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1 School of Biological, Earth & Environmental Sciences
2 School of Chemical Sciences
3 Bioanalytical Mass Spectrometry Facility
4 Center for Marine Biofouling & Bio-Innovation, University of New South Wales, Sydney 2052, Australia
* To whom correspondence should be addressed. E-mail: r.swanson{at}unsw.edu.au
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Abbreviations: CX, cation-exchange • F-I, floridoside-isethionic acid • GC-MS, gas chromatography–mass spectrometry • HPLC, high-performance liquid chromatography • ISTD, internal standard • MALDI-TOF MS, matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry • NMR, nuclear magnetic resonance • SSW, sterile seawater
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionLiterature Cited |
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Active habitat selection requires that larvae discriminate among potential settlement sites, which is possible through the detection of habitat-specific cues. Many laboratory experiments have confirmed that larvae from a diverse range of phyla respond both behaviorally (settlement—sinking to the bottom for substrate exploration) and morphologically (metamorphosis—ontogenesis into the benthic form) to such physical factors of a habitat as light (Maida et al., 1994), surface orientation (Raimondi and Morse, 2000), flow conditions (Mullineaux and Butman, 1991), crevices (Keough and Downes, 1982), and surface texture (Berntsson et al., 2000). Larvae can also be induced to settle and metamorphose (collectively termed settlement in this paper) by surface-bound or waterborne chemical cues, which are thought to indicate a suitable habitat for the benthic stage (Hadfield and Paul, 2001). The source of such chemical cues (inducers) may be conspecifics (Burke, 1986), host organisms (Williamson et al., 2000), prey (Hadfield and Scheuer, 1985), or biofilms (Wieczorek and Todd, 1998).
The chemical cues for larval settlement that have been isolated from natural sources within the habitat appear to be diverse; however, most have been only partially characterized. These include small peptides (the sand dollar Dendraster excentricus—Burke, 1984; the oyster Crassostrea virginica—Zimmer-Faust and Tamburri, 1994; the jellyfish Cassiopea xamachana—Fleck and Fitt, 1999), uncharacterized low-molecular-weight water-soluble compounds (the nudibranch Phestilla sibogae—Hadfield and Pennington, 1990; the cephalaspidean Haminaea callidegenita—Gibson and Chia, 1994), carbohydrates (the coral Agaricia humilis—Morse and Morse, 1996; the ascoglossan Alderia modesta—Krug and Manzi, 1999), and glycoproteins (the barnacle Balanus amphitrite—Clare and Matsumura, 2000).
In contrast to the numerous partially characterized inducers, there are only a few examples in which the chemical structure of a settlement cue isolated from a natural source has been determined. Delta-tocopherols from Sargassum tortile induced settlement of the hydroid Coryne uchidai (Kato et al., 1975), jacarone isolated from the red alga Delesseria sanguinea induced settlement of the scallop Pecten maximus (Yvin et al., 1985), narains and anthosamines A and B isolated from marine sponges induced settlement of ascidian larvae (Tsukamoto et al., 1994, 1995), and lumichrome isolated from conspecifics induced settlement of larvae of the ascidian Halocynthia roretzi (Tsukamoto et al., 1999). In most cases, the ecological relevance of these compounds in situ is not clear, either because the source of the settlement cue is not necessarily related to the recruitment patterns of the organism (Yvin et al., 1985; Tsukamoto et al., 1994, 1995), or because the availability of the cue to settling larvae has not been demonstrated (Tsukamoto et al., 1999).
A naturally occurring characterized settlement cue that appears to strongly affect the demography of the sea urchin Holopneustes purpurascens Agassiz 1872 (Temnopluridae: Echinodermata) was recently reported by Williamson et al. (2000). H. purpurascens is an endemic Australian echinoid that lives in shallow subtidal habitats in the canopy of macroalgae, particularly Delisea pulchra Greville (Montagne) 1844 and Ecklonia radiata (C. Agardh) J. Agardh 1898 at Bare Island, Sydney (Williamson et al., 2000,2004). Although abundant on both host plants, the smaller size classes of H. purpurascens were most abundant on D. pulchra, with the smallest size class (test diameter 
5 mm) found only on that species. This suggested that D. pulchra might produce a settlement cue for larval H. purpurascens (Williamson et al., 2000). Fresh pieces of D. pulchra (but not E. radiata) and seawater collected in situ near D. pulchra plants induced settlement in larvae of H. purpurascens. The water-soluble cue was subsequently isolated and characterized as a complex between the sugar floridoside and isethionic acid (F-I complex; Williamson et al., 2000).
