Multi-messenger innervation of the male sexual system of Lymnaea stagnalis.

The male copulation behaviour of the hermaphrodite pond snail Lymnaea stagnalis is under the control of five groups of central neurons that produce a variety of neuropeptides and a classical transmitter, 5-hydroxy tryptamine (5HT). In this article, we describe how the male sexual organs of this snail are innervated by axons from these central neurons. We carried out immunocytochemistry with antisera against the tetra peptide Ala-Pro-Gly-TRP-NH2 (APGWamide), the Lymnaea form of neuropeptide tyrosine (LNPY), conopressin, pedal peptide, the FRMFamide copeptide SEEPLY, the GDPFLRFamide co-peptide DEILSR, myomodulin, Lymnaea inhibitory peptide, and 5HT on tissue sections of the following male sexual organs that receive input from the penis nerve: the prostate gland, vas deferens, preputium, and penis. The results demonstrate that the axons of the separate muscle systems contain particular combinations of transmitters. In addition, two networks of peripheral neurons were revealed. In the tip of the everted preputium lies what appears to be a network of conopressin-containing sensory neurons, which is possibly involved in probing; probing is the part of copulation behaviour in which the male searches for the female genital pore. The other network of peripheral neurons surrounds the most proximal part of the vas deferens and is most likely involved in the pacemaker control of vas deferens motility. On the basis of the data obtained, we hypothesize how the preputium and penis are everted during copulation and which transmitters and central neurons might be involved.

Alternative splicing generates diversity of VD1/RPD2 alpha peptides in the central nervous system of Lymnaea stagnalis.

1. Two giant peptidergic neurons, VD1 and RPD2, of the visceral ganglion and right parietal ganglion of Lymnaea stagnalis, respectively, play an important role in the modulation of complex physiological and behavioral adjustments that occur as a result of changes in O2 availability. 2. By cDNA cloning, we have identified two types of VD1/RPD2 transcripts expressed in VD1 and RPD2. In addition, these transcripts are also expressed in other neurons. 3. Both transcripts encode distinct yet related VD1/RPD2 preprohormones that may be cleaved to yield distinct but overlapping sets of neuropeptides. 4. Using the polymerase chain reaction technique, we could show the existence of additional splice variants. 5. Analysis of the organization of the VD1/RPD2 gene indicates that the alpha peptide coding region is interrupted by a number of introns. 6. We concluded that the mRNA segment encoding the alpha peptide domain of the VD1/RPD2 preprohormones is alternatively spliced, thus generating different alpha peptides.

Molecular cloning of a cDNA encoding a member of a novel cytochrome P450 family in the mollusc Lymnaea stagnalis.

We isolated a cDNA encoding a cytochrome P450 from the mollusc Lymnaea stagnalis. The mRNA is 2.1 nucleotides long and contains an open reading frame encoding a protein of 545 amino acids. A conserved home-binding domain, characteristic of cytochrome P450 proteins, is present in the deduced amino acid sequence. The Lymnaea cytochrome P450 protein shares less than 40% positional identity with any known member of the cytochrome P450 superfamily, and therefore, represents a separate family, which we propose to name CYP10. The CYP10 mRNA is shown to be uniquely and abundantly expressed in the female gonadotropic hormone producing dorsal bodies of L. stagnalis.

Biosynthesis and axonal transport of multiple molluscan insulin-related peptides by the neuroendocrine light green cells of Lymnaea stagnalis.

The neuroendocrine light green cells (LGCs) control body growth and metabolism of the freshwater snail, Lymnaea stagnalis. These cells, located in the cerebral ganglia, use the peripheries of the median lip nerves (MLNs) as the neurohemal area. The LGCs express four members of a gene family encoding the precursors of distinct though related molluscan insulin-related peptides (MIPs). The molecular characterization and biosynthesis of MIPs have been investigated. MLN extracts were first size fractionated using gel permeation chromatography, and a major peak that coeluted with bovine insulin was further resolved using reverse phase high performance liquid chromatography. Amino acid composition analysis revealed that the four MIPs were present, as encoded by the MIP genes. Biosynthesis and axonal transport of the MIPs were studied in vitro with LGC systems in a series of pulse-chase incubations in combination with gel permeation chromatography analysis. During the pulse period of 20 min MIP precursors were formed in the LGC cell bodies. During the subsequent chase period these precursors were converted to MIPs and C peptides, which were transported to the LGC axon terminals in the MLNs for storage and release. The MIP precursors seem to be processed similarly to the insulin precursor in pancreatic islets.

