h.

2.4. NCA species experiment In this experiment, larvae had a choice between three species of NCA; Sporolithon durum, Mesophyllum engelhartii and Hydrolithon rupestre. One piece of each species was placed in each of six glass jars and settled larvae were counted after 3, 14, 17, 24 and 38 h. It was clear from the previous experiment that larvae settled on S. durum. Consequently, this experiment was designed to test if after 24 h the amount of settled larvae on S. durum differed significantly from the two other species occurring in this habitat and whether settlement on the two other species (M. engelhartii and H. rupestre) differed from each other. In addition, we tested if the species response differed between 14 and 24 h after the experiment had started.

2.5. Identification of NCA and frequency of bacteria and diatoms Pieces of all NCA species were subsampled randomly at the end of the experiment to verify their identification and to estimate the frequency of bacteria and diatoms on the surface. Subsamples were prepared as described in Woelkerling and Harvey (1993) and

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identified from keys in Womersley (1996). Microsections of reproductive structures were compared with those from voucher specimens (LTB 20405-20409) taken at the same locality. For scanning electron microscopy (SEM) studies, the subsamples were air dried and mounted on aluminium stubs with ‘Fotobond’ acrylic adhesive (Agfa-Gevaert). The stubs were sputter-coated with gold before viewing in a Siemens ETEC Autoscan microscope at 20 kV. For each sample, the presence or absence of bacteria and diatoms on the NCA surface was recorded at a magnification of 1200 3 in 20 randomly chosen fields of view. The mean percentage frequency of bacteria and diatom occurrence was calculated.

2.6. Growth-form experiments Larvae were offered a choice between two different growth-forms of Sporolithon durum. One piece of each growth-form was placed in each of six glass jars. The growth-form effect was also tested in a ‘no-choice’ experiment where only one piece of Sporolithon durum (encrusting or lumpy growth-form) was placed in each of 12 jars. In both experiments settled larvae were counted after 24 and 48 h.

2.7. NCA species and cell damage experiment To test whether the released cellular compounds and exposed cell walls affect larval settlement, pieces of all three species of NCA were purposely damaged. The Sporolithon durum and Mesophyllum engelhartii species with lumpy and warty growth-forms respectively had four protuberances on each piece removed with a razor blade. The encrusting Hydrolithon rupestre was scratched four times, ¯ 1–2 mm deep, across each piece of thallus. Approximately the same amount of thallus surface cells was damaged on each piece. A split-plot experimental design was used. One damaged piece and one undamaged piece of the same species (first factor) were placed in a jar so that larvae were offered a choice between damaged and undamaged pieces of the same species. Three species of NCA were tested, using six jars for each species (3 3 6 5 18 jars). The second factor (species) was applied across the blocks so species could be tested separately. Settled larvae were counted after 24 and 48 h.

2.8. Sporolithon durum versus photosynthetic pigments experiment To test the effect of photosynthetic pigments on larval settlement, S. durum with and without pigments were collected. Sporolithon durum has a very thick thallus and only approximately the first 5 mm of the thallus is pigmented. The whole thallus of pieces without pigments have a white appearance. Six replicates of S. durum without photosynthetic pigments and six replicates of pigmented S. durum were placed in separate glass jars. Settled larvae were counted after 24 and 48 h.

2.9. Effect of conspecifics experiments To test whether abalone larvae settle preferentially near conspecifics, one piece of

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Sporolithon durum with post-larvae (20–30 conspecifics) and one piece without postlarvae were placed in each of six glass jars. This experiment was replicated in six jars for each group of conspecifics of different ages. In four separate experiments, larvae maintained on pieces of S. durum for 2, 4, 17 h and 7 days were used. Settled larvae were counted after 24 h, and only additional settlers were recorded.