During further research on this system, we obtained inductive fractions that contained isethionic acid but not floridoside, and we were also unable to reproduce a synthetic F-I complex that induced settlement of larval H. purpurascens. Subsequently, we hypothesized that the F-I complex was not a natural settlement cue for this urchin. This paper identifies the true nature of this chemical cue from D. pulchra for settlement of H. purpurascens larvae, correcting the previous finding of Williamson et al., (2000). In addition, we quantify the settlement cue in host and non-host algae of H. purpurascens—the first time that a natural settlement cue has been quantified in the habitat of a marine organism.
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Materials and Methods |
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Preparation of the polar extract of Delisea pulchra
The results of Williamson et al. (2000) indicated that any settlement cues were contained within the polar fraction of the crude extract of D. pulchra. A polar extract of D. pulchra was thus prepared from 1.0 kg (wet weight) of algae collected from Bare Island. Epibiota were removed, the plants blotted dry, and the thallus exhaustively extracted in methanol (OmniSolv, EM Science). The methanol extract was filtered (Whatman #1), dried by rotary evaporation in vacuo at 40 °C, and partitioned between dichloromethane (OmniSolv) and Milli-Q water. The Milli-Q phase was filtered (Whatman #1) and dried in vacuo at 40 °C. The dried crude polar extract was dissolved in absolute ethanol three times, pooling each extract, and dried in vacuo at 40 °C to yield the polar extract.
Isolation of the settlement cue in Delisea pulchra by bioassay-guided fractionation
High-performance liquid chromatography.
The polar extract of D. pulchra was fractionated using reversed-phase high-performance liquid chromatography (HPLC—Adsorbosil C18 column, 5-µm particle size, 250 mm x 4.6 mm, Waters R410 RI-detector) (100% Milli-Q water at 1 ml · min–1). The polar extract was dissolved in Milli-Q water (50 mg · ml–1), filtered (0.22 µm), and manually injected (20 µl). HPLC resolved two major peaks, peak 1 with a retention time (rt) of 2.7 min, and peak 2 with rt = 3.4 min (Fig. 1A). Each peak fraction was collected from multiple injections and dried by rotary evaporation in vacuo at 40 °C. Peak fractions were tested for bioactivity in settlement assays and analyzed by 1H- and 13C-nuclear magnetic resonance (NMR) spectroscopy (Bruker DMX 500).
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Identification of isolated settlement cue
Nuclear magnetic resonance spectroscopy.
Bioassay-guided fractionation of the polar extract of D. pulchra by cation-exchange chromatography yielded one active fraction (CX-fraction 5, F5). The inducing compound in F5 was identified by 1H and 13C-NMR experiments (D2O), and a high-field two-dimensional 1H-15N HMBC NMR experiment (d4 MeOH, Bruker DMX 500). To confirm the putative structure of F5 as histamine, 3 mg of F5 was dissolved in D2O and analysed by 1H-NMR spectroscopy. Synthetic histamine (3 mg) was added to F5 and the sample re-analysed. The 1H-NMR spectra of the unspiked F5 sample and the spiked F5 sample were then compared.
Gas chromatography–mass spectrometry.
NMR spectroscopy analyses identified the isolated settlement cue as histamine, and this was confirmed by gas chromatography–mass spectrometry (GC-MS). Putative (naturally isolated) histamine (1 mg) and synthetic histamine (1 mg) were derivatized with heptafluorobutyric anhydride (Aldrich) and then acetic anhydride (Aldrich), using the method of Barancin et al. (1998). Derivatized samples were diluted 100-fold in ethyl acetate before analysis. A Zebron ZB-5 column (15 m, 0.25 µm x 0.25 mm ID; Phenomenex) was used on aHewlett Packard (HP) 5980 series II gas chromatograph equipped with an HP5971A or HP5972 mass selective detector. Injections (2 µl) were in the splitless mode with an inlet pressure of 170 kPa. The injection port was held as 290 °C and the interface at 300 °C. The gas chromatograph was held at 90 °C for 2 min and ramped at 10 °C · min–1 to 200 °C, then at 50 °C · min–1 to 310 °C and held for 2 min (17.2 min per run). Helium was used as the carrier gas. The mass selective detector was operated in scan mode (m/z 50–550). The average retention times of derivatized putative histamine and derivatized synthetic histamine were recorded from five injections of each sample (mean ± SD, n = 5). The electron impact ion-spectra of derivatized putative histamine and derivatized synthetic histamine were compared.
Matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry.
The elemental formula of putative histamine was determined by matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF MS) (Bucknall et al., 2002). A Perseptive Voyager DE STR (Perseptive Biosystems, Framingham, MA) MALDI-TOF MS was operated in both positive-ion linear delayed-extraction mode and reflector delayed-extraction mode for accurate mass analysis. The test samples were prepared in acetonitrile/Milli-Q water (50:50) and contained either 100 ng · µl–1 of putative histamine or synthetic histamine. 
-Cyano-4-hydroxycinnamic acid (5 mg · ml–1) prepared in acetonitrile/Milli-Q/trifluoroacetic acid (80:20:0.02) was used as the matrix. Glycine (500 ng · µl–1) and [sarcosine-15N-methyl-d3]creatinine · HCl (5 ng · µl–1, Cambridge Isotope Laboratories #DNLM-2171) were added as internal mass calibrants for accurate mass determinations. An accurate mass for the putative protonated histamine molecular ion [M+H]+ was determined by 10 repeat analyses of each sample. The mean molecular weight was calculated for these mass spectra and compared with both the theoretical molecular weight for histamine and the molecular weight measured for synthetic histamine using the same analytical technique. The standard deviation for these mass measurements was taken as an estimate of the mass measurement error.
Settlement assays
H. purpurascens larvae were cultured as previously described (Williamson et al., 2000). Larvae reached competency (i.e., become developmentally ready for settlement) within 6 days, as recognized by the presence of five well-developed tube feet. All settlement assays were done at 19 °C with a 12-h-light/12-h-dark regime, in 40-mm petri dishes and 5 ml of sterile seawater (SSW). Replicates were randomly assigned among treatments, with 10–15 replicates per treatment and one 6-day larva per replicate dish. We were unable to use multiple larvae per dish in these assays because this species is a "dribble" spawner and generally yields low numbers of larvae (settlement is not gregarious; Williamson et al., 2000). Larvae were added once all petri dishes were prepared, and percent settlement (i.e., percent metamorphosed) was recorded at set time intervals.
HPLC peak fractions.
Peak 1 and peak 2 fractions were tested against larvae to determine the presence of a settlement cue. Peak fractions were dissolved in Milli-Q water (10 mg · ml–1 stock solution) and aliquots of each stock solution were added to assigned petri dishes for final test concentrations of 25 µg · ml–1 of peak 1 and 51 µg · ml–1 of peak 2. A floridoside-isethionic complex sample ("F-I complex") from the previous study (Williamson et al., 2000) was also tested in the assay at a final concentration of 76 µg · ml–1. Pieces of fresh D. pulchra (
10 mg) were used as a positive control, and Milli-Q water and SSW were used as the negative controls. Percent settlement was scored after 18 h (n = 12 replicates per treatment).
Cation-exchange fractions.
Each CX-fraction (F) was tested against larvae to determine the presence of a settlement cue. F1, F2, F3, F4 and the polar extract of D. pulchra (used as a positive control) were dissolved in Milli-Q water at 5 mg · ml–1. Aliquots of the appropriate fraction were added to the petri dish to give final test concentrations of 50 µg · ml–1 for each treatment. F5 was dissolved in Milli-Q water at 100 µg · ml–1, and aliquots were added to assigned petri dishes for final test concentrations of 0.1–1.0 µg · ml–1 (much lower concentrations of F5 were tested because of a low yield in F5). Initial settlement assays showed that only F5 induced settlement; therefore, CX-control-fraction 5 (CF5) was tested in future settlement assays as the procedural control. CF5 was dissolved in Milli-Q water at 100 µg · ml–1 and tested at 1.0 µg · ml–1. Milli-Q water and SSW were used as the negative controls. Percent settlement was scored after 1 h (n = 10 replicates per treatment).
