Nervous system of the snail Helix aspersa

Summary The fine structure of vascular channels and amebocytes associated with the sheath of the infraesophageal ganglion of Helix aspersa, is described. The extracellular stroma of the sheath, together with the hemocoel and blood vessels, forms an interconnected system of pathways which appears to be involved in the transport of metabolites, amebocytes, hemocyanin and experimentally introduced opaque tracers. The hemocoel, blood capillaries and precapillaries are lined by a discontinuous layer of single muscle cells whose luminal aspect is covered by a lamina of extracellular material named the vascular coat. This coat consists of a ground substance that forms a basement membrane and filamentous elements some of which are collagenous. Gaps in the blood vessel wall seem to provide the main routes for the movement of cells and large molecules to the hemocoel. Tracer experiments have given support to the idea that a diffusion barrier may be absent at the sheath-ganglion junction. Amebocytes have phagocytic properties; they appear associated in groups or scattered singly within the extracellular space of the sheath and the lumen of blood vessels. Single amebocytes have features of mobile cells and may function in the transport of hemocyanin as well as other proteins.This work has been supported by the Rockefeller Foundation and grants NB 06662 (from the U.S. Public Health Service) and N-105 (from Conicyt, Santiago, Chile). The continuous advice and encouragement of Drs. R. W. Guillery and D. B. Slautterback are gratefully acknowledged.     

Gastrin-cholecystokinin immunoreactivity in the central nervous system of Helix aspersa during rest and activity

The immunostaining pattern for the peptide gastrin/cholecystokinin 8 (gastrin/CCK8) in the molluscan central nervous system has been considered. The changes in the distribution of gastrin/CCK8 immunoreactivity were analyzed in the neurons of different areas of the cerebral ganglia (mesocerebrum and metacerebrum) and in the buccal ganglia of the terrestrial snail Helix aspersa, during rest and active phases. During the period of inactivity and after one day of activity, there were several immunoreactive neurons in the mesocerebrum and metacerebrum of the snails and in the buccal ganglia, whereas after 7 days of activity the number of labeled neurons decreased. Data suggested a storage of gastrin/CCK8 in the neurons when behavioral activities in which the peptide is involved (such as feeding-related behavior) are suppressed or reduced. The different percentage of gastrin/CCK8 immunoreactive neurons in the left and right mesocerebrum provides information about the activities controlled by these neurons, which could be related to the adaptive evolution and plasticity of the brain in terrestrial pulmonates.     

Distribution of alpha CDCP-immunoreactive neurons in the central nervous system of the snail Helix aspersa.

alpha CDCP is a neuropeptide produced by the caudodorsal cells of Lymnaea stagnalis and encoded by the genes of the egg-laying hormone (ELH). The use of a polyclonal antiserum raised against alpha CDCP resulted in the detection of about 800 immunoreactive neurons in the parietal ganglia and a small population (60 cells) in the cerebral ganglia of Helix aspersa. As the genes of ELH are well conserved among the gastropod species, these data designate the parietal ganglia as a putative source for the egg-laying hormone in Helix aspersa.     

D-Mannitol oxidation in the land snail, Helix aspersa

Respiration by mitochondrial preparations from hepatopancreas tissue of the land snail Helix aspersa is stimulated by D-mannitol. The rate of mannitol-stimulated respiration is approximately one-half that given by succinate, the most effective substrate thus far tested with these preparations. Mannitol-stimulated respiration is cyanide-insensitive but is not inhibited by salicylhydroxamate. The product of the membrane-bound mannitol-oxidizing activity was shown to be D-mannose by thin layer chromatography, high voltage electrophoresis of the germanate and borate complexes, gas chromatography of the trimethylsilyl derivative, low resolution mass spectrometry of the trimethylsilyl derivative, and by an enzymatic method dependent upon phosphomannose isomerase. The reaction mannitol + O2 leads to mannose is stoichiometric; however, it is not known whether O2 is the immediate electron acceptor. The activity in Helix mitochondria is thus unique among most alditol-oxidizing enzymes in not being pyridine nucleotide linked and in acting on carbon 1 rather than carbon 2.     

