Molluscan Insulin-related Peptides Research Articles

Molecular and structural analysis of Hdh-MIRP3 and its impact on reproductive regulation in female Pacific abalone, Haliotis discus hannai

Molluscan insulin-related peptides (MIRP) play a crucial role in various biological processes, including reproduction and larval development in mollusk species. To investigate the involvement of MIRP in the ovarian development of Pacific abalone (Haliotis discus hannai), the Hdh-MIRP3 was cloned from cerebral ganglion (CG). Hdh-MIRP3 cDNA was 993 bp long, encoded a 13.22 kDa peptide, comprising 118 amino acids. Fluorescence in situ hybridization confirmed the localization of Hdh-MIRP3 in the CG and ovary. Molecular docking revealed that Hdh-MIRP3 binds to the N-terminal region of Hdh-IRP-R. Tissue expression analysis showed the highest Hdh-MIRP3 expression in the CG, followed by ovarian tissue. Hdh-MIRP3 expression was significantly upregulated in the CG and ovary during the ripe stage of seasonal ovarian development and in effective accumulative temperature conditioned abalone. Furthermore, siRNA silencing of Hdh-MIRP3 significantly downregulated the expression of four reproduction-related genes, including Hdh-GnRH, Hdh-GnRH-R, Hdh-IRP-R, and Hdh-VTG in both the CG and ovary, and Hdh-MIRP3 as well. These results indicate that Hdh-MIRP3 acts as a regulator of ovarian development in Pacific abalone. Additionally, expression analysis indicated that Hdh-MIRP3 plays a role in embryonic and larval development. Overall, the present findings elucidate the role of Hdh-MIRP3 in reproductive development in female Pacific abalone.

Molecular characterization of four genes encoding abalone insulin-related peptides and their roles in regulation of hemolymph glucose in the Pacific abalone Haliotis discus hannai

Members of the insulin superfamily play key roles in metabolism, growth, reproduction, and longevity via insulin/IGF signaling pathway. The presence of insulin and insulin-related peptides are found among animals. In Mollusca, molluscan insulin-related peptides (MIPs) are involved in their body and shell growth. Hence, this study aimed to understand if there was difference in these peptide levels of fast and slow growing Pacific abalones, Haliotis discus hannai. To this end, four abalone insulin-related peptides (AIPs) are mined from cerebral ganglia transcriptomes of fast-growing and slow-growing abalones: AIP1, −2, −3 and −6. Notably, these AIPs possess 8 cysteine residues in their predicted insulin A and B chains, potentially supporting four disulfide bridges. Further sequence analysis based on the phylogenetic tree and multiple alignment methods indicated that AIPs have sequence features that are conserved among MIPs in many gastropods. The tissue distributions of four AIPs showed ubiquitous and similar expression in the following tissues including the cerebral ganglion, hepatopancreas, gonad, and mantle of both genders. However, there is some difference in other tissue distribution. Since the food consumption is intimately associated with the hemolymph glucose and animal growth, relative AIP1, −2, −3 and − 6 expression levels in the cerebral ganglion were measured following seaweed supplementation. The animals fed with brown seaweed showed increased these AIPs by 1.6–4.7 folds from 3 h after feeding, which lasting to 12 h, coinciding with the peak of hemolymph glucose. Based on these results, we propose that a physiological glucose regulatory mechanism involving AIPs exists in the cerebral ganglion of the H. discus hannai neuroendocrine system.

In silico prediction of neuropeptides from the neural ganglia of Pacific abalone Haliotis discus hannai (Mollusca: Gastropoda)

Neuropeptides play a central role in the regulation of reproduction, growth, development, and various other physiological processes. In our study, 26 neuropeptide precursors were identified from the pleuropedal and cerebral ganglia of Pacific abalone using transcriptome analysis. Of them, two (neuromacin and neuroglian) were potentially novel and two (myomodulin and FMRFamide) were involved in the reproductive regulation of mollusks. A BLAST search indicated that most of these neuropeptides exhibited the highest homologies with neuropeptides of other protostomian species. Myomodulin, FMRFamide, and molluscan insulin-related peptides shared 87%, 72%, and 88% sequence identities with homologous peptides of the tropical gastropod mollusk, Haliotis asinina. In silico analysis revealed that these identified neuropeptide precursors were likely to be extracellular secreted proteins. Alignment of multiple cerebrin, FFamide, and insulin-related peptide sequences illustrated that most of their biologically active residues were highly conserved with other invertebrate homologous neuropeptide residues. Three-dimensional (3D) structures of adipokinetic hormone, allatostain (A, B, and C), allatotropin, cerebrin, clionin, conopressin, elevenin, and neuromycin precursors in H. discus hannai exhibited a helix-loop-helix structure. Based on phylogenetic analysis, buccalin A1 and A2, allatostain B, and FCAP neuropeptide precursors of Pacific abalone formed a clade with other gastropod and bivalve mollusks neuropeptide precursors. This study provides a novel insight for further functional studies of abalone and other gastropod mollusks.