Natural versus synthetic histamine.
Settlement assays were used to compare the responses of larvae to (i) natural histamine isolated using CX chromatography. (ii) synthetic histamine, and (iii) synthetic histamine run through the same procedure used to isolate natural histamine. Stock solutions of 900 µM of each histamine treatment were prepared in Milli-Q water, and aliquots of the appropriate stock solution added to assigned petri dishes for final test concentrations of 0.9–9.0 µM. Pieces of fresh D. pulchra (10 mg) and 50 µg · ml–1 of the polar extract D. pulchra were used as the positive controls, and Milli-Q water and SSW were used as the negative controls. Percent settlement was scored after 1 h (n = 12 replicates per treatment).
Delisea pulchra treated with antibacterial agents.
Because some marine bacteria produce histamine (Fujii et al., 1997), the identification of histamine as the settlement cue (see Results) raises the possibility that the bacterial biofilm on the surface of D. pulchra may be the source of the cue. To test this, the ability of D. pulchra to induce settlement after various antibacterial treatments was examined in a settlement assay. Antibacterial treatments were adapted from previous studies in which treatments were shown to be effective in reducing surface bacteria (Xue-Wu and Gordon, 1987; Aguirre-Lipperheide and Evans, 1993; Johnson and Sutton, 1994). Seven plants of D. pulchra were collected from Bare Island and brought back to the laboratory, where portions of each plant were allocated to each of seven treatments. There were six antibacterial treatments and a procedural control. All antibacterial treatments included a 5-min soak in a 10% betadine-SSW solution, followed by three rinses in SSW and a 24-h treatment in either (1) SSW (the "soak" treatment); (2) SSW containing 20 mg · l–1 streptomycin (Aldrich), 10 mg · l–1 penicillin G (Aldrich), and 10 mg · l–1 kanamycin (Aldrich; "SPK" treatment); (3) SSW containing 10 mg · l–1 ciprofloxacin (Bayer, "ciprofloxacin" treatment); (4) SSW after pieces of D. pulchra were gently wiped across an agar plate, before and after the 24-h soak, to physically remove bacteria ("wipe" treatment); and the combination treatments (5) "wipe + SPK", and (6) "wipe + ciprofloxacin." The procedural control was a 24-h soak in SSW without the initial betadine soak ("soak control" treatment). The next day, subsections of several D. pulchra plants were collected as a "fresh control" treatment and used in the settlement assay on that day. Pieces of D. pulchra (10 mg) from each treatment were added to assigned sterile petri dishes, and percent settlement was scored after 20 h (n = 15 replicates per treatment).
Quantitative analysis of histamine in various algae
If histamine is a natural settlement cue for this urchin, we would expect D. pulchra, the primary host plant of new recruits of H. purpurascens, to have higher levels of histamine than other algae in the habitat. To test this, we quantified the histamine content of six species of algae from the habitat of H. purpurascens. The two primary host plants (D. pulchra and E. radiata) and four other prominent species of algae (Amphiroa anceps, Corallina officinalis, Homeostrichus olsenii, and Sargassum vestitum) were collected from Bare Island in January 2003. Five replicates of each alga were analyzed, with each replicate consisting of three small sections taken from different parts of one thallus, which were then pooled into a single sample for analysis (2–4 g wet-weight). A polar extract of each algal sample was prepared as described above. Polar extracts were dissolved in Milli-Q water (200 µl) and acidified with 50 µl of glacial acetic acid. [, , ß, ß-d4]Histamine · 2HCl (1 µg, Cambridge Isotope Laboratories, #DLM 2911) was added to each sample as the internal standard (ISTD). Strong cation-exchange solid-phase extraction cartridges (50 mg, Alltech) were equilibrated with Milli-Q water (5 ml) at a flow rate of 1 ml · min–1, and the sample was loaded. Unbound compounds were eluted in 2 ml of Milli-Q water (fraction 1) and another 2 ml of Milli-Q water (fraction 2). All retained compounds were eluted in 1 ml of 30% NH4OH w/w (fraction 3) and dried in a speed vac. Standards that contained 1-µg ISTD and either 0.1, 0.5, 1.0, 5.0, or 10 µg of synthetic histamine were prepared. Standards and fraction 3 samples were derivatized with heptafluorobutyric anhydride and acetic anhydride using the method of Barancin et al. (1998).
