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The capacities of earthworms to heal wounds and to destroy allografts are modified by polychlorinated biphenyls (PCB).

Earthworms (Lumbricus terrestris) were maintained at 15 degrees C and exposed on filter paper to 10 micrograms/cm2 of the polychlorinated biphenyl (PCB) Aroclor 1254 for 5 days prior to surgical treatments which consisted of wounds, autografts, and allografts. At 1 day after surgery, we observed a higher percentage of healing defects and a significantly greater number of early signs of allograft rejection in exposed worms. Observations for 25 days post-transplantation revealed no response to autografts, but an acceleration of the allograft rejection process in exposed earthworms. We postulate that Aroclor modified host coelomocytes and/or their interactions associated with antigen recognition and inflammation.

Animals↗

Isolation and characterization of alarm pheromone from electric shock-induced earthworm secretion.

Electric stimulation of earthworms, Lumbricus terrestris, causes secretion of a yellow mucus which has alarm properties for conspecifics and chemoattractive properties for garter snakes. An alarm pheromone was isolated and purified to homogeneity from the mucus by means of permeation, thin-layer and high performance liquid chromatographies. The purified substance was highly active as an alarm chemosignal to earthworms (L. terrestris), but it did not elicit alarm responses from either sandworms (Nereis virens) or bloodworms (Glycera debranciata). It was not a snake chemoattractant. The alarm pheromone could not be retained with 1 kDa cut-off dialysis tubing, and it was eluted from a Bio-gel P2 column ahead of p-nitrophenol. These data suggest an apparent mass greater than 139 Da but less than 1 kDa. The order of solubility of this alarm pheromone is H2O greater than DMSO greater than MeOH greater than 2-propanol greater than acetone. It was thermostable, and it fully retained activity after heating at 100 degrees C for 1 hour. This alarm pheromone fluoresced under u.v. light, and it showed an optimal excitation wavelength of 420 nm and emission wavelength of 465 nm.

Animals↗

The fate of protein antigen in earthworms: study in vitro.

Earthworm coelomocytes digest protein antigen in vitro. Proteolytic activity was detected both in cell-free coelomic fluid and in cell cultures of free coelomocytes which are effectors of earthworm immunodefense mechanisms. Antigen is cleaved either intracellularly or by proteolytic enzymes released by coelomocytes into the medium. Proteolysis was observed both in non-stimulated and antigen-stimulated cultures. Since significantly higher proteolysis was shown in supernatants from cultures of antigen-stimulated coelomocytes, we can assume that the release of proteolytic enzymes was inducible.

Animals↗

Killing of intrafamilial leukocytes by earthworm effector cells.

When Lumbricus and Eisenia coelomocytes are cultured together in intrafamilial xenogeneic combinations, significant cytotoxicity occurs at 24 h but not at 5 nor 72 h, as shown by trypan blue assay. In a 4.5-h assay, measuring 51Cr release, using an effector/target ratio of 25:1, unpooled cells from a single Lumbricus killed Eisenia cells at levels of 6% and 14%. However, Eisenia coelomocyte survival was high and identical in either cell-free xenogeneic (Lumbricus) coelomic fluid or in artificial medium. In this 1-way assay, earthworm (Lumbricus) coelomocytes act as effector cells that kill non-self target cells, even those of other earthworms. Comparisons with previous results reveal greater reliability and consistently repeatable results when the 51Cr release assay is used to measure cytotoxicity regardless of the targets.

Animals↗

Accumulation of heavy metals from polluted soils by the earthworm, Lumbricus rubellus: can laboratory exposure of 'control' worms reduce biomonitoring problems?

This paper compares the patterns of metal (Pb, Zn, Cd, Cu) accumulation in nine populations of the epigeic earthworm, Lumbricus rubellus, native on metalliferous soils, with the patterns of metal accumulation in batches of L. rubellus sampled from an uncontaminated site and maintained on the nine contaminated soils for 31 days under laboratory conditions. The primary findings were: (1) the Pb, Zn and Cd concentrations in the 'native' worms were significantly higher in most cases than in the 'introduced' worms; (2) multiple regression analyses indicated that the relationships between tissue and soil metal concentrations were similar for 'native' and 'introduced' worms; (3) high soil organic matter content reduced the bioavailability of Pb, but low pH increased Pb bioavailability. It was concluded that, although no phenotypic evidence of metal-tolerant ecotypes was obtained, the exposure of earthworms from uncontaminated soils to contaminated soils under laboratory conditions can provide meaningful integrative data concerning metal bioavailability in soils which, for biomonitoring purposes, often present formidable sampling problems.

Journal Article↗

Sperm count in earthworms (Lumbricus terrestris) as a biomarker for environmental toxicology: effects of cadmium and chlordane.

