MARINE ECOLOGY PROGRESS SERIES Published July 6 P- - -- Mar Ecol Prog Ser
A proline-rich peptide originating from decomposing mangrove leaves is one natural metamorphic cue of the tropical jellyfish Cassiopea xamachana
'Lehrstuhl fiir Spezielle Zoologie, Ruhr-Universitit. D-44780 Bochum. Germany 'Institute of Ecology, University of Georgia, Athens. Georgia 30602-2602. USA 3Complex Carbohydrate Research Center, University of Georgia, Athens. Georgia 30602-4712, USA
ABSTRACT: Planula larvae of the scyphozoan Cassiopea xamachana settle and n~etamorphose on de- grading mangrove leaves of Rhizophora mangle that lie submerged In shallow water mangrove ecosys- tems. Our prior study (Fleck & Fitt 1999; J Exp Mar Biol Ecol 234:83-94) indicated that marine bacteria are involved in the release of at least 1 peptidic compound from such leaves. The goal of our present study was to isolate and purify at least 1 natural peptidic cue originating from deteriorating leaves by means of ultrafiltration, gel filtration and reversed phase HPLC and subsequently obtain characteristic data of this cue. The ultrafiltrate (110 kD) of the homogenate of decaying mangrove leaves was sub- jected to gel filtration on a Sephadex G 25 column, resulting in 3 fractions which were tested for their capacity to induce metamorphosis of planula larvae in bioassays performed in the laboratory. Fraction I (25 kD) was most effective in inducing metamorphosis of 75 % of planulae at 1 mg freeze-dried mater- ial ml-' seawater within 24 h. Fractions I1 and I11 (both 25 kD) resulted In metamorphosis of only 1 % of larvae or less within 72 h when applied at 5 mg rnl-' Isochratic HPLC separation of Fraction I with 24 % methanol yielded 2 biologically active fractions. One fraction (A/B), which induced 41 % of the larvae to metamorphose at 0.9 mg lyophilized material ml-' seawater within 24 h, consisted of a mixture of at least 2 subfractions and was not further analyzed. The other fraction (C) effected metamorphosis of 85 % of larvae at a concentration of 0.5 mg rnl-' within 24 h. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry of this fraction revealed a molecular weight of approximately 5.8 kD. Automated amino acid analysis showed that Fraction C was rich in proline (ca 44%) and glycine residues (ca 16%), corresponding to characteristic proline-rich cell wall proteins of plants. Automated sequencing of the natural inducer failed due to a blocked amino terminus. The results of our present study suggest that metamorphic inducers for C. xamachana may emerge nonspecifically as a byproduct of bacterial degradation of deteriorating, proteinaceous plant tissue in their habitat.
KEY WORDS: Natural metamorphic inducer - Cnidaria . Peptides . Settlement . Scyphozoa . Mangrove leaves - Proline-rich proteins
INTRODUCTION tion about the small steps involved in a complex net- work of signal transmissions in larval cells (for review Inducers of marine invertebrate larvae metamorpho- see Pawlik 1992, more recent papers by e.g. Pechenik sis have become a useful tool for studying possible sig- et al. 1995, Wendt & Woolacott 1995, Hassel et al. 1996, nal transduction mechanisms underlying initiation of Henning et al. 1996, Walther et al. 1996, Woolacott & larval settlement and recruitment. Most researchers in Hadfield 1996, McCauley 1997, Thomas et al. 1997, this field have used artificial triggers to obtain informa- Berking 1998, Carpizo-Ituarte & Hadfield 1998, Pechenik & Qian 1998, Froggett & Leise 1999). During the past decade a number of studies have focussed on
0 Inter-Research 1999 Resale of full article not permitted 116 Mar Ecol Prog Ser 183 115-124, 1999
the identification of natural metamorphic cues in an Research into cnidarians has shown a variety of set- attempt to test whether models established in the labo- tlement inducers. As reported above diterpenoid chro- ratory may work in nature, as well as to explain distri- manols induce planula larvae of the hydroid Coryne bution of individuals in certain specific habitats (e.g. uchidai to settle on brown algae of the family Sar- Hadfield & Pennington 1990, Morse & Morse 1991, gassaceae (Nishihira 1968, Kato et al. 1975). A lipid Tamburri at al. 1992, Leitz & Wagner 1993, Rittschof originating from the marine bacterium Alterornonas 1993, Gibson 1995, Forward et al. 1996, Lambert et al. espejiana was shown to trigger metamorphosis of Hy- 1997, Walters et al. 1997, Wieczorek & Todd 1997, dractinia echinata (Hydrozoa; Leitz & Wagner 1993). Avila 1998). However, progress has been slow, and to This bacterium covers mollusc shells inhabited by the date most cues originating from substrata in the envi- crab Eupargurus sp. that are occupied by colonies of ronment have only been partially characterized (e.g. Hydractinia echinata (Miiller 1969, Leitz & Wagner Morse & Morse 1991, Pawlik 1992, Leitz & Wagner 1993). Larvae