Germinal and non-germinal lines in the ovotestis of Helix aspersa: a survey

Summary The study of gonadal organogenesis and differentiation by means of light and electron microscopy suggested the following in Helix aspersa: (1) the distal parts of the acini have components of mesodermal origin, whereas the neck and efferent duct comprise ectodermal elements; (2) a segregation of a germinal line occurs early, during the embryonic life; (3) in juvenile and adult animals, male and female cells arise from a germinal ring located at the base of the acinar neck. Apart from developing oocytes, the epithelium lining the distal region of the acini consists of somatic cells (Sertoli and follicle cells).     

On orderly domains of particles associated with cytomembranes during spermatogenesis in Helix aspersa

Summary In an electron microscopic study of the maturation of the spermatid of the snail Helix aspersa, it was found that: 1. there is persistence and utilization of the mitochondrial architecture in the formation of the middle piece, and 2. particles originate in orderly domains within the pseudomatrix of the mitochondria. Other orderly domains of cytomembranes (Golgi system and peripheral tubules of Grassé) are discussed.Dedicated to Professor Friedrich Wassermann with admiration and affection on the occasion of his eightieth birthday.Work supported by U.S. Atomic Energy Commission.     

Surface specializations of the epithelial cells at the tip of the optic tentacle,dorsal surface of the head and ventral surface of the foot in Helix aspersa

Summary Morphologically the surface specializations of the epithelium covering the dorsal head and ventral foot regions in Helix aspersa consists either of cilia or microvilli respectively. The epithelium at the tip of the optic tentacle is a simple one. Each epithelial cell has a number of cilia-like projections from their free surfaces. These projections usually branch at their tips into two or three slender, microvilli-like structures. From the bases of the cilia-like projections arise numerous, tubular processes which form a thick, spongy layer interspersed between these projections. The microvilli-like structures are immersed in a fine, fibrous mat; unlike the fibrous mats on the dorsal head and ventral foot epithelia this material does not autofluoresce. It is suggested that it arises from the collar cells and not from typical mucocytes. The functional relationship between these surface specializations of the optic tentacle epithelium and the abundance of sensory axons in this region is discussed. These epithelial cell projections on the tentacle probably function not only as a protective covering but also to create a fluid trap for odours in the ambient air. The various contacts between epithelial cells serve to maintain the integrity of the epithelium while allowing for stretching due to protrusion of the tentacle.This work has been supported by the Australian Research Grants Committee.     

Two types of plasticity in the tentacle withdrawal reflex of Helix aspersa are dissociated by tissue location and response measure

The tentacle withdrawal reflex of the terrestrial snail Helix aspersa was studied in vitro. The reflex is evoked by mechanical stimulation of the nose. Lesion experiments showed that 45% to 75% of the response amplitude is attributable to peripheral pathways alone. The central contribution increases with increasing stimulus intensity.Repeated stimulation produced pure habituation at low stimulus strengths, and habituation mixed with intrinsic sensitization (warm-up effect) at high stimulus strengths. The simultaneous occurrence of habituation and sensitization is consistent with the dual process theory of plasticity. Additional results differentiate the two processes. Habituation can occur without the CNS, whereas intrinsic sensitization requires the CNS. Also, the two processes are differentially effective in their influences on response amplitude and duration: habituation is more effective in determining response amplitude, while sensitization is more effective in determining response duration.Although the establishment of sensitization requires the CNS, 81% of the memory for intrinsic sensitization was localized to the periphery, by lesion experiments. Extrinsic sensitization, caused by stimulation of the medial lip nerve, had similar behavioural effects and a similar memory locus. Both types of sensitization appear to be caused by neuromuscular facilitation mediated by a central pathway.Abbreviations CNS central nervous system - PNS peripheral nervous system - S-R stimulus-response - TRM tentacle retractor muscle     

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.     

