induction of larval attachment and metamorphosis in the abalone haliotis diversicolor (reeve)

LJournal of Experimental Marine Biology and Ecology,223 (1998) 39–51

Induction of larval attachment and metamorphosis in theabalone Haliotis diversicolor (Reeve)

*Patrick J. Bryan, Pei-Yuan QianDepartment of Biology, Hong Kong University of Science and Technology, Clearwater Bay, Kowloon,

Hong Kong, China

Received 6 June 1996; received in revised form 2 May 1997; accepted 3 June 1997

Abstract

The attachment and metamorphic responses of veliger larvae of the abalone, Haliotisdiversicolor (Reeve) to potassium chloride (KCl), gamma-aminobutyric acid (GABA) and naturalcues were determined. Attachment and metamorphosis of H. diversicolor were two distinctlydifferent responses. Attachment was characterized by larval contact with a substratum followed bycrawling with the larval foot firmly attached to the surface of the substratum. This behavior beganapproximately 48 h post-fertilization at 228C. Larvae could detach from the substratum andcontinue to swim for over 96 h after attachment behavior began. Metamorphosis was irreversibleand could occur in larvae that were 96 h post-fertilization. The neurotransmitter GABA, stimulated

25attachment behavior and induced low levels of normal metamorphosis at a concentration of 1023 24M, but was toxic to larvae at concentrations of 10 and 10 M. KCl at 10, 20 and 30 mM

concentrations above normal seawater salinity stimulated attachment behavior, but became toxic at30 and 40 mM. Low levels of metamorphosis were induced at excess KCl concentrations of 20and 30 mM. Single films of mucus, diatoms and three species of bacteria were not as stimulatoryas a combination film of diatoms and mucus in dish assays. Survival of larvae settled in dishescoated with a film of diatoms and mucus was higher than that in dishes containing either diatom ormucus films alone. The combination of diatom film and conspecific mucus may produce a novelcue that is not present in either film alone. Observations suggest that the condition of the adultsmay influence cues present in mucus. Moreover, when given a choice of vertical attachment sites,H. diversicolor larvae attached more frequently to slides filmed with both diatoms and mucus thanto clean or diatom-filmed slides. A combination film may increase numbers of attached larvae andreduce mortality in commercial aquaculture facilities. 1998 Elsevier Science B.V.

Keywords: Attachment; Metamorphosis; Gastropod; Conspecific cue; Artificial inducer; Diatomfilm

*Corresponding author. Tel.: 1 852 2358 7331/7336; fax: 1 852 2335 1477; e-mail: [email protected]

0022-0981/98/$19.00 1998 Elsevier Science B.V. All rights reserved.PII S0022-0981( 97 )00156-1

40 P.J. Bryan, P. Qian / J. Exp. Mar. Biol. Ecol. 223 (1998) 39 –51

1. Introduction

Larvae of many marine gastropods settle and metamorphose in response to pharma-ceutical agents such as gamma-aminobutyric acid (GABA) (Morse et al., 1979a), choline(Hirata and Hadfield, 1986; Todd et al., 1991), excess potassium (Yool et al., 1986;Pechenik and Heyman, 1987; Pechenik and Gee, 1993). Natural settlement cues oflarvae of benthic marine invertebrates may originate from conspecific adult (Crisp andMeadows, 1962; Seki and Kan-no, 1981; Burke, 1984; Pawlik, 1986; Bonar et al., 1990;Pearce and Scheibling, 1990; Slattery, 1992; Zimmerfaust and Tamburri, 1994), diatomand bacterial films (Kirchman et al., 1982; Maki et al., 1989; Fitt et al., 1990;Zimmerfaust and Tamburri, 1994), and prey items (Steneck, 1982; Morse and Morse,1984; Todd, 1985; Hadfield and Pennington, 1990; Pearce and Scheibling, 1991). Morseand co-workers (1979a) found that settlement and metamorphosis of the larvae ofabalone Haliotis rufescens was induced by compounds available at the surface ofcrustose red coralline algae. GABA and GABA-mimetic compounds from the crustosecoralline algae induce settlement and metamorphosis of larvae of H. rufescens (Morse etal., 1979a). Larvae of H. rufescens also attach to the substratum and metamorphose inthe presence of diatoms and conspecific mucus (Slattery, 1992). Larvae of anotherspecies of Haliotis, H. discus hannai, attached to surfaces filmed by diatoms andconspecific mucus (Seki and Kan-no, 1981; Y. Yang, pers. com.).

