Isolation and initial characterization of myelin-like membrane fractions from the nerve cord of earthworms (Lumbricus terrestris L)

We report here the isolation of fractions enriched in components of the myelin-like membranes surrounding the giant axons of the earthworm.Lumbricus terrestris L. The composition and purity of the fractions have been assessed using SDS-protein electrophoresis, Western immunoblots, and electron microscopy. Preliminary enzyme assays indicated that the mitochondrial marker, succinate dehydrogenase, has a similar specific activity distribution in earthworm nerve cord and in mouse liver sedimentation velocity fractions, however, the distribution of the total units of activity among the fractions seems to indicate the existence of smaller mitochondria in earthworm nerve cord compared with mouse liver mitochondria. In earthworm nerve cord fractions, Na⁺/K⁺ ATPase and Ca²⁺/Mg²⁺ ATPase were found to be enriched exclusively in the fraction containing large plasma and myelin-like membranes, while in the mouse liver fractions, the total units of these two enzymes were found to be distributed broadly among fractions. 5-Nucleotidase activity in the earthworm nerve cord seemed to be restricted to the microsomal fractions (endomembrane network), with a very low activity associated with the large plasma and myelin-like membrane fraction. We have established the presence of keratins or prekeratins in the myelin-like membranes, probably in the form of tonofilaments. However, we could not show that the desmosome-like structures, characteristic of these membranes, are composed of those proteins described for vertebrate epithelial desmosomes.     

Acrosome morphogenesis in Lumbricus terrestris

Summary Acrosome morphogenesis commences in the juxtanuclear cytoplasm at the posterior end of spermatids of Lumbricus terrestris. A dense rod-shaped structure and the Golgi apparatus together participate first in forming an acrosome vesicle that contains the acrosome granule, and somewhat later shape the conical base of the acrosome in the cytoplasm beneath the vesicle. Cytoplasmic flow may account for the migration of the immature acrosome to the apical surface of the nucleus of the spermatid. Manchette microtubules play a key role in the final modelling of the acrosome. Sheathed by the manchette the acrosome elongates to 3–4 times its pre-attachment length. The conical base of the acrosome then extends anteriorly to enclose the acrosome vesicle. A dense rod emerging from the rod-shaped granule occupies an indentation of the base of the acrosome vesicle. The mature acrosome of Lumbricus is an extremely complex structure about 5–7 microns long and is bounded by the plasmalemma of the spermatozoon.This study was supported by a research training grant GM-00582-07 from the Public Health Service.     

Influence of Lumbricus terrestris earthworms on the structure of the yeast community of forest litter

The taxonomic structure of yeast communities was studied in forest litter and soil, as well as in substrates transformed by the activity of Lumbricus terrestris earthworms (tree waste from the hole mouths, the gut contents, and coproliths). The activity of L. terrestris has a weak effect on the total yeast abundance but results in substantial changes in their taxonomic composition. The share of ascomycetous yeasts is significantly higher in the substrates associated with the activity of earthworms. The teleomorphic ascomycetes Williopsis saturnus were isolated from the gut contents. The effect of earthworms on the composition of the yeast community in the process of forest litter destruction is more markedly pronounced than seasonal changes.     

Resistance of earthworms Lumbricus terrestris and Allolobophora turgida to the fungicide captan 50 W.P

Two species of earthworms were kept in Muck, Chicot and Ste-Sophie soils treated with captan 50 W.P. solutions of 700, 1 400 and 2 800 ppm. After a 42-day treatment period, L. terrestris had a 95% survival and A. turgida 100% survival. Using a gas chromatograph method, no captan was detected in tissue extracts of exposed earthworms. Based on the survival rate and the undetected presence of captan in earthworm tissues, we concluded that soil residues of this product are unlikely to be harmful to the animal's ecology.     

Scanning electron microscopy of the gut microflora of two earthworms: Lumbricus terrestris and Octolasion cyaneum

Scanning electron microscopy was used to investigate the presence of microorganisms, probably bacteria, on the gut surface of earthworms. The washed surfaces of the intestines of two earthworms, Lumbricus terrestris and Octolasion cyaneum, were examined. Numerous organisms resembling bacteria were observed throughout the gut, some in situations suggesting attachment. Compared with similar investigations in other invertebrates, there were fewer bacteria, showing less morphological diversity, on the earthworm gut surface. The majority of organisms viewed were coccoid, some were filamentous, and a few rod-shaped cells were observed. Cocci, often in chains, were seen in the foregut of both species. Although cocci were also numerous in the midgut region, particularly in the typhlosole, in O. cyaneum tufts of segmented, filamentous organisms were also seen with some segments resembling spores. Fewer organisms were found in the hindgut, but in L. terrestris there were segmented, filamentous organisms, attached to the epithelium by way of a socket-like structure, similar to that by which segmented, filamentous bacteria (SFBs) are attached to the ileum of rats and mice. Transmission electron microscopy of the hindgut of L. terrestris was undertaken to explore the structure and attachment of SFBs to the gut epithelium. However, although a few rod-shaped bacteria were observed, no SFBs were located. The observations reported here provide evidence that earthworms have an attached gut microflora of filamentous microorganisms which are probably indigenous, and as far as we are aware this is the first published report of such findings in these invertebrates. Offprint requests to: J.M. Jolly.     