Electroretinographic and ultrastructural study of the regenerated eye of the snail cryptomphallus aspersa

The electroretinographic responses of regenerated eyes of snails (C. aspersa) were studied by means of suction electrodes and single or repetitive flash stimulation. The eyes were fixed and observed under light and electron microscopy. The results indicate that the electroretinographic response of regenerated eyes does not differ from control eyes after dark adaptation. However, the repetitive stimulation of the regenerated eyes induced an earlier fatigue of the response, evident after the fifth stimulus. This fatigue is a function of light intensity. Ultrastructural features of the regenerated eyes are similar to those of the control eyes but regenerated eyes show smaller rhabdomeres, fewer photic vesicles, and fewer paracrystalline bodies. It is suggested that the regenerated eye lability to the repetitive stimulation might be due to the decrease in the amount of photic vesicles and paracrystalline bodies, to the decrease in membrane surface of the rhabdomeres, or to some other metabolic failure not distinguished at electron microscopic level, like the membrane ionic regulation. The appearance of photic vesicles in the axon cones of regenerated and stimulated cells also suggests a possible failure in the mechanism of transport of vesicles.     

Electron microscopic study of the optic tentacle collar cells of the snail Helix aspersa and immunolocalization of methionine enkephalin-like material in their granular content

Summary— An ultrastructural and immunocytological study was carried out on the collar cells of the optic tentacle of Helix aspersa. These cells are supposed to be the source of a reproduction controlling hormone. The immunocytological study was performed using an anti-methionine enkephalin antibody obtained from rabbits in our laboratory. The collar cells are characterized by an enlarged rough endoplasmic reticulum, numerous mitochondria and Golgi bodies surrounded by secretory vesicles, suggesting an intense synthesizing activity. Their principal feature consists of numerous various-sized granules where methionine enkephalin immunoreactivity is localized. No classical neurosecretory granules are observed while synapse-like structures are often encountered. The cells should not be regarded as neurosecretory cells but rather as glandular cells which could ensure different functions, one in relation to reproduction, and another in relation to perception processes, particularly as they contain methionine enkephalin-like material.     

Ontogeny of the fused tentacles in three species of ommastrephid squids (Cephalopoda, Ommastrephidae)

Abstract. The tentacles of ommastrephid squids fuse during embryonic development and remain fused as they grow through hatching, but eventually separate to become two fully functional adult tentacles. The external anatomy of individuals at several post‐hatching ontogenetic stages of three species of ommastrephid squids (Ommastrephes bartramii, Sthenoteuthis oualaniensis, and Hyaloteuthis pelagica) was examined using scanning electron microscopy and morphometrics. The fusion of the transverse muscle mass of the tentacles was examined using light microscopy. Five ontogenetic stages of tentacle separation were defined based on landmark features such as the extent of the fusion and the presence of suckers or sucker buds at the distal tip. The total tentacle length and fused tentacle length reached a maximum when the dorsal mantle length (ML) equaled 3–4 mm (H. pelagica) or 4–6 mm (O. bartramii, S. oualaniensis), and then decreased with increasing ML. The average split length (measured from the base of the tentacles to the point of tentacle fusion) increased gradually with increasing ML, and the separate tentacle diameter was roughly half the diameter of the fused portion at all sizes. In all three species, separation of the fused tentacles began earlier in development (2–3‐mm ML) and was more advanced at smaller sizes than previously reported. The sizes presented here are conservative because excess epithelium at the location of the split may disguise the actual site of separation. Post‐separation tentacles were much shorter than the arms, and the carpal region appeared torn in 2 of the 4 specimens of S. oualaniensis examined. Finally, none of the original distal tip suckers were retained on the post‐separation tentacles of S. oualaniensis. These observations are consistent with the hypothesis that the tentacles separate gradually then rupture at the “wrist” (presumptive carpus), and argue against the possibility of prey capture by the fused tentacles.     