Both Haliotis rufescens and H. discus hannai are cold water species. Larvae ofdifferent species of Haliotis from different habitats may respond to the same cues in adifferent manner. Our work focused on H. diversicolor (Reeve), which lives in warmwaters around the southern part of China, including Taiwan. The objectives of this studywere to (1) determine the effects of GABA and excess KCl on larval attachment andmetamorphosis, (2) determine the larval attachment and metamorphosis in response tofilms of bacteria, diatoms and conspecific mucus and (3) determine the larvaeattachment on vertical settling plates to assay larval attachment behavior of the abaloneH. diversicolor.

2. Materials and methods

2.1. Larval culture

Larvae of Haliotis diversicolor were obtained from the abalone culture facility in NanAu, Shenzheng, China. Adult abalone were induced to spawn utilizing ultravioletradiation (Kikuchi and Uki, 1974). The sperm and eggs of a mass spawning (ca. 2 malesand 4 females) were mixed for a period of 15 min. The fertilized eggs were washedtwice in 0.22-mm filtered seawater (FSW) to remove excessive sperm and raised to theveliger stage at 228C in FSW. Larvae were held at a concentration of 4 to 5 larvae per mlin 4-l static culture tubes and maintained for 2 days at 228C. Larvae exhibitingexploration and orientation behavior as well as sense organ development (indicatingcompetence) were utilized in the following settlement experiments. Larvae were 2 daysold at the onset of all experiments.

P.J. Bryan, P. Qian / J. Exp. Mar. Biol. Ecol. 223 (1998) 39 –51 41

No antibiotics were used during the larval culture of H. diversicolor or in any of the21assays performed in this study. Concentrations of streptomycin at 50, 10 and 5 mg ml

narcotized veliger larvae, causing them to fall to the bottom of culture containers (P.Bryan and L. Gosselin, pers. obs.). Up to ca. 75% of larvae died when exposed tostreptomycin, thus application of this antibiotic was discontinued for the experimentsreported in this study.

2.2. Larval response to GABA and KCl

Competent larvae were tested for attachment and metamorphic responses to GABAand elevated KCl in polystyrene petri dishes. A stock solution of GABA was prepared in

22FSW at a concentration of 10 M. Serial dilutions of GABA with FSW were prepared23 24 25 26to assay concentrations of 10 , 10 , 10 and 10 M as in Baloun and Morse

(1984). KCl was added to the seawater by preparing a solution of 0.5 M KCl in filteredseawater and preparing dilutions in FSW. Assay concentrations were 10, 20, 30 and 40

1mM K concentration in 34‰ seawater. As a natural control, conspecific adults wereplaced in (5-cm diameter) assay petri dishes (Falcon 1006) with a 6-h old diatom film(Navicula sp.) and allowed to crawl for 6 h. The filmed dishes (DCF) were dipped threetimes in FSW and filled with 5 ml of FSW for assay.

Approximately 30 abalone veliger larvae were transferred from the batch culture toeach polystyrene petri dish containing 5 ml of FSW and incubated at room temperature(ca. 228C). The number of attached, metamorphosed and dead larvae were recorded at 1,6, 12, 18, 24, 36, 48, 60, 72, and 96 h after addition to the dishes. Attached larvaedisplayed firm attachment of their foot to the substratum. Metamorphosed larvae losttheir cilia and began formation of the juvenile shell. Dead larvae (indicated bydeterioration of the outer cuticle and inactivity) were removed from the dishes after theywere counted. There were five replicate dishes of each treatment. Data were checked fornormality and homogeneity and then analyzed with a one-way ANOVA utilizing apost-hoc Fisher’s PLSD test to compare difference among treatments.

