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PubMed1996

Monoclonal antibodies to proteins of the myelin-like sheath of earthworm giant axons show cross-reactivity to crayfish CNS glia: an immunogold electron microscopy study.

Monoclonal antibodies were generated to the proteins in myelin-like membranes isolated from the nerve cords of the earthworm, Lumbricus terrestris. One of these showing cross-reactivity to 30-32 and 40 kDa proteins was shown by immunofluorescence microscopy and immunogold electron microscopy to be bound primarily to glial cell process and their membranes and the myelin-like layers. This antibody cross-reacted with proteins of 60-65, 42, and 40 kDa in crayfish (Procambarus clarki) nerve cord homogenates. Localization by immunoelectron microscopy showed the antibody to be bound exclusively to the membranes of the glial processes ensheathing the axons in the crayfish nerve cord. Thus, the proteins in earthworm and crayfish glial cell membranes have some epitopes in common. We suggest that this may represent an evolutionary conservation of these proteins.

Animals↗

PubMed1992

Lipids of the earthworm Lumbricus terrestris.

The lipid composition of the earthworm Lumbricus terrestris has been reexamined under conditions intended to avoid enzymatic and chemical alterations during storage, extraction, and fractionation procedures. The simple lipids included aliphatic hydrocarbons, steryl esters, glycerides, and at least nine different sterols, all thought to be derived from the diet. Free fatty acids, previously considered to be major components of worm lipids, comprised only 0.3% of the total lipid weight. Phospholipids included (in order of relative abundance) phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, as well as sphingomyelin. Glycolipids included cerebrosides and sulfatides containing both glucose and galactose, and gangliosides containing glucosamine and sialic acid. The fatty acid compositions of these lipid classes appeared to be a mixture of what are considered typical plant, bacterial, and animal acids. Several fatty acids found in the worms, including cis-vaccenic and eicosapentaenoic acids, were essentially absent from the dietary components, and it is concluded that these acids were synthesized in the worms. The earthworm derives much of its lipid adventitiously, but exerts at least some control over its tissue lipid composition.

Animals↗

PubMed1995

Characterization of the limited specificity of antigen recognition in earthworms.

Earthworms respond to parenteral stimulation with a protein antigen by the formation of an antigen-binding protein. Earthworms were parenterally stimulated with different proteins and the protein binding was estimated in vitro on both humoral and cellular levels. The binding was significantly higher when the same protein was used for in vivo stimulation. The degree of specificity of the antigen-binding protein after the secondary in vivo challenge increased, but even so it was considerably lower than that of vertebrate immunoglobulins.

Adjuvants, Immunologic↗

PubMed1999

Characterization of the limited specificity of antigen recognition in earthworms.

Parenteral administration of foreign proteins results in earthworms in significantly increased levels of the antigen-binding protein (ABP). The earthworms (Lumbricus terrestris and Eisenia foetida; Oligochaeta, Annelida) were stimulated with different proteins and the ABP response was followed with an anti-ABP monoclonal antibody and in ligand assay with biotin conjugates of all proteins used for stimulation. ABP levels are increased after the stimulation with different proteins irrespective of the size and extent of glycosylation. Molar mass of the ABP molecules was always the same, 56 kDa in L. terrestris and 60 kDa in E. foetida. The level of the specificity is considerably lower in comparison with immunoglobulins since ABP reacts not only with the protein used for stimulation but also, though to lesser extent, with related proteins.

Animals↗

PubMed1998

Fibrinolytic activity of earthworms extract (G-90) on lysis of fibrin clots originated from the venous blood of patients with malignant tumors.

u-PA is secreted by the most malignant tumors. As a response to u-PA synthesis surrounding cells synthetize inhibitors of plasminogen activators for tissue protection. Plasminogen activators were found also in earthworm tissue. From the tissue homogenate of earthworm Eisenia foetida the glycolipoprotein mixture named G-90 was isolated. It contains two serine proteases (P I, P II) with fibrinolytic and anticoagulative activities. The fibrinolytic activity of G-90, P I and P II was tested in an in vitro euglobulinic test applied to fibrin clot from blood plasma of patients suffered from malignant tumors. G-90 and above-mentioned proteases applied in this study showed euglobulinic time proportionally with the concentrations of added substances. The influence of G-90 on the fibrinolysis rate does not depend only on its concentration, but depends too on histological type of tissue (organ) where the malignant tumors are located. Enzyme P I and P II do not show this activity.

Adult↗

PubMedDate not supplied

Cadmium-induced metallothionein in earthworms (Lumbricus terrestris).