Characterization of yellow-pigmented and motile enterococci isolated from intestines of the garden snail Helix aspersa

AIMS: Enterococci associated with garden snails (Helix aspersa) were studied in order to obtain reliable species identification and characterization. METHODS AND RESULTS: Twelve yellow-pigmented and motile enterococci, isolated from the intestines of garden snails, were phenotypically close to Enterococcus casseliflavus, but they showed certain unusual biochemical characteristics. tRNA intergenic length polymorphism analysis (tDNA-PCR) divided all strains studied into two groups, in full agreement with biochemical test results. 16S rDNA sequencing, DNA base composition analysis and DNA-DNA hybridization results showed unambiguously that the enterococci studied belonged to the species Ent. casseliflavus. The representative strains of described ecovars were deposited in the Czech Collection of Microorganisms (CCM) as Ent. casseliflavus CCM 4868, 4869, 4870 and 4871. CONCLUSIONS: Enterococcus casseliflavus associated with garden snails can be subdivided into groups. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterococcus casseliflavus differs from other enterococcal species in that it is typically associated with plants, soil, water and invertebrate animals. The different groups that can be found in these widely occurring bacteria are possibly source-specific ecovars, as exemplified by the Ent. casseliflavus inhabiting the intestines of snails.     

Localization of a substance P-like material in the central and peripheral nervous system of the snail Helix aspersa

A monoclonal antibody against substance P was used for immunocytochemical staining of the central ganglia of the snail Helix aspersa and several peripheral tissues including the gut, reproductive system, cardiovascular system, tentacle and other muscles. Within the central ganglia many neurons, and many fibres in the neuropile and the nerves entering the ganglia, were stained for the SP-like material. The largest numbers of reactive cell bodies were in the pleural ganglia and on the dorsal surfaces of the pedal ganglia. A group of cells was also found, surrounding the right pedal-cerebral connective, that did not fluoresce, but were enveloped by reactive processes terminating directly onto the neurone somata. Specific staining was observed in all peripheral tissues examined and always appeared to be concentrated in nerve terminals. Most particularly these occurred in the heart and aorta, the pharyngeal retractor muscle and the tentacle. Although mostly present in muscular tissues, some fluorescence was also observed in the nervous layer surrounding the retina. The tentacular ganglion also contained immunoreactive cell bodies.     

Localization of a substance P-like material in the central and peripheral nervous system of the snail Helix aspersa

Summary A monoclonal antibody against substance P was used for immunocytochemical staining of the central ganglia of the snail Helix aspersa and several peripheral tissues including the gut, reproductive system, cardiovascular system, tentacle and other muscles.Within the central ganglia many neurones, and many fibres in the neuropile and the nerves entering the ganglia, were stained for the SP-like material. The largest numbers of reactive cell bodies were in the pleural ganglia and on the dorsal surfaces of the pedal ganglia. A group of cells was also found, surrounding the right pedal-cerebral connective, that did not fluoresce, but were enveloped by reactive processes terminating directly onto the neurone somata.Specific staining was observed in all peripheral tissues examined and always appeared to be concentrated in nerve terminals. Most particularly these occurred in the heart and aorta, the pharyngeal retractor muscle and the tentacle. Although mostly present in muscular tissues, some fluorescence was also observed in the nervous layer surrounding the retina. The tentacular ganglion also contained immunoreactive cell bodies.     

