ELSEVIER Journal of Experimental Marine Biology and Ecology

196 (1996) 29-52

JOURNAL OF EXPERIMENTAL MARINE BIOLGGY AND ECOLOGY

Induction of metamorphosis in queen conch, Strombus gigas Linnaeus, larvae by cues associated with red algae

from their nursery grounds

Anne A. Boettcher”, Nancy M. Targett

University of Delaware, Graduate College of Marine Studies, Loves, DE 19958, USA

Received 2 February 1995; revision received 1.5 May 1995; accepted 6 June 1995

Abstract

Strombus gigas Linnaeus larvae are induced to metamorphose by a selection of substrata from their nursery grounds. The most effective inducers are cues associated with red algae, specifically Laurencia poitei (Lamouroux) Howe and the epiphyte Fosliellu sp. (Foslie) found on Thalassia testudinum KGenig detritus. Larvae metamorphose in response to these intact rhodophytes and to aqueous extracts of these species. The cues associated with Laurencia poitei and Fosliella sp. are water soluble and of low molecular size (cl kDa). They are stable over time (- 12 months) and their activity is not altered by heat treatment (10 min of boiling). Although the larvae respond to aqueous extracts of the red algae, their response in the presence of whole plants appears to be contact or near surface dependent. Unlike many invertebrate larvae that are induced to metamorphose by red algal species, Strombus gigas do not respond to the neurotransmitter y-aminobutyric acid (GABA). The responses of the larvae to the various nursery ground substrata and to cues isolated from their extracts is discussed in relation to temporal and spatial variability in the suitability of seagrass beds as sites for conch metamorphosis.

Keywords: Gastropod; Larvae; Metamorphosis; Queen conch; Red algae; Strombus gigas

1. Introduction

Marine invertebrate larval settlement and metamorphosis can be influenced by biological, physical, and chemical factors in the environment (Crisp, 1974, 1976,

1984; Burke, 1983; Cameron, 1986; Morse, 1990; Morse, 1992; Pawlik, 1992).

* Corresponding author. Fax: (1) (302) 645-4007.

0022.0981/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0022-0981(95)00101-S

30 A.A. Boettcher. N.M. Targett I J. E.K~. Mar. Bid. Ecol. I96 (1996) 29-52

Chemical cues associated with substrata in the environment are considered to be the primary stimuli initiating the settlement and metamorphosis of many marine invertebrate larvae because of their specificity (Burke, 1983; Morse, 1990; Pawlik, 1992). However, in most cases the exact nature of the chemical cues are unknown (Burke, 1983; Hadfield, 1986: Pawlik, 1992). Chemical cues that act in the induction of settlement and metamorphosis are generally classified as either aggregative (cues associated with conspecifics) or associative (cues associated with non-conspecific-hosts, prey, or biofilms) (Pawlik, 19Y2). Associative cues pro- duced by marine algae, particularly rhodophytes. have been shown to be important in the settlement and metamorphosis of a variety of larval invertebrates including echinoids, asteroids, polychaetes, gastropods, corals, and hydroids (Morse, 1992). Associative cues from red algae have been well studied for the abalone Haliotis rufescens, and the coral Agaricia humilis (Morse et al., 1979, 1980, 1988, 1994; Morse et al., 1984; Morse & Morse. 1984a,b; Morse & Morse, 1991; Morse, 1992). Larvae of Haliotis rufescens respond to a water soluble peptide found at the surface of non-geniculate coralline red algae. They also respond to y-aminobutyric acid (GABA). and it has been suggested that the natural inducer may share structural characteristics with this neurotransmitter (Morse et al., 1979; 1980: Morse & Morse, 1984a,b; Morse et al., 1984). Agaricia

humilis respond to a water insoluble cue associated with cross-linked polysac- charides in the cell wall of non-geniculate coralline red algae (Morse et al., 1988, 1994; Morse & Morse, 1991). The coral larvae do not respond to GABA or to the GABA mimetic peptide that elicits a response from Haliotis rufescens (Morse et al., 1988). Not all larvae exhibit as specific a response as H. rufescen.y and Agaricia

humilis. Other larval invertebrates known to respond to cues from red algae also respond to other substrata from their environment (Morse, 1992). For example, the urchin Strongylocentrotus droebachiensis responds with a high rate of metamorphosis to coralline reds and to high concentrations of GABA, but it also responds to brown and green algae, as well as, microbial films (Pearce and Scheibling, 1990, 1991). The apparent association between some invertebrates and coralline reds may be due to the presence of specific bacteria present on the surfaces of the alga rather than to cues from the alga itself (Johnson et al., 1991a,b; Johnson & Sutton. 1994).

The queen conch, Stromhus gigas Linnaeus (Strombidae), is found in seagrass beds (Thalassia testudinum Koenig and Syringodium ,fil(forme) and sand flats throughout the Caribbean, including Bermuda and the Florida Keys. USA (Randall, 1964; Brownell & Stevely. 1981). Juvenile conch are typically found in medium shoot density seagrass beds and in sandy areas surrounding these beds (Wicklund et al., 1991; Sandt & Stoner, lYY3: Stoner & Ray. 1993: Ray & Stoner, 1994; Stoner et al., 1994). They feed on the macrophytes (detrital blades of the seagrass Thalassia testudinum. the red alga Laurencia sp., and the green alga Batophora oerstedii J. Agardh) or the epibionts which settle on macrophytes found in their nursery grounds (Ray & Davis. 1989: Stoner & Sandt, lY91: Stoner bi Waite, 1991). Recent work by Davis (lYY4a) and Davis & Stoner (1994) has shown that factors associated with nursery ground macrophytes or their epibionts may be the natural inducers of settlement and metamorphosis. These studies have

A.A. Boettcher, N.M. Targett I J. Exp. Mar. Biol. Ecol. 196 (1996) 29-52 31

also shown that conch larvae do not respond to cues from conspecifics or to controls containing only filtered seawater, indicating that the larvae do not undergo aggregative or spontaneous metamorphosis (Davis, 1994a; Davis & Stoner, 1994). In addition, work by Mianmanus (1988) has shown that, in general, biofilms formed in either flowing or static seawater (filtered and unfiltered) do not elicit settlement and metamorphosis of conch larvae without the addition of an extract of the red alga Laurencia obtusa. Settlement and metamorphosis of Strombus gigas larvae in culture have been induced using water soluble extracts of Laurencia poitei (Lamouroux) Howe, the crustose coralline reds Lithothamnium sp. and Goniofithon sp., and high concentrations of KCl, but the specific nature of the cues which elicit settlement and metamorphosis are not known (Siddall, 1983; Spotts, 1987; Mianmanus, 1988; Heyman et al., 1989; Davis et al., 1990; Hensen, 1991; Davis, 1994b; Davis & Stoner, 1994).