of another cindarian, the scleractinian 1993, Zimmer-Faust & Tamburri 1994, Lambert et al. coral Agaricia humilis Anthozoa, metamorphose upon 1997). contact with crustose coralline red algae (Morse et al. At present, the exact chemical structure of only a few 1988). The chemical nature of this natural cue was naturally occuring metamorphic inducers of marine shown to be a complex carbohydrate associated with invertebrate larvae has been determined. Urochor- the cell walls of Hydrolithon boergesenli or its associ- damine A, a compound found in a lipophilic extract of ated microflora (Morse & Morse 1991). the tunic of the tunicate Ciona savignyi, induced meta- Synthetic oligopeptides were the first artificial com- morphosis of its larvae in laboratory experiments pounds found to trigger metamorphosis of the scypho- (Tsukamoto et al. 1993). Diterpenoid chromanols ex- zoan Cassiopea spp. (Fitt & Hofmann 1985). Laboratory tracted from brown algae of the family Sargassaceae experiments showing that bacterial cleavage of the were shown to effect metamorphosis of the hydroid proteins collagen and casein resulted in biologically Coryne uchidai (Kato et al. 1975). Bonellin, an alky- active fragments provided the basis for the hypothesis lated chlorin isolated from the female proboscis of the that in nature marine bacteria may be involved in the echiurid Bonellia viridis, induced masculinisation of production of metamorphic inducers by degradation of larvae of the same species (Pelter et al. 1978, Jaccarini biogenic substrata like some species of marine algae et al. 1983). Larvae of the bivalve Pecten maxlmus (Hofmann et al. 1978, Neumann 1979, Hofmann & were reported to metamorphose in response to Jacara- Brand 1987). Very recent findings support ths hypoth- none, a compound isolated from the red alga Delesse- esis. Larvae of C. xamachana were found to settle on ria sanguinea (Yvin et al. 1985). However, some of the black, decomposing leaves of the red mangrove Rhi- findings reported above are not absolutely conclusive zophora mangle in the environment but not on newly and the role of these substances as natural inducers fallen green or red leaves (Fitt 1991, Fitt & Costley remains questionable (see also the critical review by 1998, Fleck & Fitt 1999). One natural cue originating Pawlik 1992). from these leaves was crudely characterized as at least A variety of free fatty acids originating from the sand 1 water-soluble peptide smaller than 12 kD (Fleck & of the tubes of the polychaete Phragmatopoma lapi- Fitt 1999). Marine bacteria were found to be involved dosa lapidosa (western Atlantic) and Phragmatopoma in the production of this inducer (Fleck & Fitt 1999). lapidosa californica were shown to trigger settlement In the present study we show that a peptide, most of their larvae (Pawlik 1986, 1988. Pawlik & Faulkner likely derived from the cell walls of decomposing parts 1986). Contradictory results were presented by other of plants, can serve as a natural metamorphic cue for researchers (Jensen & Morse 1984, 1990, Jensen et al. cnidarian larvae. We provide the molecular weight and 1990).These examples show the difficulties underlying the amino acid composition of one such compound the isolation and characterization of natural metamor- originating from degrading mangrove leaves of Rhi- phic inducers once the natural source of the cues has zophora mangle that induces larvae of Cassiopea xam- been elucidated: they have to be accessible for isola- achana to settle and metamorphose. tion, they have to be purified from a huge variety of sometimes chemically overlapping substances, and they have to be subjected to suitable and unambiguous MATERIAL AND METHODS methods of characterization with only small amounts of the natural cue often available. Finally it has to be Cassiopea xarnachana. Eggs from brooding female demonstrated that the identified and fully character- medusae of Cassiopea xamachana were collected ized inducer is naturally released, i.e. the results directly in a shallow lagoon in Grassy Key, Florida obtained in the laboratory can be transferred into the Keys, Florida, USA. The eggs were transferred to glass field. dishes in the laboratory containing seawater with anti- Fleck et a.1.:Tropical jellyfish metamorphosis 117