Unpredictable responses of garden snail (Helix aspersa) populations to climate change

We studied the impact of climate change on the population dynamics of the garden snail (Helix aspersa) in the Ecotron controlled environment facility. The experimental series ran for three plant generations, allowing the snails to reproduce. We investigated the isolated and combined effects of elevated CO₂ (current + 200 μmol mol^–1) and warming (current + 2ºC) in three consecutive runs (CO₂, Temperature and Combined). In the CO₂ Run, the number of juvenile snails recorded at the end of the experiment did not differ between ambient and elevated CO₂, whereas in the Temperature Run, fewer juveniles were found at elevated temperatures. An opposite response was observed in the Combined Run, where significantly more juveniles were found in elevated temperature and CO₂ compared to elevated CO₂ on its own. Within each run, juvenile emergence was not affected by treatments but juvenile presence was first observed about 70 days earlier in the Combined Run than in the Temperature Run. The differences in snail performance in the different runs were not correlated with differences in community structure or leaf quality measured as C:N ratios and neither with the abundance of the most preferred host plant species, Cardamine hirsuta. The abundance of this species, however, was significantly altered in all runs. The results illustrate clearly the degree of difficulty in making predictable generalisations about the consequences of climate change for certain species.     

Ontogenesis of the snail,Helix aspersa: Embryogenesis timetable and ontogenesis of GABA-like immunoreactive neurons in the central nervous system

Late stages of embryogenesis in the terrestrial snail Helix aspersa L. were studied and a developmental timetable was produced. The distribution of gamma-aminobutyric acid-like immunoreactive (GABA-ir) elements in the CNS of the snail was studied from embryos to adulthood in wholemounts. In adults, approximately 226 GABA-ir neurons were located in the buccal, cerebral and pedal ganglia. The population of GABA-ir cells included four pairs of buccal neurons, three neuronal clusters in the pedal ganglia, two clusters and six single neurons in the cerebral ganglia. GABA-ir fibers were observed in all ganglia and in some nerves. The first detected pair of GABA-ir cells in the embryos appeared in the buccal ganglia at about 63–64% of embryonic development. Five pairs of GABA-ir cell bodies were observed in the cerebral ganglia at about 64–65% of development. During the following 30% of development three more pairs of GABA-ir neurons were detected in the buccal ganglia and over fifteen cells were detected in each cerebral ganglion. At the stage of 70% of development, the first pair of GABA-ir neurons was found in the pedal ganglia. In the suboesophageal ganglion complex, GABA-ir fibers were first detected at about 90% of embryonic development. In the posthatching period, the quantity of GABA-ir neurons reached the adult status in four days in the cerebral ganglia, and in three weeks in the pedal ganglia. In juveniles, transient expression of GABA was found in the pedal ganglia (fourth cluster).     

Surfactant in the gas mantle of the snail Helix aspersa

Surfactant occurs in cyclically inflating and deflating, gas-holding structures of vertebrates to reduce the surface tension of the inner fluid lining, thereby preventing collapse and decreasing the work of inflation. Here we determined the presence of surfactant in material lavaged from the airspace in the gas mantle of the pulmonate snail Helix aspersa. Surfactant is characterized by the presence of disaturated phospholipid (DSP), especially disaturated phosphatidylcholine (PC), lavaged from the airspace, by the presence of lamellated osmiophilic bodies (LBs) in the airspaces and epithelial tissue, and by the ability of the lavage to reduce surface tension of fluid in a surface balance. Lavage had a DSP/phospholipid (PL) ratio of 0.085, compared to 0.011 in membranes, with the major PL being PC (45.3%). Cholesterol, the primary fluidizer for pulmonary surfactant, was similar in lavage and in lipids extracted from cell homogenates (cholesterol/PL: 0.04 and 0. 03, respectively). LBs were found in the tissues and airspaces. The surface activity of the lavage material is defined as the ability to reduce surface tension under compression to values much lower than that of water. In addition, surface-active lipids will vary surface tension, increasing it upon inspiration as the surface area expands. By these criteria, the surface activity of lavaged material was poor and most similar to that shown by pulmonary lavage of fish and toads. Snail surfactant displays structures, a biochemical PL profile, and biophysical properties similar to surfactant obtained from primitive fish, teleost swim bladders, the lung of the Dipnoan Neoceratodus forsteri, and the amphibian Bufo marinus. However, the cholesterol/PL and cholesterol/DSP ratios are more similar to the amphibian B. marinus than to the fish, and this similarity may indicate a crucial physicochemical relationship for these lipids.     

