Metals and phosphate in the chloragosomes of Lumbricus terrestris and their possible physiological significance

Summary The chloragog cells of the earthworm Lumbricus terrestris contain numerous granules, chloragosomes, which were analyzed for metals and phosphate by histochemistry, by use of an electron microscope X-ray microprobe (EMMA), and by chemical analysis of chloragosome preparations. Inorganic and organic phosphate each accounts for about 3% of the chloragosome dry mass, Ca for 2–3%, Zn for 1–3% and Mg for 0.2–0.4%. Carbonate is not present in chloragosomes. The average molar ratios CaMgZntotal PO₄ are 10.10.31. The CaPO₄ ratio is fairly constant (correlation coefficient 0.99), while the ZnPO₄ ratio varies considerably. It is concluded that Ca is bound in the form of inorganic CaHPO₄ and organic ROPO ₃Ca (or possibly Ca-polyphosphate complexes). Mg may also be phosphate-bound, while Zn probably is not. Chemical analysis of the calciferous glands revealed a high concentration of Ca, small amounts of Mg and phosphate, but no Zn. It is concluded that Zn is not excreted through the calciferous gland.Storage of Ca in the chloragocytes and excretion of CaCO₃ by the calciferous gland may be physiologically linked. Regulation of the concentrations of Ca and HCO₃ ^–ions in the blood and coelomic fluid may assist in equalization of osmotic pressures during dehydration and rehydration. This regulation may be a major function of the chloragosomes.The chloragosomes were discussed in relation to the spherites of various arthropods and molluscs and to the cytosomes of anoxia-tolerating molluscs.I wish to thank Theodore A. Hall for the use of the EMMA-4 microprobe facility, T.C. Norrnann for help in performing the microprobe analysis, Bruno Hansen for the improved zinc determination method and Annette Prentø for performing the chemical analyses. This work was supported by the Danish Natural Science Research Council (grant 511-3522). The EMMA-4 facility is supported by a grant from the British Science Research Council     

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.     

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.     

Cytochemical observations of coelomocytes from the earthworm, Lumbricus terrestris.

Synopsis Coelomocytes of the earthworm,Lumbricus terrestris, were stained by cytochemical techniques to determine the biochemical composition of the seven different cell types and subtypes. The enzymes acid phosphatase and -glucuronidase are present in all types of coelomocytes, but are especially abundant in basophils and neutrophils; the differences in enzyme amounts correlate well with the differences in phagocytic activity of the various cell types. No peroxidase is present. The cytoplasmic basophilia of basophils is due primarily to ribonucleic acid. Basophils also contain large deposits of glycogen, with neutrophils and chloragogen cells containing somewhat lesser amounts. The predominant granules of the two types of acidophils and of granulocytes are composed of a basic protein and a neutral mucopolysaccharide or glycoprotein. A second granule population, present in low numbers in acidophils and granulocytes, but in larger numbers in basophils and neutrophils, is small in size and lipid-positive and may, in part, represent lysosomes.Lipid is especially abundant in the vesicles and granules of the two types of chloragogen cells. Some granules of chloragogen cells also contain ferrous and ferric iron and a substance with pseudoperoxidase activity. The cytoplasm contains protein, glycogen, and a neutral mucopolysaccharide. In addition, acid mucopolysaccharides are variably present in the cytoplasm of chloragogen cells, the only coelomocytes to contain this class of substances.     

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     

Intersegmental variation of afferent pathways to giant interneurons of the earthworm,Lumbricus terrestris L.

Summary We have investigated the connectivity of four classes of mechanosensory afferents to giant interneurons in the earthwormLumbricus. Three of these classes of afferents change their specification for connection to medial giant (MGF) and lateral giant (LGF) fibers along the length of the animal. Near the caudal end, stimulation of touch, pressure and small tactile fibers generates excitatory post-synaptic potentials, epsp's, in the two LGF's but not in the MGF. Near the rostral end these afferents produce much smaller epsp's in the LGFs but produce large epsp's in the MGF. In the middle region of the animal an overlap region exists where both giant fibers receive approximately equal inputs from these afferents. The amplitude of these inputs are reduced compared to the maxima seen at either end. The fourth class of sensory afferents investigated, the stretch neurons, have no synaptic effect on the giant fibers anywhere in the nerve cord.These results explain at least part of the basis, in neuronal connectivity, for the differences in response to tactile stimulation of the head and tail segments previously characterized in terms of behavior and giant fiber impulse activity. In this system developmental mechanisms generating synaptic connectivity patterns have coded certain classes of homologous afferent neurons and interneurons to make different connections in different segments.Abbreviations MGF medial giant fiber - LGF lateral giant fiber - SN1 first segmental root - SN2 second segmental root - SN3 third segmental root - RIN giant interneuron     

Neurosecretion. XII. The formation of neurosecretory granules in the earthworm,Lumbricus terrestris L.

