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.     

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.     

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 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.     

Non-Susceptibility of Earthworm Eisenia fetida to the Rhabditid Nematode Phasmarhabditis hermaphrodita, a Biocontrol Agent of Slugs

The rhabditid nematode Phasmarhabditis hermaphrodita is a lethal parasite for slugs that is sold commercially in Europe under the trade name Nemaslug™. We evaluated the effects of P. hermaphrodita on the earthworm Eisenia fetida (Savigny). Adults of E. fetida were exposed in one-liter glass beakers to P. hermaphrodita at three concentrations (1×, 10× and 50× of the field recommended rate of 3×10⁹ billion nematodes/ha) during a 14-day period in an artificial soil substrate. The average body weight, burrowing behavior, mortality and other clinical signals of the earthworms were recorded at 0, 7, and 14 days after exposure to the treatments. In addition, injured earthworms (posterior ends removed) were exposed to the 10× field recommended rate of the commercial formulation. Neither intact nor injured E. fetida showed susceptibility to the slug-parasitic nematode P. hermaphrodita during the 14 days of exposure even at concentrations 10 and 50 times higher than the label dose. However, the worms in the attenuated control (autoclaved formulation) had higher mortality and lost less weight compared to the other treatments. Under the conditions of the test, we conclude that the use of the commercially available strain of P. hermaphrodita is safe to E. fetida.     

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.     

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.     

蚯蚓中抗肿瘤蛋白组分的提取分离及其抗肿瘤活性

以赤子爱胜蚓为原料 ,通过蛋白质的丙酮沉淀和凝胶过滤 ,分离得到一组抗肿瘤活性蛋白成分 (简称蚯蚓抽提物 ) ;这组成分富含Fe、Zn、Cu以及Se等微量元素 ,蛋白含量为 6 0 4 3± 2 36 %(n =5 ) .体外实验中 ,通过MTT法和SRB法测定了蚯蚓抽提物对多种人癌细胞株 (HCT 116、SY5Y、K5 6 2、MGc80 3和HeLa)以及正常细胞株 (HEK2 93和COS 7)的抑制杀伤率 .结果表明 ,蚯蚓抽提物对癌细胞的杀伤有一定的选择性 ,受试人癌细胞达到 5 0 %生长抑制率所需作用浓度约 6 0～110mg L ;但是 ,10 0℃煮沸 5min后 ,该抑制活性完全消失 .通过纤维蛋白平板法 ,测得蚯蚓抽提物同时具有纤溶酶和纤溶酶原激活酶的活性 .体外测得丝氨酸蛋白酶抑制剂aprotinin和PMSF能显著抑制蚯蚓抽提物的细胞杀伤活性 .体内实验中 ,蚯蚓抽提物能有效延长荷瘤 (S180 )小鼠的生存时间 ,并使其身体机能得到明显改善 .腹膜内注射剂量为 2 8mg kg和 36mg kg时 ,生命延长率分别为135 3%和 12 3 5 % ,和标准药物 (环磷酰胺 )治疗后结果 (76 5 % )相比 ,有显著性差异     

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.     

阿苯哒唑对蚯蚓的生态毒理效应

采用人工污染土壤法测定了不同浓度阿苯哒唑对蚯蚓生长、生存、繁殖以及超氧化物歧化酶（SOD）、谷胱甘肽-硫转移酶（GST-S）、乙酰胆碱酯酶（AchE）活性的影响.结果表明:低浓度阿苯哒唑（5 mg·kg^-1）对蚯蚓的繁殖有显著的抑制作用（P<0.05）;当阿苯哒唑含量达到7 mg·kg^-1时,将明显影响蚯蚓的生长和生存（P<0.05）;当其含量达到10 mg·kg^-1时,蚯蚓的死亡率为53%.阿苯哒唑浓度与蚯蚓的生长、繁殖间存在明显的剂-效关系.阿苯哒唑染毒21 d后,对蚯蚓SOD、GST-S的活性有明显影响,药物浓度与效应间没有明显的剂-效关系,对AchE活性没有表现出明显的影响.阿苯哒唑对土壤动物显示毒性作用,具有潜在的生态风险.     

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.     

Cadmium and Lead Accumulation in the Earthworm Eisenia fetida (Savigny) and its Impact on Cholinesterase and Metabolic Pathway Enzyme Activity

The uptake of cadmium and lead was studied in the earthworm Eisenia fetida (Annelida: Oligochaeta) using an artificial soil exposure. Although cadmium and lead are bioconcentrated in Eisenia fetida tissue, bioaccumulation is not shown for concentrations below 100 ppm for lead, individuals eliminating as much metal as they ingest or the interactions between lead and organic matter in our substratum reduce the bioavailability of lead at low concentration. The cholinesterase activity was not inhibited when individuals were exposed for 8 weeks to either 8 or 80 ppm of cadmium or 100 or 2,000 ppm of lead. Results are different from those reported in another species Eisenia fetida andrei (= E. andrei) showing an inhibitory effect of lead on ChE activity; thus, differences in cholinesterase inhibition reflects the existence of two separate species. No effect of cadmium and lead on the activity of esterases, malate dehydrogenase, phosphoglucomutase and glutamate oxalate transferase was found in our experimental conditions, but we observed the disappearance of the fast moving band after electrophoretic separation for phosphoglucose isomerase.     

Aged Eisenia fetida earthworms exhibit decreased reproductive success

The effect of age on male and female reproductive activities was examined in 1–6-year-old age-matched populations of the simultaneous hermaphroditic earthworm, Eisenia fetida, (Annelida; Oligochaeta). As earthworm ages increase from 3 to 6 years, cocoon and hatchling production gradually decreased. Sperm counts from 2-year-old and 5-year-old earthworms did not differ. Old earthworms successfully inseminated young virgin earthworms. Thus, female functions associated with successful cocoon production diminish before the male function. These results indicate that E. fetida earthworms spend up to one-third of their adult life with limited reproductive capabilities.     

用蚯蚓溶酶体作为检测土壤污染的生物标志物

蚯蚓体腔细胞内的溶酶体能积累中性红染料.当受到环境胁迫时,溶酶体膜容易破裂,染料就逐步泄露到细胞质中,使细胞染成红色.中性红的保持时间(neutral red retention time,NRRT)可反映溶酶体受损情况.用不同浓度乙草胺和不同浓度Se处理赤子爱胜蚓(Eisenia fetida),观察体腔细胞溶酶体中性红染色的保持时间.结果表明,随乙草胺和Se处理浓度的增高和处理时间的延长,NRRT缩短;NRRT与土壤中乙草胺残留的浓度呈明显的负相关,与蚯蚓体内富集的Se也有明显的负相关.可见,蚯蚓溶酶体中性红染色保持时间是测定土壤污染物的生物标志物,在环境风险评价和土壤监测中具有稳定性和准确性,可作为早期预警生物标志物.