Fine structure of isolated fibrils from the cuticle of Lumbricus sp.

A method has been developed to isolate and purify cuticular fibrils of Lumbricus. Polarizing microscopy confirms the collagenous nature of the isolated fibrils. Study in the electron microscope of isolated fibrils, negatively or positively stained, shows that they are cylindrical, unbranching and without periodic structure. Enzymatic treatment of cuticles with alpha-amylase and trypsin results in a more or less complete dissociation of the fibrils which appear clearly to be made up of helically wound bundles of filaments (30-40 A). The biophysical data and compared to the ultrastructural organization of other periodically cross-banded fibrils.     

Ultrastructural localization of monoamines in the epidermis of Lumbricus terrestris (L.)

Summary Receptor cells in the epithelium and the basiepithelial nerve net of the prostomium of Lumbricus terrestris were investigated with electron microscope with special regard to the presence of monoamines. The receptor cells are found in groups of about 40 intermingled with supportive cells. After pretreatment with -methyl-noradrenaline and fixation with potassium permanganate a few receptor cells in each group and some nerve fibres in the basiepithelial nerve net contain small granular vesicles (about400 Å) characteristic for monoaminergic neurons. The distribution and relative number of these receptor cells and nerve fibres coincide well with previous reports on fluorescent receptor cells and varicose fibres. That the monoamine-storing small granular vesicles not are visualized until pretreatment with -methyl-noradrenaline is in accordance with recent microspectrofluorometric analysis, which shows that dopamine is the only primary monoamine present in the epithelium.In the epithelium there are occasional receptor cells and nerve fibres containing large vesicles (1000–1800 Å) which resemble the neurosecretory vesicles in the central nervous system. Photoreceptor cells having an intracellular cavity with microvilli and cilia have infrequently been observed at the base of the epithelium.No synapses on the mucous cells have been noticed. Nor have any synaptic specializations been observed in the basiepithelial nerve net. The morphological conditions necessary for the existence of possible axo-axonal synapses are briefly discussed.This work was supported by grants from the Helge Ax: son Johnson Foundation and the Magn. Bergvall Foundation.     

Histochemical localization of production and secretion of trypsin in the earthworm Lumbricus terrestris

Summary The origin of trypsin in the digestive tract of Lumbricus terrestris was localized by using the chromogenic tryptic substrate carbobenzyloxy-L-arginine--napthylamide HCl (CANA) coupled with the azo dye Fast Garnet GBC. The specificity of earthworm trypsin toward the naphthylamide substrate was confirmed by disc gel electrophoresis of homogenates of the digestive tract and of intestinal fluid. Eluted fractions were assayed for tryptic activity using the synthetic substrates benzoyl-DL-arginine-p-nitroanilide (BAPA) and p-toluenesulfonyl-L-arginine methyl ester (TAME). The peak of activity toward these substrates corresponded in electrophoretic mobility to the band of CANA activity and all activities were abolished in the presence of the tryptic inhibitor N-tosyl-L-lysyl chloromethane HCl (TLCK).In frozen sections of the pharynx, esophagus, crop, gizzard and intestine, tryptic (CANA) activity was localized consistently only in the ventral and lateral fold epithelium of the pharynx. The chromogenic reaction was completely inhibited by pre-incubation of frozen sections with TLCK.Tissues from three regions of the pharynx were fixed and studied in an attempt to correlate electron microscopic observations with the histochemical results. Whereas mucousproducing cells are generally distributed in pharyngeal epithelium, heterogeneous spherical granules were found only in the ventral and lateral fold epithelium. Observations concerning the spherical secretory granules closely paralleled those of the histochemical reaction product suggesting that the spherical secretory granules may contain the tryptic enzyme.The authors wish to thank Ruth D. Merz and Sandra K. Elson for their technical assistance. The investigation was supported in part by funds to the Instrumentation Laboratories and from the Faculty Research Committee of Miami University.     

