Presence of Culturable Bacteria in Cocoons of the Earthworm Eisenia fetida

Viable bacteria were found to coexist with developing embryos in egg capsules (cocoons) of the earthworm Eisenia fetida. Earthworms were reared under standardized conditions, and bacterial densities were measured in distinct batches of cocoons collected weekly for 10 weeks. Cocoons weighing 12 mg contained a mean viable bacterial population of approximately 10⁸ CFU/g of cocoons. No difference was found in viable counts obtained from cocoons incubated at 15°C and cocoons incubated at 24°C. Viable bacterial numbers increased with cocoon age, while acridine orange direct counts of microbial cells were stable at approximately 10⁹ cells per g of cocoons. Bacteria isolated from cocoons were used to develop antisera in rabbits for the production of strain-specific fluorescent antibodies. Fluorescent antibody and selective plating techniques were used to monitor populations of these bacteria in earthworm bedding and to determine whether cocoons acquire bacteria from the environment in which they are formed. Cocoon isolates were readily recovered from cocoons formed in inoculated bedding at densities of 10⁸ CFU/g of cocoons. Bradyrhizobium japonicum USDA 110 and UMR 161 added to bedding were also recovered from cocoons, but at lower densities than cocoon isolates. Escherichia coli K-12(pJP4) inoculum was recovered from bedding but not from cocoons. The bacterial complement of Eisenia fetida cocoons is affected by inoculation of selected bacterial isolates in the worm growth environment.     

Microbiomic Comparison of the Intestine of the Earthworm Eisenia fetida Fed Ergovaline

Tall fescue toxicosis and ergot alkaloids cost U.S. livestock producers approximately one billion dollars in annual livestock production loss annually. Ergovaline (EV) is the tall fescue alkaloid primarily responsible for clinical disease in livestock. Since native ruminal microorganisms have not been attributed to the detoxification of EV, finding detoxifying microbes from other environments is desirable. One possible source for potential microorganisms that can degrade EV is the anaerobic gut of the earthworm, Eisenia fetida. This study describes a comparative microbial analysis of earthworm digestive tracts receiving 10,000 ppb EV (E+ treatment) when compared with a control treatment with no detectable amounts of EV (E− treatment). An HPLC assay determined a 25% loss of EV from the E+ treatment was microbial in nature. A community microbiomic approach of constructing 16S-rRNA gene clone libraries was used to compare the microbes affected by the two treatments. RDPII tools such as Classifier and Libcompare were used in the analysis of 16S sequences. DOTUR analysis was used to examine the richness and diversity of the two microbial populations in these experiments. The results indicate there are few significant differences in the microbial community structure between the two microbiomes.     

从蚯蚓中联合提取抗氧化酶SOD、CAT的方法研究

本研究采用闪式提取技术,固液比为1:4(m/V)的2.5 mmol/L pH 7.0磷酸缓冲液,提取转速5500 rpm,提取时间2 min,从蚯蚓体内提取出SOD、CAT,并通过羧甲基纤维素CM-22离子交换层析实现SOD和CAT的联合提取分离,SOD、CAT的活性回收率分别达到88.23%和69.5%。在纯化工艺中经过丙酮沉淀和柱层析技术得到蚯蚓SOD纯品,比活达到9352 U/mg,产物在SDS-PAGE上为单一条带,其亚基分子量约为17 kD;通过柱层析纯化了蚯蚓CAT,比活达到22606 U/mg。     

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.     

Toxicity of A Novel Neonicotinoid Insecticide Paichongding to Earthworm Eisenia fetida

Traditional neonicotinoid insecticides are used worldwide. Paichongding (IPP), as a novel neonicotinoid pesticide, has been widely used in China. However, the ecotoxicity of IPP to non-target invertebrates in soil ecosystem has not been reported yet. In this study, acute toxicity of IPP to earthworm Eisenia fetida, as well as the antioxidant response after IPP exposure, was evaluated. In the filter paper contact test, the LC₅₀ at 24 hr and 48 hr for IPP were 14.98 μg/cm² and 7.59 μg/cm², respectively. In artificial soil test, the LC₅₀ (lethal concentration) at 14 days and 28 days for IPP were 541.07 mg/kg and 238.51 mg/kg, respectively. The LC₅₀ of IPP is much higher than that of traditional neonicotinoid insecticides. However, earthworm body weight assessment demonstrated that the growth of earthworm was inhibited by extended exposure to IPP at sublethal doses. The activities of antioxidative enzymes superoxide dismutase and catalase in earthworms were significantly induced after IPP exposure. Malondialdehyde, a biomarker of lipid peroxidation, was also increased after IPP exposure. Although the results indicated that IPP had potentially adverse effect on earthworms, its toxicity was much lower than traditional neonicotinoids.     