Purification and sequencing of molluscan insulin-related peptide II from the neuroendocrine light green cells in Lymnaea stagnalis.

The growth-controlling neuroendocrine light green cells of the freshwater snail, Lymnaea stagnalis, express a family of genes encoding structurally related, yet distinct, molluscan insulin-related peptides (MIPs). In the present study one of these peptides, MIP II, has been isolated and structurally identified. MIP II is a heterodimer of A and B chains connected by disulfide bonds. Both chains are N-terminally blocked with pyroglutamate. After cleaving of the A and B chains and deblocking with pyroglutamate amino-peptidase their sequences have been determined as: A chain: pQRTTNLVCECCFNYCTPDVVRKYCY and B chain: pQSSCSLSSRPHPRGICGSNLAGFRAFICSNQNSPS. In comparison with the MIP II sequence based on complementary DNA studies, it is clear that the two C-terminal amino acid residues of the B chain are posttranslationally removed. In addition, the glutamic acid residue in A chain was recovered in very low yields during Edman degradation, suggesting that the residue may be posttranslationally modified.

Characterization of a cDNA clone encoding molluscan insulin-related peptide V of Lymnaea stagnalis.

A cDNA clone encoding molluscan insulin-related peptide V (MIP V) was isolated from a cDNA library of the central nervous system (CNS) of the freshwater snail, Lymnaea stagnalis, using a heterologous screening with a previously identified MIP II cDNA. The MIP V cDNA encodes a preprohormone resembling the organization of preproinsulin, with a putative signal sequence, and an A and B chain, however, in this case connected by two distinct C peptide, C alpha and C beta, instead of one single C peptide. This phenomenon, which is shared by the MIP II precursor, represents a new development in the prohormone organization of peptides belonging to the insulin superfamily. The A and B chains of MIPs V, I and II, differ remarkably in primary structure; in contrast, the C alpha peptide domains are almost identical. MIP V has only limited sequence similarity with insulins and related peptides. Both MIP V and I exhibit structural features, which make them a unique class of the insulin superfamily. The MIP I, II and V genes are expressed in a single type of neuron: the growth controlling neuroendocrine light green cells of the Lymnaea CNS.

Purification and sequencing of molluscan insulin-related peptide I (MIP I) from the neuroendocrine light green cells of Lymnaea stagnalis.

The body growth controlling cerebral neuroendocrine light green cells of the freshwater snail, Lymnaea stagnalis, express various members of a gene family encoding different though related prepromolluscan insulin-related peptides. In the present study, molluscan insulin-related peptide I (MIP I) together with the corresponding connecting peptide, C alpha peptide, have been isolated and structurally identified. MIP I is a heterodimer of A and B chains bonded by disulphide bridges. Two isoforms of MIP I could be discerned. Mass spectrometry revealed that of one form both the A and B chains have N-terminal pyroglutamyl residues, whereas of the other form only the B chain has such residues. After removal of the pyroglutamyl residues with pyroglutamate aminopeptidase, followed by disulphide bond cleavage and pyridylethylation of cysteine residues, the sequences of MIP I have been determined using Edman degradation as: A chain: (p)QGTTNIVCECCMKPCTLSELRQYCP; B chain: pQPSACNINDRPHRRGVCGSALADLVDPACSSSNGPA. The C alpha peptide has also been isolated and its sequence was determined as NAETDLDDPLRNIKLSSESALTYLY. These sequences are in agreement with those predicted by a cDNA sequence encoding preproMIP I, with the exception that the two C-terminal amino acids of the B chain are posttranslationally removed.

Evolution of the vasopressin/oxytocin superfamily: characterization of a cDNA encoding a vasopressin-related precursor, preproconopressin, from the mollusc Lymnaea stagnalis.