A DB-5MS column (15 m, 0.25 µm x 0.25 mm ID, J & W Scientific) and a packed liner (3% SP-2250, Supelco; Smythe et al., 2002) were installed on the GC-MS instrument previously described, and the same run conditions were used. The mass selective detector was operated in selected ion monitoring mode using ions characteristic of the analyte (derivatized histamine—m/z 94, 307, 349) and the ISTD (m/z 97, 311, 353). Extracted ion chromatograms were used to manually integrate the area under each ion peak (which is proportional to the amount of analyte in the sample). For each standard and sample, the areas of the analyte ions (m/z 94, 307, 349) were added together and the areas of the ISTD ions (m/z 97, 311, 353) were added together. The ratio of the combined areas of analyte:ISTD in standards was used to generate a standard curve. The histamine content of the samples was calculated by reference to the standard curve and expressed in terms of micrograms per gram (wet weight) of algal tissue (µg · g–1).
After checking that the data met the assumptions of the test, the histamine content of different algae was transformed [ln(x + 1)] and compared by using a one-factor analysis of variance. We excluded A. anceps and C. officinalis from the analysis because no histamine was detected in these species. Bonferroni’s post hoc test was used to determine which species differed in their histamine contents (SYSTAT ver. 7.0). We were concerned that one high value for D. pulchra might be unduly influencing our analysis, but the outcome was unchanged when we repeated the analysis with this value omitted. Therefore, we report the results of the initial analysis.
Reanalysis of samples from Williamson et al. (2000)
Samples remaining from the study published in Williamson et al. (2000) were analyzed by GC-MS for the presence of histamine. Any histamine in the old samples was isolated using cation-exchange solid phase extraction cartridges, as outlined previously for isolating algal histamine, and then derivatized with heptafluorobutyric anhydride and acetic anhydride, using the method of Barancin et al. (1998) for quantitative GC-MS analysis.
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-D-galactopyranosyl-(1-2)-glycerol] (Karsten et al., 1993). Therefore, the isethionic acid and floridoside components of the F-I complex eluted separately, in peak 1 and peak 2, respectively. Peak 1 induced settlement of Holopneustes purpurascens larvae in settlement assays, but peak 2 did not (Fig. 2). Four batches of peak 1 (25 µg · ml–1) induced 80%–100% settlement in five assays, whereas neither of two batches of peak 2 (51 µg · ml–1) induced settlement in two assays (representative data shown in Fig. 2). These data suggested that the F-I complex is not a settlement cue for H. purpurascens and that peak 1 (which lacked floridoside) contained the settlement cue.
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Cation-exchange fractions—settlement assay.
The settlement cue could not be isolated as a pure fraction using HPLC, so the polar extract of D. pulchra was fractionated using CX chromatography (Fig. 1B). Five CX-fractions (F) were obtained and tested in settlement assays; only F5 induced settlement of larvae of H. purpurascens (Fig. 3). F5 at a concentration of 1.0 µg · ml–1 induced 100% settlement in larvae after 1 h, 0.5 µg · ml–1 induced 70% settlement, and 0.1–0.25 µg · ml–1 did not induce settlement. There was no settlement in the control fraction CF5 (1.0 µg · ml–1) and SSW treatments (Fig. 3).
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2.76 (2H, t, J 7.0 Hz, H2), 3.03 (2H, t, J 8.2 Hz, H1), 6.86 (1H, s 2H, imidazole H), and 7.57 (s, 1H, imidazole H). The 13C-NMR (D2O) and DEPT spectra of F5 showed carbon signals at 25.9, 39.5 (CH2); 116.4, 136.5 (CH) and 134.0 (quaternary C). These signals supported the assignment of F5 as histamine (2-[1H-imidazol-4-yl]-ethylamine, MW 111.15). The structure of F5 was further confirmed by a high-field two-dimensional 1H-15N HMBC NMR experiment in which the methylene triplet at 2.76 ppm showed two three-bond correlations to the ethylamine NH2 group and the imidazole nitrogen. The identity of F5 was further confirmed by a spiking experiment. All F5 signals increased in intensity and no additional signals were detected, confirming the identity of F5 as histamine.
