Toxicity of coelomic fluid of the earthworm Eisenia foetida to vertebrates but not invertebrates: probable role of sphingomyelin

The coelomic fluid (CF) of the earthworm Eisenia foetida exhibits a wide variety of biological activities. We found that the CF was not toxic to 42 species, belonging to seven invertebrate phyla, almost all in aquatic adults and larvae exposed to CF. Eleven teleostean species tested died in 0.2-1% CF mostly between 10 and 120 min and the effects were dose-dependent. Tadpoles of the toad Bufo japonicus formosus died in 0.4-2% CF between 80 and 225 min depending upon size, with larger tadpoles surviving longer. Before dying, all experimental tadpoles developed curled and shrunken tails. The Okinawa tree lizard, soft-shelled turtle, Japanese quail, mouse and rat all died after i.v. injection of CF (above 20 microl/kg). Thus, CF was not toxic to invertebrates, but toxic to vertebrates. After heating, CF lost its toxicity to fish, tadpoles and mice. Both CF and lysenin incubated with sphingomyelin-liposomes (SM-liposomes) were no longer toxic, suggesting the involvement of SM in the toxicity. Lysenin, which is a constituent of CF and known to bind specifically to sphingomyelin, exhibited toxicity similar to that of CF. Thus, lysenin in CF is probably responsible for the toxic effects of CF by binding to SM in vertebrate tissues. The bodies of invertebrates might contain little or no SM, while those of vertebrates do contain SM. The coelomic fluid of the earthworm Pheretima communissima has no toxicity to mouse.     

Sites of expression of mRNA for lysenin, a protein isolated from the coelomic fluid of the earthworm Eisenia foetida

Lysenin is a 33-kDa protein of 297 amino acids that was originally purified from the coelomic fluid of the earthworm Eisenia foetida. It binds specifically to sphingomyelin. In this study, we attempted to identify the site of synthesis of lysenin in the earthworm. We detected the expression of mRNA for lysenin and the presence of immunoreactive lysenin in the large coelomocytes and in the free large chloragocytes present in the lumen of the typhlosole, a depression in the dorsal wall of the intestine. These coelomocytes and chloragocytes seemed to be mature and separate from the chloragogen tissue that lined the typhlosole. The free large chloragocytes in the typhlosole contained numerous vacuoles. The nuclei were small and irregular in shape, and glycogen granules and mitochondria were occasionally found between vacuoles. The chloragocytes of the chloragogen tissue that surrounded the coelomic side of the intestine and the dorsal blood vessel did not react with the lysenin antiserum and no expression of lysenin mRNA was detected in these cells. Furthermore, no evidence of the protein or of the mRNA was found in the cells of the pharyngeal gland. Our findings suggest that lysenin is produced in the free large chloragocytes in the lumen of the typhlosole.     

A novel anti-plant viral protein from coelomic fluid of the earthworm Eisenia foetida: purification, characterization and its identification as a serine protease

A novel protein showing strong antiviral activities against cucumber mosaic virus (CMV) and tomato mosaic virus (TMV) was purified from the coelomic fluid of the earthworm Eisenia foetida. The protein was characterized as a cold-adapted serine protease. Its molecular weight was estimated to be 27,000 by SDS-PAGE. The enzyme was most active at pH 9.5 and 40–50 °C. The protease activity at 4 °C was 60% of that obtained at the optimal temperature. The activity was suppressed by various serine protease inhibitors. Partial N-terminal amino acid sequence of the enzyme showed homology with serine proteases of earthworms, E. foetida and Lumbricus rubellus previously studied. Our results suggest that the enzyme can be applicable as a potential antiviral factor against CMV, TMV, and other plant viruses.     