Ultrastructural localization of monoamines in the central nervous system of Lumbricus terrestris (L.) with remarks on neurosecretory vesicles

Summary The cerebral ganglion and the ventral nerve cord of Lumbricus terrestris have been studied with the electron microscope. The results are as follows: In the neuropile small granular vesicles (300 to 500 Å) occur in some varicose nerve fibres after fixation with potassium permanganate. This indicates the presence of noradrenaline. Sometimes only a few of the vesicles produce a positive reaction. After incubation with -methyl-noradrenaline the numbers of nerve terminals with small granular vesicles greatly increase, indicating the presence of dopamine and/or 5-hydroxytryptamine. In this case the reaction is now complete. The number of small granular vesicles is largest in the terminal swellings.These findings are consistent with histofluorescence, chemical, and microspectrofluorometric analyses, which have demonstrated noradrenaline, dopamine, and 5-hydroxytryptamine in neurones in the central nervous system.Large granular vesicles (600 to 900 Å) are to be found in some perikarya, not identical with neurosecretory cell bodies. In this case the granular vesicles in the axon are smaller and fewer. This indicates a simultaneous proximo-distal transport and gradual decrease in size of the granular vesicles. The intraneuronal distribution of the vesicles is in agreement with the distribution of the fluorophores in the fluorescent neurones.Neurosecretory neurones are found most likely not to contain monoamines.This work was supported by grants from the Helge Axison Johnson Foundation, the Magn. Bergvall Foundation, and the Roy. Physiographic Society at Lund.I am greatly indebted to Mrs. Lena Eriksson, Miss Rita Jönsson, Miss Inger Norling, Mrs. Lena Svenre, and Mr. Henryk Keff for their excellent technical assistance.     

Fine structural analysis of the basal epidermal receptor cells in the earthworm (Lumbricus terrestris L.)

Summary A fine structural investigation was performed on receptor cells lying at the base of the epidermis in the earthworm, Lumbricus terrestris. Two types of receptor cells with many similarities, but also with major differences, were discriminated.One receptor is of the microvillar receptor type, that appears to be identical with the photoreceptor cell described earlier by Röhlich et al. (1970). Proximal to the nucleus is a large vacuole (phaosome, Binnenkörper) with many daughter cavities containing a large number of microvilli and several cilia with the 9 × 2 + 0 microtubular pattern. The intracellular cavity has no connection with the surface membrane, in contrast to that in hirudineans (White and Walther, 1969) and pogonophores (Nørrevang, 1974).The other receptor is the ciliated receptor type, that is presently described for the first time. This receptor also has a comparatively large uniform cavity, few microvilli and about 20 cilia with the 9 × 2 + 2 microtubular pattern. The cilia leave the cell in the proximal part through a wide opening, make a turn of 180 °, and proceed toward the epidermal surface. Receptors of a similar type have been described by Golding and Whittle (1975) in the cerebral ganglion of four limicole oligochaete annelids; they presumed that these cells have an osmoreceptor function. The new epidermal receptor type described in the present investigation probably has a chemoreceptor function of hitherto unknown kind.This investigation was supported by the Royal Physiographic Society at Lund, Sweden. The author would like to express his thanks to Mrs. Lena Sandell for skilful technical assistance     

Immunocytochemical demonstration of vertebrate neuropeptides in the earthworm Lumbricus terrestris (Annelida,Oligochaeta)

Summary The localisation and distribution of 10 vertebrate-derived neuropeptides in the earthworm, Lumbricus terrestris, have been determined by an indirect immunofluorescence technique. The peptides are pancreatic polypeptide (PP), peptide tyrosine tyrosine (PYY), neuropeptide Y (NPY), glucagon (C-terminal), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), gastrinreleasing peptide (GRP), calcitonin gene-related peptide (CGRP), neurotensin (NT), and met-enkephalin. For 6 of the peptides — PYY, NPY, PHI, glucagon, GRP and CGRP — this is the first demonstration of their presence in any annelid, and NT has not previously been described in an oligochaete. Cell bodies and nerve fibres immunoreactive to the 10 peptides occur throughout the CNS. In the PNS, epidermal sensory cells displayed immunoreactivities to PP and PYY, and PP-, PYY-, NPY-, PHI- and GRP-like immunoreactivities occurred in nerve fibres supplying the main body muscles. Nerve fibres immunoreactive to PP and PYY are also associated with the innervation of the gut (pharynx, oesophageal glands, and mid and posterior regions of the intestine). No endocrine cells immunoreactive for any of the antisera tested could be identified in the gut epithelium, suggesting that dual location of peptides in the brain and gut epithelium is a phenomenon that occurred at a later stage in evolution. No immunoreactive elements were detected in any of the organs and ducts of the reproductive and excretory systems.     

