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[Effects of avermectin B1a on soil microorganism and earthworm (Eisenia fetida)].

Avermectins are the pesticides widely applied in agriculture. This paper studied the effects of different concentration avermectin B1a (AVM B1a) on soil bacteria and fungi, soil respiration, and soil earthworm (Eisenia fetida). The results showed that in 4 test soils, bacteria were significantly inhibited when the AVM B1a concentration was higher than 83.3 mg x kg(-1), while fungi were less impaired. Soil respiration was also inhibited by high concentration AVM B1a, which differed with soil types. The half lethal dosage (LD50) of AVM B1a to soil earthworm was estimated as 4.63 microg x cm(-2) in filter paper contact test, and as 24.13 and 17.06 mg x kg(-1), respectively after treated 7 and 14 days in artificial soil.

Animals↗

A new species of Monocystis stein, 1848 (Protista: Apicomplexa: Eugregarinida) from the Indian earthworm, Amynthas Hawayanus Rosa, 1891 (Annelida: Oligochaeta).

As a part of an ongoing biodiversity survey of aseptate gregarine fauna of oligochaete hosts of West Bengal, an expedition was carried out in the Darjeeling district of West Bengal and most of the earthworms collected were found to be infested with a species of Monocystis Stein, 1848. The monocystid species was collected from the seminal vesicles of the earthworm and was identified as a new species, Monocystis amynthae sp. nov. The gamont of the new species is characterized by having an elongated body with broad anterior end, separated from the narrow posterior end by a prominent constriction measuring 49.0-77.0 (66.0+/-1.3) microm x 32.0-41.0 (37.0+/-2.8) microm. The gametocysts are oval-shaped, measuring 40.0-65.0 (58.0+/-2.1) microm. The oocysts are navicular, measuring 8.0-12.0 (10.5+/-1.1) microm x 4.0-6.0 (5.5+/-1.1) microm.

Animals↗

Riboflavin content in autofluorescent earthworm coelomocytes is species-specific.

We have recently shown that a large proproportion of earthworm coelomocytes exhibit strong autofluorescence in some species (Dendrobaena veneta, Allolobophora chlorotica, Dendrodrilus rubidus, Eisenia fetida, and Octolasion spp.), while autofluorescent coelomocytes are very scarce in representatives of Lumbricus spp. and Aporrectodea spp. Riboflavin (vitamin B2) was identified as a major fluorophore in Eisenia jetida coelomocytes. The main aim of the present experiments was to quantify riboflavin content in autofluorescent coelomocytes (eleocytes) from several earthworm species through a combination of flow cytometric and spectrofluorometric measurements. Spectrofluorometry of coelomocyte lysates showed that riboflavin was non-detectable in the coelomocytes of Aporrectodea spp. and Lumbricus spp., but was a prominent constituent of lysates from species with autofluorescent eleocytes. In the latter case, riboflavin content was the highest in E. fetida, followed by Octolasion spp. > A. chlorotica > D. rubidus. The riboflavin content of coelomocytes correlates positively with eleocyte autofluorescence intensity measured by flow cytometry and visible with fluorescence microscopy.

Animals↗

Prolyl hydroxylase of earthworms. Substrate specificity of an enzyme from the subcuticular epithelium.

A relatively crude enzyme preparation derived from the subcuticular epithelium of earthworms catalyzed the formation of 4-hydroxyproline from prolyl residues in unhydroxylated natural collagens and in several synthetic collagen-like polypeptides. The specificity of hydroxylation differed from that of all vertebrate polyl hydroxylases in that (Gly-Pro-Ala)n was a much better substrate than (Gly-Ala-Pro)n. In contrast, however, only the so-called Y position proline (Gly-X-Y) was hydroxylated in Gly-Pro-Pro sequences derived either from natural collagen or from synthetic polypeptides; specificity of hydroxylation for the latter sequence is identical with that of the vertebrate enzymes. Little or no formation of 3-hydroxyproline could be demonstrated in preparations of the enzyme active as a 4-hydroxylase. In contrast with an earlier report from another laboratory, using a crude extract of earthworm body wall, we were unable to demonstrate either significant 3-hydroxyproline formation or efficient 4-hydroxylation of X position prolyl residues in synthetic polypeptides with the internal sequence Gly-Pro-Pro.

Animals↗

Hemagglutinins and bacterial agglutinins of earthworms.