Molecular evolution and functional characterisation of insulin related peptides in molluscs: Contributions of Crassostrea gigas genomic and transcriptomic-wide screening

Insulin Related Peptides (IRPs) belong to the insulin superfamily and possess a typical structure with two chains, B and A, linked by disulphide bonds. As the sequence conservation is usually low between members, IRPs are classified according to the number and position of their disulphide bonds. In molluscan species, the first IRPs identified, named Molluscan Insulin-related Peptides (MIPs), exhibit four disulphide bonds. The genomic and transcriptomic data screening in the Pacific oyster Crassostrea gigas (Mollusc, Bivalvia) allowed us to identify six IRP sequences belonging to three structural groups. Cg-MIP1 to 4 have the typical structure of MIPs with four disulphide bonds. Cg-ILP has three disulphide bonds like vertebrate Insulin-Like Peptides (ILPs). The last one, Cg-MILP7 has a significant homology with Drosophila ILP7 (DILP7) associated with two additional cysteines allowing the formation of a fourth disulphide bond. The phylogenetic analysis points out that ILPs may be the most ancestral form. Moreover, it appears that ILP7 orthologs are probably anterior to lophotrochozoa and ecdysozoa segregation. In order to investigate the diversity of physiological functions of the oyster IRPs, we combine in silico expression data, qPCR measurements and in situ hybridization. The Cg-ilp transcript, mainly detected in the digestive gland and in the gonadal area, is potentially involved in the control of digestion and gametogenesis. The expression of Cg-mip4 is mainly associated with the larval development. The Cg-mip transcript shared by the Cg-MIP1, 2 and 3, is mainly expressed in visceral ganglia but its expression was also observed in the gonads of mature males. This pattern suggested the key roles of IRPs in the control of sexual reproduction in molluscan species.

Function of insulin in snail brain in associative learning.

Insulin is well known as a hormone regulating glucose homeostasis across phyla. Although there are insulin-independent mechanisms for glucose uptake in the mammalian brain, which had contributed to a perception of the brain as an insulin-insensitive organ for decades, the finding of insulin and its receptors in the brain revolutionized the concept of insulin signaling in the brain. However, insulin's role in brain functions, such as cognition, attention, and memory, remains unknown. Studies using invertebrates with their open blood-vascular system have the promise of promoting a better understanding of the role played by insulin in mediating/modulating cognitive functions. In this review, the relationship between insulin and its impact on long-term memory (LTM) is discussed particularly in snails. The pond snail Lymnaea stagnalis has the ability to undergo conditioned taste aversion (CTA), that is, it associatively learns and forms LTM not to respond with a feeding response to a food that normally elicits a robust feeding response. We show that molluscan insulin-related peptides are up-regulated in snails exhibiting CTA-LTM and play a key role in the causal neural basis of CTA-LTM. We also survey the relevant literature of the roles played by insulin in learning and memory in other phyla.