2.10. Data analysis If X larvae settle on substratum A1, and 100 larvae were added to each jar, only 100 2 X larvae are available to settle on substratum A2. In addition, the experiment on gregariousness was designed to test the assumption of independence for ANOVAs. The attractiveness of the substratum changed with the presence of conspecifics, so that repeated observations of the same substrata can not be regarded as independent. Consequently, data from all ‘choice’ experiments and changes in larval response over time were analysed using paired t-tests. ‘Choice’ experiments were chosen because we considered them to be closer to the natural situation. In the natural environment larvae experience some degree of choice between different substrata. Statistical analyses were done with the STATISTICA (Statsoft, 1995) computer package. Numbers of settled larvae (not percentages) were analysed. Data from the experiment on damage cells of NCA species were log ( y 1 1) transformed to achieve homogeneity. Cochran’s test revealed that data sets from all other experiments were homogeneous in variances (P . 0.05). One-way ANOVAs were used for all ‘no-choice’ experiments. The dependence between substrata was particularly obvious when three species of NCA were tested. Larvae started settling earlier on S. durum (A1) than on the other two species (A2, A3). For this experiment paired t-tests were done on (1) the difference between A1 and the average of A2 and A3 (A2 1 A3 / 2) within each jar and (2) the difference between A2 and A3 (A2 2 A3) within each jar. The 24-h measurements were used here because most larvae had completed settlement at this stage. A paired t-test was also done on the difference from two measurements after 14 and 24 h (T2 2 T1) for the data in (1) above to test if the preference for S. durum changed significantly over time. Relationships between frequencies of bacteria and diatoms (number of fields in which bacteria or diatoms were present) and the settlement of larvae were examined with simple linear correlations for each species of NCA. The Pearson correlation coefficients are reported. For the species and cell damage experiment, the total settlement after 24 h (number of larvae on damaged and on undamaged pieces) of each species were analysed with a one-way ANOVA. The difference between undamaged and damaged pieces were analysed with a paired t-test for each species.

3. Results Abalone larvae showed typical behaviour (see Morse et al., 1980; Hooker and Morse, 1985) by repeatedly contacting the surface of the algae before shedding their velum to complete settlement. In all experiments, settlement started earlier on Sporolithon durum

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Table 1 Settlement of Haliotis laevigata in response to Sporolithon durum and sterilised seawater Source DF MS Haliotis laevigata settlement after 24 h, one-way ANOVA, n 5 6

F

P

Treatment Error

8.55

, 0.042

1 4

7562 884

than on the other substrata. Less than 1% of larvae settled in the control jars of the ‘choice’ experiments and, therefore, control data are not presented. Larvae in jars with sterilised seawater only (the first experiment and control jars in subsequent experiments) kept swimming for at least 72 h, indicating that larvae were in healthy condition and did not settle when no inducer was present.

3.1. Sporolithon durum versus sterilised seawater experiment There was a significant difference between Sporolithon durum and the seawater control (Table 1; P , 0.05). Up to 89% of all larvae added to the jars with S. durum settled and completed metamorphosis within 48 h (Table 2). In contrast, only 2% of larvae added to the jars without any inducer went through metamorphosis, while most larvae were still swimming after 72 h.

3.2. NCA species experiment The number of settled larvae was highest on Sporolithon durum throughout the experiment. Settlement started early on Sporolithon durum and increased strongly compared to the other two species (Fig. 1). A total settlement rate of 6% was calculated after 3 h and increased to 41% after 38 h. Eighty-five percent of settled larvae chose Sporolithon durum, 10% Mesophyllum engelhartii and 5% Hydrolithon rupestre at the end of the experiment. All counted larvae had completed metamorphosis after 24 and 38 h. Data from the count after 24 h were analysed. There was a significant difference between S. durum and the other two species (t 5 5.71, P , 0.01). There was also a significant difference between M. engelhartii and H. rupestre (t 5 2.66, P , 0.05). In addition, there was a significant difference between counts after 14 and 24 h (t 5 3.94, P , 0.05). Table 2 Settlement of Haliotis laevigata larvae in response to Sporolithon durum and sterilised seawater Treatment

Sporolithon durum Sterilised seawater

Total % settlement After 24 h

After 48 h

57613.2 160.5

8969.7 260.7

Values (6standard error) are the mean percentages of settled larvae (n 5 6 jars).

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Fig. 1. Percentage of settled Haliotis laevigata larvae when offered a choice between three species of non-geniculate coralline red algae 3, 14, 17, 24 and 38 h after larvae were added. Vertical bars indicate the standard error, n 5 6 replicates.