The biological roles of invertebrate agglutinins have been and remain an unresolved subject of controversy. Classical studies on agglutinins, beginning with the pioneer work of Noguchi (1903) on Limulus polyphemus and Homarus americanus have emphasized their hemagglutinating properties, an approach that has been criticized for its lack of biological relevance. While erythrocyte agglutination has proven useful for determining various properties of invertebrate agglutinins, it does not address the question of their natural function. More recently, invertebrate agglutinins have been investigated for their ability to interact with pathogenic agents such as bacteria (for review, see Pistole, 1982), yeast (Van der Knapp et al., 1982; Renwrantz and Stahmer, 1983) and parasitic protozoans (Ingram et al., 1984). In addition, the possible relationship of agglutinins to defense mechanisms of both vertebrates and invertebrates has been indicated by the observation that limulin, the major agglutinin of Limulus polyphemus, bears a number of similarities to vertebrate C-reactive proteins (Robey and Liu, 1981). In annelids, there have been no studies on bacterial agglutinins prior to our work with Lumbricus (Stein et al., 1985; Stein et al., submitted). Earthworms are particularly appropriate for studying bacterial agglutinins since their coelomic fluid contains constant low levels of bacteria and fungal spores, and their agglutinins are both naturally occurring and inducible. Although our initial studies on Lumbricus agglutinins were directed toward their hemagglutinating properties, our recent observations using bacteria have allowed us to reach the following conclusions: 1) Lumbricus coelomic fluid normally contains agglutinins against both erythrocytes and bacteria. After injecting worms with either erythrocytes or bacteria, agglutinin titers increase in coelomic fluid. This increase appears to be due to both an increase in numbers of agglutinins as well as levels of specific agglutinins. 2) Absorption studies, temperature effects and sugar inhibition analyses suggest that agglutinins which bind to erythrocytes are identical to bacterial agglutinins, but there are additional agglutinins capable of reacting only with bacteria. 3) The inducibility and bacterial binding properties of Lumbricus agglutinins suggest that they serve an immune function by participating in the earthworm's defense against bacterial infection. In this sense, the agglutinins serve as a humoral surveillance system that entraps and prevents the multiplication of pathogenic bacteria.

Agglutinins↗

Leukocyte activity during earthworm inflammatory reactions.

This inclusive review describes essentially the findings of the author and his colleagues on the responses of earthworm coelomocytes to wounding and parasite infection and the responses of the earthworms themselves to second-set tissue grafts.

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Second-set allograft responses in the earthworm Lumbricus terrestris. Kinetics and characteristics.

Second-set Lumbricus terrestris allografts from the same donor to the same recipient undergo accelerated rejection if transplanted less than 10 days after first-set allografts. In contrast, second-set allografts, transplanted at longer intervals (20-90 days) show no accelerated rejection. Accelerated rejection of allotransplants under these kinetic conditions agrees with earlier intrafamilial xenotransplant results obtained in earthworms affirming an essential experimental variable: the time of second-set transplantation. Accelerated rejection, weak specificity, and short-term "memory" are three characteristics of the earthworm's allogeneic cellular defense/immune system. These reactions probably result from intense responses (although of short duration) of coelomocytes activated by first transplants that are still present at the time of second-set grafting but absent or inactive at later periods.

Animals↗

[Neural control of somatic muscle function in the earthworm, Allobophora longa, and in the leech, Hirudo medicinalis].

Studies have been made on the electrical activity of the segmentary nerves and connectives of the abdominal nervous chain in the earthworm and leech. It was shown that the electrical activity of the isolated piece of the abdominal chain of the leech is manifested of periodic outbursts of impulsation. Presumably this central periodicity accounts for the discharge-like pattern of muscle rhythmic activity which was revealed in our earlier investigations. The electrical activity in the central nervous system of the earthworm depends on afferent influences which pass to the ganglia from the peripheral sensory nervous cells. Stimulation of the abdominal nervous chain did not result in extra discharges of muscle activity, but only affected some of the parameters of the latter.

Afferent Pathways↗

[Gastrointestinal bacteriology of the earthworm Eisenia foetida grown in composted broiler litter].