Patterns of serotonin-immunoreactive neurons in the central nervous system of the earthworm Lumbricus terrestris L.

Summary The distribution patterns of serotonin-immunoreactive somata in the cerebral and subpharyngeal ganglion, and in the head and tail ganglia of the nerve cord of Lumbricus terrestris are described from whole-mount preparations. A small number of serotonin-immunoreactive neurons occurs in the cerebral ganglion, in contrast to the large population of serotonin-immunoreactive neurons that exists in all parts of the ventral nerve cord. From the arrangement of serotonin-immunoreactive somata in the subpharyngeal ganglion, we suggest that this ganglion arises from the fusion of two primordial ganglia. In head and tail ganglia, the distribution of serotonin-immunoreactive somata resembles that in midbody segments. Segmental variations in the pattern and number of serotonin-immunoreactive somata in the different body regions are discussed on the background of known developmental mechanisms that result in metameric neuronal populations in annelids and arthropods.Abbreviations CG1, CG2 cerebral soma group 1, 2 - CNS central nervous system - GINs giant interneurons - 5-HT 5-hydroxytryptamine, serotonin - 5-HTi 5-HT-immunoreactive - N side nerve - SG19 subpharyngeal soma group 1–9 - SN segmental nerve     

Parvalbumin-immunoreactive material in the earthworm, Lumbricus terrestris

Parvalbumin-like material was localized using an immunoeytochcmieal method, in neurons of the central nervous system and in cells intermingled in the skin of the earthworm. Lumbricus terreslris L. Parvalbumin-immunoreactive material was found in the cytoplasm of perikarya and neutrites, not in the nucleoplasm. In contrast to vertebrates, Lumbricus musculature did not contain parvalbumin-immunoreactive material.     

Formation of filopodia in earthworm (Lumbricus terrestris) coelomocytes in response to osmotic stress

Coelomocytes, the immunodefense cells of the earthworm Lumbricus terrestris, are exposed to changing osmotic pressures as the worm's coelomic fluid responds to fluctuating wet-dry conditions of the surrounding soil. Using light and fluorescence microscopy combined with actin and tubulin disrupting drugs, we determined the effects of changing osmotic pressure on coelomocyte morphology. The coelomocytes from L. terrestris respond to an increase in environmental osmotic pressure from isotonic conditions (170 mOsm) to hypertonic conditions (715 mOsm) by changing from a round/petalloid morphology to a filopodial morphology. Cytoskeletal fluorescent staining studies indicate that for filopodia to form, the actin cortical ring, present in most coelomocytes in isotonic conditions, must be disrupted. Breakdown of the actin ring by exposure to a hypertonic environment or actin disrupting drugs allows the formation of actin or tubulin-based filopodia. The filopodia, or podial-like extensions formed by earthworm coelomocytes, may enable the cells to better explore their environment.     

Multisegmental cobalt filling of the dorsal giant fibers in the nervous system of the earthworm,Lumbricus terrestris

Summary The anatomical organization of the two dorsal giant fiber systems of the earthworm Lumbricus terrestris is demonstrated in whole mounts and serial-section reconstructions based on backfillings of the ventral nerve cord with cobalt chloride. Both the medial and lateral fiber systems can be labeled selectively over more than ten body segments. They show a characteristic segmental pattern of collaterals with some modification in tail segments and of dorsal plasma protrusions in the unpaired medial giant fiber presumably representing openings in the myelin sheath. We found no multisegmental cobalt transport in other large neurons of the nerve cord. Cobalt passes through the segmentai septa between consecutive axonal elements of the metameric giant fibers and presumably also through commissural contacts between specific collaterals of the lateral giant fibers. Since these sites of contact are known to represent electrical synapses, cobalt coupling may, in L. terrestris, correlate with functional electrotonic coupling.Abbreviations CL collateral of lateral giant fiber - CM collateral of medial giant fiber - GIN giant interneuron - LGF lateral giant fiber - MGF medial giant fiber - SN segmental nerve     

Immunoreactivity to molluskan neuropeptides in the central and stomatogastric nervous systems of the earthworm, Lumbricus terrestris L.