biotic~(ABS, 100 mg penicillin, 100 n~gneomycin and detection was carried out with a 15 pt S-G filter at a 130 mg streptomycin dissolved in 1 1 of artificial sea- laser energy of 16.57 pJ and a vacuum of 1.1 X 10-5 torr. water [Instant Ocean], salinity 35 ppt). All larvae A mass filter of 200 D was used. The voltage of the hatched within 3 d and were transferred to a clean dish detector was -4.8 kV. Polarity was positive. with fresh ABS afterwards. Amino acid analysis. Aliqouts of 0.5 pg of the most Preparation of leaf homogenate. A total of 210 sub- bioactive HPLC fraction were subjected to acid hydrol- merged, dark and degrading leaves of Rhizophora ysis with 6 N HCl. Automated amino acid analysis was mangle were collected in a lagoon inhabited by Cas- then carried out with equipment from Applied Biosys- siopea xamachana in Grassy Key. The leaves were tems: 420 A Derivatizei-, 130 A Separation System and wiped of debris. Fractions of 30 leaves each were cut AB1 920 A Data Analysis Module. The Procise Protein into small pieces with a razor blade on ice and trans- Sequencing System was used to obtain sequence data ferred to 80 m1 ice-cold distilled water. The fragments of the sample. were then homogenized with a Polytron (Brinkmann Biological assays. Each purification step was fol- Instruments) on ice. The homogenate was centrifuged lowed by a test for metamorphic induction. The lyo- for 5 min at 24 000 x g at 4OC. Supernatant and pellet philized fractions resulting from ultrafiltration and the were separated. G 25 gel filtration pools were tested in concentrations Ultrafiltration. Ultrafiltration of the supernatant of ranging from 250 pg to 5 mg freeze-dried material ml-' each leaf homogenate containing 30 leaves was car- ABS in 24 well tissue culture plates. Freeze-dried ried out on ice through Amicon YM 10 (retention limit HPLC fractions were assayed in 100 p1 aliquots of stock 10 kD) membranes in an Amicon pressure filtration cell solutions with concentrations of 0.5 mg ml-' ABS or at a pressure of 450 kPa. The filtrate containing com- 0.9 mg ml-' ABS in microtiter plates. A defined number pounds 510 kD and the sediment remaining on the fil- of larvae of Cassiopea xamachana ranging from at ter membrane (substances 210 kD) were lyophilized. least 10 to maximally 15 was used per concentration. Gel filtration. Aliquots of dried ultrafiltrate were re- The number of planulae settled and n~etan~orphosed dissolved in an appropriate volunle of distilled water to was screened after 24, 48, and 72 h. At least 6 repli- obtain a concentration of 13 mg ml-l (a typical sample cates were performed per concentration. As a control, size was 130 mg lyophilized ultrafiltrate in 10 m1 distilled larvae were exposed in 1 m1 of ABS (at least 12 repli- water) and subjected to gel filtration on a Sephadex G 25 cates). The assays were performed at a constant tem- column (Pharmacia, exclusion limit 5 kD, size 2.5 X perature of 25°C at a 12 h light:12 h dark cycle. 49 cm). The column was calibrated with Blue Dextran Additional control experiment. Twenty-five red, and standardized with Vitamin B12 (1.3 kD), Aprotinin recently fallen leaves of Rhizophora mangle were (6.5 kD), and Cytochrom C (12.4 kD). Equipment used homogenized in 70 m1 ice-cold distilled water and sub- was from LKB: 2132 Microperpex Peristaltic Pump jected to the same purification procedure as described (flow rate 1.3 m1 min-l), 2238 Uvicord S I1 spectro- above for degrading mangrove leaves (homogeniza- photometer, 21 11 Multirac fraction collector, and a 2210 tion, ultrafiltration, gel filtration and HPLC). Again 2 Channel-Recorder. Samples were eluted with distilled each purification step was accompanied by a biological water and collected in aliquots of 4.3 ml. The eluate was assay as described above to check on the capacity of pooled into 3 fractions and lyophilized. the obtained fractions to induce metamorphosis of HPLC. Samples of 0.5 mg of the dried gel filtration planula larvae of Cassiopea xamachana. Various con- fractions were re-dissolved in 0.5 m1 distilled water centrations were tested, each in at least 2 replicates. and separated by HPLC on a Sephasil C 18 reversed Statistics. Metamorphosis data were subjected to phase column (Pharmacia, size 4.6 X 100 mm). The statistical analysis using l-way ANOVA followed by sample was eluted with 24 % methanol at a flow rate of the Bonferroni test to establish the significance. Data 0.25 m1 min-'. A LKB 2152 HPLC Controller and 2 LKB were considered significant at p < 0.05. 2150 HPLC pumps were used in addition to the equip- ment mentioned above. Fractions resulting from HPLC separation were pooled and freeze-dried. RESULTS Mass spectrometry. The molecular weight of the fraction that contained the highest biological activity Leaf fractions ranging from 5 to 10 kD induce after HPLC purification was determined using matrix- metamorphosis assisted laser desorption ionization (MALDI) mass spectrometry (LDI-1700 Mass Monitor, Linear Scien- Ultrafiltration of the supernatant of black leaf homo- tific). The sample was dissolved in 50 1-11HPLC quality genate resulted in 2 crude fractions: one containing water. For mass spectrometry 1 p1 of the sample was molecules 5 10 kD (filtrate, total amount 4.22 g) and the added to 10 p1 of the matrix (sinapinic acid). Peak other one containing the substances 2 10 kD (sediment 118 Mar Ecol Prog Ser 183: 115-124, 1999