Gas chromatography–mass spectrometry.
The identity of putative histamine (F5) isolated from D. pulchra was confirmed using GC-MS. The retention times (rt) of the heptafluorobutyrlacyl derivative of putative histamine (rt = 9.728 ± 0.0045, mean ± SD, n = 5) and synthetic histamine (rt = 9.732 ± 0.0045, mean ± SD, n = 5) were nearly identical, suggesting that they were the same compound. The electron-impact ion spectra of both derivatized compounds displayed the same major fragment ions (m/z—54, 69, 81, 94, 138, 169, 226, 307, 349) and overall fragmentation pattern, confirming that they were the same compound. The electron-impact ion spectra for derivatized histamine matched that reported in the literature (Barancin et al., 1998).
Matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry.
The elemental formula of putative histamine isolated from D. pulchra was confirmed by accurate mass measurements using MALDI-TOF MS. The measured accurate mass of the putative protonated histamine molecular ion [M+H]+ was 112.08878 ± 0.0026 (n = 10, mean ± SD), and the measured mass for synthetic histamine was 112.08853 ± 0.0025 (n = 10, mean ± SD). The measured masses of the two samples were different by only 2.2 ppm. These values were different from the calculated monoisotopic mass for protonated histamine (112.08692—elemental formula C5H10N3) by only 15 ppm for synthetic protonated histamine and 17 ppm for putative protonated histamine. This is most likely due to measurement bias introduced by the very different chemical properties of histamine, glycine, and creatinine (the internal calibrants).
An elemental calculator was used to generate all possible elemental formulas with a mass of approximately 112.08878. The nearest other candidate was C6H10NO at 112.07569 with a difference of 117 ppm from the measured mass of putative protonated histamine. This difference was much higher than 17 ppm (difference of measured mass for putative histamine relative to calculated mass for C5H10N3), confirming that the putative protonated histamine had the elemental formula of C5H10N3.
The response of Holopneustes purpurascens larvae to natural and synthetic histamine
Natural histamine isolated from D. pulchra by using CX chromatography, synthetic histamine, and synthetic histamine eluted from CX resin all resulted in very similar responses in larvae when assayed concurrently (Fig. 4). More than 80% of the H. purpurascens larvae settled within an hour of incubation in 4.5 and 9 µM natural and synthetic histamine. The lowest test concentration of synthetic histamine that consistently induced rapid settlement of all larvae was 4.5 µM (in 10 separate assays). Larvae exhibited a more variable response to 0.9 and 2.3 µM histamine, both within and across different batches (Fig. 4). Up to 80% of larvae settled in response to 0.09–0.45 µM synthetic histamine, but only after long incubation times (up to 96 h) or as larval age increased to 13–21 days (data not shown).
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Williamson et al. (2000) reported on one such putative characterized cue, a metabolite complex isolated from the red algal host Delisea pulchra that induced settlement in larvae of the sea urchin Holopneustes purpurascens. At Bare Island (Sydney, Australia), H. purpurascens is found primarily on two algal hosts, D. pulchra and Ecklonia radiata, with the smallest size class (test diameter 5 mm) only found on D. pulchra. Larvae metamorphosed in response to pieces of D. pulchra and the polar extract, but not to pieces or extracts of E. radiata (Williamson et al., 2000). A water-soluble cue was implicated when seawater collected near D. pulchra plants in situ also induced settlement of larvae. The settlement cue in D. pulchra was isolated and characterized as the floridoside-isethionic acid (F-I) complex (Williamson et al., 2000).
