Glycosylated trypsin-like proteases from earthworm Eisenia fetida

Although groups of earthworm proteases have been found by several laboratories, it is still unclear how many of the isolated trypsin-like fibrinolytic enzymes are in glycosylated form. Here, eight glycosylated fibrinolytic proteases (EfP-0-1, EfP-0-2, EfP-I-1, EfP-I-2, EfP-II-1, EfP-II-2, EfP-III-1 and EfP-III-2) were isolated from an earthworm species (Eisenia fetida) through a stepwise-purification procedure: ammonium sulfate precipitation, affinity chromatography on a Sepharose-4B column coupled with soybean trypsin inhibitor (SBTI), and ionic chromatography with a DEAE-Cellulose-52 column. Among the eight purified trypsin-like glyco-proteases, EfP-0-2 and EfP-II-2 were newly isolated isozymes. Glycoprotein staining of the proteases on native-PAGE with a Schiff's reagent (sodium meta-periodate) revealed that the eight proteases were glycoproteins. Measurements of the glycan content with sodium meta-periodate and glycoprotein-test reagent showed that these proteases had different carbohydrate contents. Dot-blotting assay with ConA suggested the oligosaccharides were composed of mannose residues.     

Transmission of nephridial bacteria of the earthworm Eisenia fetida

The lumbricid earthworms (annelid family Lumbricidae) harbor gram-negative bacteria in their excretory organs, the nephridia. Comparative 16S rRNA gene sequencing of bacteria associated with the nephridia of several earthworm species has shown that each species of worm harbors a distinct bacterial species and that the bacteria from different species form a monophyletic cluster within the genus Acidovorax, suggesting that there is a specific association resulting from radiation from a common bacterial ancestor. Previous microscopy and culture studies revealed the presence of bacteria within the egg capsules and on the surface of embryos but did not demonstrate that the bacteria within the egg capsule were the same bacteria that colonized the nephridia. We present evidence, based on curing experiments, in situ hybridizations with Acidovorax-specific probes, and 16S rRNA gene sequence analysis, that the egg capsules contain high numbers of the bacterial symbiont and that juveniles are colonized during development within the egg capsule. Studies exposing aposymbiotic hatchlings to colonized adults and their bedding material suggested that juvenile earthworms do not readily acquire bacteria from the soil after hatching but must be colonized during development by bacteria deposited in the egg capsule. Whether this is due to the developmental stage of the host or the physiological state of the symbiont remains to be investigated.     

Distribution and action of some putative neurotransmitters in the stomatogastric nervous system of the earthworm,Eisenia fetida (Oligochaeta,Annelida)

Brain Cell Biology - The chemical neuroanatomy of the stomatogastric nervous system of the earthworm, Eisenia fetida, has been investigated, using antibodies raised against serotonin, tyrosine...     

Size-assortative mating in the earthworm Eisenia fetida (Oligochaeta, Lumbricidae)

In many species of simultaneous hermaphrodites, body size correlates with fecundity, and larger partners are preferred to small ones. Since sperm exchange is usually reciprocal, small individuals may be rejected by larger partners resulting in size-assortative mating. We studied the mating patterns in a natural population of the simultaneous hermaphroditic earthworm Eisenia fetida (Oligochaeta, Lumbricidae). We found that size-assortative mating processes existed, with variance in body weight within pairs lower than between pairs in mating earthworms. This non-random mating pattern probably reveals the existence of mate selection in this species, which lives at elevated densities with high availability of potential mates.     

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.     

Self-assemblage and quorum in the earthworm Eisenia fetida (Oligochaete, Lumbricidae)

Despite their ubiquity and ecological significance in temperate ecosystems, the behavioural ecology of earthworms is not well described. This study examines the mechanisms that govern aggregation behaviour specially the tendency of individuals to leave or join groups in the compost earthworm Eisenia fetida, a species with considerable economic importance, especially in waste management applications. Through behavioural assays combined with mathematical modelling, we provide the first evidence of self-assembled social structures in earthworms and describe key mechanisms involved in cluster formation. We found that the probability of an individual joining a group increased with group size, while the probability of leaving decreased. Moreover, attraction to groups located at a distance was observed, suggesting a role for volatile cues in cluster formation. The size of earthworm clusters appears to be a key factor determining the stability of the group. These findings enhance our understanding of intra-specific interactions in earthworms and have potential implications for extraction and collection of earthworms in vermicomposting processes.     