develop tentacles thus indicating that-as a consequence of the homogenization proce- dure-the ultrafiltrate contained not only metamorphic inducers but also compounds that had a toxic effect on the larvae. This was in accordance with observations made in bioassays with synthetic peptides and phar- maceuticals that did not have any metamor- phic effect on the planulae but showed such strong toxic impact in higher concentrations that the larvae were deformed and only meta- morphosed in low numbers even though highly potent peptide inducers were simulta- neously present in the test solutions that nor- mally would have induced 100% settlement and metamorphosis within a few hours (Fleck 45 h 72 h unpubl.). The lowest concentration tested Fig. l. Induction of metamorphosis of planula larvae of Cassiopea xam- (250 pg ml-') effected metamorphos~s at a achana by fractions 510 and 210 k~ resulting from ultrafiltration of the non-significant rate of 8 + 13 %. The sediment supernatant of homogenate of degrading Rhizophora mangle leaves. remaining on the filter triggered metamor- Columns show means *SD of 12 replicates. NO column = 0% metamor- phosis up to 9 + 10 % of larvae at 5 mg rnl-1 phosis. *Significantly different (p < 0.05) from antibiotics (ABS) control after 72 h but this rate was not significantly different from the ABS control (Fig. 1). on filter, total amount 0.87 g). Biological tests revealed Application of the filtrate to a Sephadex G 25 column that the filtrate induced metamorphosis of up to 24 + resulted in a typical profile shown in Fig. 2. The eluate 23 % of larvae of Cassiopea xamachana within 72 h was divided into 3 fractions. Fraction I contained com- (Fig. 1). However, the concentration used was high pounds 25 kD (Fig. 2).The pool with substances 15 kD (5 mg ml-') and the rate of metamorphosis turned out to was divided into 2 subfractions: Fraction I1 and Frac- be quite variable (Fig. 1). Likewise, settled and meta- tion 111 (Fig. 2). Fraction 1, which was eluted from the morphosed planulae were often deformed and did not column at a total amount of 49.6 mg, proved to be a
Flg. 2. Representative chromato- gram of the separation of ultrafil- trate (510 kD) of the supernatant of homogenized, degrading mangrove leaves of Rhizophora mangle on a 0 10 2 0 3 0 4 0 5 0 6 0 7 0 8 0 Sephadex G 25 column. Fraction I contains compounds 25 kD, Frac- Fraction tions I1 and 111 55 kD Fleck et al.:Tropical lellyflsh metamol-phosis 119
was not completely separated from Peak B (Fig. 4). Elution of the column with 100% methanol following isochratic elution with 24 % methanol produced an additional 15 peaks but none of the corresponding fractions proved to contain meta- morphic inducers. The total amount of the freeze-dried powder of the fractions represented by Peak NB and Peak C was determined with an analytical balance and found to be 13.6 mg for Fraction NBand 5.1 mg for Fraction C. In order to perform the bioassays parts of both fractions were resuspended in ABS to obtain stock solutions with concentrations of 0.9 mg lyophilisate ml-' ABS (Fraction A/B) or 0.5 mg lyophilisate ml-' ABS (Fraction C). Freeze- dried powder of Fraction C induced 85 +- 17% metamorphosis when applied at 0.5 mg ml-'
within 24 h, and lyophilisate- of Fraction A/B 47 * U - 24 h 48 h 72 h 24 % (at 0.9 mg ml-'; Fig. 5). This rate increased to 89 + 13 % for Fraction C and to 68 * 28 % for Frac- Fig. 3. Induction of metamorphosis of planula larvae of Cassiopea tion NB after 72 h (Fig. 5). Other concentrations xamachana by Fraction I (25 kD) and Fraction 111 ( HPLC separation of the metamorphosis inducing fraction 0 2 4 6 8 I0 12 14 16 IS 20 Fraction I was applied to reversed phase HPLC for Time [mini further purification. The best resolution of the peaks that contained metamorphosis inducing capacity was Fig. 4. Representative HPLC chrornatogram of the separation obtained with isochratic elution of the column with of Fraction I (25 kD) obtained by gel filtration of the ultrafil- 24 % methanol. ~~~di~~telution of the column with 0 trate (S10 kD) of the supernatant of homogenized, degrading leaves of Rhizophora mangle on a reversed phase column to '0°% provided poorer of the (SephasjJ C 18, constant elution with 24% Two bioactive peaks. Three peaks resulted from constant fractions emerged: Fraction A/B, consisting of at least 2 sub- elution with 24 % methanol: A, B and C (Fig. 4).Peak A fractions, and Fraction C 120 Mar Ecol Prog Ser 183: 115-124, 1999 Characterization of Peak C MALDI mass spectrornetry of Peak C, representing the most potent natural inducer purified from degrad- ing mangrove leaves, revealed a molecular weight of exactly 5783.1 D (Fig. 6). Amino acid analysis showed that Peak C contained a peptide that was rich in proline. This peptide mainly consisted of 26.2 proline residues, followed by 9.7 glycine residues and 5.8 glutamic acid residues (Fig. 7). Chemical sequence analysis, starting