The high animal concentration in intensive production brings about problems due to the volume of excreta and environmental pollution with the consequent risk for animal and human health. Therefore, previous to the application of any strategy of organic wastes recycling it is necessary to know possible sanitary risks. Farm organic wastes can be recycled through the intensive breeding of earthworms from species Eisenia foetida (Annelida, Oligochaeta) which can break down the consumed organic substance into humus. E. foetida is able to accelerate biodegrading wastes decomposition. It has been selected for recycling because its self productivity, its capacity to live in high concentrations, its feeding habits as well as its resistance to stress. The aim of this work is to study the microbial flora potentially pathogenic and their survival in the intestinal tract in sexually mature earthworms cultivated in composted broiler litter. Total coliforms, faecal coliforms, Salmonella, Pseudomonas and other enteric bacteria were sought in accordance with the recommended methodology. In E. foetida no positive results were obtained in the MPN/g (most probable number) of total coliforms, faecal coliforms, Salmonella, Pseudomonas and other Enterobacteriaceae family bacteria. These results suggest an antimicrobial response upon Gram negative bacteria from the gizzard and through the intestinal tract.

Animal Husbandry↗

Nebulin-like protein in the earthworm Eisenia foetida. Immunocytochemical electron microscopic study and western blot analysis of several muscle cell types.

Nebulin is a giant protein (500-900 kDa), which has been reported only in the skeletal muscle (not in cardiac muscle) of vertebrates. The possible presence and distribution of nebulin-like proteins in obliquely striated muscles (body wall and inner muscular layer of the pseudoheart) and smooth muscle (outer muscular layer of the pseudoheart) from the earthworm Eisenia foetida have been examined by means of Western blotting analysis and immunoelectron microscopy, using antibodies against mouse nebulin. The results were compared with those obtained in skeletal, cardiac and smooth muscles of the mouse. In the mouse, immunoreaction to nebulin was observed only in the skeletal muscle and extended along the length of the thin filament. In the earthworm, immunoreaction to a nebulin-like protein was found in the muscle of the body wall and the inner muscular layer of the pseudoheart, but not in the outer muscular layer of the pseudoheart. By electron microscopy, immunolabeling to this protein was observed along the whole length of the thin filament. Western blotting analysis of this nebulin-like protein showed a single band at an estimated molecular mass between 350 and 450 kDa that is slightly lower than that of mouse skeletal muscle nebulin.

Animals↗

Earthworm immune responses.

The knowledge of the immunity in annelids started with the use of earthworms as biomarkers indicating changes caused by environmental pollution. Defence strategies effectively protect earthworms against bacterial infections and parasitic invasion. A natural immunity formed by anatomical and chemical protective barriers prevents damage of the underlying tissues, body fluid losses, and microbial infections of the body cavity. The internal defence mechanisms of annelids involve phagocytosis, nodule formation and encapsulation, blood coagulation and wound repair, and antibacterial immune proteins. The antibacterial activity of coelomic fluid associated with lysozyme-like substances and inducible humoral molecules support haemocytic reactions in the annelid defence system.

Animals↗

Some observations on the fine structure of the giant nerve fibers of the earthworm, Eisenia foetida.

Sectioned dorsal giant fibers of the earthworm Eisenia foetida have been studied with the electron microscope. The giant axon is surrounded by a Schwannian sheath in which the lamellae are arranged spirally. They can be traced from the outer surface of the Schwann cell to the axon-Schwann membranes. Irregularities in the spiral arrangement are frequently observed. Desmosome-like attachment areas occur on the giant fiber nerve sheath. These structures appear to be arranged bilaterally in columns which are oriented slightly obliquely to the long axis of the giant fiber and aligned linearly from the axon to the periphery of the sheath. At these sites they bind together apposing portions of Schwann cell membrane comprising the sheath. Longitudinal or oblique sections of the nerve sheath attachment areas are reminiscent of the Schmidt-Lantermann clefts of vertebrate peripheral nerve. Septa of the giant fibers have been examined. They are symmetrical or non-polarized and consist of the two plasma membranes of adjacent nerve units. Characteristic vesicular and tubular structures are associated with both cytoplasmic surfaces of these septa.

Animals↗

Lethal and non-lethal responses of spermatozoa from a wide variety of vertebrates and invertebrates to lysenin, a protein from the coelomic fluid of the earthworm Eisenia foetida.