Polyclonal antisera against two related command neuropeptides (CNP2 and CNP4) described in neurons of the terrestrial snail Helix were used in a study of the nervous system of the earthworm Lumbricus. The CNP-like peptides belong to the same neuropeptide subfamily and bear a C-terminal signature sequence Tyr-Pro-Arg-X. The distribution patterns of immunoreactive (IR) neurons were studied in the central nervous system (CNS), skin, and stomatogastric nervous system of the earthworm. IR neurons were found in all CNS ganglia, the patterns being similar for both antibodies used. Several clusters of IR cells were observed in the cerebral and subesophageal ganglia. In the ventral cord ganglia, the number of IR cells decreased in the rostro-caudal direction, and the IR cells sent their fibers mostly into the median fiber bundle. Segmental nerves contained no IR fibers. After injury of the worm body, the number of IR neurons in the CNS significantly increased. In the skin, IR sensory neurons were present in sensory buds. The stomatogastric ganglia only contained IR fibers. Numerous scattered IR neurons were found in the inner subepithelial layer of the esophagus and formed the enteric plexus in which the cell bodies displayed a segmentally repeated pattern. Possible involvement of CNP-like-IR neurons in central integratory processes, sensory processes, and the regulation of feeding is discussed.This work was supported by INTAS (grant 01-2117), CRDF (grant RB1-2321-MO-02), and the Russian Foundation for Basic Research (grants 05-04-48724 and 03-04-48179).     

Occurrence of ceramide-glycanase in the earthworm, Lumbricus terrestris

We have detected the presence of ceramide-glycanase in the earthworm, Lumbricus terrestris. We have also devised a simple method for the preparation of this enzyme from the earthworm. This enzyme cleaved the linkage between the ceramide and the glycan chain in LacCer, GbOse3Cer, GbOse4Cer, GbOse5Cer, GM3, GM2, GM1 and GD1a. By using tritium-labeled GM2 as substrate, the optimum pH of this enzyme was found to be between pH 4 and 4.5. In the earthworm, the ceramide-glycanase was mainly found in the muscle. The intestine was found to contain a very low level of this enzyme. Because of their easy availability, earthworms should become a convenient source for the preparation of ceramide-glycanase.     

White-tailed deer (Odocoileus virginianus) fecal pellet decomposition is accelerated by the invasive earthworm Lumbricus terrestris

Exotic European earthworms have expanded into worm-free forests of the United States. Concurrently, populations of the white-tailed deer, Odocoileus virginianus, have also increased. During winter, deer use hemlock stands for cover while browsing elsewhere, creating a net organic matter flux into these stands. Deer fecal pellets can provide annual inputs of 48.1 kg C, 1.4 kg N, and 1.3 kg Ca per hectare. We tested the hypothesis that these pellets were readily consumed by invading earthworms. The litter-feeding anecic earthworm Lumbricus terrestris redistributed fecal pellets and accelerated mass and nutrient loss rates. These losses are likely due to the combination of enhanced fragmentation and decomposition as earthworms drag pellets into their burrows for consumption. This nutrient subsidy may be an important source of high quality “litter” input to hemlock stands, which may in turn facilitate the invasion of these stands by earthworms under high deer densities.     

Fine structure of the sensilla of Peripatopsis moseleyi (Onychophora)

Summary Three types of sensilla occurring on the lips and on the antennae of Peripatopsis moseleyi have been investigated by scanning and transmission electron microscopy. On the lips sensory spines can be found which contain numerous cilia originating from bipolar receptor cells. They reach the tip of the spine where the cuticle is modified. The perikarya of the sensory cells, a large supporting cell with a complicated surface and a second type of receptor, form a bud-like structure and are surrounded by a layer of collagen fibrils. The second receptor cell bears apical stereocilia as well as a kinocilium which are directed towards the centre of the animal — thus the cell appears to be turned upside down. The sensilla of the antennae are 1) sensory bristles containing two or three kinds of receptor cells, one of which bears an apical cilium and one kind of supportive cell and 2) sensory bulbs located within furrows consisting of receptor cells with branched cilia and two kinds of supportive cells which are covered by a modified thin cuticle. According to the electron microscopical findings the sensory spines on the lips are presumably chemoreceptors. The sensory bristles on the antennae can be regarded as mechanoreceptors and the sensory bulbs as chemoreceptors.Supported by the Deutsche Forschungsgemeinschaft (Sto 75/3)     

The fine structure of ciliated sensory cells in the epidermis of the earthworm Lumbricus terrestris

An ultrastructural study of the earthworm body wall has revealed three types of sensory cells. Two, the multiciliate and uniciliate sensory cells, are found only in the discrete sense organs and their cilia pass vertically through the cuticle. The third type-isolated multiciliate sensory cells-are scattered throughout the epidermis and never grouped together. However, their cilia do not pass through the cuticle, but run horizontally over the outer surface of the epidermal cells. The structure of the sensory cells is described and compared with that of ordinary epidermal cells and the supporting cells found in the sense organs. Their possible physiological roles are discussed.     