Nucleotidase activities in membrane preparations of nervous ganglia and digestive gland of the snail Helix aspersa

Nucleotide-metabolizing enzymes play an important role in the regulation of nucleotide levels. In the present report, we demonstrated an enzyme activity with different kinetic properties in membrane preparations of the nervous ganglia and digestive gland from Helix aspersa. ATPase and ADPase activities were dependent on Ca2+ and Mg2+ with pH optima approximately 7.2 and between 6.0 and 8.0 in digestive gland and nervous ganglia, respectively. The enzyme activities present in membrane preparations of these tissues preferentially hydrolyzed triphosphate nucleotides. In nervous ganglia, the enzyme was insensitive to the classical ATPases inhibitors. In contrast, in digestive gland, N-ethylmaleimide (NEM) produced 45% inhibition of Ca(2+)-ATP hydrolysis. Sodium azide, at 100 microM and 20 mM, inhibited Mg(2+)-ATP hydrolysis by 36% and 55% in digestive gland, respectively. The presence of nucleotide-metabolizing enzymes in these tissues may be important for the modulation of nucleotide and nucleoside levels, controlling their actions on specific purinoceptors in these species.     

The composition of intracellular granules from the metal-accumulating cells of the common garden snail (Helix aspersa).

Certain cells in the hepatopancreas of the common garden snail (Helix aspersa) contain intracellular granules that are sites of metal-ion accumulation. These granules have been extracted and investigated by u.v. and i.r. spectroscopy, atomic-absorption spectroscopy, X-ray microanalysis, thermogravimetric analysis, enzymic assay and microanalysis. The deposits contain about 18% (w/w) water, 5% (w/w) organic matter and 76% (w/w) inorganic material of which the main components are Ca2+, Mg2+ and P2O7(4)-. The possible origin of these granules is discussed, as is their role in detoxifying heavy-metal ions.     

Isolation of nematode inhibitor from hemolymph of the snail, Helix aspersa

Hemolymph plasma of the snail Helix aspersa which inhibits maturation and reproduction of its mantle cavity-inhabiting nematode, Rhabditis maupasi, was separated biochemically for the active proteinaceous component. Isolation of the active inhibitor was performed using ion-exchange chromatography in combination with subsequent gel filtration. The isolated peaks were bio-assayed in vitro on nematode larvae. The fractions harboring inhibitory protein suppressed larval growth and adult reproduction in vitro. The isolated fraction was purified by gel filtration and characterized on the basis of a single band on starch zone electrophoresis and positive reaction only with folin-phenol reagent.     

Transport of pinocytic vesicles in the eye of a snail,Helix aspersa

The heads of small adult snails, Helix aspersa, were injected with horseradish peroxidase (HRP) for one to five hours before extirpating the eyes and preparing them cytochemically for electron microscopy. There was internalization of tracer by pinocytic vesicles (pinosomes) at the bases of types-I and -II sensory cells, ganglion cells and, in lesser amounts, by pigmented supportive cells. Vesicles and vacuoles filled with HRP were transported in two directions: lensward as far distad as the ends of the cells (retrograde) and brainward down the optic nerve (anterograde). We believe that the numerous reacted vacuoles in the cell somata are formed by fusion of vesicles, tubules and C-shaped organelles filled with tracer; we present evidence that they become secondary lysosomes. Sensory cell type II possesses more HRP-reacted vacuoles distally than the other retinal cells. Other vesicles are also described. There was no uptake of tracer by the distal ends of the retinal cells following injection HRP into the hemolymph. The swelling of the optic nerve, immediately behind the eye, contains more HRP-filled pinosomes and vacuoles than does the nerve below the dilatation. The significance of endocytosis and transport of pinosomes within the eye and down the optic nerve is discussed.