Earthworms, Lumbricus terrestris, exposed in artificial soil to sublethal concentrations of technical chlordane (6.25, 12.5, and 25 ppm) and cadmium nitrate (100, 200, and 300 ppm) exhibited significant reduction in spermatozoa from testes and seminal vesicles. The onset time of reduction varied with exposure concentration, but absolute depression in sperm count was independent of exposure concentration or exposure duration after reduction was first manifested, demonstrating a threshold effect. Earthworm sperm counts show potential as a rapid-measurement endpoint biomarker for measuring sublethal effects of chemical pollutants on reproduction.

Journal Article↗

A new source of biologically active compounds--earthworm tissue (Eisenia foetida, Lumbricus rubelus).

1. Earthworms possess immunological recognition and memory as well as high regenerative abilities. Coelomic fluid was used as a source of biologically active compounds. 2. A biologically active glycolipoprotein extract from a whole earthworm tissue homogenate was isolated and named G-90. 3. G-90 forms precipitation arcs in gel with different animal and human sera. 4. It alters murine cell growth rate in vitro in serum in a dose-dependent manner and slows murine tumor growth in vivo. 5. G-90 does not contain mutagens or carcinogens.

Animals↗

Glutamate-induced deporalization in earthworm ventral nerve cord.

We investigated glutamate-induced neuron response in the ventral nerve cord of the earthworm (Eisenia foetida) using a voltage-sensitive dye-imaging technique. Isolated earthworm ganglia were stained by fluorescence voltage-sensitive dye, RH414, and voltage image was acquired by a conventional charge-coupled device (CCD) technique or a confocal laser scanning microscope. The fluorescence images before and during the glutamate stimulation was acquired and the relative fluorescence change was imaged as pseudo-color. Bath-applied glutamate (1 mM) depolarized many neurons on the ventral side, and in the three giant fibers on the dorsal side.

Animals↗

Effects of anesthesia on the electrolyte concentrations in blood and coelomic fluid of the earthworm, Pheretima communissima.

Sodium, potassium, calcium and magnesium concentrations in blood and coelomic fluid of Pheretima communissima under various methods of anesthesia or in the unanesthetized condition were determined. All electrolyte concentrations determined in ethanol-anesthetized earthworms were not significantly different from those in unanesthetized earthworms. Thermal (warming, cooling) anesthesia, urethane and chloretone brought about increases or decreases in some electrolyte concentrations in the blood or coelomic fluid. However, not all electrolytes responded similarly to a method of anesthesia.

Anesthesia↗

Noninducibility of cytochrome P-450 in the earthworm Dendrobaena veneta.

Cytochrome P-450 has been measured in the earthworm Dendrobaena veneta (Rosa) in a direct spectrophotometric procedure. The P-450 was found not in the dense microsomal fraction, but in the less dense overlying fraction often referred to as buffy coat. Earthworm P-450 was not induced by 3-methylcholanthrene or phenobarbitol.

Animals↗

Immunochemical evidence for substance P-like peptide in tissues of the earthworm Lumbricus terrestris: action on intestinal contraction.

The presence of a substance P-like peptide in intestinal and body wall tissues, ventral nerve fiber and seminal vesicles of the earthworm Lumbricus terrestris has been demonstrated by means of a radioimmunoassay technique. The greatest substance P-like immunoreactivity was measured in intestinal tissues where it stimulates the rate of spontaneous contraction. This effect is inhibited by the substance P antagonist (D-pro2, D-trp7,9)-SP suggesting a possible involvement of receptor mechanisms. Dual localization of substance P-like immunoreactivity in earthworm intestinal and nerve tissues follows the pattern observed of peptidal hormones in vertebrates which are common to both endocrine and non-endocrine tissues.

Acetylcholine↗

Cadmium binding studies to the earthworm Lumbricus rubellus metallothionein by electrospray mass spectrometry and circular dichroism spectroscopy.

The earthworm Lumbricus rubellus has been found to inhabit cadmium-rich soils and accumulate cadmium within its tissues. Two metallothionein (MT) isoforms (1 and 2) have been identified and cloned from L. rubellus. In this study, we address the metalation status, metal coordination, and structure of recombinant MT-2 from L. rubellus using electrospray ionization mass spectrometry (ESI-MS), UV absorption, and circular dichroism (CD) spectroscopy. This is the first study to show the detailed mass and CD spectral properties for the important cadmium-containing earthworm MT. We report that the 20-cysteine L. rubellus MT-2 binds seven Cd(2+) ions. UV absorption and CD spectroscopy and ESI-MS pH titrations show a distinct biphasic demetalation reaction, which we propose results from the presence of two metal-thiolate binding domains. We propose stoichiometries of Cd(3)Cys(9) and Cd(4)Cys(11) based on the presence of 20 cysteines split into two isolated regions of the sequence with 11 cysteines in the N-terminal and 9 cysteines in the C-terminal. The CD spectrum reported is distinctly different from any other metallothionein known suggesting quite different binding site structure for the peptide.