In this study, the response of competent Strombus gigas larvae to potential chemical inducers of metamorphosis isolated from a variety of macrophytes and epibionts found in juvenile conch habitat are examined using a bioassay guided approach. Initially, inducing activity is examined in specific whole substrata from conch nursery grounds, as is the contact dependency of the responses to these substrata. The responses of conch larvae to crude and partially purified extracts of these substrata are then examined in order to further characterize the nature of the chemical stimuli triggering the metamorphosis of Strornbus gigas larvae. Finally, the effects of possible seawater contaminants, the temporal and thermal stability of the extracts, the size range of the active components of the extracts, and the efficiency of extraction are examined. The effect of the neurotransmitter GABA on conch larvae is also reported.

2. Material and methods

2.1. Metamorphosis assays

All metamorphosis assays were carried out at the Caicos Conch Farm, Providenciales, Turks and Caicos, BWI; using competent Strombus gigas larvae (19-24 days post-hatch) provided by the Caicos Conch Farm. Competent larvae, approximately 1 mm shell length (SL), were identified by morphological changes that included the development of six velar lobes of equal length and a change in the foot pigmentation from orange to green (Davis, 1994b). Techniques for the culture of the conch larvae were as described in Davis (1994b). Metamorphosis assays were run as static, no-choice experiments with five replicates per treatment. For each replicate in each treatment, 15 larvae were placed in 500 ml polyethylene containers with 300 ml ultraviolet (UV) sterilized, 10 pm filtered seawater (unless otherwise noted) and the treatment substrate or extract. A positive control (the commercial inducer, an extract of Laurencia poitei at 0.01-0.02 g wet wt algae. mll’ s.w., Davis, 1994b) used as a measure of competency, and a negative control (seawater only) used as a test of spontaneous metamorphosis, were included in

32 A.A. Boettcher, N.M. Targett I J. Exp. Mur. Biol. Ecol. lY6 (lYY6) 2YC.52

each assay. To avoid toxicity, larvae in the positive control were only exposed to the L. poitei extract for 5 h, then they were placed in fresh seawater for the remaining 19 h (Davis, 1994a; Davis & Stoner, 1994). Experiments were run at ambient temperature (2%29°C) and salinity (-39%) and under natural light conditions (~12 h light:12 h dark). Percent metamorphosis was determined after -24 h, and was calculated as total number of larvae metamorphosed. total number recovered ’ (P earce & Scheibling, 1990). Larvae were considered to have undergone metamorphosis when they lost their velar lobes and began to crawl using their foot (Davis, 1994a).

All substrata used in the metamorphosis assays were collected in the Turks and Caicos, BWT. The red alga L. poitei was collected off Pine Cay: all other substrates, seagrass Thalassia testudinum detritus, the red alga Laurencia intricata

Lamouroux, the coralline red alga Amphiroa rigida var. antillana Boergesen, the green alga Batophora oerstedii, and sediment samples, were collected from conch nursery habitat in near shore shallow seagrass beds adjacent to the Caicos Conch Farm. Substrates were rinsed and held in aerated seawater (UV sterilized, 10 ,um filtered), and were used within 24 h of collection.

2.2. Statistical analysis

Mean percent metamorphosis for each treatment in each experiment was determined. Mean percent metamorphosis among treatments in each experiment was compared using a Model 1 ANOVA and Tukey’s multiple comparisons test (N = 0.05). Treatments in which percent metamorphosis was equal to zero for all replicates were not included in the statistical analyses.

2.3. Whole substratum assays

Two assays were run to test the effects of specific substrata (macrophytes, their associated epibionts, and surface sediments) from juvenile conch habitat on larval metamorphosis, expanding the range of substrata used by Mianmanus (1988) and Davis & Stoner (1994). In each experiment, 3 g wet weight of substratum were added to each treatment container (0.01 g substratum. ml-’ s.w.). In the first experiment, whole nursery habitat macrophytes (Thalassia testudinum detritus, Laurencia poitei, L. intricata, Amphiroa rigida var. untillana, and Batophora oerstedii) and sediment samples collected at the surface of the seagrass beds were tested. Because of the high response of the larvae to Thalassia testudinum detritus, the second experiment examined specific components of the detrital T. testudinum (whole T. testudinum detritus, detrital blades with epibionts removed, and isolated epibionts from the detritus). Epibionts were removed and collected as described in Davis & Stoner (1994).

2.4. Contact vs. distance assays

The focus of subsequent experiments was primarily on Laurencia poitei and Thalassia testudinum detritus because of the high response of larvae to these two

A.A. Boettcher, N.M. Targett I .I. Exp. Mar. Biol. Ecol. 196 (1996) 29-52 33

substrata. In order to determine if, under static conditions, the cue readily diffused from intact substratum, and/or if actual larval contact with the sub- stratum or substratum surface layer was necessary for metamorphosis, two experiments were run. In the first, the effects of substratum vs. substratum conditioned seawater were examined. There were three types of treatments for each of two substrata (T. testudinum detritus and Laurencia poitei): whole substratum, seawater in which the substratum had soaked overnight coarse filtered through a Kimwipe EX-L (Kimberly-Clark Corporation, Roswell, GA, USA) to remove the detritus, and seawater in which the substratum had soaked overnight filtered through a 0.40 pm nucleopore filter (Costar Corporation, Cambridge, MA, USA), (0.01 g substratum 1 ml-’ s.w.). Two filtered seawater controls were also included in this experiment: one coarse filtered (Kimwipe) and one 0.40 ,um filtered. In the second experiment there were two types of treatments for each of two substrata (Thalassia testudinum detritus and Laurencia

poitei): whole substratum and whole substratum in a mesh covered container (0.01 g substratum . ml -’ s.w.), in addition, a control covered container only treatment was included. The mesh covered containers were formed from a ring of polyvinyl chloride (PVC) (volume = 36 ml). The side in contact with the bottom of the test container was sealed with Parafilm M-laboratory film (American National Can, Greenwich, CT, USA). The top of the PVC ring was covered with 100 pm mesh screening. This set-up allowed the substratum contact with the surrounding water, allowed the larvae free movement around the mesh covered container, but did not allow the larvae direct contact with the substratum.

2.5. Crude extract assays

The cues were further characterized by examining the effects of aqueous and organic extracts of substrata from juvenile conch nursery grounds. Four experi- ments were conducted to examine the effects of seawater, distilled water and methanol extracts of the substrata on larval conch metamorphosis, as well as the effects of partitioning the crude aqueous extracts with chloroform. Extracts were prepared by chopping or breaking the substratum and/or epibiont into small pieces and grinding it using a mortar and pestle or glass tissue homogenizer with either UV sterilized, 10 pm filtered seawater, distilled water, or Burdick and Jackson high purity grade methanol (0.60 g substratum. mll’ solvent). The samples were centrifuged for 5-10 min. Where noted, the aqueous extracts were subsequently partitioned with an equal volume of Burdick and Jackson high purity grade chloroform. Aqueous extracts were held on ice prior to use, then added directly to the treatment containers. The concentration of extract used in these and all subsequent experiments was 0.02 ml extract. ml-’ S.W. (= 0.01 g substratum. ml-’ s.w.) which is within the range of the concentrations used for the positive control and equivalent on a per weight basis to the amount of whole substratum used in the previous experiments. The methanol extracts were coated onto filter paper at similar concentrations, allowed to air dry, and the coated filter paper placed in the test containers. A methanol control was included in the experiments with methanol treatments. Methanol only was coated onto filter