with the first amino terminal amino acid, failed in 6 indepen- dent samples of the natural cue indicating that the amino end of the peptide was blocked. Fraction A/B which consisted of a mixture of at least 2 compounds was not further analyzed but our previ- ous work (Fleck & Fitt 1999) makes it very likely that this fraction likewise contained bioactive peptides. Fig. 5. Induction of metamorphosis by Fraction A/B and Frac- Non-degraded leaves do not contain biologically tion C resulting from HPLC separation of gel filtration Frac- active compounds tion I (see Fig. 4). Columns show means i SD of 6 replicates. No metamorphosis was induced by the ABS control (n = 12). *Significantly different (p < 0.05) from ABS control None of the fractions prepared from red, more recently fallen mangrove leaves induced metamorpho- sis of planulae at a significant rate. Though the HPLC Fig. 6. Mass spectru.m of HPLC Fraction C. Sum of 16 shots. For details see text Fleck et a1 Tropical jellyfish metamorphosis 121 ture (Fitt & Hofmann 1985, Fitt et al. 1987, Hofmann & Brand 1987, Fleck & Hofmann 1990, 1995, Bischoff et al. 1991, Fleck & Bischoff 1993, Hofmann et al. 1996, Fleck 1997, Fleck 1998, Walther & Fleck 1998). Like the char- acterized natural cue originating from mangrove leaves, the most effective syn- thetic peptidic inducers were mainly composed of proline and glycine resi- dues (Fitt & Hofmann 1985, Hofmann & Brand 1987, Fleck & Hofmann 1990, Fleck & Bischoff 1993, Hofmann et al. 1996, Walther & Fleck 1998). We sus- pect that at least one other partially characterized inducer (Fraction A/B) in our study is also a peptide. Peptides have also been found to 0 5 10 15 20 25 30 naturally trigger metamorphosis in numberof amjno acid residues larvae of the sand dollar Dendraster excentricus (Burke 1984), the oyster Fig. 7. Amino acid composition of one natural metamorphic cue (molecular Crassostrea virginica (Tamburri et al. weight 5.8 kD)of Cassiopea xamachana originating from degrading mangrove 1992), the barnacle Balanus amphi- leaves of Rhizophora mangle. The anaIyzed inducer correspondsto Fraction C of the HPLC chromatogram (see Fig. 4) trite (Tegtmeyer & Rittschof 1989), and the nudibranch Adalaria proxinla (Lambert et al. 1997). None of these peak pattern of a sample of the gel filtration fraction studies, however, provides the exact molecular weight, 25 kD of red leaf ultrafiltrate (510 kD) was similar to the amino acid composition and/or sequence data of that of degrading leaves (Fig.4) when eluted with 24 % the natural cue. Chemical sequencing of the purified methanol no significant metamorphic induction of natural inducer of Cassiopea xamachana has thus far planula larvae of Cassiopea xamachana was obtained, failed with the methods applied because the amino ter- Concentrations tested corresponded to those of Frac- minus was blocked by a yet unidentified group. tion A/B and Fraction C of the decomposing leaves What is the source of the peptidic cue derived from (Fig. 5). Red mangrove leaves are rarely settled by the degrading mangrove leaves? A recent study by polyps in the habitat and effected settlement and Fleck & Fitt (1999) suggests that bacteria are involved metamorphosis at non-significant rates when tested in in the production of natural inducers. Bacteria have the laboratory (Fitt & Costley 1998, Fleck & Fitt 1999). been shown to take part in metamorphic induction of several marine invertebrates (see review by Johnson et al. 1997). Based on laboratory experiments dealing DISCUSSION with bacterial cleavage of collagen, Hofmann & Brand (1987) proposed that bacteria may provide potential Our study demonstrates that peptides represent at inducers for metamorphosis of Cassiopea spp. by least 1 of the natural metamorphic inducers for the up- cleaving deteriorating, proteinaceous cues in the side-down jellyfish Cassiopea xamachana found in de- ocean. Our study strongly indicates that degrading grading leaves of the red mangrove. In the present study mangrove leaves may represent such suitable cues. we have isolated and characterized at least 1 peptidic Proline-rich proteins (PRPs) consisting of amino acid cue from black, degraded mangrove leaves of Rhi- compositions corresponding to that of the natural zophora mangle that provide 1 of the substrates settled metamorphic inducer of C. xamachana isolated from by larvae in the mangove environment (Fitt 1991, Fitt & Rhizophora mangle are common elements of plant Costley 1998, Fleck & Fitt 1999).This natural inducer has cell walls (e.g. Ye et al. 1991, Nicholas et al. 1993, a molecular weight of approximately 5.8 kD, contains Kieliszewski & Lamport 1994, Rodriguez & Cardemil about 59 amino acids and is dominated by proline and 1994, Williamson 1994, Yu et al. 1996, Wojtaszek et al. glycine residues (ca 61 % in total). We have therefore 1997, Davies et al. 1997). Bacterial