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Department of Biology, University of California, Los Angeles, California 90095-1606
* To whom correspondence should be addressed. E-mail: z{at}biology.ucla.edu
Abbreviations: ASW, artificial seawater • CRC, chemical-releasing collector • GGH, glycyl-glycyl-L-histidine • GGR, glycyl-glycyl-L-arginine
Settlement rates and distributions of planktonic larvae are critical determinants of population dynamics in marine and freshwater benthic communities. On the basis of the principles of solute diffusion from a porous material, chemical-releasing collectors (CRCs) were engineered and tested in an estuary. Significantly more barnacle larvae (Balanus amphitrite) were found to colonize collectors emitting trace amounts of the synthetic peptide analog, glycyl-glycyl-L-arginine (5 x 10-8 M), than those emitting either seawater or an organic enrichment (glycyl-glycyl-L-histidine) control. The inductive compound is similar in structure to peptide signal molecules that have been shown to elicit settlement under laboratory conditions and are naturally released by adult barnacles and oysters. The potent effects of subtle changes in seawater chemistry may thus warrant careful attention as putative agents mediating habitat colonization.
For a half-century or more, the pervasive perception that small planktonic organisms are passively transported by flow has fostered the notion that active larval behaviors can be important only at the time of settlement and in response only to surface-adsorbed chemical cues (1,2,3,4,5). Circumstances exist, however, where larval responses to dissolved chemical cues in the water column could indeed enhance or determine settlement. The numerical model of Gross et al. (6), for example, indicates that hydrodynamic processes directly control larval encounter rate with the seabed and, indirectly, control settlement rate, assuming there is a high probability of larvae accepting the encountered substratum. That is, if larvae move downward (sinking or swimming) in response to a waterborne cue, their vertical distribution will become bottom-skewed, and thus more larvae can be swept into contact with the seabed by fluid flow. Settlement rate may increase dramatically (7).
Whereas settlement is presumed mediated through substratum-adsorbed compounds, induction by dissolved substances is typically regarded as a novelty demonstrated only under strictly controlled laboratory conditions (but see 8,9,10). Field studies of the effects of waterborne chemical agents on settlement are needed to evaluate the potencies and performances of these molecules. Although considerable efforts have been made to identify the natural cues that stimulate settlement, no waterborne inducer has been isolated and fully characterized (11,12). In the absence of purified natural inducers, synthetic analogs are valuable tools for quantitative field studies.
Recently, experiments involving chemical isolations of seawater indicated that low-molecular-mass peptides with carboxy-terminal arginine or lysine residues were released by adult conspecifics and elicited settlement of barnacle (Balanus amphitrite) and oyster (Crassostrea virginica) larvae in the laboratory (13,14). Synthetic peptides were screened; whereas glycyl-glycyl-L-arginine (GGR) was identified as a particularly potent analog for the natural inducers in these two species, glycyl-glycyl-L-histidine (GGH) was without effect (15,16).
We used the mathematical principles of solute diffusion from a porous material to design a gel for the controlled release of peptides from larval collectors in the field (17,18,19). Acrylamide was chosen over agar for its relative lack of microbial intrusion, its ease of use, and its small pore sizes; this latter point is particularly important with small solutes. Free-radical crosslinking of an 8.0% acrylamide solution in ASW (artificial seawater made from Forty Fathoms Marinemix salts in deionized water [dH2O]; pH = 8.0, salinity = 30
, 0.45-µm filtered) with N,N'-methylene-bis(acrylamide) (bis; 2.4% of the total acrylamide concentration) was initiated with 0.05% ammonium persulfate (APS; w/v, final) and catalyzed with 0.05% tetramethylenediamine (TEMED; v/v, final). These methods resulted in polymerization within 30 min and pore diameters of about 39 Å (20).
 | (1) |
 | (2) |
 | (3) |
is the "association" parameter of H2O (=2.6); and V is the molal volume of the solute at its normal boiling point (370 cm3 g mol-1 for disodium fluorescein).
Through computer simulations, the rates of solute release from acrylamide were determined by applying Eq. 1 to cylindrical gels with lengths ranging from 0.1 to 10 cm for up to 5 days of field exposure. Fluorescein was chosen as a tracer because its molecular mass (and, hence, diffusion coefficient) is close to that of the small peptides used in the settlement assays (330 g mol-1 versus 269 and 288 g mol-1 for GGH and GGR, respectively). The diffusion coefficients for fluorescein in 8.0% acrylamide (Dp) and H2O (D0) were calculated from Eq. 2 and Eq. 3, respectively, to be 2.9 x 10-6 and 4.4 x 10-6 cm2 s-1 at 27 °C. From these simulations, it was clear that at lengths greater than 4–5 cm, gels release very little (
50%) of their included organic compounds in 3–5 d (Figs. 1 and 2). If the gel is too thin (<0.8 cm), however, more than 90% of the solute is lost within 0.5 d.
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6 months) from the local estuary were prepared for use as settlement substrata by scrubbing free all sediment, flora and fauna, bathing for 5 min in 2%–3% NaOCl, rinsing 12 h in continuously running H2O; and autoclaving to sterilize. These shells were found to be superior to preliminary systems containing scoured glass or PVC substrata (data not shown). Monofilament mesh (0.6-cm pore size) was secured over the shells with cable ties tightened around the cylinder portion of each CRC.
