J. Joosse

Trichobilharzia ocellata: Physiological characterization of giant growth, glycogen depletion, and absence of reproductive activity in the intermediate snail host, Lymnaea stagnalis

Giant growth, depletion of energy stores, and inhibition of reproductive activity are striking effects of many trematode parasites on their intermediate snail hosts. Two hypotheses have been put forward to explain these phenomena: (1) host and parasite compete for energy rich and other essential nutrients, with the parasite as the winner, and (2) the parasite intervenes in the endocrine control of reproduction of the snail. These hypotheses were tested in the present study with the Trichobilharzia ocellata/Lymnaea stagnalis association. The snails were infected at a juvenile stage, and release of cercariae started on Day 55 after exposure. It was shown that enhanced growth of infected snails is not paralleled by a greater increase in dry weight, but hemolymph volume does increase, being 35% greater than in the noninfected controls. Control snails, on the other hand, showed an increase in the percentage body dry weight during sexual maturation. The conclusion is that infected snails retain an essentially juvenile body structure. In control snails, glycogen was depleted from the mantle store at the start of egg laying but the onset of cercariae production marked a severe glycogen depletion from the headfoot and the mantle in infected snails, being nearly complete on Day 68 after exposure. The hemolymph glucose concentration was only slightly lower in infected than in control snails and it did not change (in both groups) during glycogen mobilization. This suggests that glycogen mobilization does not result from the snail and the parasite competing directly for metabolites within the hemolymph. Infection inhibited the maturation of the accessory sex organs: there was no increase in the relative wet weights nor in the amounts of DNA and secretion products in the albumin and prostate glands. Infected snails did not lay eggs. It is presumed that the parasite produces one or more agents which intervene in the action of the gonadotrophic hormones. The release of these agents commences at an early stage of infection.

Fixation increases sensitivity of India ink staining of proteins and peptides on nitrocellulose paper

The detectability by India ink staining of proteins and peptides dot-blotted on nitrocellulose paper was assessed before and after fixation. Fixation considerably increased the detectability of proteins and peptides. Denaturation by KOH treatment or baking at 100 degrees C for 15 min gave the best results. Precipitation by isopropanol/acetic acid gave intermediate results, whereas crosslinking with glutaraldehyde improved the detectability of small peptides, but not of proteins. Ferridye and Aurodye were also tested after baking. Both dyes were more sensitive and stained more proteins and peptides than India ink. In all cases the detectability of peptides smaller than Mr 1500 was poor.

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.

Molecular properties of various snail peptides from brain and gut

Attention is focused on the similarities in primary structure of the egg-laying neurohormone of the pulmonate Lymnaea stagnalis and of the opisthobranch Aplysia californica which both consist of 36 amino acid residues. FMRFamide-like peptides have now been isolated and sequenced from six molluscan species. Besides FMRFamide, two closely related peptides were isolated from the central nervous system of L. stagnalis and sequenced. This indicates that a family of FMRFamide-like peptides exist not only in the molluscs, but also within one species. A molluscan growth hormone, isolated from the brain of L. stagnalis, has been characterized. This small peptide hormone stimulates in vitro a receptor-adenylate cyclase system of mantle edge cells and in vivo the Ca2+-incorporation in the shell edge. The biochemical characterization of three vertebrate-like peptides of L. stagnalis, resembling oxytocin, Arg-vasopressin, and insulin, confirms the immunological findings that gastropods contain peptides which are structurally closely related to mammalian peptides.

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.

Schistosomin: a pronase-sensitive agent in the hemolymph of Trichobilharzia ocellata-infected Lymnaea stagnalis inhibits the activity of albumen glands in vitro

The schistosome parasite, Trichobilharzia ocellata, nearly completely inhibits the reproductive activity of its intermediate host, Lymnaea stagnalis. The synthetic activity of albumen glands of infected snails at day 35 postinfection (p.i.) is only 1% of the control value. The parasite acts by humoral means. We tested the hypothesis that (a) specific humoral agent(s) is (are) involved and refer to this (these) agent(s) as schistosomin. The presence of schistosomin in the hemolymph of infected snails was investigated by using galactogen synthesis in albumen glands as an in vitro bioassay. The synthetic activity of albumen glands of noninfected snails decreased by about 50% during a 1-h incubation in the hemolymph of infected snails. This inhibition is attributed to schistosomin. Based on these results, with the present bioassay schistosomin appears in the hemolymph between days 28–36 p.i. onwards. Schistosomin is heat-stable (100‡ C) and pronase-sensitive, and therefore it might have a peptide nature. Schistosomin suppresses the stimulating action of the female, gonadotrophic dorsal body hormone at relatively low doses, which suggests that it may compete with this hormone for the same receptors. The development of two other bioassays for schistosomin in our laboratory is discussed.

The molecular basis of the neuro-endocrine control of egg-laying behaviour in Lymnaea

The biosynthesis, axonal transport and release of multiple peptides by the egg-laying controlling caudodorsal cells (CDC) of Lymnaea stagnalis were studied. High performance gel permeation chromatography was used to resolve newly synthesized peptides after pulse-chase experiments with radioactive amino acids. The precursor is a 35 kd polypeptide which is produced in the CDC somata. It gives rise to intermediate products (20 kd, 10 kd and 7 kd) and a number of end products which include a approximately 4.5 kd (the ovulation hormone, CDCH), a 1.5 kd peptide (the autotransmitter) and other peptides (6 kd, 3.5 kd and 2 kd). The end products are transported in neurosecretory granules to the CDC axon terminals in the cerebral commissure where they are released into the medium during electrical discharges of the CDC system. A cDNA clone encoding part of the CDCH precursor was isolated from a Lymnaea CNS cDNA library by differential hybridisation and use of synthetic oligonucleotide probes. Examination of the deduced amino acid sequence indicates that the precursor contains additional peptides, besides CDCH.

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.

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.

Neuroendocrine control of glycogen mobilization in the freshwater snail Lymnaea stagnalis

In the freshwater snail Lymnaea stagnalis the anterior mantle region, which mainly consists of large numbers of special glycogen-storing cells, is an important depot for the energy reserves of this snail. In organ culture experiments the central nervous system (CNS), in contrast to other tissues, both inhibits glycogen synthesis (measured as incorporation of [14C]glucose into glycogen) and stimulates glycogen breakdown in anterior mantle tissue (measured as a decreased retention of prelabeled glycogen). These effects are dose dependent, with saturation at the highest doses tested. High-potassium Ringer solution stimulates the secretion of a CNS factor which induces glucose release by anterior mantle tissue. This glucose-release-stimulating effect of the CNS is also dose dependent, but saturation of the response was not achieved. It is concluded that inhibition of glycogen synthesis and stimulation of glycogen breakdown and glucose release are probably effects of a single neurohormone which controls glycogen mobilization from the storage cells in the mantle. Like similar factors in other animal phyla, this putative neurohormone is referred to as a hyperglycemic factor.