Short communications

This issue of JEB sees the first three articles in a new journal section: Short Communications. Introduced in response to author feedback, this brief (<2500 words) style of article aims to provide a venue for high-quality, hypothesis-driven, self-contained pieces of original research. The articles should not be preliminary reports or merely incremental studies but should augment the body of knowledge and be of significant and broad interest to the field of comparative physiology. Additionally, they may be opinionated and challenge existing theories and/or propose new theories or concepts based on existing research.The three Short Communications in this issue cover the breadth of fields published in the journal, ranging from the repeatability of metabolic rate to the way ants use visual information to navigate to the role of insulin-related peptides in memory formation.In the first of this new style of report, Craig White, Natalie Schimpf and Phillip Casey (p. 1763) conduct a meta-analysis to compare the contrasting results of two previously published JEB papers on metabolic measurements. In the first study, Nespolo and Franco (J. Exp. Biol. 210, 2000-2005) concluded that metabolic rate is generally a repeatable trait and that repeatability is not affected by the time between measurements. However, in a later study, Norin and Malte (J. Exp. Biol. 214, 1668-1675) found that repeatability of metabolic rate for a single species (brown trout) was high in the short term but declined to essentially nothing in the long term. White and colleagues carried out their own meta-analysis of all data for metabolic rate published to date, demonstrating that the repeatability of metabolic rate decreases with time for both ectotherms and endotherms. Confirming the generality of Norin and Malte's findings, this analysis has implications for studies testing for phenotypic associations among metabolic rate and other traits, because variation among individuals is the raw material on which natural selection acts, and inter-individual differences must be both consistent and genetically based for traits to respond to selection.Challenging the perceived dogma about the information that ants store about their surroundings, Antoine Wystrach and colleagues propose an alternative method by which ants use visual information to navigate complex idiosyncratic routes to a discrete goal. Since the seminal work on ant navigation published by Cartwright and Collett in 1983 (J. Comp. Physiol. A 151, 521-543), the commonly held view is that ants rely on visual cues and store multiple ‘snapshot memories’ at discrete locations that are retrieved sequentially to break routes into sections. However, in their article on p. 1766, Wystrach and colleagues employ a computational model they have recently developed (Baddeley et al. PLoS Comput. Biol. 8, e1002336) that uses a neural network to gather information from all experienced views and compile it into a single ‘holistic memory’, which can then be used to determine the most familiar heading at a given location. By applying this familiarity-based algorithm, the team was able to replicate behaviours observed in key behavioural studies and thus show that whatever information-processing architecture insects use for navigation, it does not require the tricky problem of learning, storing and using discrete snapshots sequentially.In the last of our inaugural Short Communication studies, Etsuro Ito and colleagues continue their analysis of the role of insulin-related peptides in memory formation. Using the ability of the pond snail Lymnaea to learn taste aversion and to consolidate this learning into long-term memory (conditioned taste aversion), the international team has previously shown that molluscan insulin-related peptides (MIPs) are upregulated in snails exhibiting conditioned taste aversion and that exogenous application of MIPs to the isolated central nervous system evokes long-term synaptic enhancement at the synaptic connection between the cerebral giant interneuron and the B1 buccal motor neuron – key neurons in memory formation. Following on from this work, Jun Murakami and co-workers (p. 1771) now show that the synaptic enhancement – hypothesized to be the neural correlate of behavioural memory – is due to changes on the postsynaptic B1 neuron, emphasizing that both postsynaptic and presynaptic changes needs to be considered when studying the neuronal basis of memory formation in invertebrates. In addition, the study demonstrates that the cognitive enhancements brought about by insulin can be studied at the level of single neurons in invertebrates, which may help to further understand the function of insulin in the more complex mammalian brain.Having launched this new article type for the communication of concise, self-contained studies, we look forward to receiving more submissions in this area. Further information about submitting a Short Communication to JEB can be found at http://jeb.biologists.org/site/author/article_types.xhtml#short.

Involvement of Insulin-Like Peptide in Long-Term Synaptic Plasticity and Long-Term Memory of the Pond SnailLymnaea stagnalis

The pond snail Lymnaea stagnalis is capable of learning taste aversion and consolidating this learning into long-term memory (LTM) that is called conditioned taste aversion (CTA). Previous studies showed that some molluscan insulin-related peptides (MIPs) were upregulated in snails exhibiting CTA. We thus hypothesized that MIPs play an important role in neurons underlying the CTA-LTM consolidation process. To examine this hypothesis, we first observed the distribution of MIP II, a major peptide of MIPs, and MIP receptor and determined the amounts of their mRNAs in the CNS. MIP II was only observed in the light green cells in the cerebral ganglia, but the MIP receptor was distributed throughout the entire CNS, including the buccal ganglia. Next, when we applied exogenous mammalian insulin, secretions from MIP-containing cells or partially purified MIPs, to the isolated CNS, we observed a long-term change in synaptic efficacy (i.e., enhancement) of the synaptic connection between the cerebral giant cell (a key interneuron for CTA) and the B1 motor neuron (a buccal motor neuron). This synaptic enhancement was blocked by application of an insulin receptor antibody to the isolated CNS. Finally, injection of the insulin receptor antibody into the snail before CTA training, while not blocking the acquisition of taste aversion learning, blocked the memory consolidation process; thus, LTM was not observed. These data suggest that MIPs trigger changes in synaptic connectivity that may be correlated with the consolidation of taste aversion learning into CTA-LTM in the Lymnaea CNS.

