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Annetocin, an annelid oxytocin-related peptide, induces egg-laying behavior in the earthworm, Eisenia foetida.

Annetocin, an oxytocin-related peptide which we isolated from the earthworm Eisenia foetida, induced a series of egg-laying-related behaviors in the earthworms. These stereotyped behaviors consisted of well-defined rotatory movements, characteristic body-shape changes, and mucous secretion from the clitellum. Each of these behaviors is known to be associated with formation of the cocoon in which eggs are deposited. In fact, some of the earthworms injected with annetocin (> 5 nmol) laid eggs. Such egg-laying-related behaviors except for oviposition were also induced by oxytocin, but not by Arg-vasopressin and some other bioactive peptides isolated from E. foetida. Furthermore, annetocin also induced these egg-laying-like behaviors in the leech Whitmania pigra, but not in the polychaete Perinereis vancaurica. These results suggest that annetocin plays some key role in triggering stereotyped egg-laying behaviors in terrestrial or fresh-water annelids that have the clitella.

Amino Acid Sequence↗

Genotoxicity of a polluted river system measured using the alkaline comet assay on fish and earthworm tissues.

Monitoring genotoxicity in the environment by using endemic organisms as sentinels requires sensitive assays. In this study the genotoxic properties of water and sediment collected from the Noyyal River, which is polluted with industrial effluent and sewage, was determined in fish (Cyprinus carpio) and earthworms (Eisenia foetida) using the alkaline comet assay. Upon electrophoresis, extensive DNA damage, measured as the DNA length:width ratio of the DNA mass, was observed in erythrocytes, liver, and kidney cells of fish exposed to polluted water samples and the amount of damage increased with the duration of exposure. Similarly, the mean DNA length:width ratio was significantly higher in the coelomocytes of earthworms placed in sediment samples. The highest levels of DNA damage were obtained with samples taken at and immediately downstream of urban centers. The results of this study indicate that the Noyyal River system is contaminated with substances that are genotoxic to fish and earthworms and that the comet assay has sufficient sensitivity to detect the genotoxicity.

Animals↗

Earthworm leukocyte interactions during early stages of graft rejection.

The earliest phase of graft rejection in earthworms, the recognition of foreign tissue antigens, has been subjected to analysis by confronting host leukocytes with foreign erythrocytes. Only rabbit and rat erythrocytes significantly prevented healing of allografts when grafts were transplanted and erythrocytes injected simultaneously. In contrast, autografts and allografts transplanted on worms injected 1 or 2 days before grafting were never affected. Since earthworms readily produce higher titers of erythrocyte agglutinins at 24 h postinjection than at later times, we propose a hypothetical scheme of earthworm leukocyte interactions that may occur during the early phases of graft healing and of agglutinin synthesis.

Animals↗

The relative toxicities of insecticides to earthworms of the Pheretima group (Oligochaeta).

An artificial soil test was used to determine the LC50 values of carbaryl, chlorpyrifos, imidacloprid, cyfluthrin and fipronil against earthworms of the Pheretima group. For a 24-h interval, carbaryl was the most toxic to earthworms (LC50 = 77 mg kg-1), followed by imidacloprid (155 mg kg-1), cyfluthrin (351 mg kg-1), chlorpyrifos (390 mg kg-1) and fipronil (> 8550 mg kg-1) as the least toxic. For the 48-h and 7-day intervals, imidacloprid was the most toxic to earthworms (LC50 = 5 mg kg-1 and 3 mg kg-1 respectively), followed by carbaryl (16 mg kg-1; 9 mg kg-1), cyfluthrin (128 mg kg-1; 110 mg kg-1), chlorpyrifos (330 mg kg-1; 180 mg kg-1) and the least toxic was fipronil (> 8550 mg kg-1 both intervals). The surface application rates required to achieve these values are compared with the rates recommended for the control of turfgrass pests.

Animals↗

The effects of repeated applications of the molluscicide metaldehyde and the biocontrol nematode Phasmarhabditis hermaphrodita on molluscs, earthworms, nematodes, acarids and collembolans: a two-year study in north-west Spain.