3.3. Frequency of bacteria and diatoms on NCA surface There was not much variation in the frequency of bacteria of the thallus surfaces between the three species (Table 3). A higher frequency of diatoms was, however, observed on Mesophyllum engelhartii than the other two species. The frequency of bacteria did not correlate with the settlement of larvae in any of the three species (Sporolithon durum: r 5 0.23, Mesophyllum engelhartii: r 5 2 0.43, Hydrolithon rupesTable 3 Mean frequency of bacteria and diatom occurrence (%) on six replicates of each NCA species used in the settlement experiments NCA species

Bacteria frequency (%)

Diatom frequency (%)

Sporolithon durum Mesophyllum engelhartii Hydrolithon rupestre

19.263.96 13.363.07 14.262.39

1.761.05 9.262.71 5.861.54

Values (6standard error) are the mean frequency (n 5 6) of bacteria and diatoms recorded by SEM in 20 randomly chosen fields of view.

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Table 4 Settlement of Haliotis laevigata larvae in response to each of two growth-forms of Sporolithon durum a Design

‘Choice’

Growth-form of

Total % settlement

S. durum

After 24 h

After 48 h

Encrusting Lumpy

1766.1 48614.3 65

2468.5 54613.7 78

Encrusting Lumpy

1563.9 3463.5

2063.7 3465.5

Total ‘No-choice’ a

Percentage of settled larvae (6standard error) in n 5 6 replicates.

tre: r 5 2 0.55; P . 0.05). There was also no significant correlation between the number of diatoms and the settlement of larvae (Sporolithon durum: r 5 2 0.65, Mesophyllum engelhartii: r 5 0.19, Hydrolithon rupestre: r 5 2 0.69; P . 0.05, respectively).

3.4. Growth-form experiments In the ‘choice’ experiment, a settlement rate of 65% was calculated after 24 h and increased to 78% between 24 and 48 h (Table 4). After 24 h, a significantly greater number of abalone larvae settled on the lumpy growth-form of Sporolithon durum than on the encrusting growth-form when larvae had the choice (t 5 3.15, P , 0.05) and when the growth-forms were tested separately in a ‘no-choice’ experiment (Table 5, P , 0.05). When the growth-forms were tested separately, the settlement rate on the encrusting growth-form increased from 15 to 20% between 24 and 48 h, while a constant settlement rate of 34% was calculated throughout the experiment on the lumpy growth-form (Table 4). Consequently, the growth-form effect was no longer significant after 48 h (Table 5, P . 0.05).

3.5. NCA species and cell damage experiment The total number of settled larvae was significantly greater on Sporolithon durum than

Table 5 Settlement of Haliotis laevigata in response to each of two different growth-forms of Sporolithon durum, (a) after 24 h, (b) after 48 h Source

DF

MS

F

P

(a) Haliotis laevigata after 24 h, ‘no-choice’ experiment, ANOVA (n 5 6) Treatment 1 1102 813.06 , 0.047 Error 10 84 (b) Haliotis laevigata after 48 h, ‘no-choice’ experiment, ANOVA (n 5 6) Treatment 1 533 4.09 , 0.07 Error 10 130

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Table 6 Settlement of Haliotis laevigata in response to each of three species of NCA (damaged and undamaged) Source DF MS F P Haliotos laevigata total settlement after 24 (damaged and undamaged, n 5 6) Treatment Error

2 15

773 91

8.48

, 0.003

on the other two species of NCA (Table 6, P , 0.05). After 24 h, 28% of added larvae settled on S. durum (on average 1163.3 on the undamaged and 1762.5 on the damaged pieces), 9% on Mesophyllum engelhartii (on average 461.9 on the undamaged and 562.1 on the damaged pieces) and 6% on Hydrolithon rupestre (462.5 on the undamaged and 362.1 on the damaged pieces) (Fig. 2). Therefore, more larvae settled on damaged pieces of Sporolithon durum and Mesophyllum engelhartii but fewer on Hydrolithon rupestre compared to undamaged pieces (Fig. 2). The difference between damaged and undamaged pieces of the same species, however, was not significant.

3.6. Sporolithon durum versus photosynthetic pigments experiment There was a significant difference between the intact Sporolithon durum and the pieces without photosynthetic pigments (Table 7; P , 0.05). Up to 89% of added larvae

Fig. 2. Percentage of settled Haliotis laevigata larvae on undamaged and damaged pieces of three species of non-geniculate coralline red algae 24 h after larvae were added, n 5 6 replicates.