Damage to the gills,skin and other tissues by lysenin and the coelomic fluid of the earthworm Eisenia foetida in two teleosts,Tanichthys albonubes and Oreochromis mossambicus

Lysenin is a 33-kDa protein found in the coelomic fluid (CF) of the earthworm Eisenia foetida. Purified lysenin binds specifically to sphingomyelin (SM). In the present studies, we found that the white cloud mountain minnow Tanichthys albonubes and the Mozambique tilapia Oreochromis mossambicus died in solutions of lysenin (at concentrations above 2.5 microg/ml) and CF (0.6%, v/v) within 2 h. The gills of both species of fish were damaged similarly by lysenin and by CF. Most gill lamellae became irregularly bent or curled, with swelling of the epithelial cells of the lamellae. Red blood cells in the lamellar vascular sinuses, in the central venous sinuses, and in the blood vessels of the entire body became swollen and lysed, choking the sinuses. Epithelial cells in the skin were also damaged. When fish of both species were treated with lysenin or CF that had been incubated with SM-liposomes, they did not die. Their behavior remained normal and there was no damage to any cells or tissues. These findings suggest that SM might be involved in the lethal effects of lysenin and CF. It is likely that purified lysenin and lysenin in CF bound to SM in the cell membranes of the tissues mentioned above, damaging the cells. The presence of SM in the gills and skin was confirmed, supporting this hypothesis. The damage to gills and hemolysis might have resulted in lethal respiratory problems. Damage to the skin might disturb the exchange of ions through the skin, hastening death. Damage by lysenin and CF to epithelial cells of the cornea and the wall of the oral cavity was also recognized, but there was no such damage to the intestine.     

Purification of a novel antibacterial short peptide in earthworm Eisenia foetida

Earthworms live in an environment with abundantpathogens. These pathogens are, firstly, bacteria living inwater or soil that are ingested during feeding or introducedinto the body following injury. Parasites, particularlylarval forms, which represent the dissemination phase,are another important group of potentially pathogenicagents. During the course of evolution, earthworms havedeveloped defense strategies against these living patho-gens [1,2]. Earthworms lack true antibodies and hence anada…     

Lethal and non-lethal responses of spermatozoa from a wide variety of vertebrates and invertebrates to lysenin, a protein from the coelomic fluid of the earthworm Eisenia foetida

Lysenin, a novel protein that we isolated from the coelomic fluid of the earthworm Eisenia foetida, binds specifically to sphingomyelin (SM) among various phospholipids found in cell membranes, and causes cytolysis. The plasma membrane of mammalian spermatozoa is known to contain SM at relatively high levels and we therefore examined the effects of lysenin on the spermatozoa of various animals. Lysenin had lethal effects on spermatozoa of 5 of 33 species of invertebrates tested and on spermatozoa of 30 of 39 species of vertebrates. We postulated that plasma membranes of the spermatozoa of most invertebrates might not contain SM whereas those of most vertebrate species might contain SM. These possibilities were supported by our failure to detect SM chemically in the testes of three species of invertebrates, in none of which spermatozoa responded to lysenin. In contrast, we detected SM in the testes of all 25 vertebrate species examined, irrespective of a negative or positive response of spermatozoa to lysenin. None of the six species of Protista examined was affected by lysenin. Our survey suggests that, in general, the spermatozoa of animals can be grouped into two categories, invertebrate and vertebrate, depending on the absence or presence of SM in their plasma membrane. The incorporation of SM into spermatozoa seems first to have occurred in protochordates during the course of evolution. Discussions about the exceptional responses to lysenin observed in the spermatozoa of five species of invertebrates and of nine species of vertebrates are made from phylogenetic and reproductive viewpoints. J. Exp. Zool. 286:538-549, 2000.     