Although the nonapeptide hormones vasopressin, oxytocin, and related peptides from vertebrates and some nonapeptides from invertebrates share similarities in amino acid sequence, their evolutionary relationships are not clear. To investigate this issue, we cloned a cDNA encoding a vasopressin-related peptide, Lys-conopressin, produced in the central nervous system of the gastropod mollusc Lymnaea stagnalis. The predicted preproconopressin has the overall architecture of vertebrate preprovasopressin, with a signal peptide, Lys-conopressin, that is flanked at the C terminus by an amidation signal and a pair of basic residues, followed by a neurophysin domain. The Lymnaea neurophysin and the vertebrate neurophysins share high sequence identity, which includes the conservation of all 14 cysteine residues. In addition, the Lymnaea neurophysin possesses unique structural characteristics. It contains a putative N-linked glycosylation site at a position in the vertebrate neurophysins where a strictly conserved tyrosine residue, which plays an essential role in binding of the nonapeptide hormones, is found. The C-terminal copeptin homologous extension of the Lymnaea neurophysin has low sequence identity with the vertebrate counterparts and is probably not cleaved from the prohormone, as are the mammalian copeptins. The conopressin gene is expressed in only a few neurons in both pedal ganglia of the central nervous system. The conopressin transcript is present in two sizes, due to alternative use of polyadenylylation signals. The data presented here demonstrate that the typical organization of the prohormones of the vasopressin/oxytocin superfamily must have been present in the common ancestors of vertebrates and invertebrates.

Purification and chemical characterization of caudodorsal cell hormone-II from the egg-laying controlling caudodorsal cells of Lymnaea stagnalis.

Caudodorsal cell hormone-I (CDCH-I), a 36 amino acid peptide synthesized by the caudodorsal cells (CDCs), is one of the major neuropeptides present in the cerebral ganglia of the freshwater snail, Lymnaea stagnalis. This peptide induces ovulation and egg-mass formation, and, in addition, acts on neuronal circuits involved in the control of overt egg-laying behavior. cDNA cloning studies revealed that there is a CDCH gene family comprising two genes coding for CDCH-I and the related yet distinct CDCH-II, respectively. In the present study, using sequential high performance gel permeation and reversed phase high performance liquid chromatography, we have isolated and sequenced the peptide CDCH-II. This neuropeptide has the following sequence: Ser-Ile-Thr-Asn-Asp-Leu-Arg-Ala-Ile-Ala-Asp-Ser-Tyr-Leu-Tyr-Asp-Gln-His- Lys-Leu - Arg-Glu-Gln-Gln-Glu-Glu-Asn-Leu-Arg-Arg-Arg-Phe-Tyr-Glu-Leu-Ser-Leu-Arg- Pro-Tyr - Pro-Asp-Asn-Leu. The sequence differs from those predicted by the cDNA sequence encoding preproCDCH-II at two positions, suggesting the polymorphism of CDCH-II exists in L. stagnalis.

Primary structure and origin of schistosomin, an anti-gonadotropic neuropeptide of the pond snail Lymnaea stagnalis.

In the pond snail Lymnaea stagnalis infected with the schistosome parasite Trichobilharzia ocellata, a peptide called schistosomin is released from the central nervous system, which counteracts the bioactivity of a number of gonadotropic hormones. This leads to inhibition of the reproductive activities of the infected snail. In order to determine the structure of schistosomin, the neuropeptide was purified from the central nervous system using gel-permeation chromatography and reverse-phase h.p.l.c. The complete primary structure of the peptide was determined by N-terminal sequencing and peptide mapping. Schistosomin is a single-chain molecule of 79 amino acids with a molecular mass of 8738 Da. The peptide contains eight cysteine residues which may give rise to four intramolecular disulphide bridges that fold the peptide into a stable globular structure. A database search did not reveal any known peptides that show significant sequence similarity to schistosomin. By means of immunocytochemistry, the peptide was shown to be localized in the growth-controlling neurosecretory light green cells, which are located in the cerebral ganglia of the central nervous system of Lymnaea. In addition to schistosomin, these neurons are known to produce various insulin-related peptides.

Characterization of a cDNA clone encoding molluscan insulin-related peptide II of Lymnaea stagnalis.