34 A.A. Boettchrr. N.M. Targett I J. Exp. Mar. Bid. Ecol. I96 (1996) 29-S2

paper for these controls. In the first experiment, the effects of seawater vs. methanol extracts of Thafassia testudinum detrital epibionts were compared. Treatments included whole T. testudinum detritus, a seawater extract of the detrital epibionts, a methanol extract of the epibionts. and a methanol control. In the second experiment the responses of larvae to seawater and distilled water extracts of Laurencia poitei and the detrital epibionts were compared to assure that further analysis could be carried out on either seawater or distilled water samples. In the third experiment the effects of a chloroform partitioning step were examined. The treatments were: a seawater extract of Thalassia testudinum detrital epibionts, the aqueous fraction of a seawater chloroform partitioned epibiont extract, a control treatment of the aqueous fraction of a similar volume of chloroform partitioned seawater, the chloroform fraction of the seawater chloroform partitioned epibiont extract. and a chloroform control. The chloro- form fraction and chloroform control were run on filter paper, in the same manner as the methanol treatments described above. In the fourth experiment, the effects of the aqueous fraction of seawater chloroform partitioned extracts of various substrata from conch nursery grounds were examined (T. testudinum detrital blade with epibionts removed, T. testudinum detrital epibionts, Laurencia poitei, L. intricata, Amphiroa rigida var. antillana, and Batophora oerstedii), in order to

better clarify the results of the tests with the whole substrata.

2.6. Characterization of the inducer

Further examination of the chemical cues inducing metamorphosis of conch larvae focused on the characterization of the aqueous (seawater and distilled water) extracts of Laurencia poitei and the Thalassia testudinum detrital epibionts. The effects of possible contaminants in the seawater, the stability of the extracts, the size range of the active component of the extracts, and the effects of extracts partially purified via reverse-phase chromatography were examined.

The effect of possible contaminants in the seawater on larval metamorphosis was tested in an experiment in which distilled water epibiont and Laurencia poitei extracts were added to seawater that had been UV sterilized, treated with activated carbon to remove organics, and 0.40 pm filtered to remove micro- organisms ( = treated seawater). The treatments were: epibiont extract, 0.40 pm filtered epibiont extract in treated seawater, L. poitei extract, 0.40 pm filtered L. poitei extract in treated seawater, and a treated seawater control.

The temporal and thermal stability of the extracts were examined in two experiments. In the first, the effects of freshly prepared seawater epibiont and L. poitei extracts were compared to those that had been held for 1 yr at -20°C. In the second, the effects of boiling aqueous epibiont and L. poitei extracts were examined. The treatments in this experiment were: epibiont extract, epibiont extract boiled for 10 min. L. poitei extract, and L. poitei extract boiled for 10 min.

The molecular size of the active fraction of each of the extracts was determined in two experiments. Seawater epibiont and L. poitei extracts were fractionated

A.A. Boettcher, N.M. Targett I J. Exp. Mar. Biol. Ecol. 196 (1996) 29-52 35

into nominal molecular size components using a 400 ml Amicon (Amicon Division, W.R. Grace, Denvers, MA, USA) stirred cell with 76 mm membranes. Amicon Diaflo ultrafiltration membranes (YMlO, 3, 1) were used to separate the extracts into fractions with nominal molecular sizes of >lO kDa, 3-10 kDa, l-3 kDa, and <l kDa for the L. poirei extracts and >lO kDa, l-10 kDa, and <l kDa for the epibiont extract. Fractions were held on ice during separation. The response of the larvae to each of the fractions and to a seawater control of <l kDa were examined. In an additional experiment, the effect of boiling the <l kDa fraction of the L. poitei extract was also examined. The treatments in this experiment were: L. poitei <l kDa extract, L. poitei <1 kDa extract boiled for 10 min, L. poitei extract, L. poitei extract boiled for 10 min.

Partial purification of seawater and distilled water extracts of L. poitei and the epibionts was performed using Millipore Sep-Paks (Waters Division, Millipore Chromatography, Millford, MA, USA): C-18 for separation of a seawater epibiont extract, C-8 for a seawater L. poitei extract, and CN for a distilled water L. poirei extract. Distilled water and Burdick and Jackson high purity grade methanol and chloroform were used to rinse the column. The effects of partially purified extracts on larval metamorphosis were examined in three experiments. In the first, the treatments were: seawater epibiont extract, seawater epibiont extract passed through a C-18 Sep-Pak, a distilled water rinse, a methanol rinse, and a chloroform rinse of the Sep-Pak. Seawater controls prepared in a similar manner as the substrate extracts were included for the pass through the Sep-Pak and for each of the rinses. In the second experiment, the effects of a seawater L. poitei extract passed through a C-S Sep-Pak were examined. The treatments were similar to those in the first Sep-Pak experiment. In the third experiment, the effects of a distilled water L. poitei extract passed through a CN Sep-Pak were examined. The treatments were similar to those in the first and second Sep-Pak experiments.

In order to determine the efficiency of extraction and to determine if substrates maintain their activity after extraction, the effects of repeated extraction of the detrital epibionts and L. poitei with distilled water were examined. The substrata were initially chopped and ground with distilled water as described in the section on crude extracts, centrifuged, and the supernatant removed and saved. The pellet was then re-extracted three additional times, each with 25 ml distilled water. There were five treatments for each of two substrata (detrital epibionts and L. poitei): the initial extract, the first re-extraction, the second re-extraction, the third re-extraction, and the remaining pellet (ground and extracted substratum).

Many invertebrate larvae that are induced to metamorphose by cues associated with red algae can also be induced to metamorphose by the neurotransmitter GABA, possibly due to structural similarities between GABA and the natural inducers (Morse, 1992). To better compare the responses of S. gigus larvae to the responses of other larval invertebrates, particularly those that respond to cues produced by red algal species, the responses of Strombus gigas larvae to three concentrations of GABA (1 X 10ph M and 1 X 10m4 M, and 5 X 10d4 M) were examined.

36 A.A. Boettcher, N.M. Targett I J. Exp. Mar. Bid. Ed. 196 (1996) 29-52

3. Results

3.1. Whole substrate

There was no metamorphosis (0%) in the seawater only treatment for all except one of the experiments (~5% metamorphosis) indicating that the larvae did not appear to undergo spontaneous metamorphosis. Strombus gigas larvae responded in varying degrees (mean = 43.45%, SD = 11.31 to mean = 90.10%, SD = 6.50) to all of the substrata tested from the juvenile conch habitat (Fig. la). The highest rates of metamorphosis were in response to Thalassia testudinum detritus (mean = 90.19%, SD = 6.50), Batophora oerstedii (mean = 80.50%) SD =

13.13), and Laurencia poitei (mean = 68.21%. SD = 29.49). These were not sig- nificantly different than the response to the positive control, L. poitei extract (mean = 84.73%, SD = 11.88). The responses to L. intricata (mean = 43.45%, SD =

13.13) and Amphiroa rigida var. antillana (mean = 50.23%, SD = 12.82) were significantly lower than those to the positive control. The response to the seagrass bed sediment was significantly lower than all other treatments (mean = 8.71%. SD = 8.56). The epibiont fraction of the Thalassia testudinum detritus appeared to contain the active component from the detritus (Fig. lb). The larval responses to the whole detritus (mean = 78.47%) SD = 8.00) and the epibiont component (mean = 82.19%, SD = 13.27) were not significantly different from one another or from the positive control (mean = 89.26%, SD = 11.24), but were significantly higher than the response to the cleaned detrital blades (mean = l&69%, SD = 5.58) (Fig. lb). The dominant macroepibiont on the detrital blades was the non- geniculate coralline red alga Fosliellu sp. (Foslie) Chamberlain (pers. obs.). Fosliella sp. was also found on Laurencia poitei, L. intricata, and Batophora oerstedii, however it did not coat these substrates as it did the seagrass detritus. In the case of B. oerstedii, there also appeared to be numerous other epibionts including several filamentous and foliose red algae (pers. obs.).