degradation of such confirmed that laboratory studies using peptides as proteins of decomposing plant tlssue may therefore metamorphic triggers, indeed mimick what occurs in na- result in a variety of peptides, some of them containing 122 Mar Ecol Prog Ser 183: 115-124, 1999 a specific signal sequence enabling them to trlgger not multitude of peptidic inducers originating from man- only metamorphosis of Cassiopea spp, but also of other grove leaves may act together thereby inducing meta- marine invertebrate larvae. Hurnic acids, representing morphosis of planulae at lower and environmentally another complex mixture of compounds of degrading more relevant concentrations of each single peptide vegetation, accelerated metamorphosis of postlarvae compared to the relatively high concentration necessary of the blue crab Callinectes sapidus to the first crab for 1 single inducer, e.g. Fraction C resulting from HPLC stage (Forward et al. 1997). in the present study, which likewise may be subject to The size of the natural peptidic cues (5 to 10 kD) is further bacterial degradation in nature. Laboratory ex- much larger than the size of the synthetic peptides periments carried out with optimal concentrations (100 % used in the laboratory for metamorphosis studies in metamorphosis within 24 h) of single synthetic peptide Cassiopea spp. ( molecule (Fleck 1994). Thus we conclude that natural Ruhr-Universitat Bochum, Bochum (in German with Eng- metamorphic cues for Cassiopea spp. may emerge lish summary) from bacterial degradation of proteinaceous tissue of Fleck J (1997) Phosphatidylinositol (PI) signaling and subse- quent events in metamorphosis induction of cnidarian lar- plants assuring recruitment into nutrient-rich habitats vae. Proc 8th Int Coral Reef Symp 2:1225-1229 where juveniles are likely to grow and strobilate into Fleck J (1998) Chemical fate of a metamorphic inducer in lar- reproductively active adults. vae-like buds of the cnidarian Cassiopea andromeda. Biol Bull 194:83-91 Fleck J, Bischoff A (1993) Proteln kinase C is possibly Acknowledgements. We thank Steve Miller, University of involved in chemical induction of metamorphosis in Cas- North Carolina at Wilmington, and the National Undersea sloped spp. (Cnidaria: Scyphozoa). Proc 7th Int Coral Reef Research Center on Key Largo, Flonda, for providing lab Symp 1:456-462 space and nitrogen for the ultraf~ltration. The introduction to Fleck J, Fitt WK (1999) Degrading mangrove leaves of Rhi- mass spectrometry by Ron Orlando and his group from the zophora mangle provide a natural metamorphic cue for Complex Carbohydrate Research Center, University of Geor- the upside down jellyfish Cassiopea xamachana. J Exp gia, Athens, Georgia, is gratefully acknowledged. Thanks are Mar Biol Ecol234:83-94 also due to Barbara Potempa, now at the University of Fleck J, Hofmann DK (1990) The efficiency of metamorphosis Krakow, Poland, for helping us to determine the amino acid inducing oligopeptides in Cassiopea species (Cnidaria: composition and the sequence of the isolated cue and to D. K. Scyphozoa) depends on both primary structure and ~ofmannfor his continuing advice and support. We are grate- amino- and carboxy terminal substituents. Verh Dtsch ful for the useful comments of 4 anonymous reviewers. A Zool Ges 83:452-453 Feodor-Lynen-fellowship from the ~lexandervon Humboldt- Fleck J, Hofmann DK (1995) In vivo binding of a biologically Stiftung, Bonn, Germany, to J.F. provided the basis for this active oligopeptide in vegetative buds of the scyphozoan cooperative research. Contnbutlon No. 011 from the Key Cassiopea andromeda: den~onstration of receptor-medi- Largo Marine Research Laboratory ated induction of metamorphosis. Mar Biol 122:447-451 Forward RB Jr, DeVries MC, R~ttschof D, Frankel DAZ, Bischoff JP, Fisher CM, Welch JM (1996) Effects of envi- LITERATURE CITED ronmental cues on n~etamorphosis of the blue crab Call- inectes sapidus. Mar Ecol Prog Ser 131:165-177 Avila C (1998) Competence and metamorphosis in the long- Forward RB Jr, Tankersley RA, Blonde1 D, Rittschof D (1997) term planktotrophic larvae of the nudibranch mollusc Metamorphosis of the blue crab Callinectes sapidus: Hermissenda crassicornis (Eschscholtz, 1831). J Exp Mar effects of humic acids and ammonium. Mar Ecol Prog Ser Biol Ecol 231:81-117 157:277-286 Berking S (1998) Hydrozoa metamorphosis and pattern for- Froggett SJ, Leise EM (1999) Metamorphosis in the marine mation. Curr Top Dev Biol 38:81-131 snail Ilyanassa obsoleta, yes or NO? Biol Bull 196:57-62 Bischoff A, Fleck J, Hofmann DK (1991) Phorbol esters induce Glbson G (1995) Why be choosy? Temporal changes in larval nletamorphosis in Cassiopea androrneda and Cassjopea sensitivity to several naturally-occuring metamorphic xamachana (Cnidaria, Scyphozoa). Verh Dtsch Zool Ges inducers in the opisthobranch Haminaea callidegenita. 