Sherry D. Painter

Molecular cloning of a cDNA encoding the neuropeptides APGWamide and cerebral peptide 1: Localization of APGWamide-like immunoreactivity in the central nervous system and male reproductive organs of Aplysia

While much is known about the neural and endocrine mechanisms that control egg laying in the gastropod mollusk Aplysia, relatively little is known about the regulation of male reproductive activity in this simultaneous hermaphrodite. In the present study, we have cloned and sequenced a cDNA that encodes a precursor protein, the predicted posttranslational processing of which presumably generates nine copies of the neuropeptide Ala‐Pro‐Gly‐Trp‐NH2 (APGWamide), five connecting peptide sequences, and a C‐terminal peptide. The sequence of one connecting peptide is identical to the previously characterized cerebral peptide 1. Northern blot analysis identified two major APGWamide mRNA transcripts (∼1.3 kb, ∼2.4 kb), which were present in central nervous system ganglia, but were most abundant in the right cerebral and right pedal ganglia. Immunohistochemical studies using sexually mature Aplysia demonstrated that the vast majority of APGWamide‐like immunoreactivity was localized in 30–40 neurons along the anterior and medial margins of the right cerebral ganglion and in a cluster of 15–20 neurons in the right pedal ganglion. A total of only about ten immunoreactive neurons were located in other ganglia. Immunohistochemistry also demonstrated that APGWamide was present in the reproductive organs that participate in the storage or transport of sperm, including the small hermaphroditic duct (site of sperm storage before mating), the white hemiduct (also known as the copulatory duct), and penial complex. As a group, these data suggest that APGWamide may play a role in regulating male reproductive function in Aplysia, as it does in other gastropods. J. Comp. Neurol. 387:53–62, 1997.

Induction of copulatory behavior in Aplysia: Atrial gland factors mimic the excitatory effects of freshly deposited egg cordons

Egg laying in the marine mollusc Aplysia is induced and coordinated by peptide products of the egg-laying hormone (ELH) gene expressed in the neuroendocrine bag cells of the central nervous system. At least three structurally related genes, belonging to the ELH family but distinct from the ELH gene, are expressed in the atrial gland, an exocrine organ of unknown function that secretes into the oviduct of Aplysia. The experiments described in this report were designed to test the hypothesis that the atrial gland gene products serve a pheromonal function for the animal, coordinating reproductive behavior among individuals. Our studies showed that there was a significantly shorter latency to copulation when an Aplysia was paired with an animal that was actively laying eggs than when it was paired with a sexually mature but nonlaying animal. Moreover, the addition of extracts or homogenates of the atrial gland to the seawater surrounding two nonlaying animals reduced the latency to mating compared to animals exposed only to seawater or to homogenates of other regions of the reproductive tract, including oviduct. These results suggest that atrial gland products, secreted onto the egg cordon as it passes through the oviduct, may play a pheromonal role and induce mating behavior between individuals. Experiments are in progress to determine whether the active atrial gland factor(s) are products of the ELH-family genes expressed in the gland.

Molecular cloning of a cDNA encoding a potential water-borne pheromonal attractant released during Aplysia egg laying

Recently deposited egg cordons are a source of water-borne pheromones that attract the marine mollusk Aplysia into breeding aggregations and coordinate male and female reproductive behavior within the aggregation. A potential pheromonal attractant has been isolated from egg cordon eluates and the peptide partially characterized [S.D. Painter, B. Clough, X. Fan, G.T. Nagle, Soc. Neurosci. Abstr., Vol. 22 (1996) 837]. Using this information, we have cloned an Aplysia albumen gland cDNA that encodes a precursor protein containing a single copy of the full-length peptide, and demonstrated that there are abundant levels of pheromone mRNA transcripts (0.8 and 2.5 kb) in the albumen gland. This is consistent with the reported function of the gland (i.e. packaging the eggs into a cordon for deposition), with behavioral studies showing that the albumen gland is a potential source of attractants, and more recent biochemical studies in which the full-length peptide has been isolated from the albumen gland. This is the first candidate peptide pheromone in mollusks and the first in invertebrates. The pheromonal regulatory system in Aplysia may provide a model system for examining the structural characteristics of peptide pheromones.

Aplysia Attractin: Biophysical Characterization and Modeling of a Water-Borne Pheromone

Attractin, a 58-residue protein secreted by the mollusk Aplysia californica, stimulates sexually mature animals to approach egg cordons. Attractin from five different Aplysia species are approximately 40% identical in sequence. Recombinant attractin, expressed in insect cells and purified by reverse-phase high-performance liquid chromatography (RP-HPLC), is active in a bioassay using A. brasiliana; its circular dichroism (CD) spectrum indicates a predominantly alpha-helical structure. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) characterization of proteolytic fragments identified disulfide bonds between the six conserved cysteines (I-VI, II-V, III-IV, where the Roman numeral indicates the order of occurrence in the primary sequence). Attractin has no significant similarity to any other sequence in the database. The protozoan Euplotes pheromones were selected by fold recognition as possible templates. These diverse proteins have three alpha-helices, with six cysteine residues disulfide-bonded in a different pattern from attractin. Model structures with good stereochemical parameters were prepared using the EXDIS/DIAMOD/FANTOM program suite and constraints based on sequence alignments with the Euplotes templates and the attractin disulfide bonds. A potential receptor-binding site is suggested based on these data. Future structural characterization of attractin will be needed to confirm these models.