Paired pulse ratio analysis of insulin-induced synaptic plasticity in the snail brain

Insulin's action in the brain can directly alter cognitive functioning. We have recently shown that molluscan insulin-related peptides are upregulated following a conditioned taste aversion (CTA) training procedure. In addition, when mammalian insulin is superfused over the isolated Lymnaea stagnalis central nervous system, it elicits long-term synaptic enhancement at the monosynaptic connection between the cerebral giant cell and the buccal 1 (B1) motor neuron. This synaptic enhancement is thought to be a neural correlate of CTA. Here, we examined whether the observed changes in synaptic plasticity were the result of presynaptic and/or postsynaptic alterations using the paired pulse procedure. The paired pulse ratio was unaltered following insulin application, suggesting that insulin's effects on synaptic plasticity are mediated postsynaptically in the B1 motor neuron. Thus, it was suggested that postsynaptic changes need to be considered when insulin's actions on synaptic plasticity are examined.

Insulin and memory inLymnaea

The pond snail, Lymnaea stagnalis, is capable of learning conditioned taste aversion (CTA) and consolidating this CTA into long-term memory (LTM). The DNA microarray experiments showed that some of molluscan insulin-related peptides (MIPs) were up-regulated in snails exhibiting CTA-LTM. On the other hand, the electrophysiological experiments showed that application of secretions from the MIPs-containing cells evoked long-term potentiation (LTP) at the synapses between the cerebral giant cell (a key interneuron for CTA) and the B1 motoneuron (a buccal motoneuron). We thus hypothesized that MIPs and MIP receptors play an important role at the synapses, probably underlying the CTA-LTM consolidation process. To examine this hypothesis, we applied the antibody, which recognizes the binding site of mammalian insulin receptors and is thought to cross-react MIP receptors, to the Lymnaea CNS. Our present data showed that an application of the antibody for insulin receptors to the isolated CNS blocked LTP, and that an injection of the antibody into the Lymnaea abdominal cavity inhibited LTM consolidation, but not CTA formation.

Complement receptor 3-like immunoreactivity in the light green cells and the canopy cells of the pond snail, Lymnaea stagnalis

We observed CR3-like immunoreactivity in the central nervous system (CNS) and its surrounding peripheral nerves of the pond snail, Lymnaea stagnalis. In the CNS of L. stagnalis, the immunoreactivity presenting meshwork-like structure was detected in some neurosecretory cells, which are the light green cells (LGCs) and the canopy cells (CCs), both controlling the body growth. The immunoreactivity was also observed along the edges of median lip nerves. The immunoreactive regions in the median lip nerves appeared to form the axonal plates, from which the LGCs and the CCs release molluscan insulin-related peptides (MIPs) into the blood. By contrast, no immunoreactivity was detected in other neurosecretory cells or their release sites, for example the caudodorsal cells and the cerebral commissure, which release ovulation hormones. The present findings, therefore, suggested that CR3 expresses only in the neurosecretory cells releasing MIPs in L. stagnalis.

MIPs-containing cells in terrestrial snails: comparison of immunostaining and silver intensification

The cerebral ganglia neurons containing molluscan insulin-related peptides were studied in two species of terrestrial snails—Helix lucorum L. and Eobania vermiculata L. by means of immunostaining and sulfide-silver intensification. The neurons stained immunopositively with the antibody raised against the C-peptide of the molluscan insulin-related peptide were labelled by a sulfide-silver staining as well. It suggests the presence of an endogenous heavy metal in molluscan insulin-containing cells. Analytical procedures suggest the presence of zinc in the cells which is known to be involved in insulin release in mammals. Surprisingly, only immunopositive but not sulfide-silver stained cells were revealed in the brain of juvenile animals suggesting the presence of an age-related difference in the metabolism of molluscan insulins. © 1997 John Wiley & Sons, Ltd.