Studies are continuing to compare the common earthworm (Lumbricus terrestris) to rodents by their reactions to toxic levels of metals. A recent study suggests that LD50 of selenium compounds were about ten times less in worms than in rodents, but the toxicity of cadmium was of the same order of magnitude between these diverse species. An interesting finding is that the common earthworm produces a Cd-bound protein, metallothionein, after the injection of cadmium salts, and, similar to rodents, two Cd-bound isoproteins can be isolated by a Sephacryl S-200 column and monitored by both atomic absorption and liquid scintillation spectroscopy.

Animals↗

PubMed1988

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.

Animals↗

PubMed1998

Dose dependency of earthworm powder on antithrombotic and fibrinolytic effects.

The freeze-dried powder of Lumbricus rubellus earthworm was administered orally to rats and its fibrinolytic and antithrombotic effects were investigated. The fibrinolytic activity of plasma was determined by measuring the plasmin activity of the euglobulin fraction and was increased to two-folds of the control at a dose of 0.5 g/kg/day and five times with 1 g/kg/day after 4-day administration. The antithrombotic effect was studied in an arterio-venous shunt model of rats. The thrombus weight decreased significantly from 43.2 mg to 32.4 mg at a dose of 0.5 g/kg/day after 8-day treatment. The level of fibrinogen/fibrin degradation product (FDP) in serum was elevated in a dose-dependent manner during the treatment period. On the 8th day after administration, the FDP value was increased to 7.7 micrograms/ml compared with the control value of 3.3 micrograms/ml. These results support that earthworm powder is valuable for the prevention and/or treatment of thrombotic conditions.

Administration, Oral↗

PubMed2002

Expression of nerve growth factor-like polypeptides and immunoreactivity related to the two types of neurotrophin receptors in earthworm tissues.

Nerve growth factor (NGF) belongs by sequence homology to the neurotrophins, a family of proteins binding the same p75 receptor and closely related members of the Trk family of receptor tyrosine kinases. Fundamental in the vertebrate nervous system, neurotrophin signals have also been suggested as essential for relatively complex nervous systems occurring in invertebrate species that live longer than Caenorhabditis elegans and Drosophila melanogaster. Mammalian neurotrophins have been found to influence invertebrate neuronal growth. However, there are only a few data on the presence of molecules related to neurotrophin signalling components in invertebrates. Our studies provide evidence that analogues of neurotrophins and neurotrophin receptors are expressed in Eisenia foetida earthworms. In particular, NGF-like and Trk-like immunoreactive proteins are both expressed in the nervous system, whereas p75-like positivity identifies tubular structures associated with dorsal pores that are involved in the earthworm response to mechanical irritation or stress.

Animals↗

PubMed2002

TNT, RDX, and HMX decrease earthworm (Eisenia andrei) life-cycle responses in a spiked natural forest soil.

Sublethal and chronic toxicities of 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) on earthworm Eisenia andrei in a sandy forest soil were assessed. Various reproduction parameters of fecundity (total and hatched number of cocoons, number of juveniles, and their biomass) were significantly decreased by TNT (> or = 58.8 +/- 5.1 mg/kg dry soil), RDX (> or = 46.7 +/- 2.6 mg/kg), and HMX (> or = 15.6 +/- 4.6 mg/kg). These effects occurred at much lower concentrations than those reported earlier using artificial soil preparations. Growth of adults was significantly decreased in the TNT-spiked natural soils at 136.2 +/- 25.6 mg/kg dry soil, the highest concentration having no significant mortality. In contrast, survival and growth were not significantly reduced at relatively high measured concentrations of RDX (167.3 mg/kg) and HMX (711.0 mg/kg). Although TNT, RDX, and HMX share a common life-cycle response ( i.e., decreased juvenile counts), a number of differences related to other reproduction parameters (e.g., productivity of cocoons) was observed. These results indicate that the tested explosives do not support a common mechanism of toxicity, at least in the earthworm, probably due to differences in their physical-chemical properties as well as metabolites formed during exposure.

Animals↗

PubMed2005

Quantitative ultrastructure of metal-sequestering cells reflects intersite and interspecies differences in earthworm metal burdens.