Transmission of a bacterial consortium in Eisenia fetida egg capsules

The earthworm Eisenia fetida harbours Verminephrobacter eiseniae within their excretory nephridia. This symbiont is transferred from the parent into the egg capsules where the cells are acquired by the developing earthworm in a series of recruitment steps. Previous studies defined V. eiseniae as the most abundant cell type in the egg capsules, leaving approximately 30% of the bacteria unidentified and of unknown origin. The study presented here used terminal restriction fragment length polymorphism analysis together with cloning and sequencing of 16S rRNA genes to define the composition of the bacterial consortium in E. fetida egg capsules from early to late development. Newly formed capsules of E. fetida contained three bacterial types, a novel Microbacteriaceae member, a Flexibacteriaceae member and the previously described V. eiseniae. Fluorescent in situ hybridization (FISH) using specific and general rRNA probes demonstrated that the bacteria are abundant during early development, colonize the embryo and appear in the adult nephridia. As the capsules mature, Herbaspirillum spp. become abundant although they were not detected within the adult worm. These divergent taxa could serve distinct functions in both the adult earthworm and in the egg capsule to influence the competitive ability of earthworms within the soil community.     

Biological Responses in the Earthworm Eisenia fetida Exposed to Soils near a Typical Lead Acid Battery Plant

There are increasing concerns over the environmental implications involved in battery plant due to the elevated metal concentrations in the surrounding soils. Earthworms are regarded as a model organism for evaluation of terrestrial environmental pollution. The biochemical responses in the earthworms, Eisenia fetida, exposed to polluted soils were investigated. Soil samples at 0, 50, 100, 200, 500 and 1000 m from a battery plant were collected and used to assess the relationship between sampling distance and biochemical responses in E. fetida. Biochemical alterations were carried out on 1, 3, 7 and 14 d of exposure. The metallothionein (MT), malondialdehyde (MDA) and antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD)) were measured. The results demonstrated that concentrations of heavy metals, especially Pb, decreased sharply with increase in distance, which may be due to the leaching and accumulation of Pb in soil during production processes. The concentrations of MT and MDA increased with exposure (dosage and time), indicating that E. fetida presented dose and time dependence after heavy metal exposure. The activities of SOD and CAT increased after heavy metal induction, while the activity of POD was lower than that of the control group. The SOD, CAT and POD present a cooperative effect for the protection of lipids and enzymes due to the pollution. The soils around the battery plant were polluted with metals giving rise to adverse biological effects.     

Oxidative Stress,Cytotoxicity and Genotoxicity in Earthworm Eisenia fetida at Different Di-n-Butyl Phthalate Exposure Levels

Recognized as ubiquitous contaminants in soil, the environmental risk of phthalic acid esters (PAEs) is of great concern recently. Effects of di-n-butyl phthalate (DnBP), an extensively used PAE compound to Eisenia fetida have been investigated in spiked natural brown yellow soil (Alfisol) for soil contact test. The toxicity of DnBP to E. fetida on the activity of superoxide dismutase (SOD) activity, peroxidase (POD), reactive oxygen species (ROS) content, and the apoptosis of coelomocytes and DNA damage at the 7th, 14th, 21st and 28th day of the incubation have been paid close attention to. In general, SOD activity and ROS content were significantly induced, opposite to total protein content and POD activity, during the toxicity test of 28 days especially under concentrations higher than 2.5 mg kg^-1. The reduction in neutral red retention (NRR) time along with the increase of dead coelomocytes as the increasing of DnBP concentrations, indicating severe damage to cell viability under varying pollutant stress during cultivation, which could also be proved by comet assay results for exerting evident DNA damage in coelomocytes. DnBP in spiked natural soil could indeed cause damage to tissues, coelomocytes and the nucleus of E. fetida. The key point of the apparent change in different indices presented around 2.5 mg DnBP kg^-1 soil, which could be recommended as the threshold of DnBP soil contamination, so that further investigation on threshold values to other soil animals or microorganisms could be discussed.     

