Characterization of Aplysia Enticin and Temptin, Two Novel Water-borne Protein Pheromones That Act in Concert with Attractin to Stimulate Mate Attraction

Abstract

Mate attraction in Aplysia involves a long-distance water-borne signal (attractin) that is released during egg laying. Other pheromones are predicted to be released during egg laying that act in concert with albumen gland attractin to stimulate attraction, but their identities are unknown. To identify other candidate water-borne pheromones, we employed differential library screening of an albumen gland cDNA library, Northern blot analysis, purification, characterization, cloning, and expression of albumen gland proteins, matrix-assisted laser desorption ionization mass spectrometry, pheromone secretion assays, behavioral bioassays, immunolocalization studies, and comparative genomics. Four genes, Alb-23, Alb-24, Alb-69, and Alb-172, were highly expressed in Aplysia californica albumen glands and encoded novel proteins. The products of the Alb-24 ("enticin") and Alb-172 ("temptin") precursors were soluble and highly abundant in albumen gland extracts, whereas Alb-23 and Alb-69 were membrane-associated proteins. A comparative analysis showed that the predicted Aplysia brasiliana enticin and temptin proteins were 90 and 91% identical, respectively, to their A. californica homologs. T-maze attraction bioassay studies have previously demonstrated that egg cordons alone are attractive to Aplysia but that attractin alone is not. In the present study, however, the combination of attractin, enticin, and temptin was found to be significantly attractive to potential mates and doubled the number of animals attracted to this stimulus compared with control animals. The combined data strongly suggest that enticin and temptin are novel candidate waterborne protein pheromones that act in concert with attractin to attract Aplysia to form and maintain egglaying and mating aggregations.Chemical communication is the most ancient form of communication and is used by most, if not all, animals examined, including ciliated protozoans (1), yeast (2), insects (3-5), mollusks (6 -10), worms (11, 12), fish (13), amphibians (14 -16), rodents (17, 18), and humans (19).Aplysia are simultaneous hermaphrodites that do not normally fertilize their own eggs. Field studies (20 -23) have shown that they are solitary animals that move into breeding aggregations during the reproductive season. The aggregations usually contain both mating and egg-laying animals and are associated with masses of recently deposited egg cordons, often deposited one on top of another. Most of the egg-laying animals mate simultaneously as females, even though mating does not trigger reflex ovulation (24), suggesting that egg laying precedes mating in the aggregation and that egg laying may release pheromones that establish and maintain the aggregation (25,27,28). The pheromonal factors seem to be derived from the egg cordon rather than the egg layer and some are waterborne (29).One of these water-borne pheromonal attractants (attractin) has been isolated from eluates of Aplysia californica egg cordons and characterized. Attractin is a 58-residue N-glycosylated protein with ...

Packaging of chemicals in the defensive secretory glands of the sea hareAplysia californica

“…Escapin, aplysianin-A and their homologues have been shown to have antibacterial, cytolytic, haemagglutinating, and/or anti-heparin activities (e.g. Kamiya et al, 1986;Cummins et al, 2004;Yang et al, 2005), but we believe it unlikely that this is the primary function of these proteins. Rather, we believe that escapin and its homologues function as antipredatory chemical defenses, though we are not aware of any reports of such activity.…”

“…The high specificity of the antibody is demonstrated from the fact that there is no escapin-like immunoreactivity in the albumen gland and albumen gland complex of adults, which contains aplysianin-A precursor protein, a homologue of escapin with 60% identity to escapin (Cummins et al, 2004).…”

“…Ink of many species of sea hares has been shown to contain a protein of ~60·kDa Kamiya et al, 1989;Yamazaki, 1993;Nistratova et al, 1993;Melo et al, 2000;Rajaganapathi and Kathiresan, 2002;Petzelt et al, 2002;Yang et al, 2005). In A. californica, this protein is called escapin (Yang et al, 2005), and it has a homologue in the albumen gland, called aplysianin-A (Kamiya et al, 1986;Cummins et al, 2004). Escapin, aplysianin-A and their homologues have been shown to have antibacterial, cytolytic, haemagglutinating, and/or anti-heparin activities (e.g.…”

Cloning, characterization and expression of escapin, a broadly antimicrobial FAD-containing l-amino acid oxidase from ink of the sea hare Aplysia californica

“…D83255; Kamiya et al, 1986) and A. californica (Accession no. AY161041; Cummins et al, 2004), respectively. Escapin also had 48% identity with achacin precursor, an antibacterial protein isolated from a land snail Achatina fulica (Accession no.…”

“…These studies differ in their findings as to whether the proteins are bacteriostatic or bactericidal (Yamazaki et al, 1990;Otsuka-Fuchino et al, 1992;Melo et al, 1998;. In addition to the interesting biological roles these proteins may play for their host organisms, some may also have practical industrial or medical uses, yet none have been successfully expressed in high output systems such as bacteria (Obara et al, 1999;Torii et al, 2000;Petzelt et al, 2002;Butzke et al, 2004;Cummins et al, 2004).…”