Investigations of the Diurnal Activity of the Endocrine Dorsal bodies in Helix aspersa

Summary

The activity of the endocrine dorsal bodies (DB) of the adult land snail Helix aspersa living in the field shows striking fluctuations during a 24 hr cycle. Quantitative electron microscopical data revealed that the number of secretory granules, the volume of the Golgi apparatus and the number of Golgi saccules containing electron-dense material were maximal at 1 am and minimal at 1 pm. The use of tannic acid indicated the exocytosis of secretory material was intense around 1 pm and only moderate at 1 am. The results suggest that, under natural conditions, the DB have a diurnal rhythm of activity, packaging secretory material into secretory granules mainly during the night and releasing it during the afternoon.     

Immunocytochemical distribution of neuropeptide F (NPF) in the gastropod mollusc,Helix aspersa,and in several other invertebrates

The distribution of neuropeptide F (NPF) immunoreactivity in the snail, Helix aspersa, has been demonstrated by immunocytochemistry using 2 regionspecific antisera. One, designated NPF3, was raised against a synthetic N-terminal fragment of Helix aspersa NPF; the other, designated PP221, was raised against the C-terminal hexapeptide amide of mammalian pancreatic polypeptide (PP) but cross-reacts fully with the analogous C-terminal region of Helix aspersa NPF. The distribution of NPF immunoreactivity has also been compared with that of FMRFamide using alternate serial sections of Helix aspersa ganglia. Results showed that NPF immunoreactivity was abundant and widespread in the central and peripheral nervous systems and the pattern of immunostaining obtained using both region-specific antisera was similar. Likewise, immunocytochemistry of neural tissues of a congeneric species, Helix pomatia, and 2 prosobranch gastropods, Buccinum undatum and Littorina littorea, produced similar staining patterns with both antisera. However, in the cephalopod mollusc, Loligo vulgaris, and the cestode, Moniezia expansa, positive immunostaining was only obtained with the C-terminal PP antiserum. Immunostaining of alternate serial sections of Helix aspersa ganglia with NPF3, and an antiserum raised to FMRFamide, showed that while a few neurones were immunoreactive with one antiserum only, in the majority, both immunoreactivities were co-localised. NPF thus appears to be an important neuropeptide of widespread distribution in Helix aspersa and the differential immunocytochemical staining obtained using the 2 region-specific antisera would suggest a high degree of primary structural conservation within the gastropod molluscs, but lack of conservation of the N-terminal region of the peptide in other invertebrate groups.     

Pro-Angiogenetic Effects of Purified Extracts from Helix aspersa during Zebrafish Development

Helix aspersa is a species of land snail belonging to the Helicidae family, widespread in the Mediterranean and continental area up to Northern Europe. In some areas it is appreciated as a food, but is mostly considered a parasite of gardens and cultivated fields. The mucus of Helix aspersa has found multiple applications in the cosmetic and health fields. In the present study, we investigated for the first time the angiogenetic properties of purified extracts from Helix aspersa using a transgenic zebrafish line Tg (kdrl:EGFP). The angiogenesis induced by purified snail extracts was demonstrated by their capability to increase the three well-established parameters of angiogenesis: generation of intersegmental vessels, modeling of caudal venous plexus, and formation of sub-intestinal venous plexus. The effects appeared to be mediated by the vascular endothelial growth factor (VEGF) pathway, being prevented by pretreatment of embryos with the selective VEGF receptor antagonist SU5416, and supported by the increased VEGF mRNA levels found in snail-extract-treated embryos. Insufficient vascular supply is underlined by low VEGF signaling, primarily because of its indispensable role in preventing capillary loss. Our findings might have a pharmacological impact by counteracting VEGF hypofunction and promoting angiogenesis to maintain adequate microvascular and vascular density in normal and suffering tissues and organs.     