Elemental analysis of histochemically defined cells in the earthworm Lumbricus terrestris

Summary A method for preparation of biological specimens for electron probe X-ray microanalysis is described, that aims at retaining the original elemental distribution within the tissue at the cellular level. The tissue is without any chemical fixation, quench-frozen, and 16-m sections are prepared with a conventional cryomicrotome, transferred to a carbon specimen holder and freeze-dried.Adjacent serial sections, collected on glass slides and stained with various histological procedures, are used to correlate the data obtained by X-ray microanalysis with other histochemical information on the same cell or tissue.To demonstrate the possibilities of the method, sections of the earthworm Lumbricus terrestris were analyzed. In the chloragogenous cells, high concentrations of Ca, Zn and P were found. The inner and outer muscle layer show slightly different properties, both with regard to elemental composition and to myofibrillar ATPase activity.     

Fine structure of the abdominal epidermis of the adult mudpuppy,Necturus maculosus (Rafinesque)

Summary The ventral epidermis of adult Necturus maculosus has been studied using electron and light microscopy. Many larval characteristics of amphibian epidermal structure are retained in adult Necturus. The epidermis is a stratified epithelium consisting of four cell layers and five cell types. Major differences compared with other adult amphibians are: (1) the absence of a well defined moulting cycle together with an apparently diminished synthetic and mitotic activity in the stratum germinativum; (2) an outermost cell layer (stratum mucosum) that is unkeratinized and appears to synthesize a mucous layer; and (3) numerous large club-shaped Leydig cells which span the epidermis between the cells of the stratum germinativum and stratum mucosum. The apical region of the stratum granulosum and stratum mucosum cells shows evidence of extensive synthesis. The stratum mucosum appears to be involved in the secretion of vesicular contents onto the outermost surface of the epithelium. The external surfaces of the stratum mucosum cells possess numerous microridges which are supported by an intricate network of cytofilaments in the apical region of these cells. The significance of these features is discussed in relation to the physiology and ecology of this species.     

LTCI, a novel chymotrypsin inhibitor of the potato I family from the earthworm Lumbricus terrestris. Purification, cDNA cloning, and expression

A novel chymotrypsin inhibitor of the potato I protease inhibitor family from the earthworm Lumbricus terrestris was purified. The inhibitor, named LTCI, was isolated by methanol extraction, affinity chromatography on immobilized methylchymotrypsin, and ion exchange chromatography followed by RP–HPLC. The 7076 Da inhibitor consists of a single polypeptide chain of 64-amino-acid residues without disulfide bridges. LTCI is the first of the potato I protease inhibitors with Tyr in position P1 of the reactive site. cDNA analysis revealed that LTCI is produced as a 86-amino-acid precursor with a 22-amino-acid secretory signal peptide. RT–PCR analysis demonstrates that LTCI mRNA is expressed in body wall, intestine, and coelomocytes. The recombinant LTCI was produced in Escherichia coli as a fusion protein with intein and chitin binding domain using IMPACT™–CN system.     

Scanning electron microscopy of photoreceptor cells in the light- and dark-adapted retina of Haplochromis burtoni (Cichlidae,Teleostei)

Summary The photoreceptor layer in the retina of Haplochromis burtoni (Cichlidae, Teleostei) was studied by scanning electron microscopy. Three types of receptors were identified: rods, single-cones and double-cones. The three-dimensional arrangement of these photoreceptors is described in the light- and dark-adapted retina. The surface of the inner segment of the photoreceptor cells displays fine vertical fissures which give rise to slender processes. These so called calycal processes which are of different lengths in rods and cones, surround the beginning of the smooth-surfaced outer segment. The myoid, the contractile part of the receptor, which is located beneath the ellipsoid, was examined in the single-cones of the dark-adapted retina. It is a slender structure with surface infoldings. The myoid, studied by transmission electron microscopy, contains bundles of parallel myofilaments, which are thought to be contractile.This investigation was supported by grants of the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 51-E/10)