Over two years, six consecutive field experiments were done in which the chemical molluscicide metaldehyde and the nematode biocontrol agent Phasmarhabditis hermaphrodita (Schneider) were applied at the standard field rates to replicated mini-plots successively planted with lettuce, Brussels sprouts, leaf beet and cabbage, to compare the effectiveness of different treatments in reducing slug damage to the crops. Soil samples from each plot were taken prior to the start of the experiments, and then monthly, to assess the populations of slugs, snails, earthworms, nematodes, acarids and collembolans. The experiments were done on the same site and each plot received the same treatment in the six experiments. The six treatments were: (1) untreated controls, (2) metaldehyde pellets, (3 and 4) nematodes applied to the planted area 3 days prior to planting without or with previous application of cow manure slurry, (5) nematodes applied to the area surrounding the planted area 3 days prior to planting, and (6) nematodes applied to the planted area once (only in the first of the six consecutive experiments). Only the metaldehyde treatment and the nematodes applied to the planted area at the beginning of each experiment without previous application of manure significantly reduced slug damage to the plants, and only metaldehyde reduced the number of slugs contaminating the harvested plants. The numbers of slugs, snails and earthworms in soil samples were compared among the six treatments tested: with respect to the untreated controls, the numbers of Deroceras reticulatum (Müller) were significantly affected only in the metaldehyde plots, and the numbers of Arion ater L only in the plots treated with nematodes applied to the planted area 3 days prior to planting without previous application of manure; numbers of snails (Ponentina ponentina (Morelet) and Oxychilus helveticus (Blum)) were not affected by the treatment. The total numbers of all earthworm species and of Lumbricus spp were unaffected by the treatment, but Dendrobaena spp increased significantly in the plots treated with manure. The numbers of nematodes, acarids and collembolans in soil samples were compared between the untreated controls and the treatments with nematodes applied 3 days prior to planting to the planted area or to the surrounding area, without previous application of manure: the treatment had a significant effect on the number of nematodes in soil samples, but acarids and collembolans were unaffected.

Acetaldehyde↗

Relevance and applicability of a simple earthworm biomarker of copper exposure. I. Links to ecological effects in a laboratory study with Eisenia andrei.

A simple earthworm biomarker, neutral-red retention by coelomocyte lysosomes, was measured concurrently with ecological parameters in order to link effects at different levels of biological organization in a laboratory study. Exposure of the earthworm Eisenia andrei to an increasing range of soil copper concentrations in the laboratory indicated a threshold range for the neutral-red assay at soil copper concentrations between 40 and 80 mg Cu kg-1. This threshold coincided with the soil to worm copper bioconcentration factor decreasing from 1 to 0.3 and thus an apparent onset of a copper regulatory mechanism. Effects at the individual and population levels only occurred at soil copper concentrations beyond that of the biomarker threshold. Thus, it was possible to differentiate between exposure and toxicity. It was therefore possible to conclude that the neutral-red assay has great potential and relevance in earthworm ecotoxicity studies. The assay provides an early warning that can be linked directly to important physiological changes that were observed to precede adverse effects on individuals or populations in this laboratory study.

Analysis of Variance↗

Acute toxicity of 2,4,6-trinitrotoluene in earthworm (Eisenia andrei).

2,4,6-Trinitrotoluene (TNT) is an worldwide recalcitrant environmental contaminant and is toxic to a number of organisms including humans. This study examines the acute effects (lethal and biomass changes) of TNT on the oligochaetes species Eisenia andrei, using the 3-day filter paper, and the 7- and 14-day direct contact spiked soil (OECD artificial and forest soil) toxicity tests. Studies using the filter paper test indicated that the lethality of TNT could be detected in the range 1.5 to 14.2 microg/cm(2), with significant biomass (body weight) changes occurring at the lowest concentration. Acute effects (lethality) could not be measured when earthworms were placed on filter paper containing a saturated aqueous solution of TNT. This may indicate that with these exposure conditions, TNT may have been adsorbed to the filter paper, and that this matrix should be saturated with TNT before becoming available to the earthworms. Spiked soil toxicity tests indicated that the E. andrei lethality by TNT was >1.5 times higher when earthworms were exposed to TNT-spiked forest soil (LOEC:260 mg/kg; LC(50) 14 days 222.4 mg/kg) than to spiked OECD artificial soil (LOEC:420 mg/kg; LC(50) 14 days: 364.9 mg/kg). The sublethal effect on biomass change at the selected TNT concentrations in soil was not significant compared to controls. Results indicate that the bioanalytical methods described in this article could be used as TNT toxicity assessment tools. This soil quality test method gives valuable information for the screening of soil toxicity.