Reorganization of the GABAergic system following brain extirpation in the earthworm (Eisenia fetida,Annelida, Oligochaeta)

The reorganization of the GABAergic system was studied by means of immunohistochemistry after the symmetrical and asymmetrical (unilateral) extirpation of the brain of the annelid Eisenia fetida. GABA-immunoreactive neurons were first observed in the wound tissue on the 3rd postoperative day. Thereafter the number of labelled cells gradually increased, and by postoperative days 76-80 all GABA-immunoreactive cells (approx. 140 neurons) could be found in their final positions in the symmetrically regenerated brain. After asymmetrical brain extirpation, nearly all cells (70-75) could be detected in the regenerating hemisphere by postoperative days 50-56. In the early stages of the asymmetrical regeneration of the brain, more GABAergic cells were concentrated dorsally and laterally in the preganglion than during the symmetrical type of regeneration. In both types of regeneration, the immunoreactive neurons in the regenerated brain originated in part from undifferentiated neuroblasts situated in different parts of the body, and in part from dividing neurons localized mainly in the pharyngeal nerve plexus. Both exogenous GABA and picrotoxin, applied during the early stages (days 10- 12) of brain regeneration, inhibited the development of the wound tissue and the migration of the neuroblasts and the enteric neurons. At the same time, exogenous GABA application accelerated the proliferation of the pharyngeal neurons. No effect on the process of regeneration could be demonstrated when exogenous GABA and picrotoxin were given together.     

8-hydroxydeoxyguanosine generated in the earthworm Eisenia fetida grown in metal-containing soil

Heavy metal pollution of soil causes biological problems, such as mutagenicity to living organisms, including human beings. However, few methods have been developed to assess metal mutagenicity in soil. To avoid metal mutagenicity, an adequate bio-monitoring method is required. In the present study, to determine if the analysis of oxidative DNA damage generated in the earthworm is a useful bio-monitoring method for soil mutagenicity, the accumulation of 8-hydroxydeoxyguanosine (8-OH-dG), a major form of oxidative DNA damage, in Eisenia fetida (Savigny, 1826) treated with cadmium chloride (CdCl2) or nickel chloride (NiCl2) was analyzed. E. fetida was treated with Cd (10 or 200 microg/g soil) or Ni (10 or 200 microg/g soil) for 1, 2, and 3 weeks or 3 months. After metal exposure, the metal concentration in E. fetida was analyzed by atomic absorption spectrometry and the 8-OH-dG accumulated in E. fetida was analyzed by HPLC analyses and immunohistochemistry. Atomic absorption spectrometry revealed that Cd, but not Ni, accumulated within E. fetida. The 8-OH-dG levels in the DNA of E. fetida treated with Cd for 3 months were significantly higher than those in control E. fetida. Moreover, immunohistochemical analyses revealed that positive signals for 8-OH-dG accumulation in seminal vesicles were detected only in E. fetida treated with 10 microg of Cd for 3 months. Although some points remain unresolved, a bio-monitoring system analyzing the DNA damage generated in the earthworm might be useful for the assessment of the mutagenicity of soil contaminated with various heavy metals, such as Cd.     

Characterization of two endoglucanases for the classification of the earthworm,Eisenia fetida Waki

Eisenia fetida and Eisenia andrei are vermicomposting species that are used as model animals for testing chemical material toxicology. Eisenia spp. are grown commercially in various fields in Japan. However, these two species have not been classified because it is difficult to distinguish them morphologically; thus, all bred earthworms are called E. fetida. However, it has been proposed that these two species have different expression regulation mechanisms. Here, we classified a sample of earthworms purchased from several farms, confirming that both E. fetida and E. andrei are present in Japanese earthworm breeding programs. We also characterized two highly active endoglucanases (EfEG1 and EfEG2) from the E. fetida Waki strain, which contained strong fibrinolytic enzymes for improving human health. We confirmed that EfEG1 is 1371 bp long and belongs to GHF9. Thus, E. fetida Waki may have commercial application for biomass utilization and as a dietary health supplement.     

Embryogenesis of GABAergic elements in the nervous system of Eisenia fetida (Annelida, Oligochaeta)

The appearance and development of the GABA-immunoreactive nervous elements in the central nervous system of Eisenia fetida were studied by immunocytochemistry. The nervous system originates from the neuroectoderm situated on the ventral side of the embryo. The organization of the circumpharyngeal ring starts earlier than that of the ventral cord. In the elementary ring the first GABA-immunopositive neurons can be observed (E1 stage) around the mouth. Later the cell number gradually increases and parallel to this process the elementary ring is separeted into a superficial and a deeper portion. The brain and the subesophageal ganglion will be organized from the superficial ring, while the nervous elements of the deeper ring will give rise for the first GABA-immunoreactive elements of the stomatogastric nervous system. In the early stages of the embryogenesis the immunoreactive cells of the developing brain appear solitary, while from the stage E4 they gradually are observed in groups. According to their position, these cell groups are similar to those observed in the brain of the adult earthworms. During embryogenesis the level of the ventral cord ganglia depends on their position in the ectodermal germ bands. It means, that the more organized ganglia are near the circumpharyngeal ring, mean while less developed ganglia are situated caudally from them. By the end of the embryogenesis all ganglia of the ventral cord will be equally well organized. The nerve tracts of the ganglia are built up from contra- and ipsilateral by projected fibres. From E3 stage the medial tracts, mean while from the E4 stage the lateral tracts begin to be formed. During the next stages, more and more fibres connect to the both tracts. At hatching, the development of the central nervous system of Eisenia fetida is not completed, the process is continued during the postembryonic development.     