3.2. Contact vs. distance

Under static conditions, the inducer did not appear to be freely diffusible. The larvae apparently must contact either the substratum or substratum surface layers for high enough concentrations of the cue to be detected to initiate metamor- phosis. There was low response of larvae to whole substrata and to filtered substratum water in the experiment examining the diffusability of chemical cues (Fig. 2). There was no response to coarse filtered Laurencia poitei water, 0.40 pm filtered detritus water, coarse or 0.40 pm filtered seawater. The response of larvae to whole L. poitei (mean = 20.95%, SD = 5.42) was significantly higher than their response to 0.40 ,um filtered L. poitei water (mean = 2.76%. SD = 3.79). There was no significant difference in the response of larvae to whole detritus (mean = 9.83%, SD = 5.66) and coarse filtered detritus water (mean = 1.43%. SD = 3.19) (Fig. 2). There were significantly higher responses of the larvae to uncovered substrata (>50%) than to covered substrata (< 10% ) for both the detritus and L. poitei (Fig. 3).

MEA

N PE

RCEN

T M

ETA

MO

RPH

OSI

S

-luw

Pu1m

~wlD

o 00

0000

0000

0 I

I I

I I

I I

I I

I I

seaw

ater

only

i

Th

ala

ssia

te

stu

din

um

de

tritu

s

clea

ned

Th

ala

ssia

te

stu

din

um

de

tritu

s

detr

ital

epib

iont

s -

al

L M

EAN

PERC

ENT

MET

AM

OR

PHO

SIS

L

-locJ

+ul

a-l.J

w(0

0 00

0000

0000

0 &

x

I I

I I

I I

I I

I I

I 2 s “?

seaw

ater

on

ly

1

Th

ala

ssia

te

stu

din

um

de

tritu

s

Lau

ren

cia

poi

tei

La

ure

nci

a

intr

ica

ta

Ba

top

hor

a

oers

ted

ii

Am

ph

iroa

ri

gida

sedi

men

t

Y

A.A. Boettcher, N.M. Targett I J. Exp. Mar. Bid. Ed. I96 (1996) 29C.52

g 100 -- C

0 E 90 --

8 80 --

$ 70 --

L r

60--

2 50 --

:: 40 --

E 30 -- B

z 20 -- A _.I-

z lO-- I A A

0 GL I I I I I I I I

1 1

TREATMENT

Fig. 2. Percent metamorphosis of queen conch larvae in response to whole substrata from conch

nursery grounds and to substratum conditioned seawater. Data are mean 2 SD, n = 5. Treatment results

with the same letter above the error bar are not significantly different at p <O.OS. Absence of a data

bar ahove a treatment indicates 0% metamorphosis.

3.3. Crude extracts

Even though, under static conditions, the cues were not freely diffusible from whole substrata, the chemical nature of the inducer associated with L. poitei and the detrital epibionts was water soluble. The response of the larvae to the seawater extract of the epibionts (mean = 45.54%, SD = 17.45) was not signih- cantly different than their response to the whole detritus (mean = 72.00%, SD = 26.42), although it was lower than the response to the positive control (mean = 94.75%, SD = 8.68) (Fig. 4). The response to the methanol extract was significantly lower (mean = 5.25%, SD = 5.56) than that to the whole detritus, the seawater epibiont extract, and the positive control. There was no response to the methanol control treatment, There was no significant difference in the responses of larvae to the seawater (mean = 68.51%, SD = 15.36) or distilled water (mean = 64.88%, SD = 10.26) extracts of the epibionts or the seawater (mean = 73.24%, SD = 18.13)

A.A. Boettcher, N.M. Targett I J. Exp. Mar. Biol. Ecol. 196 (1996) 29-S2 39

cn 100

CJ 90

2 80

$ 70

5 60

= 50

z 40

z 30

E 20

TREATMENT

Fig. 3. Percent metamorphosis of queen conch larvae in response to whole substrata from conch

nursery grounds in covered and uncovered treatments. Data are mean 2 SD, n = 5. Treatment results

with the same letter above the error bar are not significantly different at p < 0.05. Absence of a data

bar above a treatment indicates 0% metamorphosis.

and distilled water (mean = 90.57%, SD = 3.57) extracts of L. poitei. Further purification steps were, therefore, carried out using either seawater or distilled

water extracts of the substrata. Partitioning the aqueous extracts of the substrata with chloroform increased the

activity of the extracts, with the activity remaining in the aqueous fraction. The response of the larvae to the aqueous fraction of the chloroform partitioned seawater epibiont extract (mean = 68.64%, SD = 4.21) was significantly greater than the response to the seawater epibiont extract (mean = 34.67%, SD = 16.60) and was not significantly different than either the response to the positive control (mean = 93.85%) SD = 8.43) or the Thalassia testudinum detritus (mean = 68.97%, SD = 26.20) (Fig. 5). There was no response to the chloroform fraction or chloroform control. There was also no response of the larvae to chloroform partitioned seawater (Fig. 5).

As with the whole nursery ground substrata, all of the aqueous fractions of seawater chloroform partitioned extracts of these substrata elicited metamor- phosis to varying degrees, however, the hierarchy established with the whole substrata did not carry through with the extracts. The responses to Laurencia

40 A.A. Boettcher, N.M. Targett I .I. Exp. Mar. Bid. Ed. 196 (1996) 29-52

BC

I

TREATMENT

Fig. 4. Percent metamorphosis of queen conch larvae in response to whole substrata and to seawater

and methanol extracts of substrata from conch nursery grounds. Data are mean + SD, n = 5. Treatment

results with the same letter above the error bar are not significantly different at p < 0.05. Absence of a

data bar above a treatment indicates 0% metamorphosis.

poitei extract (mean = 53.66%, SD = 25.47). Amphiroa rigida var. antillana extract (mean = 52.36%, SD = 38.65) and detrital blade extract (mean = 90.81%, SD =

9.34) were not significantly different than the response to the positive control (mean = 85.33%, SD = 5.58) (Fig. 6). The responses to the detrital blade extract and the positive control were significantly higher than the response to the epibiont extract (mean = 38.50%) SD = 24.44), the Laurencia intricata extract (mean = 19.83%, SD = 21.15), and the Batophora oerstedii extract (mean = 19.25%, SD =

23.45).

