Although animals in the genus Aplysia are solitary during most of the year, they form breeding aggregations during the reproductive season. The aggregations contain both mating and egg-laying animals and are associated with masses of egg cordons. The egg cordons are a source of pheromones that establish and maintain the aggregation, but none of the pheromonal factors have been chemically characterized. In these studies, specimens of Aplysia were induced to lay eggs, the egg cordons collected and eluted, and the eluates fractionated by C18 reversed-phase HPLC. Four peak fractions were bioassayed in a T-maze. All four increased the number of animals attracted to a nonlaying conspecific and were thus subjected to compositional and microsequence analysis. Each contained the same NH2-terminal peptide sequence. The full-length peptide ("attractin") was isolated from the albumen gland, a large exocrine organ that packages the eggs into a cordon. The complete 58-residue sequence was obtained, and it matched that predicted by an albumen gland cDNA. Mass spectrometry showed that attractin is 21 wt.% carbohydrate as the result of N-linked glycosylation. T-maze bioassays confirmed that the full-length peptide is attractive. Attractin is the first water-borne peptide pheromone characterized in molluscs, and the first in invertebrates.

Pheromones play a significant role in coordinating reproductive activity in many animals, including opisthobranch molluscs of the genus APLYSIA: Although solitary during most of the year, these simultaneous hermaphrodites gather into breeding aggregations during the reproductive season. The aggregations contain both mating and egg-laying animals and are associated with masses of egg cordons. The egg cordons are a source of pheromones that attract other Aplysia to the area, reduce their latency to mating, and induce egg laying. One of these water-borne egg cordon pheromones ("attractin") has been characterized and shown to be attractive in T-maze assays. Attractin is the first water-borne peptide pheromone characterized in invertebrates. In the current studies, behavioral assays were used to better characterize the attraction, and to examine whether attractin can induce mating. Although the two activities could be related (i.e., attraction occurring because animals were looking for a partner), this was not tested. T-maze assays showed that attractin works as part of a bouquet of odors: the peptide is attractive only when Aplysia brasiliana is part of the stimulus. The animal does not need to be a conspecific, perhaps explaining why multiple species may be associated with one aggregation. Native and recombinant attractin are equally attractive, verifying that N-glycosylation at residue 8 is not required for attraction. Mating studies showed that both native and recombinant attractin reduce the latency to mating. The effects are larger when hermaphroditic mating is considered: in addition to reducing latency, attractin doubles the number of pairs mating as hermaphrodites. The effect may result from attractin stimulating both animals to mate as males and would be consistent with behaviors previously seen in the T-maze. Attractin may thus be contributing to the formation of copulatory chains and rings seen in aggregations in the field. These results may be interpreted in two ways: (1). attractin has multiple activities that contribute to the establishment and maintenance of the aggregation; or (2). the induced desire to mate may make attractin attractive when it is presented in conjunction with an animal. In either case, the results open the door for cellular and molecular studies of mechanism of action.

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