Electron microscopic study of the optic tentacle collar cells of the snail Helix aspersa and immunolocalization of methionine enkephalin-like material in their granular content

An ultrastructural and immunocytological study was carried out on the collar cells of the optic tentacle of Helix aspersa. These cells are supposed to be the source of a reproduction controlling hormone. The immunocytological study was performed using an anti-methionine enkephalin antibody obtained from rabbits in our laboratory. The collar cells are characterized by an enlarged rough endoplasmic reticulum, numerous mitochondria and Golgi bodies surrounded by secretory vesicles, suggesting an intense synthesizing activity. Their principal feature consists of numerous various-sized granules where methionine enkephalin immunoreactivity is localized. No classical neurosecretory granules are observed while synapse-like structures are often encountered. The cells should not be regarded as neurosecretory cells but rather as glandular cells which could ensure different functions, one in relation to reproduction, and another in relation to perception processes, particularly as they contain methionine enkephalin-like material.     

EFFECTS OF WOUND HEALING ON ZINC DISTRIBUTION AND ALKALINE PHOSPHATASE ACTIVITY OF HELIX ASPERSA (GASTROPODA: PULMONATA)

Helix aspersa were loaded with ⁶⁵zinc and after 2 and 5 daysstarvation there was no significant reduction in the zinc concentrationof the digestive gland and shell. Wounding of the optic tentacle,foot tissue and partial shell removal of H. aspersa resultedin deposition of ⁶⁵zinc in the wound area. Alkaline phosphataseshowed no increase in concentration at the sites of wounding. (Received 15 October 1985;     

Fertilization and normal development inAscidiella aspersa (Tunicata) studied with Nomarski-optics

Normal development ofAscidiella aspersa was studied over a period of approx. 24 h at 20°C from egg insemination through metamorphosis of the tadpole larva using Nomarski-optics. Records were made of spermatozoa attaching to and passing through the cellular envelopes and the chorion of the egg. Egg shape alterations upon entry of the fertilizing sperm, which reflect the early phase of ooplasmic segregation, were monitored in intact and dechorionated eggs. The time course of normal development was recorded, and prominent stages were photographed within or deprived of the egg envelopes. The present observations are compared with recent accounts on early development in other solitary ascidian species.     

Tentacle development in dermophis mexicanus (amphibia,gymnophiona) with an hypothesis of tentacle origin

The development of the tentacle, a chemosensory and perhaps tactile structure unique among vertebrates to gymnophione amphibians is described in Dermophis mexicanus and Gymnopis multiplicata. The tentacle is associated with the vomeronasal organ and its glands, and utilizes several structures usually associated with the eye, such as the Harderian gland, the retractor and levator muscles, and their nerves. Innervation of the tentacle itself is from the trigeminal nerve. We present an hypothesis that the tentacle originated from modified eye components.     

Partial Characterization of a Novel Cardioinhibitory Peptide from the Brain of the Snail Helix aspersa

1. We report the isolation of a peptide from the brain of the snail Helix aspersa by radioimmunoassay using an antisomatostatin.2. The sequencing of an immunopositive fraction showed the presence of a new tridecapeptide, termed Helix cardioinhibitory peptide (HCIP), with the following primary structure : H-Val-Phe-Gln-Asn-Gln-Phe-Lys-Gly-Ile-Gln-Gly-Arg-Phe-NH₂. It is structurally related to the Achatina cardioexcitatory peptide (ACEP-1) and the terminal-ammo acid sequence of HCIP is identical to that of FMRFamide family peptides.3. The synthetic HCIP was tested on heart and neuronal activities and it was found to have inhibitory actions not only on the ventricle but also on visceral neurons of the central nervous system of Helix. Immunocytochemical investigation indicates its presence in visceral and parietal ganglia, in which cells taking part in the regulation of the heartbeat have been previously identified .     

