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.     

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).     

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     

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     

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.     

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     

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.     

Subunit distribution of calcium-binding sites in Lumbricus terrestris hemoglobin

The giant, 3.6-MDa hexagonal bilayer hemoglobin (Hb) of Lumbricus terrestris consist of twelve 213-kDa globin subassemblies, each comprised of three disulfide-bonded trimers and three monomer globin chains, tethered to a central scaffolding of 36–42 linkers L1–L4 (24–32 kDa). It is known to contain 50–80 Ca and 2–4 Cu and Zn; the latter are thought to be responsible for the superoxide dismutase activity of the Hb. Total reflection X-ray fluorescence spectrometry was used to determine the Ca, Cu, and Zn contents of the Hb dissociated at pH 2.2, the globin dodecamer subassembly, and linker subunits L2 and L4. Although the dissociated Hb retained 20 Ca²⁺ and all the Cu and Zn, the globin subassembly had 0.4 to 3 Ca²⁺, depending on the method of isolation, and only traces of Cu and Zn. The linkers L2 and L4, isolated by reversed-phase high-pressure liquid chromatography at pH 2.2, had 1 Ca per mole and very little Cu and Zn. Electrospray ionization mass spectrometry of linker L3 at pH 2.2 and at neutral pH demonstrated avid binding of 1 Ca²⁺ and additional weaker binding of 7 Ca²⁺ in the presence of added Ca²⁺. Based on these and previous results which document the heterogeneous nature of the Ca²⁺-binding sites in Lumbricus Hb, we propose three classes of Ca²⁺-binding sites with affinities increasing in the following order: (i) a large number of sites (>100) with affinities lower than EDTA associated with linker L3 and dodecamer subassembly, (ii) 30 sites with affinities higher than EDTA occurring within the cysteine-rich domains of linker L3 and dodecamer subassembly, and (iii) 25 very high affinity sites associated with the linker subunits L1, L2, and L4. It is likely that the low-affinity type (i) sites are the ones involved in the effects of 1–100 mM Group IIA cations on Lumbricus Hb structure and function, namely increased stability of its quaternary structure and increased affinity and cooperativity of its oxygen binding.     

Neurotensin and cholecystokinin depress motility in isolated Lumbricus terrestris crop-gizzard preparations

The effects of neurotensin (NT) and cholecystokinin (CCK) were studied on isolated crop-gizzard preparations of Lumbricus terrestris suspended in a smooth muscle organ bath. Changes in the amplitude and frequency of contractions associated with spontaneous motility were observed in response to neurotransmitters known to have an excitatory effect (acetylcholine) and an inhibitory effect (serotonin); and to the hormones NT and CCK, which in vertebrate models have both been shown to inhibit gastric motility. The overall contractile amplitude and frequency of crop-gizzard contractions were decreased in response to increasing concentrations of NT and CCK. In general, both hormone-induced responses were similar when compared at equal molar concentrations. Cholecystokinin, however, did exhibit a greater reduction in contractile frequency than NT. It is speculated that possible desensitization of earthworm NT-receptors to higher hormone concentrations resulted in a depressed maximal response in the concentration–response curve. Despite that possibility, the overall hormonal inhibition was statistically significant. These results infer that NT- and CCK-induced inhibition of crop-gizzard motility may have a modulatory role in the transport of nutrients and overall efficiency of worm metabolism.     

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.     

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.     

Zur neuronalen Regulation des Wasserhaushalts bei Lumbricus terrestris L.

Zusammenfassung Im Dehydrations-Experiment tolerieren Regenwürmer Flüssigkeitsverluste bis zu 70% ihres Körpergewichts. Hierbei ergeben sich hohe Anforderungen an die Anpassungsfähigkeit von Nephridien, Darm und Integument, die im Dienste des Wasser-und Elektrolythaushalts stehen. Um die Abhängigkeit dieser Organe von zentralnervösen Einflüssen zu studieren, wurden bei Lumbricus terrestris Ober-, Unterschlundganglion sowie ein Bauchmarkabschnitt zwischen dem 10.–15. Segment hinter dem Clitellum entfernt. In diesen Segmenten wurden 30, 60 und 120 min nach Versuchsbeginn der Schleimgehalt von Epidermiszellen, die Kerngröße von Darmepithel- und Chloragogen-Zellen sowie der innere und äußere Tubulusumfang von Nephridien cytometrisch bestimmt. In gleichen Intervallen wurde im Oberschlundganglion der Nucleinsäure- und Proteingehalt Gomoripositiver Neurone mikrospektrographisch gemessen. Aus dem Vergleich von cytometrischen und mikrospektrographischen Daten geht hervor: 1. Der nephridiale Stäbchenkanal steht am stärksten unter neuroendokrinem Einfluß. 2. Darmepithel- und Chloragogen-Zellen hängen sowohl von zentraler, neuroendokriner als auch von segmentaler, nervöser Kontrolle ab. 3. Die Schleimsekretion von Epidermiszellen wird wahrscheinlich über segmentale Nerven gesteuert; sie zeigt jedoch die ausgeprägteste Autonomie. Somit stehen die an der Regulation des Wasser- und Elektrolythaushalts beteiligten Organe von Lumbricus terrestris unter einer doppelten Kontrolle durch peptiderge sekretorische Neurone des Oberschlundganglions und segmentale Neurone des Bauchmarks.