Phoresy of the entomopathogenic nematode Steinernema feltiae by the earthworm Eisenia fetida

The free-living stage of entomopathogenic nematodes occurs in soil, and is an environmental-friendly alternative for biological control. However, their dispersal capability is limited. Earthworms improve soil characteristics, changing soil structure and influencing many edaphic organisms. Thus, earthworms could be used as vectors to introduce/disperse beneficial organisms. Nevertheless this interaction has not been studied in detail. This study presents the infectivity results of Steinernema feltiae after passing through the Eisenia fetida gut. Although entomopathogenic nematodes have no deleterious effects on earthworms, their passage through E. fetida gut seriously affected their mobility and virulence.     

Enantioselective toxicity and degradation of chiral herbicide fenoxaprop-ethyl in earthworm Eisenia fetida

The enantioselective degradation of fenoxaprop-ethyl in ecological indicator earthworm was studied and the main metabolites (fenoxaprop, 6-chloro-2,3-dihydrobenzoxazol-2-one, ethyl-2-(4-hydroxyphenoxy)propanoate, 2-(4-hydroxyphenoxy)propanoic acid) were also monitored on an enantiomeric level. The individual enantiomers of fenoxaprop-ethyl and its three chiral metabolites were prepared to study the acute toxicity to earthworm. Chiral analysis methods were set up based on HPLC–MS/MS with chiralpak IC chiral column. Fenoxaprop-ethyl was not found in earthworms, while the primary metabolite fenoxaprop was in relatively high levels indicating a quick hydrolysis degradation. Fenoxaprop was accumulated almost exclusively with R-enantiomer in earthworms and the bio-concentration factors of R-fenoxaprop and S-fenoxaprop were 1.39 and 0.17 respectively with the enantiomer fraction (EF) values about 0.99. The degradation of R-fenoxaprop in earthworms followed first-order kinetics with half-life of 1.82 day. The other metabolites could not be detected in earthworms. The calculated LC₅₀ values showed ecological indicator earthworm was more sensitive to the four metabolites than fenoxaprop-ethyl. Furthermore, earthworm was more sensitive to the R-form of the chiral metabolites than the S-form and rac-form. The results suggested metabolites and enantioselectivity should be taken into consideration to better predict the exposure concentration and apply ecological indicators in toxicological studies.     

Isolation and some characterizations of a glycosylated fibrinolytic enzyme of earthworm,Eisenia fetida

Resin coupled with m-aminophenylbornic acid was used to isolate a glycosylated component from homogenate of earthworm (Eisenia fetida). The fraction showed a single band on SDS-PAGE with a molecular weight of 34, 193 Da determined by mass spectroscopy. The N-terminal region is AQVCCPDI, different from those of earthworm fibrinolytic enzymes reported previously (Nakajima et al. 1993). This glycosylated component showed an activity on digesting both Chromozym-TH and fibrin, suggesting that it is a novel fibrinolytic enzyme.     

Multi-isomorphous replacement phasing of the earthworm fibrinolytic enzyme component A from Eisenia fetida

Thrombosis is one of the most widely occurring diseases in modern life, which often causes disability and death. Fibrinolytic enzymes degrade fibrin, the major protein component of blood clots, and eventually lead to thrombolysis. Medications using fibrinolytic enzymes are the most effective methods used in the treatment of thrombosis. A variety of fibrinolytic enzymes, such as tPA, uPA, and streptokinase, have been extensively studied and used as thrombolytic agents in clinic. However, thes…     

Protein extraction from the earthworm Eisenia fetida for 2‐DE

We identified an efficient protocol for extracting proteins from whole earthworm, Eisenia fetida, for 2‐DE. Sample preparation is a critical step in a 2‐DE proteome approach and is absolutely essential for obtaining good results. Six protein extraction protocols based on different protein precipitation agents were tested and evaluated using 2‐DE. The methods generated remarkably different 2‐DE protein spot patterns. We conclude that trichloroacetic acid (TCA)‐A eliminates interfering compounds, thus allowing for the efficient resolubilization of proteins. TCA‐A gives good distinction, more bands in 1‐DE gels, and the most number of protein spots in 2‐DE gels. It is also rapid, provides the higher protein yield, and has the less number of steps. To demonstrate the quality of the extracted proteins, we cut several protein spots that were common to four methods from 2‐DE gels, analyzed them using MALDI‐TOF/TOF MS, and tentatively identified them. The classic TCA‐A method proved to be most useful as a standard method of extracting proteins from E. fetida.     

