Chemical identity and ecological implications of a waterborne, larval settlement cue

Planktonic oyster larvae ( Crassostrea virginica ) respond behaviorally to waterborne chemical cues and rapidly settle on substratum. “Settlement” is defined here as attachment of the larval foot to the substrate. Although a topic of considerable research for the past 50 yr, the identity of these cues remains unresolved. We provide four lines of experimental evidence all pointing to the singular identity of these substances. First, molecular‐weight fractionations of seawater used to bathe adult oysters (with intact biofilms) were bioassayed, indicating the presence of waterborne settlement inducers between 500 and 1,000 Da. The inducers were degraded by proteases but not by carbohydrases or by lipase. Of several proteases we applied, only those cleaving basic amino acids (lysine and arginine) from the C‐terminal and arginase (an enzyme condensing arginine at the C‐terminal to ornithine) eliminated settlement‐inducing activity. Second, trypsin hydrolysates of casein were significantly more effective in causing larval settlement than products of either acid or pronase hydrolysis of this protein. Third, a tri‐peptide having arginine at the C‐terminal, glycyl‐glycyl‐ l ‐arginine (hereafter referred to as GGR), evoked settlement at a concentration as low as 10 −10 M. Dose‐response curves for GGR and for the active fraction (500–1,000 Da) of oyster bath water were essentially identical. Fourth, tests of 21 free amino acids identified only lysine and arginine as settlement cues. Larval settlers were far more sensitive to arginine than lysine but significantly less sensitive to arginine than to peptides with arginine at the C‐terminal. Our combined results are all consistent in identifying low‐molecular‐weight peptides with arginine at the C‐terminal as the natural, water‐soluble cues inducing oyster settlement.