2.3. Larval response to biological films

A benthic diatom and bacterial associates, three strains of bacteria previously isolatedfrom the diatom film, and adult foot mucus were tested for effects on larval settlement.A benthic diatom, Navicula sp., was maintained at 228C and in 18-h light:6-h darkphotoperiod in 2-l glass beakers containing FSW. Three bacteria species were isolatedusing standard microbiological procedures on Difco 2216 media from scrapings of thediatom film and were maintained in pure culture on Difco 2216 media. The bacteria arereferred to as isolates DB1, DB2, and DB3. All isolates were gram-negative bacilli thatproduced the enzyme catalase and oxidase. Isolates DB1 and DB3 metabolized severalsugars including glucose, fructose, sucrose and mannose, isolate DB2 did not.

Falcon polystyrene petri dishes (50 3 9 mm) were prepared for experiments byfilming with diatoms, bacteria or adult mucus. Dishes were coated with diatoms byresuspending the diatom culture in FSW for 2 days, pipetting 6 ml of the diatomsuspension into petri dishes, and incubating the culture for 6 h in light. Bacterial films


were obtained following Maki et al. (1988) procedure. Bacterial cultures were grown tostationary phase at 228C in Marine Broth 2216 and pelleted by centrifugation for 10 minat 10 000 rpm. Bacteria were washed in FSW, centrifuged and resuspended in FSW to a

6concentration of 10 cells per ml. For each strain of bacteria, petri dishes were filledwith 6 ml of the bacterial solution and incubated at 228C for 3 h. The dishes were rinsedby dipping three times in FSW to remove unattached bacteria. Adult foot mucus wasassayed by allowing an individual adult H. diversicolor to crawl around for 6 h in eachof 5 replicate unfilmed petri dishes to coat the plate in mucus (5 adults in 5 dishes intotal). Diatoms and conspecific foot mucus (DCF) were assayed together by placing asingle adult for 6 h in a petri dish which was previously filmed by the diatom. Sixunfilmed dishes served as controls for attachment behavior and spontaneous metamor-phosis. To verify competence of the larvae (n 5 6), GABA was assayed in petri dishes at

25a concentration of 10 M as a pharmacological attachment inducer.For attachment assays, 5 ml of FSW were pipetted into each of the petri dishes and

approximately 30 competent larvae were added to each dish. Larvae were observed indishes at 1, 6, 12, 24 and 48 h. The number of attached (firmly attached to thesubstratum by the foot) and dead larvae were recorded. Data for each time period wereanalyzed with a one-way ANOVA. Post-hoc Fisher’s PLSD tests were performed todetermine the degree of significance for differences among individual treatments.

2.4. Attachment site choice assay

A problem with assaying attachment behavior on horizontal surfaces is that larvaemay be narcotized by the cue being assayed. Some studies (Akashige et al., 1981;Pawlik, 1990) have addressed the fact that narcotized larvae may have been miscountedas settled in previous studies. To address this problem, criteria were set in this study toqualify larvae as attached (see larval attachment methods) and a choice assay wasperformed with filmed glass slides hung vertically in 250-ml beakers. This assay allowedlarvae present in the beaker to attach to one of several vertical substrata offered.

