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 water-borne protein pheromones that act in concert with attractin to attract Aplysia to form and maintain egglaying and mating aggregations.
Highlights
EXPERIMENTAL PROCEDURESCDNA Library Screening—The albumen gland is a large exocrine organ in the reproductive tract (31, 32) that secretes its products during egg laying
□S The on-line version of this article contains additional experimental procedures, primer sequences, tables containing cDNAs identified by library screening, predicted amino acid sequences of novel precursors, and MALDI-MS of atrial gland peptides that may correspond to Alb-23 and Alb-24
Previous studies showed that water-borne peptide/protein pheromones in the ciliated protozoan Euplotes and Aplysia are typically small (Ͻ110 residues) and that Aplysia attractin is highly represented in a cDNA library (6)
Summary
CDNA Library Screening—The albumen gland is a large exocrine organ in the reproductive tract (31, 32) that secretes its products during egg laying. For differential cDNA library screening, adult A. californica (Ͼ250 g) were obtained from Marine Research and Educational Products (Escondido, CA), total RNA was isolated from albumen and atrial glands (33) using TRIzol (Invitrogen), and poly(A)ϩ RNA was selected (Oligotex mRNA Mini Kit; Qiagen). Peptides were eluted with 50% acetonitrile (CH3CN)/ 0.1% HFBA, lyophilized, and the lyophilizate was resuspended in 0.1% HFBA; one third of the sample (33% of one gland) was purified by analytical Vydac C18 reversed phase high performance liquid chromatography (RP-HPLC) using a 2-step linear gradient (0 –10% CH3CN/0.1% HFBA in 5 min; 10 –58% CH3CN/0.1% HFBA in 170 min). Cloning of Temptin—Based on N-terminal and tryptic fragment sequence information obtained for this novel albumen gland peptide, a 3Ј-RACE probe was generated to isolate its cDNA. 20,000 plaques from the albumen gland cDNA library were screened using the 3Ј-RACE insert, and 13 positive clones were rescreened and sequenced. The identity of recombinant Alb-24 was confirmed by N-terminal microsequence analysis
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