Scanning electron-microscopic study of the involvement of coelomic cells in earthworm antibacterial defense

Summary The first steps of an antibacterial reaction in the earthworm Eisenia fetida andrei were investigated. The main cellular mediators of this activity are the chloragocytes, a class of free coelomocytes existing only in annelids. Our observations using scanning electron microscopy have shown that chloragocytes were able to agglutinate and perhaps to destroy pathogenic bacteria such as Bacillus megaterium in the same way that they agglutinate and lyse vertebrate erythrocytes. Bacteria known to be non-pathogenic for the worm, such as Acinetobacter, were not agglutinated but slowly eliminated by segregation into brown bodies. Chloragocytes maintained in vitro, lost their chloragosomes and exhibited stronger agglutination activity against pathogenic bacteria than chloragocytes in situ. From this increased efficiency of chloragocytes in vitro, we infer that, in normal living conditions, chloragocytes probably intervene in antibacterial defense mainly after their extrusion from the coelomic cavity and their spreading and degranulation at the surface of the integument.     

Comparison of precursor structures of the GGNG peptides derived from the earthworm Eisenia foetida and the leech Hirudo nipponia

Earthworm and leech cDNAs encoding the GGNG peptides, a family of myotropic peptides, were cloned and examined in this study. Both of the predicted precursor proteins are of polyprotein structure and contain several putative peptides distinct from the GGNG peptides. However, the precursors show organizations distinct from each other and no sequence similarity except for the GGNG peptides.     

Analgesic and anti-inflammatory effects of the amphibian neurotoxin, anntoxin

Anntoxin is the first gene-encoded neurotoxin identified from amphibians, which is a 60-residue neurotoxin peptide, acting as an inhibitor of tetrodotoxin-sensitive (TTX-S) voltage-gated sodium channel (VGSC). Sodium channels have been considered as therapeutic targets for pain. Several animal models of persistent inflammatory and neuropathic pain (tail-flick test, hot plate test, acetic acid-induced writhing test, formalin-induced paw licking, carrageenan-induced paw edema) were used to test analgesic functions of recombinant anntoxin (r-anntoxin). In all these animal models, r-anntoxin showed strong analgesic functions. R-anntoxin obviously inhibited secretions of both tumor necrosis factor alpha (TNF-α) and cyclooxygenase-2 (COX-2). Histopathological study indicated that r-anntoxin reduced the edematous epidermis induced by carrageenan. All these results indicate that r-anntoxin has strong analgesic and anti-inflammatory activities.     

Lipid agglutinins from coelomic fluid of the earthworm,Lumbricus terrestris

• 1.1. Coelomic fluid of the earthworm, Lumbricus terrestris, contains lipid agglutinins that are primarily glycolipids. They are present in fluid from both immunized (induced) and unimmunized (naturally occurring) worms.
• 2.2. Lumbricus agglutinins partially purified by Folch extraction followed by silicic acid chromatography, were present in all fractions but in highest concentration in acetone and methanol fractions.
• 3.3. Immunodetection revealed agglutinin activity in acetone and methanol fractions, but not in the chloroform fraction.

     

Examination of water balance and ionic regulation in the earthworm,Eisenia foetida

• 1.1. Weight change after submerging the earthworm into water varied remarkably according to the environmental humidity in which animals were placed before submergence.
• 2.2. Pretreatment with physiological saline solution before submergence in water gave stable values for the ionic concentrations of the body fluid.
• 3.3. Brain removal caused decrease of both sodium and chloride ion concentrations and increase of potassium ion concentration of the coelomic fluid when animals were submerged in water.
• 4.4. Although brain replacement failed, action of a brain hormone is suggested to regulate the decrease of both sodium and choride ions and increase of potassium ion of the coelomic fluid to normal level when animals were submerged in water.