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Table 7 Settlement of Haliotis laevigata in response to Sporolithon durum with and without photosynthetic pigments Source DF MS F P Haliotis laevigata after 24 h, balanced ANOVA (n 5 6) Treatment Error

1 10

7252 537

13.51

, 0.004

settled on the intact S. durum pieces, and up to 11% settled on the pieces without pigments (Fig. 3).

3.7. Effect of conspecifics experiments We did not observe any gregarious behaviour in response to recently-settled conspecific larvae. Significantly less larvae settled on Sporolithon durum with postlarvae 2 and 4 h post-settlement (t 5 2.94, P , 0.05; t 5 4.10, P , 0.01, respectively) (Fig. 4). There was no significant difference between Sporolithon durum with and without post-larvae 17 h post-settlement (t 5 0.22, P . 0.05). Significantly more larvae

Fig. 3. Percentage of settled Haliotis laevigata larvae on Sporolithon durum with and without photosynthetic pigments 24 and 48 h after larvae were added. Vertical bars indicate the standard error, n 5 6 replicates.

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Fig. 4. Percentage of settled Haliotis laevigata larvae in response to conspecifics on Sporolithon durum after 2, 4, 17 h and 7 days. Vertical bars indicate the standard error, n 5 6 replicates.

settled on the NCA with 7-day-old post-larvae (t 5 3.02, P , 0.05) indicating that larvae settle gregariously in response to older conspecifics.

4. Discussion

4.1. Sporolithon durum versus seawater experiment This initial experiment clearly demonstrates that there is a positive inducer present that is associated with Sporolithon durum and detectable by larvae competent for settlement.

4.2. NCA species experiment Settlement trials with H. laevigata larvae and the three species of NCA revealed a preference for Sporolithon durum (Figs. 1 and 2). The results were consistent irrespective of whether larvae were offered a ‘choice’ between the three species of NCA (this experiment) or tested separately (species and cell damage experiment). Larvae

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started to settle earlier on Sporolithon durum, indicating that the inducer was stronger and / or available in a more useable form on the surface of this species. Larvae may have preferred Sporolithon durum because of (a) the occurrence of bacteria and / or diatoms on the algal surface, (b) morphology of the thallus (growthform) (c) the presence of a chemical inducer of S. durum which is readily available to the larvae at the thallus surface and / or (d) gregarious settlement behaviour in response to conspecifics.

4.3. Frequency of bacteria and diatoms on NCA surface Preferential settlement on Sporolithon durum in this study is unlikely to have been due to the occurrence of bacteria or diatoms because no correlation was found between the number of bacteria and the settlement of larvae. We observed high variation in frequencies of bacteria and diatoms between the samples of each species; this indicates that variation might occur on a smaller level than species level. In addition, Shepherd and Daume (1996) found that bacteria were sparse and that diatoms were absent on the surface of Sporolithon durum compared to Mesophyllum spp. and Hydrolithon rupestre sampled at West Island, South Australia. In contrast, research on the settlement of Acanthaster planci larvae concluded that inducing substances are produced by epiphytic bacteria (Johnson and Sutton, 1994). Settlement was inhibited if bacteria on the NCA surface were reduced after antibiotic treatment. Johnson and Sutton (1994) found that neither bacteria nor NCA cell compounds alone would induce settlement of Acanthaster planci, indicating that bacteria require a substance from the alga to produce the inducer, or that compounds from both the algae and the bacteria are required. In the case of Haliotis laevigata settlement, the species-specific response could not be explained by this factor because the bacterial frequency did not differ significantly between the NCA species tested. It is unlikely that bacteria or diatoms may have been selectively lost during the preparation process for this SEM study because all samples were prepared at the same time using the same procedures.

4.4. Growth-form experiments The physical structure of Sporolithon durum is unlikely to be the main inducer because the contact of larvae with the algal surface, and thus the contact with inducers, occurs on a smaller scale than the morphological pattern of the alga, described here as growth-form. The physical effect of sheltering or creating a different microenvironment (e.g. pH or O 2 changes behind protuberances, enhanced microbial growth) however, may influence larval settlement (Eckman and Nowell, 1984; Kaspar, 1992). Alternatively, larvae may respond to a shading effect caused by the protuberances of the alga. Larvae of other invertebrates (corals and ascidians) are known to settle close to a light–dark margin or edge (Crisp and Ghobashy, 1971; Cloney and Torrence, 1984; Morse et al., 1988). In this study, we provided evidence that, in still water conditions, larvae initially preferred the more complex lumpy growth-form of Sporolithon durum. In the ‘nochoice’ experiment, however, this initial response was no longer significant after 48 h. This result indicates that larvae settle more readily on the suboptimal substrate after 24 h