3.4. Purijication of the inducer

The UV sterilized, 10 ,um filtered seawater did not contribute contaminants that influenced the results of the metamorphosis assays. There was no significant difference in the response of the larvae to the treated (mean = 48.00%, SD =

15.92) and untreated (mean = 53.10%, SD = 14.48) water with epibiont extract, or the treated (mean = 74.38%, SD = 16.49) and untreated (mean = 94.67%, SD =

5.58) water with Laurencia poitei extract. In addition, there was no response to either the treated or untreated seawater. The extracts were stable over time (Fig.

A.A. Boettcher, N.M. Targett I J. Exp. Mar. Biol. Ecol. 196 (1996) 29-52 41

v, 100

fs 2 90

8 80

5 70 r

& 60

t 50

F 40

L& 30

z 20

4 I

10

0 !

B

I

l-

r

TREATMENT

Fig. 5. Percent metamorphosis of queen conch larvae in response to whole substrata and aqueous and

chloroform partitioned seawater extracts of substrata from conch nursery grounds. Data are mean k

SD, n = 5. Treatment results with the same letter above the error bar are not significantly different at

p < 0.05. Absence of a data bar above a treatment indicates 0% metamorphosis.

7a). Although the epibiont extract that had been held for 1 yr had slightly less effect on the larvae than the fresh epibiont extract, the response was still >60% (Fig. 7a). The response to the L. poitei extract that had been held for 1 yr was actually higher than the response to the fresh L. poitei extract (Fig. 7a). Boiling had no effect on the activity of the epibiont or L. poitei extracts (Fig. 7b).

The inducers from both the epibionts and L. poitei have a nominal molecular size of ~1 kDa (Fig. 8a,b). There was no response to any of the other molecular size fractions of either the epibiont or L. poitei extracts. The response of the larvae to the Cl kDa epibiont extract fraction (mean = 22.67%, SD = 7.60) was lower than the response to the whole extract (mean = 38.13%, SD = 11.51) (Fig. 8a). Although full metamorphosis for both the (1 kDa and the whole epibiont extract treatments was low, partial metamorphosis (lobes not fully lost or resorbed) in both was >90%. There was no response to the (1 kDa seawater

42 A.A. Boettcher. NM. Turgrtt I .I. Exp. Mar. Bid. Ed. 196 (1996) 29-52

; 100 0 Lz 90

E 80

$ 70

L I 60

I- 50

z v 40

g 30

z 20

z 2 10

0

BC

AB

ii

t; ‘d P \i

BC

AB AB

l.-.au

TREATMENT

Fig. 6. Percent metamorphosis of queen conch larvae in response to chloroform partitioned aqueous

extracts of substrata from conch nursery grounds. Data are mean t SI). ,I = 5. Treatment results with

the same letter above the error bar arc not significantly different at p <MS. Absence of a data bar

above a treatment indicates 0% metamorphosis.

treatment in either assay. The ~1 kDa fraction of the epibiont extract was clear and colorless, the <1 kDa fraction of the L. poitei extract was clear and yellow. As in the experiment with whole L. poitei extract, boiling had no effect on the activity of the <l kDa fraction of L. poitei extract (Fig. 9).

The activity of both the epibiont extract and the L. poitei extracts was lost in all of the separations using Sep-Paks (CN. C-8, C-18). The active components were either retained on the columns or altered by some factor associated with the columns such that they were no longer recognized by the larvae. The control treatments had no effect on the larvae.

The response of larvae to extracted epibionts was as high as the response to the initial epibiont extract, with the re-extractions all being significantly lower (Fig. 10). Although the extracted L. poitei caused lower metamorphosis than the initial extract, it elicited significantly higher metamorphosis than any of the rc-ex- tractions (Fig. 10).

A.A. Boettcher. N.M. Targett I J. Exp. Mar. Bid. Ecol. 196 (1996) 29%S2 43

E 100

g E 90

6 80

I 70

;Is 2 60

5 50

Y 40

B 30

“z 20

9 10 I

0

TREATMENT

a

A

l-l

b

Fig. 7. Percent metamorphosis of queen conch larvae in response to: (a) one. year old and freshly

prepared aqueous extracts of substrata from conch nursery grounds, and (b) aqueous and boiled

aqueous extracts of substrata from conch nursery grounds. Data are mean 2 SD, n = 5. Treatment results with the same letter above the error bar are not significantly different at p < 0.05. Absence of a

data bar above a treatment indicates 0% metamorphosis.

seow

oter

only

seow

oter

, <

1 kD

o

La

ure

n&

a

poi

tei

extr

act,

< 1

kDo

La

ure

nci

a

poi

tea

extr

act,

1 -3

kDo

La

ure

nci

a

poi

tei

extr

act,

3-1O

kDo

La

ure

nci

a

poi

tez

extr

act.

> IO

kDo

La

ure

nci

a

poi

tez

extr

act

ME

AN

P

ER

CE

NT

M

ET

AM

OR

PH

OS

IS

-NuP

Lnol

Xfm

uJo

0000

0000

000

I I

I I

I I

I I

I I

----

---b

m

seaw

ater

on

ly

seow

oter

. <

1 kO

o

detr

itol

epib

iont

extr

act

detr

ltol

eplb

iont

extr

act.

< 1

kDo

detr

itol

epib

iont

extr

act,

1 -

1 O

kDo

detr

ital

eprb

iont

ex

trac

t, >

1 OkD

o

La

ure

nct

a

poi

tei

extr

act

ME

AN

P

ER

CE

NT

M

ET

AM

OR

PH

OS

IS

L L -~

w~

vIm

~rn

u)o

0000

0000

000

L5

I I

I I

I I

I I

I I

I I

I I

I I

I

A.A. Boettcher, N.M. Targett I J. Exp. Mar. Biol. Ecol. 196 (1996) 29-52 45

E g 100

2 90

8 80

3 70

4 60

+ 50

fs 2 40

k 30

z 20

4 3 10

0

TREATMENT

Fig. 9. Percent metamorphosis of queen conch larvae in response to molecular size fractionated, boiled

aqueous extracts of Lauren& poitei from conch nursery grounds. Data are mean 2 SD, n = 5. Treatment results with the same letter above the error bar are not significantly different at p =C 0.05. Absence of a data bar above a treatment indicates 0% metamorphosis.

The larval response to GABA was -6% for all three concentrations tested. At the lowest concentration (1 X 10m6 M) all larvae remained actively swimming, at

the two higher concentrations (1 X 10e4M and 5 X 10P4M) the larvae were found primarily near the bottom of the treatment container, with their lobes expanded, but not actively swimming.

