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.     

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…     

Hydrolysis of fibrinogen and plasminogen by immobilized earthworm fibrinolytic enzyme II from Eisenia fetida

Earthworm fibrinolytic enzyme II (EFE-II) from Eisenia fetida has a broad hydrolytic specificity for peptide bonds. Our experiments show that EFE-II can hydrolyze the specific chromogenic substrates of thrombin (Chromozym TH), trypsin (Chromozym TRY) and elastase (Chromozym ELA). The Michaelis–Menten constant (K_m) for Chromozym ELA (245 μM) is much higher than those for the thrombin (90 μM) and trypsin (60 μM) substrates. On the other hand, EFE-II is inhibited most strongly by soybean trypsin inhibitor (SBTI), and weakly inhibited by elastinal, suggesting that EFE-II has a trypsin-like activity. Degradation of plasminogen (PLg) and fibrinogen by EFE-II was investigated after EFE-II had been immobilized onto 1,1′-carboryl-diimidazole (CDI)-activated Sepharose CL-6B. The immobilized EFE-II has 55–60% activity of the native enzyme with a higher thermal and pH resistance. EFE-II cleaves PLg at four hydrolytic sites: Lys₇₇–Arg₇₈, Arg₃₄₂–Met₃₄₃, Ala₄₄₄–Ala₄₄₅ and Arg₅₅₇–Ile₅₅₈. The site Arg₅₅₇–Ile₅₅₈ is also recognized and cleaved by tissue plasminogen activator (t-PA) and urokinase (UK), producing active plasmin. Cleaving Ala₄₄₄–Ala₄₄₅ released mini-plasmin with secondary activity to hydrolyze fibrin. Immobilized EFE-II degrades not only the A chain of fibrinogen in the C-terminal region (like human neutrophil elastase, HNE), but also in the N-terminal region at the Val₂₁–Glu₂₂ site.     

Expression and characterization of ARSP1 from Eisenia fetida

To study the characterization of a protease ARSP1 (apoptosis-related serine protease) of Eisenia fetida, a recombinant ARSP1 was constructed. ARSP1 was produced in E. coli BL21-CodonPlus (DE3)-RIL after IPTG induction and exited in inclusion body. After refolding in vitro, the protein was purified by DEAE-Sepharose F.F. and Sephacryl S-100 chromatography in sequence. ARSP1 showed high sequence identity to other chymotrypsin-like serine proteases and the catalytic triad was His41-Asp90-Ser188. ARSP1 could degrade casein following Michaelis-Menten kinetics with a Vmax of 43.9 U/mg protein and a Km for casein of 0.83 g/l. Studies with inhibitors indicated that ARSP1 was a chymotrypsin-like serine protease. Experiments in vitro demonstrated that ARSP1 could not only hydrolyze fibrinogen and fibrin directly, but also activate plasminogen to plasmin. ARSP1 inhibited thrombin activity and ADP-induced platelet aggregation in a dose-response correlation. These results showed that ARSP1 has thrombolytic activity and also an anti-thrombus function.     

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 cells in the central nervous system of earthworm, Eisenia fetida

Glial elements in the central nervous system of Eisenia fetida were studied at light- and electron microscopic level. Cells were characterized with the aid of toluidine blue, Glial Fibrillary Acidic Protein (GFAP), S100 staining. We identified neurilemmal-, subneurilemmal-, supporting-nutrifying- and myelinsheath forming glial cells. Both neuronal and non-neuronal elements are S100-immunoreactive in the CNS. Among glial cells neurilemmal and subneurilemmal cells are S100-immunopositive. With the antibody against the S100 protein one band is visible at 15 kDa. GFA P-immunopositive supporting-nutrifying glial cells are localized around neurons and they often appear as cells with many vacuoles. GFA P-positive cell bodies of elongated neurilemmal glial cells are also visible. Western blot analysis shows a single 57 kDa GFA P immunoreactive band in the Eisenia sample. At ultrastructural level contacts between neuronal and glial cells are recognizable. Glial cell bodies and their filopodia contain a granular and vesicular system. Close contacts between neuronal cell membranes and glial filopodia create a special environment for material transport. Vesicles budding off glial cell granules move towards the cell membranes, probably emptying their content with kiss and run exocytosis. The secreted compounds in return may help neuronal survival, provide nutrition, and filopodia may also support neuronal terminals.     

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.     

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.     

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.     

An enzyme from the earthworm Eisenia fetida is not only a protease but also a deoxyribonuclease

The earthworm enzyme Eisenia fetida Protease-III-1 (EfP-III-1) is known as a trypsin-like protease which is localized in the alimentary canal of the earthworm. Here, we show that EfP-III-1 also acts as a novel deoxyribonuclease. Unlike most DNases, this earthworm enzyme recognizes 5′-phosphate dsDNA (5′P DNA) and degrades it without sequence specificity, but does not recognize 5′OH DNA. As is the case for most DNases, Mg²⁺ was observed to markedly enhance the DNase activity of EfP-III-1. Whether the earthworm enzyme functioned as a DNase or as a protease depended on the pH values of the enzyme solution. The protein acted as a protease under alkaline conditions whereas it exhibited DNase activity under acid conditions. At pH 7.0, the enzyme could work as either a DNase or a protease. Given the complex living environment of the earthworm, this dual function of EfP-III-1 may play an important role in the alimentary digestion of the earthworm.     

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.     

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).     