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if they are not offered a choice. The encrusting growth-form was still highly inductive. The result emphasises the importance of using larvae that have just developed competency for settlement and of offering larvae a choice between substrates. Bourget (1988) describes a shift in the importance of settlement cues as the larvae age from pure biological cues, such as algal cover, to primarily physical cues such as surface structures. Rittschof and Sanford Branscomb (1984) established that barnacle larvae discriminate less between substratum when they are older. Furthermore, surface characteristics are important in inducing metamorphosis of barnacle larvae but lose importance with age of the larvae. The growth-form of Sporolithon durum, and possible indirect factors resulting from it, are probably not the main factors in the settlement of Haliotis laevigata. It is possible, however, that in the natural environment, the indirect influence of different growth-forms could be stronger due to changing small scale hydrodynamics and light regimes (Crisp and Ghobashy, 1971; Cloney and Torrence, 1984; Eckman and Nowell, 1984; Morse et al., 1988).

4.5. NCA species and cell damage experiment Johnson et al. (1991b) did not find any differences in the number of settled larvae of Acanthaster planci between damaged and undamaged surfaces of the NCA Lithothamnion prolifer Foslie (as Lithothamnium pseudosorum; nom. inval. see Keats et al., 1996). In our experiment, we found some evidence that cell compounds of Sporolithon durum and Mesophyllum engelhartii can enhance the settlement of abalone larvae. S. durum was highly preferred, and this species-specific response was even stronger when the thallus was damaged. In addition, larval settlement response was quicker when exposed to S. durum compared to the other two species. It is probable that S. durum contains a chemical that is available to the Haliotis laevigata larvae in a stronger or more useable form on the surface, especially when thallus surface cells are damaged, cell compounds released, and cell walls exposed. The overall settlement rate was lowest in this experiment because larvae did not have a choice between species of NCA, and most of the larvae exposed only to Hydrolithon rupestre did not settle. None of the larvae settled on the bottom or sides of the jars in any of the above experiments. This suggests that inducers of settlement may not be soluble in water, are not released from the NCA surface or do not occur in a high enough concentration in the surrounding water or adjacent to glass surfaces, even if the thallus of NCA is damaged. It can be concluded, however, that inducers of settlement have a local effect on pieces of NCA. The larvae were observed lining up on the damaged edge of Sporolithon durum and not on adjacent surfaces like the bottom or sides of the jar or on undamaged parts of the thallus.

4.6. Sporolithon durum versus photosynthetic pigments experiment Settlement of Haliotis laevigata larvae was reduced substantially when photosynthetic pigments were absent from the thallus of Sporolithon durum. This suggests that the

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inducers are chemicals produced by the algae and are probably associated with the photosynthetic pigments of the preferred alga. Morse and co-workers (Morse et al., 1979a; Morse and Morse, 1984a,b; Morse, 1991, 1992) found that molecular inducers associated with the red photosynthetic pigment phycoerythrin chemically stimulated settlement and metamorphosis of Haliotis rufescens. It is possible that a similar molecule is involved in the induction of Haliotis laevigata. Our study confirms the conclusion of Morse and co-workers that the inducers originate from the NCA. Morse et al. (1980); Morse and Morse (1984a) showed that a variety of species of NCA induced settlement and metamorphosis of Haliotis rufescens larvae. Settlement was not induced by other red algae. They concluded that a chemical inducer must be uniquely available to the larvae at the NCA surface and suggested that the relatively thick polysaccharide surface layer of fleshy red algae may prevent physical contact between the algal inducer and the larvae (Morse and Morse, 1984a). The external part of the thallus of a variety of different species of NCA is also covered by a layer of polysaccharide material (Giraud and Cabioch, 1976). This layer might be thick enough to increase the concentration of the inducer near the surface by extending the diffusion barrier through a film of polysaccharide (Kaspar, 1992) but not too thick to prevent contact by larvae. In contrast to the findings of Morse and Morse (1984a) for Haliotis rufescens larvae, we demonstrated that the settlement of Haliotis laevigata is species-specific.