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Neuronal regulatory mechanisms of the water balance in Lumbricus terrestris L.

Summary Earthworms tolerate loss of fluids up to 70% of their total body weight. The adaptive capacity must thus be very high in organs which control the exchange of water and electrolytes in nephridia, the intestine and skin. In order to study the influence of the central nervous system on adaption, the supra- and suboesophageal ganglion and the 10th–15th segments behind the clitellum were removed. In segments deprived of ventral nerve cord the mucous content of epidermal cells, the nuclear area of intestinal epithelial cells and chloragogen cells together with the circumference of nephridial tubules were measured 30, 60 and 120 min after placing Lumbricus terrestris in a standardized dry environment. Protein and nucleic acid contents of Gomori-positive neurons in the supraoesophageal ganglion were measured at these times. Comparison of the results of cytometric and microspectrographic measurements shows that 1) the distal segment of the wide nephridial tube is under neuroendocrine control, 2) intestinal epithelial cells and chloragogen cells are influenced by segmental neurons and central secretory nervous elements, 3) mucous secretion is autonomous but may be controlled by segmental nerve impulses. Therefore, water- and electrolyte exchange in Lumbricus terrestris is regulated by secretory peptidergic neurons of the supraoesophageal ganglion and by nerve cell complexes in the ventral nerve cord.

Geschäftsführender Direktor: Prof. Dr. A. Oksche.     

Lumbricid earthworm invasion in the Carpathian Mountains and some other sites in Romania

Recent field research shows that the ubiquitous Lumbricus terrestris and Octolasion lacteum (the large parthenogenetic form) have invaded new habitats in Romania, replacing mostly endemic or rare species of the former earthworm communities which were recorded 20–30 years ago. Seven different cases of earthworm invasion are currently described. Invader species have larger ecological tolerances as compared with the endemic species.     

Fine structure of photoreceptor cells in the earthworm,Lumbricus terrestris

Summary Photoreceptor cells in the epidermis and nerve branches of the prostomium and in the cerebral ganglion of Lumbricus terrestris were investigated with the electron microscope. The photoreceptor cell is similar to the visual cell of Hirudo by having a central intracellular cavity (phaosome) filled with microvilli. Besides microvilli, several sensory cilia can also be found in the phaosome but they are structurally independent of the microvilli. A gradual branching of the phaosome cavity into smaller cavities makes its sectional profile extremely labyrinthic. Flattened smooth-surfaced cisternae in stacks of 2 to 5 are frequently observed around the phaosome. Characteristic constituents of the cytoplasm are vesicles and vacuoles filled with a substance of varying density. The photoreceptor cell is covered by glial cells or by their processes which at many places deeply invaginate the cell surface (trophospongium).

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Zusammenfassung Die Feinstruktur der Photorezeptorzellen in der Epidermis, in kleineren Nervenästen und im Zerebralganglion von Lumbricus terrestris wurde untersucht. Das Vorhandensein eines zentralen, intrazellulären Lumens (Phaosom), das mit Mikrozotten gefüllt ist, erinnert in der Struktur der Photorezeptorzelle des Regenwurms an Lichtsinneszellen von Hirudo. Außer Mikrozotten findet man im Phaosom einige Zilien vom Typ 9×2+0; solche Zilien sind von den Mikrozotten strukturell unabhängig. Durch eine Aufzweigung des Phaosoms in kleinere Buchten, die tief in das umgebende Zytoplasma eindringen, erhält es ein labyrinthartiges Aussehen.Glatte Zisternen in Gruppen von 2 bis 5 wurden oft um das Phaosom im Zytoplasma beobachtet. Charakteristische Bestandteile der Zelle sind noch Vesikel und Vakuolen, die mit einer Substanz von wechselnder Elektronendichte gefüllt sind. Die Photorezeptorzellen werden von Gliazellen und Gliafortsätzen umgeben, die an vielen Stellen die Zelloberfläche tief einstülpen (Trophospongium).