Morphometric analysis of transmission electron micrographs was used to compare the effects of metals on the multifunctional, metal-sequestering, chloragocyte cells of two epigeic earthworm species, Dendrodrilus rubidus and Lumbricus rubellus, inhabiting three field soils: a clean circumneutral reference soil (Dinas Powys); an acidic moderately Pb- and Zn-contaminated soil (Cwmystwyth); and a calcareous Cd-, Pb-, and Zn-contaminated soil (Draethen). The main findings were: (1) D. rubidus accumulated significantly higher tissue Cd and Pb and lower Zn concentrations than L. rubellus, especially at Draethen; (2) the volume fraction of chloragosomes was significantly lower and the volume fraction of debris vesicles significantly higher in D. rubidus from Draethen compared with L. rubellus at all sites and with the other two D. rubidus populations; (3) estimated relative toxicity factors, derived from soil metal concentrations and published EC50 data, suggested that the subcellular changes in chloragocytes, particularly in D. rubidus from Draethen, were caused mainly by Zn and Pb exposures; (4) scrutiny of the body burdens of each metal in both worm species across the three sites indicated that Cd was a major contributor to the structural changes observed in Draethen D. rubidus, and its impact was disproportionate to its soil and tissue concentrations in comparison with those of Pb and Zn. The apparent greater susceptibility of D. rubidus cells, compared with L. rubellus cells, to soil metal contaminants is discussed in light of differences in the quality and quantity of the metal body burdens accumulated by the two species. Further histopathalogic and morphometric studies on key organs and tissue of earthworms are required to provide biomarkers of exposure and to underpin linkage of biochemical-level changes and demography.

Animals↗

PubMed1997

Application of plant and earthworm bioassays to evaluate remediation of a lead-contaminated soil.

Earthworm acute toxicity, plant seed germination/rootelongation (SG/RE) and plant genotoxicity bioassays were employed to evaluatethe remediation of a lead-contaminated soil. The remediation involved removalof heavy metals by a soil washing/soil leaching treatment process. A portionof the soil after remediation was rinsed with water in order to simulateexposure to rainfall. The bioassay results showed that the soils beforetreatment (BT) and after treatment plus water rinsing (RT) were not toxic toearthworms in a 14-day exposure, while after treatment (AT) showedsignificant toxicity. The LC50 values for Eisenia fetida andLumbricus terrestris were 44.04 and 28.83 (as % AT soilsupplemented in artificial soil), respectively. The phytotoxicity dataindicated that all three test soils significantly inhibited lettuce SG/RE ina dose-related manner, with AT being the most phytotoxic. In oats, RT had noeffect on SG/RE and AT was more toxic than BT. For the two local site grassseeds tested (blue grama and sideoat grama), the AT soil was the mostphytotoxic followed by BT and RT. In Allium cepa (common onion), BTand AT induced similar levels of genetic damage to root tip cells, whereas RTwas not genotoxic. High salt levels generated during the remediation processappeared to be responsible for the increased toxicity of AT soil for bothplants and earthworms. The rinsing of the AT soil with water effectivelyremoved both acutely toxic and genotoxic components of the soil.

Animals↗

PubMed2001

Contribution of the Earthworm Lumbricus rubellus (Annelida, Oligochaeta) to the Establishment of Plasmids in Soil Bacterial Communities.

The contribution of the earthworm Lumbricus rubellus in spreading plasmids from a nonindigenous bacterial species to the soil microbial community was studied with Escherichia coli strains as donor organisms. The selected donor strains harbored marker-gene tagged plasmids with different transfer properties and host ranges. Prototrophic benzoate degrading indigenous bacteria were analyzed as potential recipients. In filter-mating experiments, donor strains were mixed with bacterial cell consortia extracted from earthworm casts (feces) and incubated on nutrient agar at 28 degrees C. Transfer was detected with the broad host range IncP plasmid pRP4luc; with the IncQ plasmid, pSUP104luc, but only when it was present in a mobilizing donor strain; and with the transposon delivery vector pUTlux. No transfer was detected with the nonmobilizable pUCluc and the mobilizable pSUP202luc, both of narrow host range. In microcosm studies with E. coli inoculated soil incubated at 12 degrees C, transconjugants were only detected in casts of L. rubellus but not in bulk soil, indicating that the gut passage was a precondition for plasmid transfer. Plasmid pRP4luc was transferred at higher frequencies than detected in filter mating. Results of the filter matings were confirmed except that transfer of pUTlux could not be detected. The majority of transconjugants isolated in this study lost their acquired plasmid upon further cultivation. Stable transconjugants, however, were obtained and identified at the 16S rRNA gene level as members of the b- and g-subgroups of Proteobacteria. Incubation of E. coli and selected transconjugants in soil microcosms with L. rubellus demonstrated that the gut passage resulted in a slight but significant reduction of ingested cells. In contrast to the donor strains, however, the population sizes of transconjugants in bulk soil and in casts did not decrease over time. This demonstrated that the transferred plasmids had established themselves in the soil microbial community.

Journal Article↗

PubMed2004

Earthworm leukocyte populations specifically harbor lysosomal enzymes that may respond to bacterial challenge.