New evidence presented in this paper shows that histamine, not the F-I complex, is a natural inducer of settlement in H. purpurascens. The settlement cue was isolated from the polar extract of D. pulchra by using bioassay-guided fractionation by cation-exchange chromatography. The isolated compound at 0.5 µg · ml–1 induced settlement in 80%–100% of larvae within an hour. The settlement cue was identified as histamine using NMR spectroscopy, and this was confirmed by GC-MS and MALDI-TOF MS. The response of larvae to synthetic histamine in settlement assays mirrored their response to natural histamine isolated from D. pulchra. D. pulchra, the primary plant on which new recruits of H. purpurascens are found, had the highest average histamine content (11.82 ± 6.56 µg · g–1 wet weight), approximately an order of magnitude higher than other algae surveyed. Seawater collected near D. pulchra plants in the study by Williamson et al. (2000) induced rapid settlement of larval H. purpurascens; however, those samples were used completely in bioassays and are therefore not available for histamine analysis. We have since detected histamine in seawater surrounding D. pulchra and E. radiata (at concentrations ranging from 20 to 70 nM), but not in samples 2 m away from the macroalgae. A comprehensive analysis of histamine levels in seawater will be reported in another manuscript. Although the histamine concentrations measured in seawater do not induce rapid settlement in larvae that have just attained competence, this concentration can induce settlement of H. purpurascens larvae over longer time periods and in older larvae (data not shown). In addition, the natural habitat may contain other settlement cues that if detected in conjunction with histamine, may lower the threshold concentration of histamine required for rapid induction of settlement. These findings support our proposal that histamine released from macroalgae is a natural settlement cue for H. purpurascens.
Reanalysis of samples from the study by Williamson et al. (2000) provides an explanation for the incorrect conclusion that the F-I complex is a settlement cue for larvae of H. purpurascens. The F-I complex was isolated from the polar extract of D. pulchra, using reversed-phase HPLC and methanol as the mobile phase, and eluted as a single peak (Williamson et al., 2000). 13C-NMR spectroscopy analysis of this peak showed only 13C-signals for floridoside and isethionic acid (Williamson et al., 2000). However, trace amounts of histamine were also present but not detected, because their levels were below the limit of detection for 13C-NMR spectroscopy. Histamine elutes in the first peak from reversed-phase (C18) columns regardless of the mobile phase, so any histamine in the polar extracts of D. pulchra used by Williamson et al. (2000) would have co-eluted with the F-I complex fraction. Consequently, the "F-I complex" samples contained histamine, detected here using GC-MS, and induced settlement of H. purpurascens larvae. Although a synthetic F-I complex induced rapid settlement in H. purpurascens larvae in the previous study (Williamson et al., 2000), not all batches induced settlement (R. de Nys, pers. obs.). The synthetic F-I complexes were made using natural floridoside isolated from D. pulchra and synthetic isethionic acid. The floridoside used to make the synthetic F-I complex was contaminated by histamine and thus induced settlement. Confirming this, histamine was detected by GC-MS in a floridoside sample (used for preparation of the synthetic complex) and a synthetic F-I complex sample prepared by Williamson et al. (2000). In summary, histamine was present in trace amounts in the "F-I complex" samples that induced settlement of larval H. purpurascens in the previous study, and histamine was the inductive compound in the "F-I complex" samples.
The finding that histamine is a natural settlement cue for H. purpurascens is of considerable interest in the context of linking ecological patterns with physiological mechanisms. Histamine is a biogenic amine produced by the decarboxylation of the amino acid histidine. It is one of five primary biogenic amines in invertebrates, along with serotonin, octopamine, dopamine, and tyramine (Blenau and Baumann, 2001). Biogenic amines, all decarboxylation products of amino acids, play critical roles in initiating and controlling behavior, and in the physiology of invertebrates, by acting as classical neurotransmitters, neuromodulators, and neurohormones (Katz, 1995; Beltz, 1999). For example, dopamine activates hunting behavior in an opisthobranch mollusc (Norekyan and Satterlie, 1993), and serotonin controls aggressive behavior in crustaceans (Huber et al., 1997). The photoreceptors in all classes of arthropod eyes are histaminergic; that is, they synthesize histamine and use it as their neurotransmitter (Stuart, 1999). Also, histamine is thought to be an inhibitory neurotransmitter in the stomatogastric and cardiac ganglia and the sensory system of lobsters (Claiborne and Selverston, 1984; Bayer et al., 1989; Hashemzadeh-Gargari and Freschi, 1992). Importantly, in the context of this study, histamine directly gates a chloride channel in the receptor cells of the olfactory pathway of lobsters (McClintock and Ache, 1989). Fast neurotransmitters directly gate ion channels, which leads to fast behavioral and physiological outcomes. We have observed that the settlement response of H. purpurascens to histamine is rapid, with complete metamorphosis within half an hour. This fast response is consistent with the notion that the larvae of H. purpurascens have specific receptors that bind histamine and act directly on ion channels, leading to rapid settlement.