Evidence of lipofuscin and melanin in the brown body of the earthworm Eisenia fetida andrei

By means of histochemical and electron-microscopic techniques, two pigments were identified in the brown body of the earthworm Eisenia fetida andrei. Lipofuscin, often called the aging pigment, was the more abundant pigment and the first to be synthesized. Melanin was also found but was only present in the mature brown body. A computer-assisted image analysis was used to quantify the relative abundance of the two pigments at different stages of the formation of the brown body. A previous study showing phenoloxidase activity in the coelomocytes and the present demonstration of the ability of these cells to produce reactive oxygen systems when agglutinated can be correlated with the simultaneous presence of the two pigments in the brown body. The physiological significance of pigment synthesis by brown bodies is discussed with respect to the immune response in earthworms.     

Purification,characterization and crystallization of a group of earthworm fibrinolytic enzymes from Eisenia fetida

Seven fibrinolytic enzymes were purified from the earthworm Eisenia fetida. The molecular weights of the enzymes were 24663, 29516, 29690, 24201, 24170, 23028 and 29595, and the respective isoelectric points were 3.46, 3.5, 3.5, 3.68, 3.62, 3.94 and 3.46. All the proteases showed different fibrinolytic activity on fibrin plates. Studies on substrate specificity and inhibition indicated that they belonged to different types of serine proteases. N-Terminal sequencing indicated their high homology to those from the earthworm Lumbricus rubellus. All the enzymes have been crystallized.     

Glial repair in the cultured central nervous system of an insect

Summary Insect glial cells are capable of division and repair in organ culture after selective damage with the toxin ethidium bromide. The repair is slower and less organised than seen in vivo after similar treatment and is still incomplete after one month. Granule-containing cells, which play an important role in the early stages of repair in vivo, are never seen in cultured connectives. This observation adds further support to the hypothesis that these cells are derived from haemocytes and that their presence is necessary for rapid and orderly repair. The uptake of ³H-thymidine into perineurial glial cells in vitro, both in control and ethidiumtreated connectives, shows that there is a considerable proliferation of cells in this region. Some uptake of thymidine is also seen in subperineurial glia but division alone cannot account for the large increase in the number of glial nuclei found at the early stages of repair in this region. Further, glial cells with diverse morphologies suggest that subpopulations are present. We conclude that cell migration from undamaged areas, as well as cell proliferation, is necessary for CNS repair in vitro.     

Effects of chronic gamma irradiation on reproduction in the earthworm Eisenia fetida (Oligochaeta)

Eisenia fetida were exposed continuously to (60)Co gamma radiation during two generations (F(0) and F(1)). Adult F(0) reproduction capacity (i.e., number of cocoons produced, hatchability and number of F(1) hatchlings) in controls and at five dose rates (0.18, 1.7, 4, 11 and 43 mGy/h) was measured over a 13-week exposure period. Survival, growth and sexual maturation of F(1) hatchlings were observed for 11 weeks. F(1) adults were exposed for a further 13 weeks to determine their reproduction capacity. There was no radiation-induced effect on the cocoon production rate in either F(0) or F(1). For F(0), hatchability of cocoons produced during the first 4 weeks was reduced to 60% at 43 mGy/h (98% in controls), and none of the cocoons produced at 5-13 weeks hatched. At 11 mGy/h the cocoon hatchability was reduced to 25% at 9-13 weeks. In addition, the number of hatchlings per hatched cocoon was reduced at 11 and 43 mGy/h. Correspondingly, at these dose rates, the total number of F(1) hatchlings per adult F(0) was significantly lower than in the control. This number was also reduced at 4 mGy/h, but the effect was of borderline significance. For adult F(1), the hatchability of cocoons at 11 mGy/h was reduced to 45-69% during the 13-week exposure period. The number of hatchlings (F(2)) per cocoon and the total number of F(2) individuals produced was also reduced. However, and in contrast to the results observed for F(0), hatchability increased with time, suggesting a possible acclimatization or adaptation of the F(1) individuals. In conclusion, chronic irradiation reduced the reproduction capacity of E. fetida, but extensive exposure periods (13 weeks) were needed for these effects to be expressed. The lowest dose rates at which an effect was observed were 4 mGy/h in F(0) and 11 mGy/h in F(1).