A cDNA clone encoding molluscan insulin-related peptide (MIP) II was isolated from a cDNA library of the central nervous system (CNS) of the freshwater snail, Lymnaea stagnalis, using a heterologous screening with a previously identified MIP cDNA (renamed MIP-I cDNA). The MIP-II cDNA encodes a preprohormone resembling the organization of preproinsulin, with a putative signal sequence, and A and B chains; however, in this case connected by two distinct C peptides, C alpha and C beta, instead of a single C peptide, a phenomenon which represents a new development in the prohormone organization of peptides belonging to the insulin superfamily. The A and B chains of MIP II and I differ remarkably in primary structure; in contrast, the C alpha peptide domains are fully identical. MIP II has only limited sequence similarity with insulins and related peptides. Both MIP II and I exhibit structural features, which make them a unique class of the insulin superfamily. The MIP I and II genes are expressed in a single type of neuron: the growth-controlling neuroendocrine light green cells of the Lymnaea CNS.

Neuropeptide schistosomin inhibits hormonally-induced ovulation in the freshwater snail Lymnaea stagnalis.

This study examines the interaction between the caudodorsal cell hormone (CDCH) and schistosomin, a peptide secreted by the central nervous system of the snail (Lymnaea stagnalis) infected with the avian schistosome Trichobilharzia ocellata. Non-infected snails were injected with synthetic as well as native CDCH in the absence or presence of purified schistosomin. The response to 2 pmol of synthetic CDCH was blocked for 90% by coinjection with 3.5 pmol of schistosomin. The ovulation-inducing activity of extracts of cerebral commissures (the storage area of native CDCH) was also blocked by schistosomin. The degree of inhibition (65%), however, was less than that observed with synthetic CDCH. These results show that schistosomin inhibits ovulation and egg laying in Lymnaea. This explains the decrease or absence of egg laying in schistosome-infected freshwater snails.

Characterization and evolutionary aspects of a transcript encoding a neuropeptide precursor of Lymnaea neurons, VD1 and RPD2.

We isolated and characterized a cDNA clone encoding the major prohormone of VD1 and RPD2, two electrotonically coupled identified neurons in the central nervous system of the freshwater snail, Lymnaea stagnalis. The VD1/RPD2 prohormone may be cleaved to generate a set of 4 different neuropeptides, called epsilon, delta, alpha 1 and beta peptides, as well as a single aspartate. Since VD1 and RPD2 probably are involved in O2 perception and modulation of cardio-respiratory functions, it is thought that the neuropeptides synthesized and released by these neurons coordinate the adaptive physiological and behavioural processes that occur in response to changes in O2 availability. Comparison of the Lymnaea VD1/RPD2 precursor with two related precursors, prohormones R15-1 and R15-2, identified from neuron R15 in the marine mollusc Aplysia californica revealed a similar pattern of organization of the preprohormones. The overall homology is rather low, however, detailed comparisons show a highly differential pattern of conservation of peptide regions on the precursors.

Isolation of schistosomin, a neuropeptide which antagonizes gonadotropic hormones in a freshwater snail.

The molecular mechanisms underlying parasite-induced inhibitory effects on host reproduction were studied in the freshwater snail, Lymnaea stagnalis, infected with the schistosome parasite Trichobilharzia ocellata. This combination is used as a model system for host-parasite interactions involved in schistosomiasis transmission. The female gonadotropic snail neuropeptide, calfluxin, was labelled with fluorescein isothiocyanate (FITC) and used as a ligand in receptor-binding studies on membranes of its target organ, the albumen gland. The binding of calfluxin to its receptor-guanyl-nucleotide-binding-protein (G-protein) complex was inhibited in vitro in the presence of haemolymph of schistosome-infected snails. This inhibition appeared to be established by a peptidergic factor called schistosomin. The receptor assay was used to identify schistosomin from haemolymph during subsequent purification and characterization steps. The peptide could also be purified from the central nervous systems of non-infected snails, indicating that it is produced by the snail itself and released into the haemolymph as a result of infection. Analysis by plasma-desorption mass spectrometry revealed that purified schistosomin has a molecular mass of 8780 Da.

Quantification of proteins in the subnanogram and nanogram range: comparison of the AuroDye, FerriDye, and India ink staining methods.

The usefulness of three sensitive dyes, AuroDye, FerriDye, and India ink, for the quantification of proteins and peptides bound to nitrocellulose paper has been assessed. In general, the staining intensity varies linearly with the logarithm of protein concentrations. The detection limit of small peptides (Mr less than 5000) is higher than that of large peptides and proteins, but the sensitivity is independent of the molecular weight. Oligopeptides of four or less amino acids either stain with very high detection limits or do not stain at all. The detection limit of proteins stained by AuroDye is approximately 1 ng, and in a number of cases even lower. The useful range for quantification of proteins extends to around 100 ng. The FerriDye and India ink staining methods are less sensitive and can be used to quantify proteins over a wide nanogram range. Among the methods tested, the India ink staining method has the highest protein to protein variation in sensitivity.