Animals↗

The occurrence and localisation of heavy metals and glycogen in the earthworms Lumbricus rubellus and Dendrobaena rubida from a heavy metal site.

The lead content of whole earthworms, highest in contaminated site specimens, was significantly higher in Dendrobaena rubida than Lubricus rubellus and a species difference in zinc was also recorded. Selective feeding and differential absorption are discounted and a species difference in maximum tolerance to body lead is suggested. Copper was low in all specimens. Chloragocytes and intestinal tissue showed significantly higher lead levels in contaminated earthworms than in control material. Ultrastructurally, chloragocytes from contaminated earthworms showed electron dense flecks associated with the chroragosome peripheries and within the debis vesicles. Very fine flecks occurred in the nuclei, but mitochondria and Golgi were indistingushable from control material. Preliminary X-ray microanalysis of contaminated chloragocytes revealed lead and phosphorus. The deposits within the chloragocytes might represent unbound lead precipitated by phosphate buffer; flecks being absent from contaminated, citrate buffered material and from control material. The chloragosomes are proposed as possible sites for sequestered lead. Chloragocyte and intestinal glycogen levels were significantly higher in control material where the chloragocyte cytoplasm was rich in alpha-glycogen rosettes, these being absent from lead contaminated cells. The glycogen-lead correlation suggests that the metabolism of contaminated chloragocytes is directed towards lead sequestration, though differing nutritional states cannot be ignored.

Animals↗

Metabolic fate of earthworm cadmium-binding proteins in rats.

Three different molecular weight cadmium-binding proteins isolated from the earthworm, Eisenia foetida, were injected intraperitoneally into young female rats. Cadmium bound to earthworm protein-I (estimated molecular weight, 63,000-70,000 daltons) and -II (estimated molecular weight about 7,000 daltons) was recovered from the kidney supernatants as metallothionein, which indicated that the proteins were degraded and that the cadmium liberated from the degraded proteins induced metallothionein biosynthesis in the kidneys. However, cadmium bound to earth-worm protein-III (estimated apparent molecular weight about 2,000 daltons) was recovered primarily from urine as a form bound to the protein. Compared with cadmium bound to albumin, metallothionein, and the low molecular weight cadmium-binding protein in Chlorella, the metabolic fates of cadmium bound to the three earthworm proteins were explained by the differences of molecular sizes and stability constants. Induced metallothioneins showed different chromatographic properties with time after injections on a Sephadex G-75 column and on a gel permeation column (TSK GEL SW 3,000).

Animals↗

Immunocytochemical electron microscopic study and western blot analysis of paramyosin in different invertebrate muscle cell types of the fruit fly Drosophila melanogaster, the earthworm Eisenia foetida, and the snail Helix aspersa.

The presence and distribution pattern of paramyosin have been examined in different invertebrate muscle cell types by means of Western blot analysis and electron microscopy immunogold labelling. The muscles studied were: transversely striated muscle with continuous Z lines (flight muscle from Drosophila melanogaster), transversely striated muscle with discontinuous Z lines (heart muscle from the snail Helix aspersa), obliquely striated body wall muscle from the earthworm Eisenia foetida, and smooth muscles (retractor muscle from the snail and pseudoheart outer muscular layer from the earthworm). Paramyosin-like immunoreactivity was localized in thick filaments of all muscles studied. Immunogold particle density was similar along the whole thick filament length in insect flight muscle but it predominated in filament tips of fusiform thick filaments in both snail heart and earthworm body wall musculature when these filaments were observed in longitudinal sections. In obliquely sectioned thick filaments, immunolabelling was more abundant at the sites where filaments disappeared from the section. These results agree with the notion that paramyosin extended along the whole filament length, but that it can only be immunolabelled when it is not covered by myosin. In all muscles examined, immunolabelling density was lower in cross-sectioned myofilaments than in longitudinally sectioned myofilaments. This suggests that paramyosin does not form a continuous filament. The results of a semiquantitative analysis of paramyosin-like immunoreactivity indicated that it was more abundant in striated than in smooth muscles, and that, within striated muscles, transversely striated muscles contain more paramyosin than obliquely striated muscles.