MIPs‐containing cells in terrestrial snails: comparison of immunostaining and silver intensification

The cerebral ganglia neurons containing molluscan insulin-related peptides were studied in two species of terrestrial snails—Helix lucorum L. and Eobania vermiculata L. by means of immunostaining and sulfide-silver intensification. The neurons stained immunopositively with the antibody raised against the C-peptide of the molluscan insulin-related peptide were labelled by a sulfide-silver staining as well. It suggests the presence of an endogenous heavy metal in molluscan insulin-containing cells. Analytical procedures suggest the presence of zinc in the cells which is known to be involved in insulin release in mammals. Surprisingly, only immunopositive but not sulfide-silver stained cells were revealed in the brain of juvenile animals suggesting the presence of an age-related difference in the metabolism of molluscan insulins. © 1997 John Wiley & Sons, Ltd.

Molecular sequences of two minisatellites in blacklip abalone, Haliotis rubra.

In the cloning and sequencing of growth-promoting genes of the blacklip abalone, Haliotis rubra (Leach, 1814), two DNA variable number of tandem repeats (VNTRs) were identified in abalone cDNA libraries. One contained a 33 bp repeat unit (5'-CCCAAGGTCCCCAAGGTCAGGGAGGCGAAGGCT-3') located in the 3' untranslated region of a putative growth hormone (GH) gene, and the repeat was designated as GHR. The other contained an 18 bp repeat unit (5'-ACCCGGCGCTTATTAGAG-3') located in the 3' untranslated region of a putative molluscan insulin-related peptides (MIP) gene, and was designated as MIPR. Primers flanking the two VNTR repeat regions were derived from sequence information. One hundred blacklip abalones were collected along the Victorian coastline and used in a preliminary population study. The range of GHR alleles containing the 33 bp basic unit repeat motif included 7 to 20 repeats, with allele GHR 8 not being identified. The most frequent alleles contained GHR 16 and 17 repeats (56.0% and 16.5%, respectively). Four types of alleles were identified in MIPR, viz 4, 5, 6 and 7 repeats. The alleles containing 6 and 5 repeats were the most frequent (50.0% and 41.5%, respectively). Overall, the results indicate that these two DNA minisatellites have use in abalone studies, including paternity resting, triploid testing, population genetic structure, and gene flow.

Expression and characterization of molluscan insulin-related peptide VII from the mollusc Lymnaea stagnalis.

A complementary DNA clone encoding molluscan insulin-related peptide VII was identified from a complementary DNA library of the cerebral ganglia of the CNS of the freshwater snail, Lymnaea stagnalis. The novel molluscan insulin-related peptide VII complementary DNA encodes a preprohormone resembling the organization of preproinsulin, with a putative signal sequence, and an A and B chain, and is connected by an unusual long C peptide. The A and B chains, as well as the C peptide of molluscan insulin-related peptide VII, differ remarkably in primary structure with the previously identified molluscan insulin-related peptides. The C peptide of molluscan insulin-related peptide VII shares no significant sequence identity with counterparts in other molluscan insulin-related peptides. Both molluscan insulin-related peptide VII and the other molluscan insulin-related peptides exhibit structural features which make them a unique class of the insulin superfamily. Molluscan insulin-related peptide VII complementary DNA was shown to hybridize in situ with messenger RNA present in the cerebral light green cells, neuroendocrine cells that control growth and that have previously been shown to produce molluscan insulin-related peptides I-III and V. Uniquely, the molluscan insulin-related peptide VII gene is also expressed in neurons that may form part of the feeding circuitry in Lymnaea, indicating that it may function as a neurotransmitter/neuromodulator.

Characterization of a putative molluscan insulin-related peptide receptor

In the pond snail Lymnaea stagnalis (Ls), growth and associated processes are likely to be controlled by a family of molluscan insulin-related peptides (MIP). Here we report on the cloning of a cDNA encoding a putative receptor for these MIP. This cDNA was isolated from Ls via PCR with degenerate oligodeoxynucleotides corresponding to conserved parts of the tyrosine kinase domain of the human insulin receptor and its Drosophila homologue. Many of the typical insulin-receptor features, including a cysteine-rich domain, a single transmembrane domain and a tyrosine-kinase domain are conserved in the predicted, 1607-amino acid (aa) protein. Comparison of the aa sequence of the molluscan receptor to other insulin-receptor sequences revealed strong variations in the percentage of sequence identity for the different domains, ranging from 70% sequence identity in the tyrosine-kinase domain to virtually no sequence identity in the C-terminal sequence. Striking differences are the absence of a clear tetrabasic cleavage site, and the extremely long C-terminus of 308 aa that contains seven Tyr residues. Southern blot analyses at varying stringencies, extensive screening of cDNA- and genomic libraries, and PCR experiments indicate the presence of a single putative MIP receptor. This suggests that the four different MIP may exert their functional role in Ls by binding to the same receptor.