The Earthworm Eisenia fetida Can Help Desalinate a Coastal Saline Soil in Tianjin,North China

A laboratory microcosm experiment was conducted to determine whether the earthworm Eisenia fetida could survive in a saline soil from a field site in North China, and an experiment using response surface methodology was conducted at that field site to quantify the effects of E. fetida and green waste compost (GWC) on the salt content of the soil. The microcosm results showed that E. fetida survived in GWC-amended saline soil and increased the contents of humic acid, available N, and available P in the GWC-amended soil. The data from the field experiment were described by the following second-order model:y^ =-1.76+0.091x1+0.48x2-0.00083x1x2-0.00078x12-0.022x22, where y is the decrease in soil salinity (g of salt per kg of dry soil) relative to the untreated control, x ₁ is the number of E. fetida added per m², and x ₂ is the quantity of GWC added in kg per m². The model predicted that the total salt content of the saline soil would decrease by > 2 g kg^-1 (p<0.05) when 29–90 individuals m^-2 of E. fetida and 6.1–15.0 kg m^-2 of GWC were applied. We conclude that the use of E. fetida for soil desalination is promising and warrants additional investigation.     

Cloning,Expression and Characterization of a Gene from Earthworm Eisenia fetida Encoding a Blood-Clot Dissolving Protein

A lumbrokinase gene encoding a blood-clot dissolving protein was cloned from earthworm (Eisenia fetida) by RT-PCR amplification. The gene designated as CST1 (GenBank No. {type:entrez-nucleotide,attrs:{text:AY840996,term_id:56718408,term_text:AY840996}}AY840996) was sequence analyzed. The cDNA consists of 888 bp with an open reading frame of 729 bp, which encodes 242 amino acid residues. Multiple sequence alignments revealed that CST1 shares similarities and conserved amino acids with other reported lumbrokinases. The amino acid sequence of CST1 exhibits structural features similar to those found in other serine proteases, including human tissue-type (tPA), urokinase (uPA), and vampire bat (DSPAα1) plasminogen activators. CST1 has a conserved catalytic triad, found in the active sites of protease enzymes, which are important residues involved in polypeptide catalysis. CST1 was expressed as inclusion bodies in Escherichia coli BL21(DE3). The molecular mass of recombinant CST1 (rCST) was 25 kDa as estimated by SDS–PAGE, and further confirmed by Western Blot analysis. His-tagged rCST1 was purified and renatured using nickel-chelating resin with a recovery rate of 50% and a purity of 95%. The purified, renatured rCST1 showed fibrinolytic activity evaluated by both a fibrin plate and a blood clot lysis assay. rCST1 degraded fibrin on the fibrin plate. A significant percentage (65.7%) of blood clot lysis was observed when blood clot was treated with 80 mg/mL of rCST1 in vitro. The antithrombotic activity of rCST1 was 912 units/mg calculated by comparison with the activity of a lumbrokinase standard. These findings indicate that rCST1 has potential as a potent blood-clot treatment. Therefore, the expression and purification of a single lumbrokinase represents an important improvement in the use of lumbrokinases.     

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.     

Recycling of organic wastes by employing Eisenia fetida

This paper reports the recycling of nutrients by vermicomposting of cow dung (CD), poultry droppings (PD) and food industry sludge (FIS) employing earthworms (Eisenia fetida). A total of six vermicomposting units were established and dynamics of chemical and biological parameters has been studied for 13 weeks. The waste mixture containing 50% CD+25% PD+25% FIS had better fertilizer value among studied waste combinations. At the end of experiment, vermicomposts showed decrease in pH and organic C, but increase in EC, total Kjeldhal N, total available P and total K contents. The C:N ratio of final vermicomposts also reduced to 10.7-12.7 from 22.8 to 56 in different waste combinations. The earthworms have good biomass gain and cocoon production in all vermicomposting units but CD alone and 50% CD+25% PD+25% FIS were better than other studied combinations.     

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.     

Metallothionein response following cadmium exposure in the oligochaete Eisenia fetida

We studied the metallothionein (MT) response in cadmium-exposed worms (Eisenia fetida) both at the protein level by Dot Immunobinding Assay (DIA) with a polyclonal antibody raised against the most immunogenic part of this protein and at the expression level by Northern blotting using a specific probe. MT appeared as two close isoforms. DIA results clearly demonstrated significant differences in MT level of whole worm heat-treated supernatants between E. fetida exposed to Cd concentrations as low as 8 mg Cd kg(-1) of dry soil compared to controls. Northern blotting analysis performed on whole bodies of worms revealed that a single exposure to 8 mg Cd kg(-1) of dry soil for 1 day resulted in the production of MT mRNA. This response was maintained for exposure of at least 1 month. Clear differences of MT gene expression were also observed between worms exposed to different Cd concentrations (8, 80 or 800 mg Cd kg(-1) of dry soil). Immunocytochemistry demonstrated that MT was located in the chloragogenous tissue surrounding the gut where metals are known to be accumulated. This work revealed that E. fetida MT is a sensitive and relevant biomarker of Cd exposure and especially when considering gene expression response. Further experiments have now to prove its usefulness in natural metal-contaminated soil toxicity assessments.     

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.     

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.     

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.     

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…