     

甲胺磷、乙草胺和铜单一与复合污染对蚯蚓的毒性效应研究

以赤子爱胜蚓(Eisenia foetida)为例,通过滤纸急性毒性试验法研究了东北黑土区普遍存在的3种农用化学品甲胺磷、乙草胺和Cu对蚯蚓的单一与复合毒性效应．单一实验结果表明,三者对蚯蚓均有毒性,它们的毒性顺序为乙草胺＞甲胺磷＞Cu,半致死浓度分别为：0．307、0．708和118．70mg.kg^-1;同时通过观察可知,乙草胺与Cu以皮肤渗入蚯蚓体的毒性效应较甲胺磷明显．复合毒性实验结果表明,低浓度Cu与高浓度Cu对甲胺磷的毒性均有增强作用;低浓度Cu对乙草胺的毒性有削弱作用,但高浓度Cu对乙草胺的毒性有增强作用．可见,三者对土壤生态系统生态安全性和土壤健康质量存在潜在危害,同时这几种污染物的共存进一步加大了潜在危害性,且复合毒性效应与各组浓度组合密切相关．     

Molecular cloning and characterization of cDNA encoding fibrinolytic enzyme-3 from earthworm Eisenia foetida

Earthworm fibrinolytic enzyme (EFE), a multi-com-ponent protease purified from some earthworm breeds,belongs to serine protease family with fibrinolytic activity[1]. It has been used in prevention and treatment of cardiacand cerebrovascular diseases in Ch…     

Survival of body wall autographs, allografts, and xenografts in the earthworm Eisenia foetida.

It is acknowledged that autografts of Eisenia foetida body wall are accepted by their hosts. Allografts, however, have not been found to be accepted, although macroscopic observation of these might suggest they are, possibly owing to fusion of grafts and host epidermal cells. All xenografts eventually fail. Histological studies show that both allografts and xenografts were isolated from hosts, invaded, absorbed, and replaced with collagen. Statistical analyses of results from multiple transfers of xenografts show no evidence of hosts producing a heightened response of any nature to a second challenge of the same antigen.     

Purification and characterization of novel raw-starch-digesting and cold-adapted alpha-amylases from Eisenia foetida

Novel raw-starch-digesting and cold-adapted alpha-amylases (Amy I and Amy II) from the earthworm Eisenia foetida were purified to electrophoretically homogeneous states. The molecular weights of both purified enzymes were estimated to be 60,000 by SDS-PAGE. The enzymes were most active at pH 5.5 and 50 degrees C and stable at pH 7.0-9.0 and 50-60 degrees C. Both Amy I and II exhibited activities at 10 degrees C. The enzymes were inhibited by metal ions Cu(2+), Fe(2+), and Hg(2+), and hydrolyzed raw starch into glucose, maltose and maltotriose as end products.     

9种农药对家蚕和蚯蚓的急性毒性

【背景】当前农药品种及其使用量日益增多,测试农药对环境生物的急性毒性成为农药环境安全监测的重要途径。【方法】采用食下毒叶法和药土法分别测定了甲氨基阿维菌素苯甲酸盐、阿维菌素、氰氟虫腙、螺螨酯、螺虫乙脂、嘧菌酯、苯醚甲环唑、戊唑醇、2,4-D二甲胺盐等9种农药对家蚕和蚯蚓的急性毒性,并根据其毒性等级划分标准进行分级,评价其对环境的安全性。【结果】甲氨基阿维菌素苯甲酸盐、阿维菌素、氰氟虫腙、螺螨酯、螺虫乙脂、嘧菌酯、苯醚甲环唑、戊唑醇和2,4-D二甲胺盐对家蚕的LC50(96 h)分别为2.05×10-3、8.59×10-4、2.79、250.48、11.52、272.18、2.50、1.93×10-2和534.47 a.i.mg·L-1,对蚯蚓的LC50(14 d)分别为11.05、6.29、100、100、100、100、99.13、115.31和100 a.i.mg·kg-1干土。其中,甲氨基阿维菌素苯甲酸盐、阿维菌素和戊唑醇对家蚕"剧毒",氰氟虫腙、螺虫乙脂和苯醚甲环唑对家蚕"高毒",其余农药对家蚕均为"低毒";阿维菌素对蚯蚓为"中毒",其余农药对蚯蚓均为"低毒"。【结论与意义】9种农药对家蚕和蚯蚓的急性毒性存在差异,为农药的合理使用和环境保护提供了依据。     