Lysenin, a novel protein that we isolated from the coelomic fluid of the earthworm Eisenia foetida, binds specifically to sphingomyelin (SM) among various phospholipids found in cell membranes, and causes cytolysis. The plasma membrane of mammalian spermatozoa is known to contain SM at relatively high levels and we therefore examined the effects of lysenin on the spermatozoa of various animals. Lysenin had lethal effects on spermatozoa of 5 of 33 species of invertebrates tested and on spermatozoa of 30 of 39 species of vertebrates. We postulated that plasma membranes of the spermatozoa of most invertebrates might not contain SM whereas those of most vertebrate species might contain SM. These possibilities were supported by our failure to detect SM chemically in the testes of three species of invertebrates, in none of which spermatozoa responded to lysenin. In contrast, we detected SM in the testes of all 25 vertebrate species examined, irrespective of a negative or positive response of spermatozoa to lysenin. None of the six species of Protista examined was affected by lysenin. Our survey suggests that, in general, the spermatozoa of animals can be grouped into two categories, invertebrate and vertebrate, depending on the absence or presence of SM in their plasma membrane. The incorporation of SM into spermatozoa seems first to have occurred in protochordates during the course of evolution. Discussions about the exceptional responses to lysenin observed in the spermatozoa of five species of invertebrates and of nine species of vertebrates are made from phylogenetic and reproductive viewpoints. J. Exp. Zool. 286:538-549, 2000.

Animals↗

Delaminating myelin membranes help seal the cut ends of severed earthworm giant axons.

Transected axons are often assumed to seal by collapse and fusion of the axolemmal leaflets at their cut ends. Using photomicroscopy and electronmicroscopy of fixed tissues and differential interference contrast and confocal fluorescence imaging of living tissues, we examined the proximal and distal cut ends of the pseudomyelinated medial giant axon of the earthworm, Lumbricus terrestris, at 5-60 min post-transection in physiological salines and Ca2+-free salines. In physiological salines, the axolemmal leaflets at the cut ends do not completely collapse, much less fuse, for at least 60 min post-transection. In fact, the axolemma is disrupted for 20-100 microm from the cut end at 5-60 min post-transection. However, a barrier to dye diffusion is observed when hydrophilic or styryl dyes are placed in the bath at 15-30 min post-transection. At 30-60 min post-transection, this barrier to dye diffusion near the cut end is formed amid an accumulation of some single-layered and many multilayered vesicles and other membranous material, much of which resembles delaminated pseudomyelin of the glial sheath. In Ca2+-free salines, this single and multilayered membranous material does not accumulate, and a dye diffusion barrier is not observed. These and other data are consistent with the hypothesis that plasmalemmal damage in eukaryotic cells is repaired by Ca2+-induced vesicles arising from invaginations or evaginations of membranes of various origin which form junctional contacts or fuse with each other and/or the plasmalemma.

Animals↗

Embryogenesis of the serotonergic system in the earthworm Eisenia fetida (Annelida, Oligochaeta): immunohistochemical and biochemical studies.

Organization of the serotonergic system and changes of the serotonin (5-HT) content were studied during the embryogenesis of the earthworm Eisenia fetida, using immunocytochemistry and HPLC. A gradual emergence of 5-HT immunoreactive (IR) cells and their axon projections in the several ganglia of the central (CNS) and peripheral nervous system are described in the context of a staged time-scale of development. The first 5-HT-IR neurons appear in the subesophageal ganglion at an early embryonic stage (E2), followed by neurons in some rostrally located ventral ganglia. In the cerebral ganglion, 5-HT-IR cells can be detected only from stage E5. The number of labeled cells in each ganglion of the embryo increases until hatching, when it is still considerably lower than that observed in adults. This shows that the development of the 5-HTergic system is far from complete by the end of embryogenesis. Organization of 5-HT-IR innervation of the body wall starts by stages E3 to E4. In the stomatogastric nervous system the first 5-HT-IR fibers can be detected by stage E5. By stage E9 5-HT immunopositive neurons can be observed in both the stomatogastric ganglia and the enteric plexus. Both 5-HT levels and the numbers of the labeled cells show a significant increase before hatching, which indicate a functional maturation of the 5-HTergic system. Based on the early appearance of 5-HT, we suppose that it may play a regulatory role in both the gangliogenesis and the maturation of peripheral functions necessary during postembryonic life.

Animals↗

Graft rejection in earthworms: an electron microscopic study.