CONTACT CHEMOSENSORY CUES IN EGG BUNDLES ELICIT MALE-MALE AGONISTIC CONFLICTS IN THE SQUID Loligo pealeii

Male Loligo pealeii engage in frequent agonistic bouts to gain access to female mates while aggregated at communal egg beds. Male squids are attracted to eggs in the field and in the laboratory. It was recently demonstrated that visual detection followed by physical contact with egg capsules elicited male–male aggression. We tested specific physical and chemical features of the egg capsules that may cause this strong behavioral reaction. Male squids were presented with either natural or artificial egg stimuli and scored for four selected behaviors (egg touch, egg blowing, forward-lunge grab, and fin-beating), the last two of which are highly aggressive behaviors. First, squids were presented with natural eggs versus eggs sealed in agarose-coated tubes (ESACT), which eliminated both tactile and chemical stimuli. Second, males were presented with natural eggs versus eggs sealed in agarose coated tubes containing C18 Sep-Pak-purified extracts (TCPE) from squid egg capsules, which provided chemical cues from natural eggs without the physical stimulus of the egg capsules. Third, natural eggs versus heat-denatured eggs were tested to determine whether the active factor in natural eggs is heat-labile. Squids responded aggressively when contacting natural eggs and TCPE, whereas squids did not respond after touching ESACT or denatured eggs. These results suggest that aggressive behavior is elicited by a heat-labile factor that is embedded within squid egg capsules. This chemosensory cue appears to be a contact pheromone that stimulates the agonistic interactions that characterize the mating behavior of migratory squids on inshore spawning grounds.

Behavioral Characterization of Attractin, a Water-Borne Peptide Pheromone in the Genus Aplysia

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.

Coordination of Reproductive Activity in Aplysia: Peptide Neurohormones, Neurotransmitters, and Pheromones Encoded by the Egg-Laying Hormone Family of Genes

Pheromones play a significant role in coordinating reproductive activity in the marine opisthobranch mollusk 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 recently deposited egg cordons. Behavioral studies suggest that cordon-derived pheromonal factors are primarily responsible for establishing and maintaining the aggregations. Egg-laying animals are more attractive than sexually mature, but nonlaying, conspecifics and have a shorter mean latency to mating; egg cordons and egg-cordon eluates, when placed in the surrounding seawater, enhance the attractiveness of nonlaying animals and reduce their mean latency to mating. Similar effects are observed when extracts of the atrial gland are placed in the seawater, suggesting that secretory products of this oviductal exocrine organ may function as sexual pheromones. Biochemical analyses indicate that there may be multiple attractants in atrial gland extracts, and that at least one of these (A-NTP) is a peptide encoded by the A gene. The A gene belongs to a small family of structurally related genes that are expressed in a tissue-specific manner. Another member of the family, the egg-laying hormone (ELH) gene, is expressed in the neuroendocrine bag cells. Peptide products of the ELH gene act as neurohormones and nonsynaptic neurotransmitters, initiating egg laying and coordinating its associated behaviors. Peptide products of a family of genes may thus act internally and externally to coordinate both male and female reproductive activities.

I: Aplysia californica neurons R3-R14: primary structure of the myoactive histidine-rich basic peptide and peptide I

The R3-R14 neurons of the marine mollusc Aplysia are neuroendocrine cells that express a gene encoding peptides I, II and histidine-rich basic peptide (HRBP), a myoactive peptide that excites Aplysia heart and enhances gut motility in vitro. Peptide II has been chemically characterized (35), but the complete primary structures of peptide I and HRBP have not been established by amino acid sequence analysis. HRBP, peptide I, and the prohormone (proHRBP) were therefore purified from acid extracts of Aplysia californica neural tissue using sequential gel filtration and reverse-phase high-performance liquid chromatography and chemically characterized. Amino acid sequence analysis demonstrated that HRBP was a 43-residue peptide whose sequence was: less than Glu-Val-Ala-Gln-Met-His-Val-Trp-Arg-Ala-Val-Asn-His-Asp-Arg-Asn-His-Gly- Thr-Gly - Ser-Gly-Arg-His-Gly-Arg-Phe-Leu-Ile-Arg-Asn-Arg-Tyr-Arg-Tyr-Gly-Gly-Gly- His-Leu - Ser-Asp-Ala-COOH. Compositional and sequence analyses of peptide I and proHRBP demonstrated that peptide I was a 26-residue peptide with the following sequence: NH2-Glu-Glu-Val-Phe-Asp-Asp-Thr-Asp-Val-Gly-Asp-Glu-Leu-Thr-Asn-Ala- Leu-Glu-Ser-Val-Leu-Thr-Asp-Phe-Lys-Asp-COOH. These results demonstrated that the pro-HRBP sequence predicted by nucleotide sequence analysis of a cDNA clone (24) was in fact synthesized in R3-R14 neurons. Hydrophilicity and hydrophobicity profiles of preproHRBP, combined with charge distribution profiles and predictive secondary structural analysis, showed that cleavage at dibasic sequences was strongly associated with peaks of hydrophilicity in alpha-helical regions of the preprohormone.