4. Discussion

Queen conch larvae metamorphose in response to a variety of detrital and algal substrata found in their nursery grounds, as well as to aqueous extracts of these substrata. Laurencia poitei and Thalassia testudinum detritus are two of the most dominant substrata found in conch nursery grounds (Stoner & Waite, 1991). This study shows that cues associated with Laurencia poitei and the epiphytic crustose coralline red alga Fosliella sp. from Thalassia testudinum detritus are the most effective and consistent inducers of conch metamorphosis. The cues associated

seaw

ater

on

ly

detr

ital

epib

iont

ex

trac

t

detr

ltal

epib

iont

re

extr

act

1

detr

itol

epib

iont

re

extr

act

2

detr

itol

epib

iont

re

extr

act

3

extr

acte

d de

trita

l ep

ibio

nts

La

ure

n&

a

poi

tei

extr

act

La

ure

n&

a

poi

tei

reex

trac

t 1

ME

AN

P

ER

CE

NT

M

ET

AM

OR

PH

OS

IS

-h)G

JPul

m.la

lulo

00

0000

0000

0

I I

I I

I I

I I

I I

La

ure

nci

a

poi

tei

reex

trac

t 2

La

ure

n&

a

poi

tei

reex

trac

t 3

extr

acte

d L

au

ren

cia

p

ozte

z L

au

ren

cia

p

oite

l ex

trac

t

A.A. Boettcher, NM. Targett I J. Exp. Mar. Biol. Ed. 196 (1996) 29-52 47

by the green alga Batophora oerstedii. B. oerstedii possesses a rich assemblage of epibionts including Foshefla sp. as well as several filamentous and foliose red algae. Like the cue from Thalassia testudinum detritus, the cue from Batophora

oerstedii may be derived from its associated epibiont assemblage rather than the alga itself since there was low metamorphosis in response to aqueous extracts of this alga. In the current study, specific components of B. oerstedii were not examined, however, work by Davis & Stoner (1994) which showed that the epibionts from B. oerstedii fronds elicited significantly higher percent metamor- phosis than fronds from which epibionts had been removed, supports the suggestion that it is the epibiont fraction that is active.

The cues from Laurencia poitei and the detrital Thalassia testudinum epibionts were similar to one another and to cues from other red algae that induce the metamorphosis of larval invertebrates including Haliotis spp., Strongylocentrotus

droebachiensis, and Spirorbis rupestris, in that they were all water soluble and of low molecular size (Gee, 1965; Morse, 1984, 1990; Pearce & Scheibling, 1990). However, unlike Haliotis spp. and Strongylocentrotus droebachiensis larvae, conch larvae do not respond to GABA. In addition, although the red algal cues eliciting metamorphosis of Huhotis rufescens (crude extracts) and Strongyfocentrotus droebachiensis (whole substrate) are unstable (Morse & Morse, 1984b; Pearce & Scheibling, 1990), extracts of Laurencia poitei and epibionts eliciting metamor- phosis of queen conch larvae were unaffected by boiling and were stable over time, indicating that although the cues inducing conch metamorphosis may share certain general characteristics with those for other marine invertebrates induced to metamorphose by red algal species, the specific nature of the cues differ. Partitioning the seawater extracts of the substrata increased their activity, possibly by removing compounds that masked the inducer. The larvae were unaffected by chloroform partitioned seawater, therefore, the chloroform partitioning step does not appear to introduce confounding factors.

Although queen conch larvae are able to respond to aqueous extracts of the substrata, the response to whole substrata appears to be dependent on either contact with the substratum or near surface contact. Under static conditions, low responses were elicited by substratum conditioned seawater and mesh covered substratum. Flume experiments examining the availability of the cue under flow conditions would help to demonstrate whether or not the cue is available at a distance from the substratum. It has generally been concluded that under natural conditions cues are only in high enough concentrations near or at the substratum surface (Pawlik, 1992). Recently, investigators have examined the availability of water soluble cues in the water column under flow conditions, showing that the cues eliciting metamorphosis of oyster larvae are present in sufficient con- centrations to elicit settlement at (4 mm above the substratum surface (Turner et al., 1994). In addition, Hadfield & Scheuer (1985) found that cues inducing metamorphosis of Phestiffu sibogae larvae could be found in sufficient con- centrations as far as 2-3 cm from their coral source. In the current study, repeated extraction of the epibionts and Laurencia poitei does not release substantially more water soluble cue than the initial extraction, despite the fact that the

48 A.A. Boettcher, N.M. Targett I .I. Exp. Mar. Biol. Ecol. 196 (1996) 29-SZ

extracted substratum maintains its activity. This suggests that there may be an additional non-water soluble component to the cue or that part of the response of the conch larvae to whole substratum is tactile. As is the case for the inducer of larval Huliotis rufescens metamorphosis (Morse & Morse, 1984a), mechanical disruption or sloughing of the substratum surface may be necessary for the chemical cues inducing conch metamorphosis to be released, thus linking the tactile and chemical portions of the cue.

There is a high degree of both temporal and spatial variability in the suitability of specific seagrass bed substrata as sites for conch metamorphosis. In both the current study and those by Davis (1994a) and Davis & Stoner (1994), larvae responded with a high percent metamorphosis to whole Thalassia testudinum detritus and Batophoru oerstedii. In the current study, there was also consistently a high response to both whole Laurenciu poitei and to aqueous extracts of this species. Aqueous extracts of L. poitei are used commercially to induce metamor- phosis of queen conch larvae, however, it has been noted that there is variability among populations of L. poitei in their effectiveness as inducers (pers. obs.: Dyer, pers. comm.; Davis and Stoner, 1994).Variability can also be seen in the responses of larvae to sediment from conch nursery ground (current study vs. Davis & Stoner, 1994). The substrata in these studies were collected at similar sites, but in different years, therefore the differences seen could be due to differences in the epibiont assemblages or in the concentrations of inducer in the plants among years. The responses of the larvae to substrata collected at the same site, at different times during a single year were also variable. Both Thalassia testudinum detritus and Laurencia poitei whole substrata elicited high percent metamorphosis in the current study except for the experiment examining the effects of substratum conditioned seawater, in which both Thalassia testudinum detritus and Laurencia poitei elicited low responses. Based on morphology, the larvae in all of the experiments appeared competent to metamorphose, in addition, they responded with a high percent metamorphosis to the positive control. It has, however, been shown that the competency of conch larvae to metamorphose in response to whole substratum occurs at a slightly later stage than their competency to respond to extracts of L. poitei (Davis, 1994a), therefore, some of the differences in the percentage of larvae responding to whole substrata vs. extracts of the substrata may be explained by slight differences in the developmental states of the larvae.

Spatial variability in conch response is also evident among study sites. Davis & Stoner (1994) found that substrata from seagrass beds in traditional conch nursery grounds elicited significantly higher rates of metamorphosis than similar substrata from seagrass beds in which juveniles are not usually found. In addition, tethering experiments comparing survival of juvenile conch in the nursery ground areas with those in similar non-nursery ground areas have shown that survival is higher in the nursery ground areas (Stoner & Ray, 1993; Stoner, pers. comm.). It is not clear why these differences in rates of metamorphosis and survival exist. Although the assemblage of macrophytes and epibionts appear to be similar among sites and between collection times, there may be spatial and temporal differences in the densities of the specific components of each. Differences in larval response may

A.A. Boettcher, N.M. Targett I J. Exp. Mar. Biol. Ed. 196 (1996) 29-52 49

also be due, in part, to intraspecific variability in the presence or availability of the inducer in the substratum or to differences in the production of compounds that would block or otherwise mask the inducer. Differences in water circulation patterns around nursery ground vs. those around similar non-nursery ground areas may lead to differences in nutrient cycling and food production (Stoner et al., 1995) that could also contribute to the differences seen in the species assemblages and/or the availability of the cues.