Diatom film and grazing conspecific film were assayed along with controls (cleanglass slides) in six replicate beakers. The concentration of larvae in beakers was similarto the original culture conditions (4 larvae per ml), resulting in approximately 800 larvaein 200 ml of FSW. Filmed slides were prepared in a similar fashion to filmed dishes.Diatom films resulted from 6 h of filming and diatom-conspecific films (DCF) from 6 hof crawling on a 6-h diatom film. Three slides (control, diatom, and diatom-conspecificfilm) were hung in each of six replicate beakers, such that one side was against the wallof the beaker and the other freely exposed to the water. The number of larvae attached tothe outward surface of the slides was recorded by removing slides from the beakers after

224 h and viewing under a dissecting microscope. Slide surface area was 14.25 cm . Dataare presented as number of larvae attached on a slide as a portion of all larvae attachedon slides. The lack of independence between treatments in a choice assay requiresnon-paramatric analysis to be performed, thus a post-hoc Kruskal–Wallis test wasperformed. Individual comparisons were made using a Wilcoxon signed rank test.


3. Results

3.1. Effects of GABA and KCl on larval attachment and metamorphosis

Larvae of Haliotis diversicolor in stock culture began to display attachment behaviorat approximately 2 days post-fertilization as they began to explore and orient toward thesubstratum. Twenty-four hours after these 2-day-old larvae were transferred to dishes forlarval attachment assays, approximately 20% began to crawl on the bottom of controldishes (Fig. 1A and B). Over 70% of the larvae were attached in dishes containing DCFwithin 6 h of addition to dishes and over 90% of larvae were attached after 72 h (Fig.1A and B). Levels of attachment were variable in dishes containing excess KCl and

Fig. 1. Effects of GABA and KCl on larval attachment of Haliotis diversicolor. A. Larval response to23 24 25 26gamma-aminobutyric acid (GABA) at 10 , 10 , 10 and 10 M, filtered seawater (control) and diatom

conspecific film (DCF). Values that are statistically different (P , 0.05) from that of the control at the sametime (Fisher’s PLSD test) are indicated with an asterisk. The double asterisk over some bars in the DCFcategory indicate that in addition to control seawater, the percent of attachment at this time was alsosignificantly different from GABA treatments at the same time. B. Percent attachment of larvae in response toelevated levels of KCl. Four concentrations of KCl were assayed along with filtered seawater (control) anddiatom conspecific film (DCF). Values that are statistically different (P , 0.05) from that in the control at thesame time (Fisher’s PLSD test) are indicated with an asterisk. The double asterisk over some bars in the DCFcategory indicate that in addition to control seawater, the percent of attachment at this time was alsosignificantly different from KCl treatments at the same time. In both A and B, data plotted are mean 1 standarddeviation of five replicates.


Fig. 2. Effects of GABA and KCl on larval mortality of Haliotis diversicolor. A. Influence of GABA onmortality of abalone larvae. B. Influence of elevated concentrations of KCl in seawater on mortality of larvae.Values that are statistically different at the 0.05 level from that in control (FSW) at the same time (Fisher’sPLSD test) are indicated with an asterisk. DCF was utilized as a natural control for competence. Data plottedare mean 1 standard deviation of five replicates.

23 24GABA (Fig. 1A and B). At the concentrations of 10 and 10 M, GABA induced50% of larvae to attach (Fig. 1A). However, these concentrations appeared to be toxic tolarvae as almost 100% post-attachment larvae died within 96 h of exposure (Fig. 2A).

25 26GABA at 10 and 10 M induced almost the same degree of larval attachment as23 2410 and 10 M, but was not toxic to larvae, as post-attachment larval mortality

remained as low as that in the control or in DCF (Fig. 2A) (Fisher’s PLSD test,25P 5 0.437). Only a low level of attached larvae went through metamorphosis at 10

26and 10 M after 72 h (Fig. 3A). Potassium chloride at elevated concentrations of 10and 20 mM KCl induced larvae to attach while levels of 30 and 40 mM were toxic (Fig.2B). Diatom conspecific film (DCF), induced over 50% metamorphosis by 48 h and upto 90% by 96 h (Fig. 3) which was significantly different from the control and allconcentrations of GABA and KCl (Fisher’s PLSD tests, p , 0.01). Moreover, themortality of larvae in dishes with DCF was ca. 15% after 48 h of exposure (Fig. 2)which was not significantly different from control seawater (Fisher’s PLSD test,p 5 0.496).