Summary The granules of neurosecretory cells in the supraesophageal ganglion of the earthworm, Lumbricus terrestris L., are formed by the Golgi apparatus. The process of neurosecretion is discussed in relation to this observation.This research was aided by grants (B-840, B-2145 and 2M-6418) of the United States Public Health Service.     

Elemental analysis of histochemically defined cells in the earthworm Lumbricus terrestris.

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-micron 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.     

The coelomocytes of the earthworm Lumbricus terrestris: morphology and phagocytic properties.

The coelomocytes of Lumbricus terrestris have been classified and described, based on Wright's stained preparations and on living cells. The five major categories consist of basophils, acidophils, neutrophils, granulocytes and chloragogen cells. Both the acidophil and chloragogen cell groups contain two subgroups. Granulocytes also exhibit heterogeneity with respect to staining properties of granules. Some possess acidophilic granules, some basophilic granules, and others contain both types. Granules of acidophils have been observed to be occasionally excreted from the cells. All cell types, with the exception of chloragogen cells, produce pseudopodia and are capable of phagocytosis, a vital component of the earthworm's immune response.     

Evidence of the presence of extraperoxisomal catalase in chloragogen cells of the earthworm,Lumbricus terrestris L.

Summary The DAB reactivity of the midintestine of the earthworm, consisting of epithelial layer, muscle layer, and chloragogen tissue, was examined electron microscopically. Besides the mitochondrial membranes of the examined cell types and the hemoglobin content of the blood vessels and chloragogen cells, a considerable DAB reactivity was found in the whole cytosol of the chloragocytes. The DAB reaction of the cytosol was more intensive when incubation medium for catalase, less intensive when incubation medium for peroxidase, was used and did not occur when H₂O₂ was omitted.Cytosol of the chloragogen cells was isolated and preliminary assay of catalase and peroxidase activities was made. Cytosol samples showed moderate peroxidase activity, but catalase activity measured by the decomposition of hydrogen peroxide showed a very high rate. Catalase and peroxidase activities of the cytosol were heat-sensitive and might have been inhibited by azide and cyanide, respectively. Results prove the assumption that the intensive DAB reactivity of the chloragocyte cytosol is caused by its extraperoxisomal catalase content.     

Development and characterization of novel microsatellite markers for the common earthworm (Lumbricus terrestris L.)

We developed and characterized 10 highly polymorphic microsatellite loci from an SSR‐enriched genomic DNA library of the common earthworm (Lumbricus terrestris L). Characterization of these loci using 32 individuals revealed high levels of genetic diversity, five to 18 alleles per locus and a high observed and expected heterozygosity. These loci will be used for paternity analysis and population genetic studies of the co‐evolution between L. terrestris and its parasites.     

Electron-microscopic observations of normal coelomocytes from the earthworm,Lumbricus terrestris

Summary Coelomocytes of the earthworm, Lumbricus terrestris, were studied by transmission electron microscopy. Four morphological cell types are distinguishable: lymphocytic coelomocytes, granulocytic coelomocytes, eleocytes (chloragogen cells), and inclusion-containing coelomocytes. Within these major categories, several distinct cell types differ and may represent developmental stages. The two types of lymphocytic coelomocytes are small with central nuclei and scanty cytoplasms. Two types of granulocytic coelomocytes differ greatly in shape and content; both have small dark-staining granules that resemble lysosomes. Electrocytes, derived from chloragogen tissue, contain a variety of granules, inclusions and vacuoles. Inclusion-containing coelomocytes appear as two types which may be immature and mature forms. Although these cells resemble those that have been referred to as erythroid cells in other invertebrates, the large inclusion bodies are apparently unrelated to hemoglobin; they can undergo morphologic transformation and be extruded by exocytosis. This information on lymphocytic, granulocytic and inclusion-containing coelomocytes is crucial to understanding more about cellular immunity in the earthworm.D.S.L. is supported by USPHS Training Grant AI-00453-05. E.A.S., D.H.M. and E.L.C. are supported by USPHS Grant HD-09333-03     

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.     

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).