Graft rejection in the earthworm, Lumbricus terrestris, is mediated by granulocytic coelomocytes (neutrophils, by light microscopy). Within 1--3 days post-transplantation of body-wall xenografts, granulocytic coelomocytes migrate to the transplant site, penetrate the graft matrix, and actively phagocytize viable muscle fibers. Destruction of muscle tissues may be augmented by lymphocytic coelomocytes (basophils, by light microscopy), but these cells participate in a minor way and do so very late in the rejection process (days 11--13). Xenografts, autografts and sham-operated controls display generalized inflammatory reactions, including coelomyocyte infiltration into the grafted tissues. In autografts, however, granulocytes only penetrate the inner longitudinal muscle layers and extensive infiltration, persistence and complete destruction never occur. Lymphocytic coelomocytes are not observed in autografts or sham-operated controls. Grafts rejection by leukocytes in an advanced invertebrate confirms that aspects of cell-mediated immunity evolved early in phylogenetic history.

Animals↗

Damage to the gills, skin and other tissues by lysenin and the coelomic fluid of the earthworm Eisenia foetida in two teleosts, Tanichthys albonubes and Oreochromis mossambicus.

Lysenin is a 33-kDa protein found in the coelomic fluid (CF) of the earthworm Eisenia foetida. Purified lysenin binds specifically to sphingomyelin (SM). In the present studies, we found that the white cloud mountain minnow Tanichthys albonubes and the Mozambique tilapia Oreochromis mossambicus died in solutions of lysenin (at concentrations above 2.5 microg/ml) and CF (0.6%, v/v) within 2 h. The gills of both species of fish were damaged similarly by lysenin and by CF. Most gill lamellae became irregularly bent or curled, with swelling of the epithelial cells of the lamellae. Red blood cells in the lamellar vascular sinuses, in the central venous sinuses, and in the blood vessels of the entire body became swollen and lysed, choking the sinuses. Epithelial cells in the skin were also damaged. When fish of both species were treated with lysenin or CF that had been incubated with SM-liposomes, they did not die. Their behavior remained normal and there was no damage to any cells or tissues. These findings suggest that SM might be involved in the lethal effects of lysenin and CF. It is likely that purified lysenin and lysenin in CF bound to SM in the cell membranes of the tissues mentioned above, damaging the cells. The presence of SM in the gills and skin was confirmed, supporting this hypothesis. The damage to gills and hemolysis might have resulted in lethal respiratory problems. Damage to the skin might disturb the exchange of ions through the skin, hastening death. Damage by lysenin and CF to epithelial cells of the cornea and the wall of the oral cavity was also recognized, but there was no such damage to the intestine.

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Reorganization of monoaminergic systems in the earthworm, Eisenia fetida, following brain extirpation.

The present study describes the major aspects of how monoaminergic (serotonin, dopamine) systems change in the course of regeneration of the brain in the earthworm (Eisenia fetida), investigated by immunocytochemistry, HPLC assay, and ligand binding. Following brain extirpation, the total regeneration time is about 80 days at 10 degrees C. On the 3rd postoperative day serotonin, and on the 11th postoperative day tyrosine hydroxylase-immunoreactive neurons can be observed in the wound tissue. Thereafter the number of the immunoreactive cells increases gradually, and by the 76th-80th postoperative days all serotonin- and tyrosine hydroxylase-immunopositive neurons can be found in their final positions, similarly to those observed in the intact brain. Labeled neurons located in the dorsal part of the regenerated brain appear earlier than the cells in lateral and ventral positions. Both serotonin- and tyrosine hydroxylase-immunoreactive neurons of the newly formed brain seem to originate from undifferentiated neuroblasts situated within and around the ventral ganglia and the pleura. Dopaminergic (tyrosine hydroxylase-immunoreactive) elements may additionally derive from the proliferation of neurons localized in the subesophageal ganglion and the pharyngeal nerve plexus. Following brain extirpation, both serotonin and dopamine levels, assayed by HPLC, first increase in the subesophageal ganglion; by the 25th day of regeneration, the monoamine content decreases in it and increases in the brain. Hence it is suggested that monoamines are at least partly transported from this ganglion to the regenerating brain. At the same time, (3)H-LSD binding can be detected in the regenerating brain from the 3rd postoperative day, showing a continuous increase until the 80th postoperative day, suggesting a guiding role of postsynaptic elements in the monoaminergic reinnervation of the newly formed brain.

Animals↗