Earthworm leukocytes (coelomocytes) are responsible for innate cellular immune functions such as phagocytosis and encapsulation against parasites and pathogens. Microbial killing results from the combined action of the phagocytic process with humoral immune factors such as agglutinins (e.g., lectins), lysosomal enzymes (e.g., acid phosphatase, lysozyme), and various cytotoxic and antimicrobial molecules. There is also evidence of weak adaptive immune responses against foreign transplants. This study focused on aspects of the innate immune response. First, anti-human acid phosphatase (anti-AcP) polyclonal antibody characterized different acid hydrolase patterns in coelomocytes. Second, flow cytometry identified a strongly immunoreactive coelomocyte population. Third, ultrastructural and cytochemical analyses revealed acid phosphatase in discrete granules (lysosomes) of effector hyaline and granular coelomocytes but not in mature chloragocytes. Coelomocytes were exposed to bacteria to assess how phagocytosis influences: (a) the production of acid phosphatase using Western blot, and (b) release of acid phosphatase using ELISA from cell-free coelomic fluid. Fourth, after phagocytosis, acid phosphatase levels differed between controls and experimentals. Fifth, we found a 39-kDa molecule that reacted intensely with anti-AcP. Our results suggest that effector earthworm coelomocytes may not eliminate pathogens only by phagocytosis but also by extracellular lysis.

Acid Anhydride Hydrolases↗

PubMed2006

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.

Animals↗

PubMed2000

Sites of expression of mRNA for lysenin, a protein isolated from the coelomic fluid of the earthworm Eisenia foetida.

Lysenin is a 33-kDa protein of 297 amino acids that was originally purified from the coelomic fluid of the earthworm Eisenia foetida. It binds specifically to sphingomyelin. In this study, we attempted to identify the site of synthesis of lysenin in the earthworm. We detected the expression of mRNA for lysenin and the presence of immunoreactive lysenin in the large coelomocytes and in the free large chloragocytes present in the lumen of the typhlosole, a depression in the dorsal wall of the intestine. These coelomocytes and chloragocytes seemed to be mature and separate from the chloragogen tissue that lined the typhlosole. The free large chloragocytes in the typhlosole contained numerous vacuoles. The nuclei were small and irregular in shape, and glycogen granules and mitochondria were occasionally found between vacuoles. The chloragocytes of the chloragogen tissue that surrounded the coelomic side of the intestine and the dorsal blood vessel did not react with the lysenin antiserum and no expression of lysenin mRNA was detected in these cells. Furthermore, no evidence of the protein or of the mRNA was found in the cells of the pharyngeal gland. Our findings suggest that lysenin is produced in the free large chloragocytes in the lumen of the typhlosole.

Animals↗

PubMed1989

Interactions between earthworm hemolysins and sheep red blood cell membranes.

The hemolytic activity exhibited by the coelomic fluid of the Annelid Eisenia fetida andrei is mediated by two lipoproteins of mass 40 and 45 kDa, each of them capable of hemolysis. Such an activity is not inhibited by zymosan, inulin or lipopolysaccharide (LPS), nor by hydrazine or methylamine, suggesting that earthworm hemolysins are not related to C3 or C3b complement components. Among the membrane lipids tested (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingomyelin and cholesterol) only sphingomyelin inhibited hemolysis. The analysis of E.f. andrei proteins bound to sphingomyelin microvesicles, as well as to sheep red blood cell (SRBC) membranes, revealed a polymerization of E.f. andrei 40 kDa and/or 45 kDa hemolysins. Consequently, sphingomyelin appears a likely candidate for hemolytic complex receptor. Electron microscopy observations suggested that the polymerization causes an open channel through the lipid bilayer. As demonstrated using metal ions, heparin, chondroitin sulfate, poly(L-lysine) and protamine chloride, the mode of action of earthworm hemolytic complex is not analogous to that of C9 or perforine.

Animals↗

PubMed1992

Axonal conduction and electrical coupling in regenerating earthworm giant axons.

Severed halves of medial giant axons (MGAs) and lateral giant axons (LGAs) in earthworms survive and are functionally reconnected as early as the first postoperative week. During the first 150 postoperative days, there is an increase in conduction velocity of action potentials and strength of electrotonic coupling between the severed axonal stumps across the lesion site. Electrophysiological analyses suggest that this functional reconnection occurs by transmission of action potentials through the lesion site by active propagation along neurites which make electrotonic connections rather than chemical synapses. The regenerated connections restore the original connectivity pattern for conduction of action potentials or spread of electrotonic potentials; i.e., MGA stumps reconnect with MGA stumps, and LGA stumps with LGA stumps. These and other data suggest that the mechanisms responsible for establishing appropriate functional reconnection of severed earthworm giant axons requires cell-specific matching of axons and neurites, rather than a competition between appropriate and inappropriate functional connections.

Animals↗