Bioconversion of garden waste,kitchen waste and cow dung into value-added products using earthworm Eisenia fetida

Solid waste management is a worldwide problem and it is becoming more and more complicated day by day due to rise in population, industrialization and changes in our life style. Transformation of industrial sludges into vermicompost is of double interest: on the one hand, a waste is converted into value added product, and, on the other, it controls a pollutant that is a consequence of increasing industrialization. Garden waste, kitchen waste and cow dung were subjected to recycle through vermicomposting by using the epigeic earthworm Eisenia fetida under field conditions. The pH, moisture content, total organic carbon, humus, nitrogen, phosphorous and potassium in vermicompost was analysed. It was found that moisture content, total organic carbon, humus, nitrogen, phosphorous and potassium was high in cow dung, followed by kitchen waste and garden waste. This study clearly indicates that vermicomposting of garden waste, kitchen waste and cow dung can not only produce a value added produce (vermicomposting) but at the same time reduce the quantity of waste.     

Distributions of polymorphisms among pathways of carbohydrate metabolism in the earthworm Eisenia fetida (Oligochaeta).

1. Activities and genetic banding patterns of 36 isozymes in carbohydrate metabolism were detected by spectrophotometry and starch-gel electrophoresis, respectively, in the earthworm Eisenia fetida. 2. Polymorphisms were not distributed randomly among metabolic pathways, activity levels, or gene copy numbers. 3. In glycolysis and the Krebs cycle, 16% of the loci were polymorphic and polymorphisms occurred only when multiple copies of the loci existed. 4. In other pathways, 45% of the loci were polymorphic and the distribution of polymorphisms was independent of gene copy number. 5. Polymorphisms may affect metabolic phenotypes and natural selection may have led to conserved biochemical activity in glycolysis and the Krebs cycle.     

In vitro characterization of cholinesterases in the earthworm Eisenia andrei

Assessment of pollution impact in soil ecosystems has become a priority and interest has grown concerning the use of invertebrates as sentinel organisms. Inhibition of cholinesterase (ChE) activity has a great potential as a biomarker of pesticide exposure, and we evaluated the ChE kinetic parameters in the earthworm Eisenia andrei in the presence of acetylthiocholine (ASCh), proprionylthiocholine (PSCh) and butyrylthiocholine (BSCh). The highest ChE activity was found in the presence of ASCh and PSCh (42.45 and 49.82 nmol min(-1) mg protein(-1), respectively). BSCh was hydrolyzed at a rate of 4.04 nmol min(-1) mg protein(-1), but the time course did not reach a plateau under our experimental conditions. Km values were 0.142+/-0.006 and 0.183+/-0.053 mM for ASCh and PSCh, respectively. ASCh and PSCh hydrolysis were significantly inhibited by eserine (IC50 values were 1.44 x 10(-8) and 1.20 x 10(-8) M, respectively) and by carbaryl (IC50 values of 5.75 x 10(-9) and 4.79 x 10(-9) M). The presence of different ChEs in tissues from E. andrei was assessed by using selective inhibitors for AChE (BW284c51) and BChE (iso-OMPA). BW284c51 strongly reduced ASCh and PSCh hydrolysis and slightly affected that of BSCh, while iso-OMPA was without effect in all cases.     

Isolation of genomic DNA from the earthworm speciesEisenia fetida

Our interest in detecting genotoxic exposure in earthworms led us to isolate high quality DNA from theEisenia fetida species. For that, we compared a modification of the conventional phenol-chloroform extraction procedure, usually refered to as the Maniatis procedure, to two commercially available kits reportedly eliminating multiple partitions in phenol and chloroform, namely the Qiagen and Nucleon protocols. From the 260 nm optical density values, the commercial kits extracts hinted toward higher DNA recovery with those procedures. However, the 260/280 nm ratios indicated that the quality of the DNA isolated with the modified Maniatis procedure was purer than that isolated with the commercial kits, the latter being most probably contaminated by proteins and/or RNA. The Maniatis procedure was slightly modified by the introduction of a potassium acetate step for protein precipitation and by shortening the proteinase K treatment from 12–18 h to only 2 h. The higher quality of the DNA isolated by phenol-chloroform extraction was confirmed by quantification with the fluorescent 3,5-diaminobenzoic acid assay. Preliminary results suggest that the modified Maniatis procedure herein described is not only applicable for DNA adducts studies using³²P-postlabelling techniques but is also suitable for DNA extraction from other earthworm species such asLumbricus terrestris.     

Cold-stress induced formation of calcium and phosphorous rich chloragocyte granules (chloragosomes) in the earthworm Eisenia fetida

The cytochemical and functional characteristics of chloragocytes of both 'control' and cold-stressed Eisenia fetida were examined. Flow cytometry revealed the heterogeneity of chloragocytes: the first group was characterized by low, the second one by high acid phosphatase (AcP) content. In 'control' animals the former, in cold-stressed ones the latter type were the dominant form. The elevated AcP-activity correlated with the accumulation of autophagic vacuoles (AVs) in chloragocytes. Both AVs and all small chloragosomes showed high AcP activity, while most of the large chloragosomes did not display any. Most 'control' granules (0.75-1.25 μm) contained high amounts of Ca and P, with less and variable quantities of S, Cl, K, Fe and Zn. Small chloragosomes with low Ca and P concentrations were seldom found. In cold-stressed animals the number of small granules (0.25-0.75 μm) increased up to 40% of total population. Their Ca and P contents were significantly lower; S and Fe concentrations were higher than those of large chloragosomes (1.0-1.5 μm). Our results prove that the formation and elemental composition of chloragosomes can be influenced by environmental stressors and suggest that the mature chloragosomes are tertiary lysosomes and their formation is coupled to autophagocytosis.