Acute toxicity of cadmium and zinc in the earthworm (Lumbricus terrestris)

Studies are continuing to explore the use of the earthworm (Lumbricus terrestris) for the determination of the acute toxicity of metal compounds. Worms were injected intraperitoneally with cadmium and zinc chlorides, and also zinc chloride followed by cadmium chloride to see if zinc could protect against the toxicity of cadmium. The 48 h acute toxicity (LD₅₀) values were 22 and 23 mg/kg for Cd and Zn respectively and 30 mg/kg for Cd after the worms were pretreated with Zn. It appears that the earthworm can be a useful test subject for obtaining preliminary information on metal toxicity.     

Lipid Composition of the Nervous System of Earthworms (Lumbricus terrestris)

As part of a systematic study of the evolution of the nervous system, the lipid composition of the ventral nerves of earthworms was examined. The nerve axons are wrapped in copious layers of loosely bound membrane, superficially resembling the myelin sheath of vertebrates. However, neither galactocerebroside nor sulfatide, both of which are considered to be markers for myelin, was present, and only traces of glucocerebroside, which is abundant in shrimp nerve, were detected. The lipids were rich in cholesterol (15.3 mumol/g of fresh tissue) and phospholipids (21.7 mumol/g of fresh tissue). The phospholipids were composed of phosphatidylethanolamine, -choline, -serine, and -inositol in the ratio of 41:44:8:7. Most of the ethanolamine-containing phospholipids were in the form of plasmalogens. The fatty acid moieties of these phospholipids were predominantly 18:1, 18:0, and 20:1, whereas the aldehyde moieties of plasmalogen were mostly 18:0. Sphingomyelin, which is considered a ubiquitous component of animal membranes, was not detected. How the unique structure of the membranes of earthworm nerves may be related to the function of the nervous system in this organism is discussed.     

Elektronenmikroskopische Untersuchung der Neurosekretion im Cerebralganglion des Regenwurmes (Lumbricus terrestris)

Zusammenfassung Die Feinstruktur der neurosekretorischen Nervenzellen und der Gliazellen im Cerebralganglion des Regenwurmes (Lumbricus terrstris) wurde untersucht.Die Nervenzellen zeigen verschiedenartige Erscheinungsformen. Einzelne sind mit reifen Neurosekretgranula (Durchmesser von rund 280 m) gefüllt (Speicherzellen). In anderen dominieren leere Vesikel, oder das Ergastoplasma nimmt die ganze Zelle ein. In einzelnen Fällen erweitern sich die Ergastoplasmacysternen sackartig, so daß die Zelle ein vakuolisiertes Aussehen gewinnt. Der für ein Sekret charakteristische Stoff wird zuerst in den flachen Cysternen des Golgi-Apparates und in den Golgi-Vesikeln der entleerten Zellen gefunden. Daraus kann geschlossen werden, daß der Golgi-Apparat in enger Beziehung zur Sekretbildung steht. In einigen Zellen werden reife Sekretgranula im Interzellularraum zwischen den Fortsätzen der Glia- und Nervenzellen beobachtet.Charakteristisch für die Gliazellen sind ein gut entwickelter Golgi-Apparat, Stützfilamente und einzelne Vesikelreihen. Letztere stehen vermutlich mit der Pinocytose und Phagocytose in Zusammenhang. Oft kommen in den Gliazellen — aber in geringer Menge auch in den Nervenzellen — große, dunkle Körper (Durchmesser 0,5–2,5 ) mit feinkörnigem, homogenem oder lamellärem Inhalt vor. Anscheinend bestehen zwischen diesen Körpern und den Gliamitochondrien Übergangsformen.Erweiterungen des Interzellularraumes an isolierten Abschnitten stehen aller Wahrscheinlichkeit nach mit der Entleerung des Sekretes in Verbindung. In ihnen ist ein blasser, fein präzipitierter Stoff zu finden. Die Wand der Kapillaren wkd von einer feinen Basalmembran und einer Myoendothelzellschicht gebildet. Oft sind zwischen benachbarten Endothelzellen und zwischen ihnen und der Basalmembran kleine homogene, dunkle Gebilde mit verwaschenem Umriß zu beobachten, die vielleicht mit der Entleerung der Sekretgranula in die Kapillaren in Zusammenhang stehen.     

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.     

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.     

The nitrogen excretory metabolism of Lumbricus terrestris

The use of the earthworm as a laboratory animal for studying the effects of starvation on nitrogen metabolism is discussed. Simple techniques and methods are presented allowing in vivo physiological responses to be compared with changes in the enzyme potential of gut tissue.     

Lectin histochemistry of epidermal glandular cells in the earthworm Lumbricus terrestris (Annelida Oligochaeta)

Carbohydrate residues were localized in the glandular cells of the epidermis of Lumbricus terrestris by lectin histochemistry. The following biotinylated lectins were used: ConA, PNA, WGA, UEA-I. Each lectin has a specific binding pattern in the epidermal glandular cells. The ConA binding is evident in the orthochromatic mucous cells; PNA in the metachromatic mucous cells; WGA in the neuroendocrine-like cells; UEA-I in the cuticle. The epidermal glandular cells possess specific sites for the different lectins in relation to their functional characteristics. Therefore, these sugar residues indicate different behaviours of the cells in epidermal functions related to ion transport, receptor-secretory processes and defence.     

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.