     

Neurochemical characterization of nervous elements innervating the body wall of earthworms (Lumbricus, Eisenia): immunohistochemical and pharmacological studies

The distribution and chemical neuroanatomy of nervous elements and certain pharmacological–physiological characteristics of the innervation of the body wall in earthworms are described. Solitary sensory bipolar cells can be found among the epithelial cells. These bipolar cells contain serotonin, tyrosine hydroxylase, histamine, gamma-amino-butyric acid (GABA), Eisenia tetradecapeptide, proctolin or rhodopsin in various combinations. In the body wall, the plexus submuscularis is composed of nerve fibres only, whereas the plexus subepithelialis and muscularis also contain solitary nerve cells. These cells display histamine, GABA or neuropeptide Y immunoreactivity. The fibres of the three plexuses are reactive to serotonin, histamine, Eisenia tetradecapeptide, proctolin, GABA and neuropeptide Y antibodies. FMRFamide-immunoreactive fibres of the plexus muscularis originate from the central nervous system, whereas axons containing the other studied molecules are derived from both peripheral and central structures. High pressure liquid chromatography assays have revealed serotonin, dopamine and histamine in the body wall. Contractions of the body wall musculature can be elicited with serotonin and FMRFamide. Serotonin-evoked contractions are suppressed by the application of GABA. Serotonin acts both directly on the muscle cell receptors and indirectly through initiating transmitter release from the nervous elements, whereas the FMRFamide-induced contractions seem to be mediated through the muscle cell receptors only. The pharmacological profiles of the serotonin and GABA receptors resemble those of the vertebrate 5-HT₃ and GABA_B receptor types. Our findings indicate that both the sensory and efferent system of the annelid body wall operate by means of a variety of neuroactive compounds, suggesting a complex role of signalling systems in the regulation of this organ.This work was supported by the Hungarian Scientific Research Fund (OTKA; grant nos. T 34106 and T 34160). Márta Wilhelm is in receipt of a János Bolyai Scholarship.     

Evidence for the presence of thyroid stimulating hormone, thyroglobulin and their receptors in Eisenia fetida: a multilevel hormonal interface between the nervous system and the peripheral tissues

The present study describes the localization and distribution of thyroid-stimulating hormone (TSH), thyroglobulin (TGB) and their receptors in Eisenia fetida (Annelida, Oligochaeta) as revealed by immunohistological methods. Immunopositive neuronal and non-neuronal cells are present in both the central nervous system and some peripheral organs (e.g. foregut and coelomocytes). TSH- and TGB-immunopositive neurons in the various ganglia of the central nervous system are differentailly distributed. Most of the immunoreactive cells are found in the suboesophageal ganglion. The stained cells also differ in their shapes (round, oval, pear-shaped) and sizes (small, 12–25 μm; medium, 20–35 μm; large, 30–50 μm). In all ganglia of the central nervous system, TSH-positive neurons additionally show gamma aminobutyric acid (GABA) immunopositivity. Non-neuronal cells also take part in hormone secretion and transport. Elongated TSH-positive cells have been detected in the capsule of the central ganglia and bear granules or vacuoles in areas lacking neurons. Many of capillaries show immunoreactivity for all four tested antibodies in the entire central nervous system and foregut. Among the coelomocytes, granulocytes and eleocytes stain for TSH and its receptor and for TGB but not for thyroid hormone receptor. Most of the granulocytes are large (25–50 μm) but a population of small cells (10–25 μm) are also immunoreactive. None of the coelomocytes stain for GABA. We therefore suggest that the members of this hormone system can modify both metabolism and immune functions in Eisenia. Coelomocytes might be able to secrete, transport and eliminate hormones in this system.This work was supported by the MTA-PTE Adaptation Biology Research Group and National Research and Developmental Fund (NKP 1/048/2001). M.W. is in receipt of a János Bolyai Scholarship.     

Intra- and extraganglionic nerve endings formed by neurosecretory cells of the cerebral ganglion of the earthworm (Lumbricus terrestris L.)