Little is known about the behavior and distribution of either larval or newly metamorphosed (0 + ) juvenile queen conch. Recent work near Lee Stocking Island, Bahamas has shown that early (cl5 day post-hatch) larvae are present in the water column over both traditional nursery grounds and over seagrass beds which are not traditional nursery ground areas, but have similar macrophyte assemblages (Stoner et al., 1992, 1994). Very few late stage, competent larvae have been observed anywhere in the field (Chaplin & Sandt, 1992; Stoner et al., 1992, 1994), however, it has been shown that their are higher concentrations of competent conch larvae over traditional nursery ground areas than non-nursery ground areas (Davis & Stoner, pers. comm.). There are also only a few field studies which have examined the distribution of newly set juveniles (Stoner et al., 1992, 1994). Sandt & Stoner (1993) found that in Neighbor Cay, near Lee Stocking Island, the majority of the 0 + juveniles were found in the sand flats surrounding seagrass beds and the 1 + juveniles were in aggregations within the seagrass beds. They hypothesized that there may be an ontogenetic shift in habitat from the sand flats to the seagrass beds. However, in an additional study, 12-25 mm SL juveniles were found within seagrass beds in deeper water, therefore some of the differences in distribution may be site specific (Stoner, unpub. data. in Sandt & Stoner, 1993). To fully understand the behavior and distribution of larval and juvenile conch, laboratory studies of the inducer will have to be expanded into the field to examine the responses of conch larvae to the specific cues under natural conditions.

Acknowledgements

We thank the Caicos Conch Farm for use of both larvae and laboratory space and C. Dyer for assistance in field collections. We thank M. Davis, A. Stoner, and A. Morse for helpful discussion, and J. Duffy, D. Martin, T. Arnold and two anonymous reviewers for critical review of the manuscript. Research was sup- ported by grants from the Lerner-Gray Fund for Marine Research to AB. Additional support was provided by NSF Grant #OCE9314351 to NMT.

References

Brownell, W.N. & J.M. Stevely, 1981. The biology, fisheries, and management of the queen conch,

Strombus gigas. Mar. Fish. Rev., Vol. 43, pp. 1-12.

so A.A. Boettcher. N.M. Targett I J. Exp. Mar. Biol. Ecol. lY6 (1996) 29%S2

Burke, R.D.. 1983. The induction of metamorphosis of marine invertebrate larvae: stimulus and

response. Can. .I. Zoo..Vol. 61. pp. 1701-1719.

Cameron, R.A., 1986. Introduction to the invertebrate larval biology workshop: a brief background.

Bull. Mar. Sci..Vol. 39, pp. 145161.

Chaplin, J. & V.J. Sandt, 1992. Vertical migration and distribution of queen conch veligers. Proc. Gulf

Cad. Fish. Inst..Vol. 42. pp. 158-160.

Crisp, D.J., 1974. Factors influencing the settlement of marine invertebrate larvae. In, Chemoreception

in marine organisms, edited by P.I. Grant & A.N. Mackie, Academic Press, New York, pp. 1777265.

Crisp, D.J.. 1976. Settlement responses in marine organisms. In, Adaptution to environment. essays on

the physiology of marine animals. edited by R.C. Newell. Butterworths, London. pp. 83-124.

Crisp, D.J., 1984. Overview of research on marine invertebrate larvae, 1940-1980. In, Marine

hiodeterioration: an interdisciplinary study, edited by J.D. Costlow and R.C. Tipper. Naval Institute

Press, Annapolis, pp. 103-133.

Davis, M., 1994a. Short-term competence in larvae of queen conch (Strombus gigas Linne): shifts in

behavior, morphology and metamorphic response. Mar. Eco. Prog. Ser.. Vol. 104, pp. 101-10X.

Davis. M., 1994b. Mariculture techniques for queen conch (Strombus gigus Linne) eggmass to juvenile

stage. In. The biology. fisheries, maricultwe und mrmngrment of the queen conch. edited by R.S.

Appeldoorn & B. Rodriguez. Fundacion Cicntitica Los Roques. Caracas, pp. 231-252.

Davis, M. & A.W. Stoner. 1994. Trophic cues induce metamorphosis of queen conch larvae (Stromhw

gigas Linnaeus). J. Exp. Mar. Biol. Ecoi., Vol. 180, pp. 83-102.

Davis. M., W.D. Heyman. W. Warvey & C.A. Withstandley, 1990. A comparison of two inducers. KC1

and Luurrnciu extracts, and techniques for the commercial scale induction of metamorphosis in

queen conch, Strombus gigas Linnaeus, 1758. larvae. J. Shrl@h Kes., Vol. 9, pp. 67-73.

Gee. J.M., 1965. Chemical stimulation of settlement of larvae of Spirorbis rupestris (Serpulidae). Anim.

Behuv., Vol. 13, pp. 181-186.

Hadfield. M.G., 1986. Settlement and recruitment of marine invertebrates: a perspective and some

proposals. Bull. Mar. SC;., Vol. 39. pp. 418-42.5.

Hadheld, M.G. & D. Scheuer, 19%. Evidence for a soluble metamorphic inducer in Phestilla:

ecological. chemical, and biological data. RLLII. Mar. Sci., Vol. 37, pp. 5566.566.

Hensen. R.R., 1991. Devclopmcnt of aquaculture in the Netherlands Antilles and Aruba. Proc. (;[~!f

Carib. Fish. Inst.. Vol. 40, pp. 3633366.

Heyman. W.D.. R.A. Dobberteen. L.A. Urry & A.M. Heyman, 1989. Pilot hatchery for the queen

conch, Strombus gigas, shows potential for inexpensive and appropriate technology for larval

aquaculture in the Bahamas. Aquuculture, Vol. 77, pp. 2777285.

Johnson. C.R. & D.C. Sutton 1994. Bacteria on the surface of crustose coralline algae induces

metamorphosis of the crown-of-thorns starfish Acanthnstrr plnwi. Mar. Biol., Vol. 120. pp. 305-310.

Johnson, C.R., D.C. Sutton, R.R. Olson & R. Giddins, 1991a. Settlement of crown-of-thorns starfish:

role of bacteria on surfaces of coralline algae and a hypothesis for dcepwater recruitment. Mar.

Ed. Prog. Ser., Vol. 71, pp. 143-162.

Johnson, C.R., D.G. Muir & A.L. Reyscnbach, 1991b. Characteristic bacteria associated with surfaces

of coralline algae: a hypothesis for bacterial induction of marine invertebrate larvae. Mar. Ecol.

Prog. Ser., Vol. 74, pp. 281-294.