3.2. Effects of biological films on larval attachment

The type of filmed substratum had a significant effect on larval attachment at each


Fig. 3. Effects of GABA and KCl on larval metamorphosis of Haliotis diversicolor. A. Percent metamorphosisof larvae exposed to gamma-aminobutyric acid (GABA). B. Percent metamorphosis of larvae exposed toelevated levels of KCl. Values that are statistically different from that in filtered seawater control at the 0.05level at the same time (Fisher’s PLSD test) are indicated with an asterisk. Diatom conspecific film (DCF) wasutilized as a natural control. A double asterisk over bars in the DCF category indicates that the percent ofmetamorphosis, at these times, was significantly different from the control and all other treatments. Dataplotted are mean 1 standard deviation of five replicates.

sampling time; (6 h ANOVA, F 5 90.25, P 5 , 0.0001), (12 h ANOVA, F 5[7, 40] [7, 40]

31.63, P 5 , 0.0001), (24 h ANOVA, F 5 21.28, P 5 , 0.0001), (48 h ANOVA,[7, 40]

F 5 17.06, P 5 , 0.0001). Ten to 20 percent of larvae attached in dishes[7, 40]25containing filtered seawater (control) over a 48-h period (Fig. 4A). GABA at 10 M

induced 30 to 40% of larvae to attach within 48 h. DCF induced the highest levels( ¯ 80%) of larval attachment after 12 h of exposure. Conspecific foot mucus aloneinduced higher levels ( ¯ 50%) of attachment than the diatom film alone (Fisher’s PLSDtest, P , 0.001) after 6 h. However, over 60% of larvae died in dishes containingconspecific mucus alone (Fig. 4B). Levels of attachment in response to films of bacteriaisolated from the diatom film were variable (Fig. 4A). There was no difference betweenisolate DB2 film and the control seawater. Bacterial isolate DB3 induced higher levels ofattachment than the control after 12 and 24 h (Fisher’s PLSD, P , 0.0001). Isolate DB1


Fig. 4. Effects of biological films and chemical compounds on larval attachment and mortality of Haliotisdiversicolor. A. Percentage of larval attachment. B. Percentage of larval mortality. Values that are statisticallydifferent (post-hoc Tukey’s test at the 0.05 level) are indicated with an asterisk. Diatom conspecific film(DCF) was utilized as a natural control. DB1, DB2, and DB3 are bacterial isolates from the diatom film. Dataplotted are mean 1 standard deviation of six replicates.

induced higher levels of attachment only after 48 h of exposure (Fisher’s PLSD,P 5 0.014). However, approximately 20% mortality was observed after 48 h in dishescontaining these two bacterial isolates (Fig. 4B).

3.3. Attachment site choice assay

There was a significant difference between the percent of larvae attached to slides atboth 24 h (Kruskal–Wallis test, H 5 15.15, P 5 0.0005) and 48 h (Kruskal–Wallis test,H 5 15.17, P 5 0.0005), but no difference between the two time periods (Kruskal–Wallis test, H 5 0.63, P 5 0.9165). When presented with a choice of three differentsubstrata [clean glass (control), diatom film (Navicula sp.), and combined diatom filmand conspecific mucus (Navicula sp. and H. diversicolor foot mucus)] verticallysuspended in the water column, larvae of H. diversicolor settled on the diatomconspecific film more frequently than either the control (Wilcoxon test, Z 5 2 2.201,P 5 0.027) or the diatom alone film (Wilcoxon test, Z 5 2 2.201, P 5 0.027). Over 60%of the larvae (attached to the slides presented) chose the slides coated with thecombination film (Fig. 5). The diatom film alone was more attractive than the unfilmedslide (Wilcoxon test, Z 5 2 2.201, P 5 0.027) but less attractive than DCF.