Summary In the cerebral (= supraesophageal, suprapharyngeal) ganglion of the earthworm, a number of neurosecretory Gomori-positive perikarya are bipolar; others are unipolar, or multipolar. Some of the neurosecretory cell processes project centrally into a fibrous zone; peripheral processes enter small nerves which leave the dorsocaudal aspect of the ganglion.In the central fibrous zone, the neurosecretory fibers form varicose Gomoripositive terminals. Here, also zinc-iodine-osmium (ZIO)-positive fibers and monoamine fluorescent fibers are found. With the electron microscope, nerve terminals containing synaptic vesicles and either large neurosecretory peptidergic granular vesicles (diameter more than 1500 Å), or smaller granular vesicles (diameter about 1300 Å, or 900 Å) are observed. These axon endings mainly form axo-dendritic synapses. Peptidergic profiles are both pre- and postsynaptic. Some of the extraganglionic peptidergic fibers appear to terminate around vessels, but most of them form terminals on the visceral muscle cells which surround the ganglion.We think that the central neurosecretory processes communicate with the fibers of the synaptic zone of the ganglion. The peripheral neurosecretory peptidergic fibers are supposed to form a primitive neurohemal area and/or to function as vasomotor nerves. The fibers innervating the visceral muscle cells may represent vegetative nerves.     

The effects of FMRFamide, serotonin, and acetylcholine on the isolated crop-gizzard of the earthworm, Lumbricus terrestris

We examined the effects of FMRFamide, serotonin, and acetylcholine on the isolated crop-gizzard of the earthworm, Lumbricus terrestris. FMRFamide caused a decrease in contraction amplitude with a threshold between 10⁻⁹ and 10⁻⁸ M and a biphasic change in contraction rate. The contraction rate increased with a threshold between 10⁻⁸ and 10⁻⁷ M and decreased with a threshold between 10⁻⁶ and 10⁻⁵ M. Serotonin decreased the contraction rate with a threshold between 10⁻⁸ and 10⁻⁷ M and the amplitude with a threshold between 10⁻⁸ and 10⁻⁷ M. Acetylcholine increased the contraction rate with a threshold between 10⁻⁸ and 10⁻⁷ M and caused a biphasic change in contraction amplitude. Amplitude rose with a threshold between 10⁻⁹ and 10⁻⁸ M and decreased with a threshold between 10⁻⁶ and 10⁻⁵ M. These results suggest that all three neurotransmitters may play a role in controlling the movement of the digestive tract in L. terrestris.     

Functional expression of an earthworm fibrinolytic enzyme in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Two cDNA fragments (lrF1 and lrF2) representing a fibrinolytic enzyme gene of F-III-2 (GenBank AB045719), without and with signal peptide coding sequence, were cloned from earthworm Lumbricus rubellus. The two fragments were inserted into bacterial expression vector pET28a (+), respectively. Subsequent expression showed that both lrF1 and lrF2 proteins were produced as an inclusion body form in E. coli BL21 (DE3) pLysE. After protein refolding and purification, the fusion lrF1 and its derivative without poly histidine tags at the N-terminus showed fibrinolytic activity on fibrin plates with relative activity of 134.3 U/mg protein and 139.7 U/mg protein, respectively, whereas the fusion lrF2 and its derivative without the tags at the N-terminus, had no fibrinolytic activity. The results indicated that the E. coli expression system could not recognize the endogenous signal peptide of F-III-2, and the effect of the histidine tags at the N-terminus on the fibrinolytic activity of the expressed protein was insignificant.     

Clostridiaceae and Enterobacteriaceae as active fermenters in earthworm gut content

The earthworm gut provides ideal in situ conditions for ingested heterotrophic soil bacteria capable of anaerobiosis. High amounts of mucus- and plant-derived saccharides such as glucose are abundant in the earthworm alimentary canal, and high concentrations of molecular hydrogen (H₂) and organic acids in the alimentary canal are indicative of ongoing fermentations. Thus, the central objective of this study was to resolve potential links between fermentations and active fermenters in gut content of the anecic earthworm Lumbricus terrestris by 16S ribosomal RNA (rRNA)-based stable isotope probing, with [¹³C]glucose as a model substrate. Glucose consumption in anoxic gut content microcosms was rapid and yielded soluble organic compounds (acetate, butyrate, formate, lactate, propionate, succinate and ethanol) and gases (carbon dioxide and H₂), products indicative of diverse fermentations in the alimentary canal. Clostridiaceae and Enterobacteriaceae were users of glucose-derived carbon. On the basis of the detection of 16S rRNA, active phyla in gut contents included Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Firmicutes, Gemmatimonadetes, Nitrospirae, Planctomycetes, Proteobacteria, Tenericutes and Verrucomicrobia, taxa common to soils. On the basis of a 16S rRNA gene similarity cutoff of 87.5%, 82 families were detected, 17 of which were novel family-level groups. These findings (a) show the large diversity of soil taxa that might be active during gut passage, (b) show that Clostridiaceae and Enterobacteriaceae (fermentative subsets of these taxa) are selectively stimulated by glucose and might therefore be capable of consuming mucus- and plant-derived saccharides during gut passage and (c) indicate that ingested obligate anaerobes and facultative aerobes from soil can concomitantly metabolize the same source of carbon.