84:484 J Exp Mar Biol Ecol 194:9-24 Burke R (1984) Pheromonal control of metamorphosis in the Hadfield MG, Pennington J'T (1990) Nature of the metamor- Paciflc sand dollar, Dendraster excentncus. Science 225: phic signal and its internal transductlon In larvae of the 442-443 nudibranch Phestilla sibogae. Bull Mar Sci 46:455-464 Carpizo-Ituarte E, Hadfield MG (1998) Stimulation of meta- Hassel M, Leitz T, Miiller WA (1996) Signals and signal-trans- morphosis in the polychaete Hydroides elegans Haswell duction systems in the control of development in Hydra (Serpulidae). Biol Bull 194:14-24 and Hydractinia. Int J Dev Biol40:323-330 Cuba TR, Blake NJ (1983) The initial development of marine Henning G, Hofmann DK, Benayahu Y (1996) The phorbol fouling assemblage on a natural substrate in a subtropical ester TPA induces metamorphosis in Red Sea coral planu- estuary. Bot Mar 26:259-264 lae (Cnidaria: Anthozoa). Experientia 52:744-749 Davies HA, Findlay K, Daniels MJ, Dow JM (1997) A novel pro- Hofmann DK, Brand U (1987) Induction of metamorphosis in line-rich glycoprotein associated with the extracellular ma- the symbiotic scyphozoan Cassiopea andromeda: role of trix of vascular bundles of Brassica petioles. Planta 202:28-35 marine bactena and of biochemicals. Symbiosis 4:99-116 F~ttWK (1991) Natural metamorphic cues of larvae of a tropi- Hofmann DK, Neumann R, Henne K (1978) Strobilation, bud- cal jellyfish. Am Zool 31:106 dlng and initiation of scyphistoma morphogenesis in the F~ttWK, Costley K (1998) The role of temperature in survival rhizostome Cassiopea andronleda (Cnidaria: Scyphozoa). of the polyp stage of the trop~calrhlzostome jellyfish Cas- Mar Biol47.161-176 siopea xamachana. J Exp Mar B101 Ecol222:79-91 Hofmann DK, Fitt WK, Fleck J (1996) Checkpoints in the life- Fltt WK. Hofmann DK (1985) Chemical induction of settlement cycle of Cassiopea andromeda: control of metagenesls and and metamorphosis of the reef-dwelling coelenterate Cas- metamorphosis in a tropical jellyfish. Int J Dev Biol 40: siopea andromeda. Proc 5th Int Coral Reef Symp 2:239-244 331-338 Fitt WK, Hofmann DK, Wolk M, Rahat M (1987) Requirement Jaccarini V, Agius L, Schembri PJ, Rizzo M (1983) Sex deter- of exogenous inducers for metamorphosis of axenic larvae mination and larval sexual interaction in Bonellia viridis and buds of Cassiopea andromeda (Cnidaria: Scyphozoa). Rolando (Echiura: Bonelliidae). J Exp Mar Biol Ecol 66: Mar Biol94:415-422 25-40 Fleck J (1994) Efficiency of modified inducing peptides, pos- Jensen RA, Morse DE (1984) lntraspecific facilitation of larval sible signal transduction mechanism and chemical fate of recruitment: gregarious settlement of the polychaete a biologically active peptide in induction of metamorpho- Phragmatopoma californica (Fewkes). J Exp Mar Biol Ecol sis in Cassiopea spp. (Cnidana, Scyphozoa). PhD thesis, 83:107-126 124 Mar Ecol Prog Ser I Jensen RA. Morse DE (1990) Chemically induced metamor- sibogde Rergh become responsive to three chemical cues phosis of polychaete larvae in both the laboratory and at the same age. J Exp hlar Biol Ecol 191:l-17 ocean environment. J Chem Ecol 16:911-930 Pelter A. Ballantine JX, Slurray-Rust P, Ferrito V, Psaila AF Jensen RA, Morse DE, Petty RL, Hovker N (1990) Artificial (1978) The structures of anhydrobonellin and bonellin, the induction of larval metamorphosis by free fatty acids. Mar physioloqically active pigment from the marine echiuroid Ecol Prog Ser 6755-71 Bonellia riridis. Tetrahedron Lett 21 1881- 1884 Johnson CR, Lewis DE, Nichols DS, Degnan BM (1997) Bacte- Rittschof D (1993) Body odors and neutral-basic peptides rial induction of settlement and metamorphosis in marine mlmics: a review of responses of marine organisms. Am invertebrates. Proc 8th Int Coral Reef Symp 2:1219-1224 2001 33:487-493 Kato T, Kumanireng AA, Ichinose I,Kitahara Y. Kakinuma Y. Rodriguez JG, Cardemil L (1994) Cell wall protei.ns in seed- Nishihira bl, Kato M (1975) Active components of Sargas- ling cotyledons of Prosopls chilensls. Phytochemistry 35: sum tortile effecting the settlement of swimming larvae of 281-286 Coryne uchidai. Experientia 31:433-434 Tamburri blN, Zimmer-Faust RK, Tamplin ML (1992) Natural Kieliszewski MJ, Lamport DT (1994) Extensin: repetitive sources and properties of chemical inducers mediating motifs, functional sites, post-translational codes, and phy- settlement of oyster larvae: a re-examination. Biol Bull logenv. Plant J 5157-172 183:327-338 Lambert WJ, Todd CD, Hardege JD (1997) Partial characteri- Tegtmeyer K. Rittschof D (1989) Synthetic peptide analogs to zation and biological activ~tyof a metamorphic inducer of barnacle settlement pheromone. Peptides 9:1403-1406 the dorid nud.ibranch Adalaria proxirna (Gastropoda. Thomas MB, Edwards NC, BaIl BE, McCauley DW (1997) Nudibranchia). Invertebr Biol 116.71-81 Comparison, of metamorphic induction in, hydroids. Inver- Leitz