Animals↗

Cocoon production, morphology, hatching pattern and fecundity in seven tropical earthworm species - a laboratory-based investigation.

Data on the reproductive biology of seven Indian species of earthworms, viz. Perionyx excavatus Perrier, Lampito mauritii Kinberg, Polypheretima elongata (Perrier), Pontoscolex corethrurus (Muller), Eutyphoeus gammiei (Beddard), Dichogaster modiglianii (Rosa) and Drawida nepalensis Michaelsen are presented. The peregrine earthworms such as Perionyx excavatus, Pontoscolex corethrurus, Dichogaster modiglianii, and Polypheretima elongata are considered to be continuous breeders with high fecundity. Native Lampito mauritii and Drawida nepalensis are semi-continuous and Eutyphoeus gammiei discrete breeders. There is a dramatic increase in cocoon production by most earthworm species of Tripura in the summer and monsoon with a corresponding peak during April and July. Cocoon production decreased or ceased during winter. Temperature affected the incubation period of cocoons. With increase in temperature, incubation period increased in the endogeic worms, Pontoscolex corethrurus, Polypheretima elongata and Drawida nepalensis and decreased in the epigeic worms, Perionyx excavatus and Dichogaster modiglianii, within a temperature range between 28-32 degrees C under laboratory conditions. There was a significant (P < 0.05) positive correlation between number of hatchlings per cocoon and incubation period in Lampito mauritii. High rate of cocoon production, short development time with high hatching success, as well as continuous breeding strategies in the epigeic species Perionyx excavatus and Dichogaster modiglianii and the top soil endogeic species, Pontoscolex corethrurus, Drawida nepalensis and Lampito mauritii, indicate their possible usefulness in vermiculture. The giant anecic worm, Eutyphoeus gammiei, which has a very long cocoon development time, discrete breeding strategy and very low rate of cocoon production, is not a suitable species for vermiculture.

Animals↗

Reconfirmation of antimicrobial activity in the coelomic fluid of the earthworm Eisenia fetida andrei by colorimetric assay.

A novel tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) was used in the assessment of antimicrobial activity in earthworm in the presence of phenazine methosulphate (PMS) as an electron coupling reagent. This activity was purified from the coelomic fluid of the earthworm (ECF), Eisenia fetida andrei (Oligochaeta, Lumbricidae, annelids) using a series of column chromatography techniques and was tested against three Gram-negative strains of Escherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila and three Gram-positive strains of Staphylococcus aureus, Bacillus megaterium, Arthrobacter sp., respectively. Only the pigment-free eluate of coelomic fluid of the earthworm (ECFPE) showed activity against B. megaterium amongst three isolated active fractions. The anion (DEAE-52) exchange effluent of the ECFPE was reported to have the strongest activity against P. aeruginosa amongst the three active fractions. The 20% acetonitrile eluate (AE) by Sep-Pak C18 cartridge was also tested and showed fair resistance against E. coli, P. aeruginosa and Arthrobacter sp., respectively.

Animals↗

Remediation of heavy metal-contaminated soils using phosphorus: evaluation of bioavailability using an earthworm bioassay.

This study investigated the effectiveness of phosphorus (P) as an in situ remediation technique in site soils collected from the Tri-State mining area. The site soils were treated with two different types (KH(2)PO(4) and Super Triple Phosphate fertilizer) and levels (600 and 5,000 mg/kg) of phosphorus. A toxicokinetic approach was used to determine whether phosphorus amendments reduced heavy metal bioavailability to the earthworm Eisenia fetida. Bioaccumulation factors were used as a measure of bioavailability. Depuration patterns of lead, zinc, and cadmium also were studied after removing earthworms from contaminated soils and then placing them in noncontaminated artificial soil. Results showed that the high phosphorus treatments significantly reduced lead, zinc, and cadmium bioavailability to the earthworms, probably due to formation of metal-phosphate complexes in the soils. Results also indicated that other soil characteristics in addition to amended phosphorus have an affect on bioavailability. Depuration experiments showed a biphasic elimination pattern for each metal. The current results indicate that phosphorus soil amendments may reduce ecological risk to soil-inhabiting invertebrates exposed to heavy metal-contaminated soils.