6种重金属对赤子爱胜蚓的急性毒性效应与风险评价

【背景】近年来,土壤重金属污染问题日益凸显,对生态环境、食品安全和人体健康构成了严重威胁,其急性毒性尚未明确。【方法】采用滤纸接触法和人工土壤法测定了铜(Cu2+)、锌(Zn2+)、镍(Ni+)、镉(Cd2+)、铅(Pb2+)和锰(Mn2+)等6种重金属对赤子爱胜蚓的急性毒性效应,并参照欧盟指令91/414/EEC标准评价了其环境风险。【结果】滤纸接触法48h测定结果表明,6种重金属对蚯蚓的LC50为3.17(2.53～3.81)～90.42(69.45～140.47)μg.cm-2,其毒性次序为Cu2+>Zn2+>Ni+>Cd2+>Pb2+>Mn2+。人工土壤法14d测定结果表明,6种重金属对蚯蚓的LC50为1347(1236～1453)～6936(6144～8930)mg.kg-1,其毒性次序为Cu2+>Cd2+>Ni+>Zn2+>Mn2+>Pb2+。风险评价结果显示,Cu2+、Zn2+、Ni+、Cd2+、Pb2+和Mn2+等6种重金属的暴露比(toxicity/exposureratio,TER)分别为3.37、4.46、8.68、1428、13.87和5.85。【结论与意义】6种重金属对土壤动物蚯蚓均具有潜在的毒性效应。Cu2+、Zn2+、Ni+、Mn2+等4种重金属对赤子爱胜蚓存在急性毒性风险,而Cd2+和Pb2+对赤子爱胜蚓的急性毒性风险水平是可接受的。该评价结果可为我国制定基于风险的土壤环境质量标准提供依据。     

Analysis of oxygen equilibria of the giant hemoglobin from the earthworm Eisenia foetida using the Adair model

Oxygen equilibrium curves of the giant hemoglobin from the earthworm Eisenia foetida were determined at various concentrations of cations. Using the Adair model of 12 oxygenation steps, we succeeded in fitting the data better than the simple concerted model (MWC model). Analysis of the Adair constants (K1 to K12) indicated that the increase in oxygen affinity occurs in the last six steps (K7 to K12) of the oxygen binding and that it is enhanced by increase in Ca2+ concentration. The Hill coefficient (nmax) at pH 7.5 attained a maximum value of 9.76 at 20 mM CaCl2. In the presence of physiological levels of Ca2+ (5 mM), the Bohr effect was similar to that seen in vertebrates. The data were consistent with the release of two Bohr protons being accompanied by the oxygen-linked binding of one Ca2+. Mg2+ and Na+ exerted a similar effect on the hemoglobin, though to a lesser extent. The stoichiometry of Ca2+ binding of the hemoglobin revealed the presence of two classes of binding sites, of which the affinities are high (Ka = 8.8 x 10(3) +/- 103 M-1) and low. The number of high affinity sites per heme was found to be 0.3, comparable to the number of oxygen-linked Ca2+ binding sites.     

赤子爱胜蚓超氧化物歧化酶的纯化和部分性质研究

采用硫酸铵分级沉淀和柱层析的方法,从赤子爱胜蚓整体细胞抽提液内分离得到纯的铜锌超氧化物岐化酶（Ｃｕ,Ｚｎ－ＳＯＤ）。每１００ｇ蚯蚓得到的ＳＯＤ制品,总活力为１１１５０Ｕ,比活力为５１３８Ｕ／ｍｇ蛋白,回收率为２０％。铜锌超氧化物岐化酶呈淡蓝绿色,最大紫外吸收波长为２７０ｎｍ。测得该酶分子量为３３０００,亚基分子量为１６５００。该酶亚基由１５６个氨基酸残基组成,不含酪氨酸。