Atrial gland cells synthesize a family of peptides that can induce egg laying inAplysia

Summary1.Endogenous peptides induced egg laying in the marine molluscAplysia in two ways: egg-laying hormone (ELH) from the neuroendocrine bag cells acts directly, causing the release of eggs from the ovotestis; peptides A and B from the atrial gland act indirectly, activating the bag cells to release ELH. Another atrial gland peptide (egg-releasing hormone; ERH) is a structural and functional hybrid of ELH and peptides A and B; it can act both directly and indirectly to induce egg laying.2.Atrial glands were incubated in a mixture of3H-amino acids for 18 h, and the biosynthetically labelled peptides isolated using sequential Sephadex G-50 column chromatography and isoelectric focusing. Radiolabelled peaks were localized and bioassayed in intact animals. Bioactive peaks were then characterized functionally using two additional assays: egg laying in bag cell-less animals (ELH-like peptides) and in vitro induction of bag cell discharge (A- and B-like peptides). ERH-like molecules are active in both assays. Homogeneity of bioactive IEF peaks was assessed by SDS-PAGE.3.Sephadex G-50 gel filtration of biosynthetically labelled atrial gland extracts reveals two major peptide peaks. Peak D (apparentMr 6,000) is strongly radiolabelled and contains most of the egg-laying activity, but has a low absorbance at 274 nm. Peak E (apparentMr 3,500) is weakly labelled and contains a small proportion of the total egg-laying activity, but has a large absorbance at 274 nm.4.Isoelectric focusing of radiolabelled peptides in peak D reveals seven distinct ELH-like species (pI 5.5, 7.5, 8.5, 8.7, 8.9, 9.1, 9.4), and two peaks (pI 5.9, 8.1) that have both ELH-like and A-/B-like activity. The pI 8.1 peak may result from the comigration of peptide A with ERH or with an unidentified ELH-like peptide. It is not yet clear whether the pI 5.9 activity results from comigration of distinct peptides or from the presence of a previously uncharacterized ERH-like molecule.5.Isoelectric focusing of radiolabelled peptides in peak E reveals five distinct ELH-like species (pI 7.3, 8.5, 8.7, 9.1, 9.4), and one peak (pI 8.9) with both ELH-like and A-/B-like activity. The pI 8.9 peak may result from the comigration of an ELH-like peptide with peptide B. Three of the ELH-like peptides (pI 8.5, 8.9, 9.1) found in peak E are probably identical to the ELH-like peptides found at the same pIs in peak D.6.The ELH-like pI 7.5 species is a peptide complex held together by a disulfide linkage and hydrophobic interactions. The complex dissociates when treated with 2-mercaptoethanol and pyridine, resulting in a reduction in apparent molecular weight, and the appearance of one basic (pI 9.4) and two acidic (pI 3.3, 3.8) peptides. The pI 9.4 peptide presumably accounts for the ELH-like activity of the parent pI 7.5 complex but differs, based on its mobility on SDS-PAGE, from the pI 9.4 ELH-like peptide(s) described above.7.These studies, utilizing a two-step purification procedure, functional bioassays, and SDS-PAGE to assess peptide homogeneity, indicate that atrial gland cells synthesize a surprisingly large number of peptides that can induce egg laying inAplysia.

Attractin, a water-borne peptide pheromone in Aplysa

Summary The opisthobranch mollusk Aplysia is a simultaneous hermaphrodite that does not usually fertilize its own eggs. It is solitary during most of the year, but moves into breeding aggregations during the summer 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 both contact and water-borne pheromones that attract animals to the area and induce or facilitate reproductive activity. Three characteristics of the Aplysia reproductive system make it ideal for the isolation and characterization of a water-borne peptide pheromone: (a) egg laying can be induced by injecting atrial gland extract into the hemocoel; (b) the resulting eggs are packaged into a long string or “cordon” which has a large surface area; and (c) the cordon is a source of water-borne pheromones. By using atrial gland extract to induce egg laying, it is possible to control the number of animals laying eggs, the timing and synchroniz...