Mianmanus, R.T., 1988. Induction of scttlemcnt and metamorphosis in larvae of Apfysiu hrasilicmtr and

Strombus gigns (MolluscaGastropoda). Ph. D. Dissertation, University of Miami, Coral Gables,

Florida.

Morse, A.N.C., 1992. Role of algae in the recruitment of marine invertebrate larvae. In. Plant-animal

interaction.\ in the marine benthos, Systematics Association Specid. Vol. 46, edited by D.M. John. S.J.

Hawkins bi J.H. Price. Clarendon Press, Oxford, pp. 385-403.

Morse, A.N.C. & D.E. Morse. 1984a. GABA-mimetic molecules from Porphyra (Rhodophyte) induce

metamorphosis of Haliotls (Gastrapoda) larvae. Hydrobiologtu. Vol. 116, pp. 15%1%.

Morse, A.N.C. & D.E. Morse. 1984b. Recruitment and metamorphosis of Hdioti.c larvae induced by

molecules uniquely available at the surfaces of crustose red algae. ./. E,rp. Mar. Biol. Ecol., Vol. 75, pp. 191-215.

A.A. Boettcher, NM. Targett I .I. Exp. Mar. Biol. Ecol. 196 (1996) 29-52 51

Morse, A.N.C., C. Froyd & D.E. Morse, 1984. Molecules from cyanobacteria and red algae that induce

larval metamorphosis in the mollusc Haliotis rufescens. Mar. Biol., Vol. 81, pp. 293-298.

Morse, D.E., 1984. Biochemical and genetic engineering for improved production of abalones and

other valuable molluscs. Aquaculture, Vol. 39, pp. 263-282.

Morse, DE., 1990. Recent progress in larval settlement and metamorphosis: closing the gap between

molecular biology and ecology. Bull. Mar. Sci., Vol. 46, pp. 645-483.

Morse, D.E. & A.N.C. Morse, 1991. Enzymatic characterization of the morphogen recognized by

Agaricia humilis (scleractinian coral) larvae. Biol. Bull. Woods Hole Mass., Vol. 181, pp. 104-122.

Morse, D.E., N. Hooker, H. Duncan & L. Jensen, 1979. y-aminobutyric acid, a neurotransmitter,

induces planktonic abalone larvae to settle and begin metamorphosis. Science,Vol. 204, pp. 4077410.

Morse, D.E., N. Hooker & H. Duncan, 1980. GABA induces metamorphosis in Haliotis, V:

stereochemical specificity. Brain Res. Bull., Vol. 5, pp. 381-387.

Morse, DE., N. Hooker, A.N.C. Morse & R.A. Jensen, 1988. Control of larval metamorphosis and

recruitment in sympatric agariciid corals. J. Exp. Mar. Biol. Ecol., Vol. 116, pp. 193-217.

Morse, D.E., A.N.C. Morse, P.T. Raimondi & N. Hooker, 1994. Morphogen-based chemical flypaper

for Agaricia humilis coral larvae. Biol. Bull. Woods Hole Mass., Vol. 186, pp. 172-181.

Pawlik, J.R., 1992. Induction of marine invertebrate larval settlement: evidence for chemical cues. In,

Ecological roles of marine natural products, edited by V.J. Paul, Comstock Publishing Associates,

Ithaca, pp. 189-236.

Pearce, CM. & R.E. Scheibling, 1990. Induction of metamorphosis of larvae of the green sea urchin

Strongylocentrotus droebachiensis by coralline red algae. Biol. Bull. Woods Hole Mass., Vol. 179, pp.

304-311.

Pearce, C.M. & R.E. Scheibling, 1991. Effect of macroalgae, microbial films, and conspecifics on the

induction of metamorphosis of the green sea urchin Strongylocentrotus droebachiensis (Muller). J.

Exp. Mar. Biol. Ecol., Vol. 147, pp. 147-162.

Randall, J.E., 1964. Contributions to the biology of the queen conch, Strombus gigas. Bull. Mar. Sci.

Gulf Carib., Vol. 14, pp. 246-295.

Ray, M. & M. Davis, 1989. Algae production for commercially grown queen conch (Strombus gigas).

Proc. Gulf Carib. Mar. Fish. Inst., Vol. 39, pp. 453-457.

Ray, M. & A.W. Stoner, 1994. Experimental Analysis of growth and survivorship at a juvenile queen

conch aggregation: balancing growth with safety in numbers. Mar. Ecol. Prog. Ser., Vol 105, pp.

47-59.

Sandt,VJ. & A.W. Stoner, 1993. Ontogenetic shift in habitat by early juvenile queen conch, Strombus

gigas: patterns and potential mechanisms. Fish. Bull. U.S., Vol. 91, pp. 516-525.

Siddall, S.E., 1983. Biological and economic outlook for hatchery production of juvenile queen conch.

Proc. Gulf Carib. Mar. Fish. Inst., Vol. 35, pp. 46652.

Spotts, D.G., 1987. Farming queen conch in the Netherland Antilles and Aruba. Aquacult. Magaz.,

Vol. 13, pp. 32-35.

Stoner, A.W. & M. Ray, 1993. Aggregation dynamics in juvenile queen conch: population structure,

growth, mortality, and migration. Mar. Biol., Vol. 116, pp. 571-582.

Stoner, A.W. & VJ. Sandt, 1991. Experimental analysis of habitat quality for juvenile queen conch in

seagrass meadows. Fish. Bull. U.S., Vol. 89, pp. 693-700.

Stoner, A.W. & J.M. Waite, 1991. Trophic biology of Strombus gigas in nursery habitats: diets and food

sources in seagrass meadows. J. Mall. Stud., Vol. 57, pp. 451-460.

Stoner, A.W., VJ. Sandt & IF. Boidron-Metairon, 1992. Seasonality in reproductive activity and larval

abundance of queen conch Strombus gigas. Fish. Bull. U.S., Vol. 90. pp. 161-170.

Stoner, A.W., M.D. Hanisak, N.P. Smith & R.A. Armstrong, 1994. Large scale distribution of queen

conch nursery habitats: implications for stock enhancement. In, The biology, fisheries, mariculture

and management of the queen conch, edited by R.S. Appeldoorn & B. Rodriguez, Fundacion

Cientifica Los Roques, Caracas, pp. 169-189.

Stoner, A.W., P.L. Pitts & R.A. Armstrong, 1995. The interaction of physical and biological factors in

large-scale distribution of juvenile queen conch populations in seagrass meadows. Bull. Mar. Sci., (in

press).

52 A.A. Boettcher, N.M. Targett I J. Exp. Mar. Biol. Ecol. 196 (1996) 29-52

Turner, E.J., R.K. Zimmer-Faust, M.A. Palmer & M.W. Luckenbach. 1994. Settlement of oyster

(Crassostrea virginica) larvae: effects of water flow and water-soluble chemical cue. Limnol. Oceanogr., Vol. 39, pp. 1579-1593.

Wickland, R.I., L.J. Hepp & G.A. Wenz, 1991. Preliminary studies on the early life history of the

queen conch, Strombus gigas, in the Exuma Cays, Bahamas. Proc. Gulf Cad. Fish. Inst., Vol. 40, pp.

283-298.