Neurotransmitters, or their precursors, have been suggested to mimic the function of natural settlement cues (Morse, 1985; Bonar et al., 1990). The best-known example is the gamma-aminobutyric acid (GABA)-mimetic peptide (or peptides), present on the surface of crustose coralline algae, which Morse and colleagues proposed as a settlement cue for abalone (Morse et al., 1979, 1984). Another example comes from oyster larvae, where L-3, 4-dihydroxyphenylalanine (L-DOPA) induced stereotypical searching behavior, while epinephrine and norepinephrine induced metamorphosis (Coon et al., 1985). Endogenous levels of neurotransmitters, and their precursors, also appear to modulate the behavioral and physiological processes accompanying settlement (Coon and Bonar, 1987; Pires et al., 2000). Our findings show that a naturally produced neurotransmitter is in fact a settlement cue for larvae, a phenomenon that may be widespread in the marine environment.
The finding that histamine, rather than the F-I complex, is a settlement cue for H. purpurascens potentially complicates the previous interpretations of the relationship between settlement cues and the demography of this sea urchin (Williamson et al., 2000, 2004). Histamine, a simple breakdown product of the amino acid histidine, may be broadly distributed across the natural habitat of H. purpurascens; for example, in algal and animal tissue, and in bacterial communities living on their surfaces. For histamine to be an ecologically relevant settlement cue, its distribution in the natural habitat must relate to the recruitment patterns of H. purpurascens. This was in fact the case. The histamine content of the algae surveyed was consistent with the recruitment patterns of the organism, with much higher levels of histamine measured in D. pulchra, the primary plant on which we find new recruits.
D. pulchra had the highest average histamine content (11.82 ± 6.56 µg · g–1 wet weight), ranging from 1.88 to 34.22 µg.g–1 wet weight. Similarly, levels of histamine varied for E. radiata, with concentrations ranging from 0 to 4.73 µg.g–1 wet weight. Since only subsections of plants (not whole plants) were extracted, these results may reflect within-plant variation, within-species variation, or both. Future histamine analyses will extract whole plants, as well as specific regions of thalli, to directly test these possibilities. The low (or absent) levels of histamine typically measured in E. radiata samples may explain why pieces and extracts of E. radiata did not induce settlement in the study by Williamson et al. (2000). However, we have observed that some pieces of E. radiata do induce settlement of H. purpurascens, which is consistent with the variation we measured in levels of histamine in the alga. Given this, and the large biomass of E. radiata kelp beds in the natural habitat of H. purpurascens, E. radiata may contribute to environmental levels of histamine, inducing the settlement of larvae in this habitat. Histamine was not detected in the turfing coralline algae Corallina officinalis and Amphiroa anceps, although they induce settlement of larvae of H. purpurascens (Williamson et al., 2000; R. Swanson, pers. obs.) and provide a habitat for new recruits (R. Swanson, pers. obs.). Larger samples of A. anceps (up to 180 g wet weight) were extracted and no histamine was detected. The coralline algae may produce a different settlement cue for H. purpurascens, or histamine may only be produced and released in situ—for example, by surface-associated bacteria.
Finally, the two possible sources of histamine in D. pulchra are the host alga or the surface-associated bacterial community (or both). D. pulchra treated with various antibacterial agents still induced high levels of settlement in H. purpurascens, suggesting that the host alga produces the histamine. A bacterial source of histamine is, however, possible, as a known histamine-producing bacterium, Photobacterium phosphoreum (Fujii et al., 1997) is a constituent of the microbial community on local algal species (M. Watson, UNSW Australia; pers. comm.). If histamine-producing bacteria are colonizing algal surfaces within the habitat, then it is possible that they produce and release histamine to seawater, which could lead to the induction of settlement of H. purpurascens.
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Current address: Department of Biological Sciences, Macquarie University, NSW 2109 Australia. 
Current address: School of Marine Biology & Aquaculture, James Cook University, QLD 4811 Australia.
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