Infections of Larval Stages of Dicrocoelium dendriticum and Brachylaima sp. in Brown Garden Snail,Helix aspersa,in Turkey

The aim of this study was to determine the presence and prevalence of larval stages of Dicrocoelium dendriticum and Brachylaima sp. in the first intermediate host, a species of land snail, Helix aspersa, in Turkey. A total of 211 snails were collected in April-May 2014 from pastures in Mersin District. Larval stages of D. dendriticum were identified under a light microscope. Hepatopancreas from naturally infected H. aspersa snails were examined histologically. The prevalence of larval stages of D. dendriticum and Brachylaima sp. in H. aspersa snails was found to be 2.4% and 1.9%, respectively, in Mersin, Turkey. Cercariae were not matured in sporocysts at the beginning of April; however, it was observed that cercariae matured and started to leave sporocysts by early-May. Thus, it was concluded that H. aspersa acts as an intermediate host to D. dendriticumin and Brachylaima sp. in Mersin, Turkey. A digenean trematode Brachylaima sp. was seen for the first time in Turkey.     

Presence of cells and fibers immunoreactive toward antibodies to different peptides or amine in the digestive tract of the snailHelix aspersa

An immunocytological study of four different parts of the gut of Helix aspersa clearly demonstrates the presence of many cells and fibers immunoreactive toward antibodies directed to vertebrate (α, β-endorphin, α, β-MSH, ACTH 1–24 and ACTH 17–39, met-enkephalin, somatostatin, insulin, glucagon, P.P., serotonin) or invertebrate (FMRF-amide) peptides. These results are evidence of the presence of different substances related to known peptides or amines in the epithelial and connective tissue cells and nerve fibers of the snail gut. Immunocytochemistry may help to elucidate the morpho-functional characteristics of the enteroendocrine cells of H. aspersa.     

Effectors of metabolic depression in an estivating pulmonate snail (Helix aspersa): whole animal and in vitro tissue studies

 We have examined metabolic depression in the land snail (Helix aspersa) during estivation, and have developed a tissue model of metabolic depression using an in vitro mantle preparation. The metabolic rate of H. aspersa is depressed by 84% in vivo within 4 weeks of onset of estivation, and this metabolic depression is accompanied by a decrease in haemolymph PO₂ and pH, and an increase in haemolymph PCO₂. The in vitro mantle preparation has a stable O₂ consumption and energy charge, and an energy charge similar to that of mantle in vivo. The in vitro mantle is an O₂-conforming tissue, with VO₂ varying curvilinearly with PO₂. Consequently, we have developed a mathematical method of calculating tissue VO₂ at any PO₂. These calculations show that under appropriate incubation conditions of pH and PO₂, the mantle from estivating animals shows a stable in vitro metabolic depression of 48% compared to mantle from control snails. The extrinsic effects of PO₂ and pH account for 70% of the total in vitro metabolic depression of mantle tissue; intrinsic effectors contribute a further 30%. Accepted April 26, 1996     

The early stages of ommatidial development in the flour beetle Tribolium castaneum (Coleoptera; Tenebrionidae)

 Using electron microscopy, the first stages of ommatidial development in the flour beetle Tribolium castaneum were analysed in relation to the cellular architecture of the adult compound eye and were compared to the corresponding patterning process in the fruit fly Drosophila melanogaster. The ommatidia of the slightly horse-shoe shaped beetle compound eye contain six peripheral and two central retinula cells. The rhabdomere of the posteriorly located central photoreceptor cell is restricted to the distal half of the rhabdom whilst that of the anterior one is restricted to its proximal half. The development of the compound eye takes place in an external eye imaginal disc. Most stages of ommatidial development, as known from Drosophila, i.e. arc-like cell groups, five-cell clusters, immature eight-cell clusters and symmetrical eight-cell clusters, are very precisely conserved between the two species. Two major differences exist: 1. In Tribolium, the cone cell precursor cells synchronously join to the immature eight-cell cluster. As a consequence, the symmetrical eight-cell cluster immediately transforms into a four-cone-cell cluster. 2. The maturing ommatidia do not undergo rotation in Tribolium. Overall, no morphological indiation for an equator in the adult Tribolium compound eye could be found. Considering the strong evolutionary conservation of early ommatidial development, homology of photoreceptor cells of distantly related insects is proposed to be inferred from their ontogenetic origin. Received: 6 November 1995 / Accepted: 9 April 1996