Animals↗

The comet assay as biomarker of heavy metal genotoxicity in earthworms.

The ubiquitous occurring earthworm species, Eisenia fetida, were exposed to nickel chloride to determine whether the heavy metal Ni caused DNA damage, as measured by the comet (single cell gel electrophoresis) assay. Primary cell cultures of earthworm coelomocytes were exposed in vitro and whole animals either in spiked artificial soil water or in spiked cattle manure substrates. Comets formed were scored using mean tail lengths as well as comparing percentages of damage in five different damage classes. The exposure concentrations used for the in vitro exposure (2, 6, and 12 microg/ml) caused the formation of comets of which the mean tail lengths differed significantly (p < 0.05) from those of unexposed controls but not from each other. Coelomocytes from worms exposed in artificial soil water at concentrations of 0.0049, 0.0078, 0.0175, and 0.025 mg/ml formed comets of which the mean tail lengths differed significantly (p < 0.05) between the exposure groups with increasingly longer tail lengths with higher concentration (dose-related response). The tail length means of the comets of the three highest exposure concentrations also differed significantly (p < 0.05) from the controls. No dose-related response was found between comet tail lengths of the three exposure concentrations (60, 240, and 480 mg/kg) used for the worms in the cattle manure substrates, but the mean tail lengths of comets from all three exposure groups differed significantly (p > 0.05) from the controls. The comets formed in cells from animals exposed in artificial soil water and in cattle manure substrates, scored within damage classes, indicated a clear shift with increasing exposure concentrations from low to high damage. Our results indicated DNA single-strand breaks in soil invertebrate cells caused by exposure to a nickel compound, verifying previous findings for mammals which indicated that this heavy metal has genotoxic potential. These results therefore suggest that earthworms may be useful indicator organisms to assess the genotoxic risks of nickel pollution to terrestrial environments and that the comet assay is a useful tool to use as biomarker of genotoxic effects on invertebrates in soil.

Animals↗

Transport of sodium and chloride across earthworm skin in vitro.

We present a new invertebrate model for the study of epithelial sodium transport in tight epithelia, the earthworm integument. Dissected segments of earthworm integument were mounted in modified Ussing chambers and perfused with either pond water (PW) or earthworm ringer solution (ERS) on the apical side. In order to investigate ion transport under near-in vivo physiological conditions, measurements were performed under current-clamp conditions by monitoring the transepithelial potential (V (T)), as well as the transepithelial resistance (R (T)). These were recorded continuously and the virtual short circuit current (I (SC)) was calculated. The integument has a high transepithelial resistance (R (T) = 9,037 +/- 502 Omega cm(2) for PW, n = 24, and 11,055 +/- 1,320 Omega cm(2) for ERS, n = 32). V (T) was -3.7 +/- 2.2 mV for PW (n = 24) and -1.5 +/- 1.0 mV for ERS (n = 32), and I (SC) was -0.57 +/- 0.30 microA/cm(2) for PW (n = 24) and -0.44 +/- 0.24 microA/cm(2) for ERS (n = 32). Only under PW, but not under ERS conditions, was there a pronounced inhibition of I (SC) by low doses of amiloride or its analogues phenamil and benzamil. The resistance of the paracellular pathway was found to be very high. The terrestrial oligochaete Lumbricus seems especially adapted to the environmental conditions because it has an ultra-tight integument and a very fast up- and down-regulation of apical Na(+) channels.

Animals↗

Genetic variation in the enzyme esterase, bioaccumulation and life history traits in the earthworm Lumbricus rubellus from a metal contaminated area, Avonmouth, England.