4.7. Effect of conspecifics experiments We reject the hypothesis that larvae of the abalone Haliotis laevigata settle gregariously in response to recently-settled conspecific larvae, and we can exclude the possibility that the species-specific response can be explained by gregarious behaviour. The number of settled larvae was significantly greater on the pieces without recentsettlers (Fig. 4: 2, 4 h after conspecifics start to settle). Thus we conclude that larvae of the abalone Haliotis laevigata were not influenced by newly settled conspecifics. Larvae settled gregariously, however, in response to older conspecifics (7 days post-settlement). Seki and Kan-no (1981) found that larvae of the abalone Haliotis discus hannai settle gregariously in response to the mucous trail of juvenile and adult abalone. Similarly, larvae of the red abalone (Haliotis rufescens) settle close to adults if the substratum has been grazed by them (Slattery, 1992). In these studies, juveniles and adults were . 1 cm and at least 1 month old. During the first three treatment levels in our study (2, 4 and 17 h; Fig. 4), larvae had not completed metamorphosis. The mouth is not formed at this stage and grazing is therefore impossible. Seven-day-old post-larvae, however, are able to graze on NCA by sweeping the algal surface with their radula (Kitting and Morse, 1997). We also demonstrated previously that the grazing of Haliotis rubra larvae has an effect on the epiphytic diatom community on the surface of the NCA Phymatolithon rupestre (Daume et al., 1997). Our results also suggest that post-larvae as young as 7 days post-settlement could be used to condition plates in abalone hatcheries and plates could be resettled with the following batch of larvae to improve densities on the plates. The number of settled

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larvae on each plate could be enhanced without too much variation in animal size, which reduces labor costs during the weaning process. Settlement in the natural environment may also be enhanced if the substratum had been grazed by young post-larvae. In this paper we demonstrated that larvae of the abalone Haliotis laevigata can distinguish between three species of non-geniculate coralline red algae. Larvae strongly preferred to settle on Sporolithon durum and avoided Hydrolithon rupestre. An intermediate response was observed on Mesophyllum engelhartii. The distribution and abundance of specific substrata within a habitat has an important influence on the settlement rate of those invertebrate larvae which have high specificity in substrata selection (Morse, 1992). Hydrolithon rupestre was the dominant NCA species at Taylor Island. The preferred species, Sporolithon durum, was present in this habitat less than half as often as Hydrolithon rupestre, indicating that the boulder habitat at Taylor Island may be not a good place for recruitment of Haliotis laevigata. Our results indicate that one of the most important factors for the success of seeding projects could be the species composition of NCA in the chosen habitats. Inconclusive results in the past concerning the feasibility of seeding on a commercial scale may have been due to inadequate knowledge of resident NCA and the demonstrated preference of Haliotis laevigata larvae for settlement on certain NCA species. It is likely that additional factors (e.g. characteristic bacteria and / or diatoms on the surface of NCA, compounds produced or transformed by the epiphytes, surface morphology of the NCA, and the microenvironment involving both NCA and epiphytes) influence the settlement of abalone larvae. In this study, however, we showed that for Haliotis laevigata, these are not the main factors in inducing larval settlement under laboratory conditions. Stoner et al. (1996) found that high settlement of queen conch larvae occurred on substrata that were preferred by post-larval conch. Our results together with the findings of Shepherd and Daume (1996) in the field indicate that recruitment patterns of Haliotis laevigata can be at least partially explained by preferential settlement. In the case of Sporolithon durum, abalone larvae preferred to settle on this species of NCA, and juveniles are still found predominantly on S. durum in the natural environment.

Acknowledgements We thank the Port Lincoln Marine Science Centre and the South Australian Research and Development Institute (SARDI) for providing laboratory facilities. We are grateful to South Australian Mariculture and South Australian Abalone Developments in Port Lincoln for the supply of abalone larvae and to Peter Preece, Brian Foureur and Kate Rodda from SARDI for their support and help with diving operations. We also thank Dr. S.A. Shepherd from SARDI for helping to improve the manuscript. Statistical advice from Dr. R. Day is also gratefully acknowledged. This study was supported by a joint ARC collaborative research grant between La Trobe University and SARDI and a La Trobe University post-graduate scholarship to the first author.

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