Fig. 5. Attachment preference of Haliotis diversicolor larvae on vertical slides. Data plotted are percent larvalattachment on three types of vertical slides [clean glass (control), diatom filmed, and diatom conspecific film(DCF) filmed] offered simultaneously to swimming 2-day-old larvae at 228C. A Kruskal–Wallis test was usedto determine statistically significant differences in the number of larvae attached to the slides. Both diatomfilmed and DCF slides had greater numbers of larvae attached than the control. Letters above bars indicate thatmeans are significantly different from each other. Data plotted are mean 1 standard deviation of six replicates.

4. Discussion

Attachment behavior in larvae of Haliotis diversicolor was observed approximately54 h post-fertilization. Competent larvae of H. diversicolor began to swim downwardtoward the substratum, periodically attached their foot to the substratum, and began tocrawl. Larvae will detach from the substratum and begin to swim if they are not attractedto the attachment site. Similar behavior has been reported for H. discus hannai (Seki andKan-no, 1981) as well as other marine benthic invertebrates (see reviews by Burke,1984; Crisp, 1984). This behavior contrasts to that described for H. rufescens, which isreported to commit to metamorphosis by exploring a substratum (Morse et al., 1979a;Yool et al., 1986; Morse, 1990). Therefore, attachment behavior of H. diversicolor issimilar to that of H. discus hannai (Seki and Kan-no, 1981; Y. Yang, pers. comm.) butdifferent from H. rufescens (Morse et al., 1979a). It is likely that different species ofhaliotid gastropods behave differently based on adaptations to their specific naturalenvironment.

Attachment and metamorphic inducers such as GABA and KCl were not dramaticallyeffective on H. diversicolor larvae. The high levels of mortality reported for larvae

23 24exposed to GABA and KCl at concentrations of 10 and 10 M and 30 and 40 mM,respectively, which agrees with reports on H. discus (Y. Yang, pers. comm.) and H.rufescens (Morse et al., 1979b; Barlow, 1990). GABA stimulated attachment behavior

25and induced low levels of normal metamorphosis at a concentration of 10 M. Levelsof attachment and metamorphic induction by GABA were significantly lower than thosefor DCF. Potassium chloride at 10 and 20 mM stimulated attachment behavior while 20and 30 mM concentrations induced low levels of metamorphosis. Once again thismetamorphosis occurred 24 h after attachment in dishes containing diatom conspecific


film. The concentrations of 30 and 40 mM were toxic to larvae after a prolongedexposure of 60 h. Similar trends were observed for larvae of the nudibranch Phestillasibogae and the prosobranch Astraea undosa (Yool et al., 1986). The drastic differencebetween the percentage of larval attachment and the percentage of successful larvalmetamorphosis induced by GABA and KCl clearly indicated that although thosecompounds can induce the larvae to go down to the bottom of dish and to attach, theyhave strong negative effects on post-settlement fitness of animals.

Diatom films induce attachment and metamorphosis in H. discus hannai (Seki andKan-no, 1981) and H. rufescens (Slattery, 1992). Our results suggest that veliger larvaeof H. diversicolor were stimulated to attach on diatom films and may also be stimulatedby certain species of bacteria within these films. Conspecific foot mucus alonestimulated attachment. The combination of a diatom film and foot mucus of aconspecific was most stimulatory for attachment and metamorphosis. Survival of larvaeafter settling on diatom conspecific mucus was higher than those on other surface films.Seki and Kan-no (1981) and Slattery (1992) also found increased survival on acombination of diatom and mucus films for H. discus hannai and H. rufescens,respectively. Induction of attachment to surfaces coated with conspecific mucus may becommon among haliotid gastropods. Compounds within the film, whether they originatefrom bacteria and/or the abalone, may be specific to the condition of the abalone.Larvae of H. discus hannai settled on mucous trails of conspecifics and three additionalspecies of haliotid gastropods, however larvae did not settle on mucous trails of threenon-haliotid gastropods (Seki and Kan-no, 1981). Larval settlement was affected by thecondition of the conspecific mucus trail. Significantly more larvae settled in mucous ofconspecifics which were grazing on diatoms or crawling than on mucus from stationaryor stressed individuals (Seki and Kan-no, 1981).