Central and peripheral expression of genes coding for egg-laying inducing and insulin-related peptides in a snail.

Egg laying in the hermaphrodite freshwater snail Lymnaea stagnalis is a highly complex activity, including a series of internal activities (ovulation, egg and egg mass formation) which are closely correlated to a pattern of behaviours (alteration of locomotion and feeding, specific postures, oviposition). In this snail egg laying is induced by the neuroendocrine caudodorsal cells (CDCs), consisting of two homogeneous clusters at a total of 100 neurons. At egg laying these neurons release their products during a 60 min period of firing. The genes coding for these products have been cloned and characterized. There are two genes, CDCH-I and -II. Each gene codes for 12 peptides; one of these is the ovulation hormone (CDCH). The genes display over 90% homology. The most striking difference is a 17 bp deletion near the carboxy-terminal region. With immunocytochemistry and in situ hybridization both CDCH genes appeared to be expressed in the CDC and in paired groups of ectopic CDC-like neurons in the pleural ganglia, while a group of small neurons in the cerebral ganglia expresses the CDCH-I gene only. In addition, a widespread expression of the CDCH genes has been demonstrated in peripheral tissues. In the female part of the reproductive tract neurons were found to express the CDCH-I gene. In the male part of the tract exocrine secretory cells express the CDCH-I or -II gene. The gene products are secreted into the male tract and transferred to the female partner during copulation. Finally, sensory neurons in the head skin and mantle edge were found to express the CDCH-I gene. The presence of insulin-related peptides has been demonstrated in the brain as well as the digestive system of Lymnaea. The brain insulin-related peptides are produced in 4 groups of 50 giant neurons each (Light green cells, LGC). These neurons are involved in various physiological activities, related to body growth and glycogen metabolism. The major gene products expressed in the LGC have been cloned and characterized. It appeared that the predicted proteins represent three types of insulin-related molecules (MIP, molluscan insulin-related peptide). In these MIPs, those elements important in the determination of the tertiary structure, have been conserved. The MIP of the digestive system has been characterized up to now only at the peptide level. The snail gut MIP is more hydrophobic compared to bovine insulin. Cells containing MIP have been identified immunocytochemically in the gut epithelium.

Isolation, characterization, and evolutionary aspects of a cDNA clone encoding multiple neuropeptides involved in the stereotyped egg-laying behavior of the freshwater snail Lymnaea stagnalis.

The cerebral neurosecretory caudodorsal cells (CDCs) of the freshwater pulmonate snail Lymnaea stagnalis control egg laying, an event that involves a pattern of stereotyped behaviors. The CDCs synthesize and release multiple peptides, among which is the ovulation hormone (CDCH). It is thought that each peptide controls a specific aspect of the processes involved in egg laying. We isolated and characterized a CDC-specific cDNA clone that encodes the ovulation hormone (CDCH). RNA blot analysis and in situ hybridization experiments demonstrated that the CDCs are the major cell groups in the cerebral ganglia that transcribe the CDCH gene. In addition to CDCH, the 259-amino acid-long CDCH preprohormone contains 11 other predicted peptides. The overall homology of the CDCH preprohormone with the egg-laying hormone (ELH) preprohormones of the marine opisthobranch snails Aplysia californica and A. parvula is very low (29 and 26%, respectively). However, a more detailed comparison revealed a highly differential pattern of conservation of peptide regions. Significant homology was found between the regions containing (1) CDCH and ELH, (2) repeated pentapeptides, (3) alpha-caudodorsal cell peptide and alpha-bag cell peptide, and (4) 2 regions representing as yet unidentified peptides. Insignificant homology was found when comparing regions containing the other predicted peptides. The conserved peptides probably control similar aspects of the egg-laying fixed action patterns in these distantly related gastropod species. The pentapeptide region exhibits the highest level of homology (75%); in addition, an extra pentapeptide has been generated on the CDCH precursor. This indicates a vital function of these peptides in Aplysia, as well as in Lymnaea species.