T, Wagner T (1993) The marine bacterium Alterornonas tebr Biol 116:277-285 espejiana induces metamorphosis of the hydroid. Hydrac- Tsukamoto S, Hirota H, Kato H, Fusetani N (1993) Urochor- tinia echinata. Mar Biol 115:173-178 damines A and B: larval settlemenVmetamorphosis-promot- McCauley DW (1997) Serotonin plays an early role in the ing, pteridine-containing physostigmine alkaloids from the metamorphosis of the hydrozoan Phialidium gregarium. tunicate Ciona savignyi. Tetrahedron Lett 34:4819-4822 Dev Biol 190:229-240 Vodenichar JS (1995) Ecological physiology of the scypho- Morse DE, Morse ANC: (1991) Enzymatic characterization of zoan Cassiopea xamachana. MS thesis, University of the morphogen recognized by Agaricia humilis (sclerac- Georgia, Athens tinian coral ) larvae. Biol Bull 181:104-122 Walters LJ, Hadfield MG, del Carmen KA (1997) The impor- Morse DE, Hooker N, Morse ANC, Jensen RA (1988) Control tance of larval choice and hydrodynamics in creating of larval metamorphos~s and recruitment in sympatr~c aggregations of Hydroides elegans (Polychaeta: Serpull- agariciid corals. J Exp Mar Biol Ecol 116:193-217 dae). Invertebr Biol 116.102-114 Muller WA (1969) Auslosung der Metamorphose durch Bak- Walther M, Fleck J (1998)Synthetic peptides inducing metamor- terien bei den Larven von Hydractinid echinata. Zoo1 Jb phosis in a tropical jellyfish. a quantitative structure-act~vity (Abt Anat Ontog Tiere) 86~84-95 relationship study. Comp Riochem Physiol A 120-655-659 Neumann R (1979) Bacterial induction of settlement and Walther M, Ulrich R,Kroiher M, Berking S (1996) Metamor- metamorphosis in the planula larvae of Cassiopea andro- phosis and pattern formation in Hydractinia echinata, a meda (Cnidaria: Scyphozoa, Rhizostomeae). Mar Ecol colonial hydroid. Int J Dev Biol40:313-322 Prog Ser 1:21-28 Wendt DE, Woollacott RM (1995) Induction of larval settle- Nicholas CD, Lindstrom JT, Vodkin L0 (1993) Variation of ment by KCL in three species of Bugula (Bryozoa). Inver- proline rich cell wall proteins in soybean lines with antho- tebr Biol 114:345-351 cyanin mutations. Plant h?ol Biol 21:145-156 Wieczorek SK, Todd CD (1997) Inhibition and facilitation of Nishihira M (1968) Br~ef experiments on the effect of algal bryozoan and ascidjan settlement by natural multi-species extracts in promoting the settlement of the larvae of biofi.lms: effects of film age and the roles of active and pas- Coryne uchidai Stechow (Hydrozoa). Bull Mar Biol Sta sive larval attachment Mar Biol 128:463-473 Asamushi 13:91-101 Willi.amson MP (1994) The structure and function of proline- Pawlik JR (1986) Chemical induction of larval settlement and rich regions in protelns Blochem J 297:249-260 metamorphosis in the reef-building tube worm Phrag- Wojtaszek P. Trethowan J, Bolwell GP (1997) Reconstitution matopoma californica (Polychaeta: Sabellariidae). Mar in vitro of the components and conditions required for the Biol 9159-68 oxidative cross-linking of extracellular proteins in French Pawlik JR (1988) Larval settlement and metamorphosis of sabel- bean (Phaseolus vulgaris L.). FEBS Lett 405:95-98 lariid polychaetes, with speclal reference to Phragmato- Woollacott RM, Hadfield MG (1996) Induction of metamor- poma lapidosa, a reef-building species, and Sabellana flon- phosis in larvae of a sponge. Invertebr Biol 115:257-262 densis, a non-gregarious species. Bull Mar Sci 43:41-60 Ye ZH, Song YR, Xlarcus A, Varner JE (1991) Comparative Pawlik JR (1992) Chemical ecology of the settlement of ben- 1ocali.zation of three classes of cell wall protelns. Plant J thic marine invertebrates Oceanogr Mar Uiol Annu Rev 1:175-183 30:273-335 Yu LX,Chamberland H, Lafontaine JG, Tabaeizadeh Z (1996) Pawlik JR, Faulkner DJ (1986) Specific free fatty acids induce Negative regulat~onof gene expression of a novel prol~ne, larval settlement and metamorphosis of the reef-building threonine-, and glycine-nch protein by water stress in tube worm Phraymatopoma californica (Fewkes). J Exp Lycopersicon chdense. Genome 39: 1185-1 193 Mar Biol Ecol 102:301-310 Yvin JC, Chevolot L. Chevolot-Magueur AX4, Cochard JC Pechenik JA, Qian PY (1998) Onset and maintenance of meta- (1985) First isolation of jacaranone from an alga, Delesse- morphic competence in the mclrine polychaete Hydroides ria sanguinea. A metamorphosis inducer of Pecten larvae. elegans Haswell in response to three chemical cues. J Exp J Nat Prod 48:814-816 Mar Biol Ecol 226:51-74 Zimmer-Faust RK, Tamburri MN (1994) Chemical identity Prchenik JA, Hddfield XTG, Eyster S (1995) Assessing and ecological implications of a waterborne, larval settle- whether larvae of the opisthohranch gastropod Phestilla ment cue. Limnol Oceanogr 39:1075-1083 Editorial responsibil~ty: Otto Kmne (Editor), Submitted: May 8, 1998, Accepted: February 19, 1999 Oldendorf/Luhe, Germany Proofs received from author(s): June 22, 1999