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.

Differential expression of four genes encoding molluscan insulin-related peptides in the central nervous system of the pond snail Lymnaea stagnalis

In the pond snail Lymnaea stagnalis, the growth regulating system consists of (1) about 200 neuroendocrine light green cells, located in four clusters in the cerebral ganglia, and (2) the paired canopy cells, located in the lateral lobes. These cells express genes encoding the molluscan insulin-related peptides (MIPs). Six MIP genes have previously been identified. Four of these (I, II, III and V) are expressed in the light green cells and the canopy cells. The MIP-VI gene is a pseudogene. In the present in situ hybridization study, using oligonucleotide probes specific to the transcripts of MIP-I, -II, -III, -IV and -V, no signal was obtained with the MIP-IV probe, indicating that gene IV is also a pseudogene. With the other four probes, two types of light green cells were distinguished. Type-A cells express all four MIP genes, whereas type-B cells do not (or only faintly) express the MIP-I gene. Gene III is relatively strongly expressed in type-B cells. Genes II and V are moderately expressed in both cell types. Type-A cells are mainly located in the periphery of the clusters, whereas type-B cells are present in the center. The canopy cell resembles type-A light green cells. The expression levels of the MIP-II and MIP-V genes are low in the canopy cell. The expression pattern of the MIP genes correlates with the staining pattern of the anti-MIP-C antibody, which has been raised to a synthetic C-fragment shared by MIP-I, -II and -V. Type-A cells stain more intensely with the antibody than type-B cells.

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.

Demonstration of insulin-related substances in the central nervous systems of pulmonates and Aplysia californica

the occurrence of insulin-related substances in the central nervous system of pulmonates and Aplysia californica was investigated by means of immunocytochemistry and in situ hybridization. Previous experiments have shown that, in Lymnaea stagnalis, the growth hormone-producing neurons in the cerebral ganglia (the so-called light green cells) express at least 5 genes that are related to the vertebrate insulin genes, i.e., they encode prohormones that are composed of a B- and A-chain and a connecting C peptide. These insulin related molecules also have the amino acids essential for their tertiary structure (viz. cysteines) at identical positions to those of the vertebrate insulins. In the investigated basommatophoran and stylommatophoran snails and slugs, neurons reacted with an antiserum raised against the C peptide of one of the molluscan insulin-related peptides. These neurons can be considered to be, based on morphological and endocrinological criteria, homologous to the light green cells of L. stagnalis. In A. californica, all central ganglia contain immunoreactive neurons. The highest number (about 50) was observed in the abdominal ganglion. The present results indicate that insulin-related substances are generally occurring neuropeptides in the central nervous system of molluscs.

Ultrastructural evidence for synthesis, storage and release of insulin-related peptides in the central nervous system of Lymnaea stagnalis

The cerebral neuroendocrine Light Green Cells of the pulmonate snail Lymnaea stagnate, which control body growth and associated processes, stain positively with an affinity-purified antiserum raised to a large part of the C-chain of pro-molluscan insulin-related peptides. At the ultrastructural level, the rough endoplasmic reticulum is immunonegative, the Golgi apparatus is slightly positive and secretory granules in the process of budding from the Golgi apparatus are strongly positive. These observations indicate that the Light Green Cells synthesize molluscan insulin-related peptides, which are processed before packing by the Golgi apparatus into secretory granules. The two morphologically distinct secretory granule types, i.e. with pale and dark contents, respectively, are equally immunoreactive with antiserum raised to the C-chain of molluscan insulin-related peptides. Secretory granules within lysosomal structures reveal various degrees of immunoreactivity, indicating their graded breakdown. The Light Green Cells release secretory material by the process of exocytosis into the haemolymph from neurohaemal axon terminals located in the periphery of the median lip nerve. The electron-dense (tannic acid method) released contents are clearly immunopositive. The same holds for secretory granule contents released from Light Green Cells axon profiles in the centre of the lip nerve. Some immunoreactivity is also present in the intercellular space between these axon profiles. It is concluded that molluscan insulin-related peptides may act in two ways, namely (1) as neurohormones via the haemolymph at peripheral targets (2) in a non-synaptic (paracrine) fashion at targets within the central nervous system.