The field experiments were conducted in the North Inlet Estuary (Town Creek; 33° 19.5' N, 79° 11.5' W) near Georgetown, South Carolina. This tidal creek is shallow and rapidly flushed, with flow speeds up to 35 cm s-1 (and shear velocities to 3.8 cm s-1) about 2200 m from the mouth of the inlet. The creek bank falls steeply, 2–3 m, to a mud bottom. Deployments occurred during August, when the mean (±SEM) water temperature and salinity were 28.3 ± 0.2 °C and 30.3 ± 0.6, respectively (P. Kenny and D. Allen, unpubl. data). The CRCs were held above the creek bed, at least 20 cm below mean low tide level, by harnesses constructed of 0.6-cm-diam. nylon rope and brass fittings attached to wooden posts embedded into the sediment.
Concentrations of diffused solute from acrylamide gel were measured using disodium fluorescein and GGR as chemical tracers in the field. The experiment employing fluorescein was performed to elucidate the effects of tracer release rate. GGR determinations were subsequently made for seawater sampled during tests of larval settlement. Three replicate 1-ml aliquots of seawater were removed via sterile syringes from 0.5, 1.1, and 3.5 cm above the gels. The samples were filtered on site with 0.45-µm syringe filter cartridges, stored in sterile vials, and placed on ice in the dark until analysis was performed (GGR samples were maintained at -80 °C upon return to the laboratory). The sampling was repeated for 12 or 4 replicate collectors at 12-h and 24-h intervals in the fluorescein and GGR experiments, respectively, for a total of 72 h. Fluorescein concentrations were determined from fluorescent output (Turner Designs field fluorometer, model 10-AU-005, excitation wavelengths 455–500 nm, emission wavelengths 510–700 nm) by comparison to a standard concentration curve. GGR was quantified by high-performance liquid chromatography (HPLC) with a Beckman System Gold 126 binary solvent module/507 autosampler and a Jasco FP-920 fluorescence detector. Briefly, the guanidinium group of the arginine on GGR reacted with benzoin under basic conditions to quantitatively form a highly fluorescent derivative (21,22). Separation through a silica-based bonded phenyl column (Waters Corp.) was induced by increasing the concentration of ethyl alcohol in the 75 mM Tris-HCl (pH 8.1) mobile phase from 25% to 85% in a series of gradients over 23 min at 0.8 ml min-1. HPLC peaks (excitation 
= 325 nm, emission = 435 nm) were identified by their retention times relative to previously run standards (guanidinium, arginine, glycyl-L-arginine, and GGR), and concentrations were calculated from the areas under the peaks (23).
Previous laboratory investigations with barnacle and oyster larvae indicated maximum settlement in response to peptides at 10-7 to 10-8 M (15,16). Concentrations greater or less than two orders of magnitude from these target doses substantially diminished the effectiveness of GGR to stimulate settlement. A gel concentration of 1 mg ml-1 fluorescein produced mean levels that were nearly constant through 72-h field deployments. These values were as follows: for shell-hash pore waters: 1.1 (±0.1 SEM) x 10-2 mg ml-1; for settlement (shell) substrata: 2.1 (±0.1 SEM) x 10-4 mg ml-1; and, for the water column at 0.5 cm above the collector: 1.0 (±0.1 SEM) x 10-6 mg ml-1. On the basis of this information, a concentration of 2 x 10-4 M peptide was established for the gels. A 3700-fold dilution led to a mean GGR concentration of 5.4 (±0.5 SEM) x 10-8 M in the settlement substrata during the field trials, in good agreement with our measurements of fluorescein release. The inclusion of the shell-hash layer above the gel resulted in near steady-state concentrations of fluorescein and GGR. Solute concentrations in shell-hash, settlement substrata, and water at 0.5 cm above the collector did not significantly change as a function of time since placing the CRCs in the field (Table 1, and one-way ANOVAs: F 1.23, P 0.37, all comparisons).
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4 m3 filtered seawater) taken with 30-cm-diam., 153-µm-mesh Nitex plankton nets. The results indicated that oyster larvae in the water column were rare (2.1 individuals m-3) during the course of the investigation, so only data for barnacle larvae (mean concentration: 40.4 larvae m-3) are presented here. The effects of ASW, glycyl-glycyl-L-arginine in ASW (GGR; settlement stimulant), and glycyl-glycyl-L-histidine in ASW (GGH; organic enrichment control) were determined by assaying eight CRCs of each chemical treatment. The exact position of each CRC was established according to a randomized block design in which eight sets of three collectors (one collector of each treatment) were placed along a 120-m transect parallel to and 4 m from shore. The CRCs were incubated in the field with the open ends oriented upward for 72 h. At the end of the incubation time, the chambers were removed from the estuary and the number of newly metamorphosed barnacle (Balanus amphitrite) juveniles on the settlement (shell) substrates were counted under a dissecting microscope.
GGR was found to stimulate substrate colonization by significantly more barnacle larvae than did either ASW or GGH (Fig. 3). Because GGR and GGH are basic peptides with nearly identical molecular weights and chemical functionalities, settlement induction cannot be attributed to stimulation by organic compounds in general but is due, specifically, to the arginine moiety at the carboxy terminus of the peptide.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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1/2 versus gel length. The nonlinear curve has been fitted to a third-order expression relating the variation in the residency time (