The chemical origin of the inductive cue is unclear. Bacteria associated with thediatoms or with the foot of abalone may be primarily responsible for induction ofattachment. Bacterial films induce attachment in other species of mollusk (Fitt et al.,1990; Zimmerfaust and Tamburri, 1994) and may affect abalone larvae as well. Thethree species of bacterial films which were assayed in this study gave variable results.Isolate DB3 was stimulatory for attachment yet caused low levels of mortality inpost-attachment stage larvae. The levels of attachment on DB3 at each sampling timewere not significantly different from the diatom film with the exception of attachedlarvae at 1 h, indicating that this bacteria may attribute to the attractiveness of the diatomfilm. However, the combination of diatoms and mucus was significantly higher thanDB3 at all time intervals sampled. Mucus alone and diatoms alone were not asstimulatory as the combination. Moreover, mucus alone caused high levels of mortalityin newly attached larvae. Microbial biotransformations of compounds resulting from theinteraction of diatoms and mucus is one possible explanation for increased attachmenton combination films. Another possible explanation is that different compounds arepresent in the foot mucus secreted by juvenile and adult abalone based on theirphysiological condition. Adults crawling on a preferred substratum may secretecompounds in their mucus which indicate favorable conditions whereas stressedindividuals may release compounds indicating unfavorable conditions (Seki and Kan-no,1981). The biochemical characterization of the inductive molecule(s) should give insight


to the source. Also, the effects of physical characters of the surfaces coated withdifferent strains of bacteria need to be examined.

The attachment of larvae on vertical slides coated with diatom and combinationdiatom and conspecific mucous films indicates that these larvae are not merelynarcotized by compounds from these sources as previously suggested (Akashige et al.,1981; Pawlik, 1990). Larvae were observed to be actively crawling on these substrataafter settling out of the water column. It seems unlikely that larvae were trapped by thecombination film as larvae were observed to attach and detach from this film in dishes.The significantly higher number of larvae attached to diatom and conspecific mucousslides compared to diatom alone and control slides suggests an attraction to thecombination film. Under natural field conditions, larvae may recruit to substratum filmedby diatoms, which they graze on as juveniles, covered with the mucus trail ofconspecifics. Slattery (1992) suggested that high levels of survival and growth in recentjuvenile H. rufescens, settled on combination films, may be due to the nutritionalbenefits derived from the mucus as well as the diatoms. Attraction to conspecifics andjuvenile food items has been documented in the field for several other invertebratespecies (reviewed by Pawlik, 1992; Rodriguez et al., 1993).

The findings of this study are important ecologically and economically. The numbersof larvae recruiting to vertical hanging plates in abalone farms could be increased byallowing conspecifics adults to crawl on these plates for a few hours. This procedurewould not only increase the numbers of settlers but also increase the number whichsurvive through metamorphosis. The use of vertical substrata to assay settlementbehavior is preferable over horizontal substrata, if possible, because there is noconfusion as to which larvae have actually attached to the substratum and which havemerely fallen out of the water column.

Acknowledgements

We thank Mr. Xin Zheng of the Shenzheng Abalone Farm Ltd. for kindly providingus with larvae. The comments of Drs. D. Rittschof, J. A. Pechenik, L. A. Gosselin andseveral anonymous reviewers helped to improve this manuscript. This project wasfunded through grant DAG/SC21 and RGC grant: HKUST651/96M from the HongKong University of Science and Technology to P.Y. Qian.

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