Earthworms, Lumbricus rubellus, were collected in three successive years, 1997, 1998 and 1999 from 5, 6 and 4 sites, respectively, along a metal gradient near a smelter at Avonmouth, England. In total 271 worms were collected, 47 in 1997, 97 in 1998, and 127 in 1999. The worms were analysed for the enzyme esterase by means of agarose electrophoresis and for internal metal concentration. At one site collected in three successive years, 64 individuals of 66 were heterozygous for the esterase enzyme. The metal concentrations (Cu and Zn) in worms were significantly correlated with the total concentration and the CaCl2 extractable fraction of the metal in the soil. One of the homozygous esterase genotypes was significantly correlated with distance from the smelter, the extractable fraction of metals from soil, and the metal concentration in the worms. The other homozygous genotype was correlated with the CaCl2 extractable Cu and the Cu concentration in the worms. The internal levels of Cd, Cu, and Zn showed a higher classification potential (Discriminant Analysis) for 50 of 127 individuals, collected in 1999, when based on the original habitat of the earthworms than on the esterase genotype. The remaining 77 specimens, representing three sites, were exposed for 30 days in the laboratory to soils from four sites. For the individuals in this transplantation experiment, parameters measured were the esterase phenotype, internal metal concentration (Cd, Cu, and Zn), mortality, wet weight change, cocoon production, and cocoon hatching success. Cocoon production was highest among the worms from the most contaminated area, while worms from the least contaminated area had the highest hatching success. Neither the metal concentrations, in soil or internal, nor the life-history parameters could fully explain the genotypic distribution of esterase along the gradient. The internal concentrations of Cd, Cu and Zn in the worms used for the transplantation experiment showed the highest classification potential (Discriminant Analysis) when the prediction was based on the original habitat of the earthworms. The classification potential was less strong when based on esterase genotype and least on the transplantation conditions. Including life history traits did not enhance the prediction potential.

Animals↗

Environmental metabonomics: applying combination biomarker analysis in earthworms at a metal contaminated site.

Earthworms were taken across an environmental gradient of metal contamination for ecotoxicology assessment. Both indigenous (Lumbricus rubellus and L. terrestris) and introduced earthworms (Eisenia andrei, exposed in mesh bags) were studied. Changes in the levels of small molecule metabolites in earthworm tissue extracts were analysed by 1H NMR spectroscopy as a means of identifying combination biomarker compounds. Principal components analysis of the NMR spectral data revealed that biochemical changes were induced across the metal contamination gradient. Native worms (L. rubellus) from the most polluted sites were associated with an increase in the relative concentration of maltose; a decrease was also seen in the concentration of an as yet unidentified biomarker compound. Introduced worms (E. andrei) did not show differences to the same extent. Direct integration of the resonances from histidine and 1-methylhistidine showed that relative histidine concentrations were elevated slightly for L. rubellus, confirming the results of earlier mesocosm studies. Conversely, the relative concentrations of both histidine and 1-methylhistidine were greatly reduced by metal contamination in L. terrestris. This study demonstrates the utility of NMR spectroscopy in detecting previously unknown potential biomarkers for ecotoxicity testing and identified maltose as a potential biomarker compound deserving of further study.

Animals↗

Isolation from earthworms of a proteinaceous chemoattractant to garter snakes.

We have isolated a potent proteinaceous chemoattractant from aqueous washes of earthworm (Lumbricus terrestris) for garter snakes (Thamnophis sirtalis) by means of a covalent chromatography. It contained free sulfhydryl groups and showed an apparent mass of 20 kDa. The chemoattractive activity of this protein could be destroyed by heating as well as by proteolysis. Its activity could also be reversibly blocked by mixed disulfide formation with dithiodipyridine, suggesting that the free sulfhydryl(s) was essential for its function as a chemoattractant. This bioactive material had a tendency to form intermolecular crosslinked aggregates during isolation, if reducing agents were not included. Some of the high-molecular-weight aggregates cochromatographed with earthworm cuticle collagen on Ultragel AcA 34 or 44 columns. In contrast to an earlier report by D. M. Kirschenbaum, N. Schulman, and M. Halpern [1986) Proc. Natl. Acad. Sci. USA 83, 1213-1216) the purified earthworm collagen showed no chemoattractive activity to garter snakes.

Animals↗