Animals↗

Nutrient changes and biodynamics of epigeic earthworm Perionyx excavatus (Perrier) during recycling of some agriculture wastes.

Potential of an oriental composting earthworm: Perionyx excavatus (Perrier) to decompose waste resources generated from agricultural practices (crop residues, farm yard manure, and cattle dung) was studied for 150days under laboratory conditions. At the end of experiment, all vermibeds showed significant decrease in their organic C content ( approximately 21-29%), while increase in total N ( approximately 91-144%), available P ( approximately 63-105%), and exchangeable K ( approximately 45-90%). P. excavatus showed maximum individual live weight (662.05mg) after 120days in MIXED (mixed crop residues+cow dung in 1:1) substrate. The maximum growth rate (mg worm(-1)day(-1)) was between 3.79+/-0.08 and 2.35+/-0.16 on different substrates. The mean number of cocoon production was between 394.3+/-23.2 and 690.7+/-23.2 for different experimental beddings. MIXED bedding showed maximum reproduction rate (0.23+/-0.004 cocoons worm(-1) day(-1)), whereas farmyard manure bedding (FYM) showed least value (0.15+/-0.002 cocoons worm(-1)day(-1)). During vermicomposting, the total mortality in worms' population was recorded between 0% (in MIXED) and 21.7% (in Jowar straw (Sorghum vulgare)+millet straw (Pennisenum typhoides)+sheep manure in 1:1:2 ratio (JMS)). The waste decomposition and earthworm production was associated strongly with the quality of the substrate, especially with their chemical as well as biological composition.

Agriculture↗

Responses of earthworms (Lumbricus rubellus) to copper and cadmium as determined by measurement of juvenile traits in a specifically designed test system.

In this study, the effects of two metals, copper and cadmium, on the growth and development of juvenile Lumbricus rubellus were measured in a toxicity test in which individuals were grown in isolation. This design had a number of advantages over traditional test systems for earthworms. Importantly, the test is specifically designed to measure two juvenile traits (survival over and length of the juvenile period) that have been shown to have a high sensitivity for determining population growth rate. The test system also maximizes replication, while allowing time-series-based monitoring of individual growth. For both metals, significant exposure-dependent effects on survival, growth, development time, and (less certainly) maturation weight were observed. Comparisons of the relative toxicity of the two metals indicated different concentration-response relationships. For copper, hormesis was found at low levels, while only at the highest soil concentration tested (10.07 micromol g(-1)) were (severe) toxic effects present. For cadmium, hormesis was also evident at the lowest concentration tested; however, at soil levels above this, a graded concentration-dependent toxic effect was apparent. These differences in the exposure response patterns can be (tentatively) explained in terms of the mechanisms for handling copper (an essential metal for earthworms) and cadmium (a putative nonessential element). The applicability of the test for routine measurement of chemical effects on ecologically relevant juvenile traits is also outlined and future developments are discussed.

Animals↗

Sublethal soil copper concentrations increase mortality in the earthworm Aporrectodea caliginosa during drought.

Simultaneous effects of drought and a sublethal concentration of copper on the earthworm Aporrectodea caliginosa were investigated in the laboratory. Experiments were performed with exposure to 150 mg Cu kg(-1) dry soil and different levels of drought during a 3-wk period. Soil water potentials from pF 1.5 (wet) to pF 5 (very dry) were obtained. The soil water potential resulting in 50% mortality was significantly reduced from pF 4.48 in worms not exposed to copper to pF 4.09 in copper-exposed worms, thus demonstrating synergy-like effects of drought and copper. Development of estivation cells was significantly depressed in copper-exposed worms compared to worms not exposed to copper. For all drought levels (except the highest, where 100% mortality occurred), copper-exposed earthworms with no estivation cells had more than twice as high mortality and, at severe drought exposure, also lower water content than those which had developed the estivation cells. With increasing drought level, the whole-body burden of copper increased from about 40 microg Cu g(-1) dry weight to about 90 microg Cu g(-1). When the worms were exposed to drought, the osmolality in their body fluids increased. This was due to a greater concentration of already existing solutes resulting from extensive loss of water, and not to mobilization or synthesis of additional solutes.

Animals↗

Pesticide-induced surface migration by lumbricid earthworms in grassland: life-stage and species differences.

Pesticide-induced changes in surface migration by earthworms in grassland were investigated using trapping and the fungicide benomyl. Traps were tended daily for 15 days after spraying, resulting in 2152 earthworms, five species, and juvenile predominance which reflected species/life-stage composition in the soil. Significant increases in migration (all worms) occurred already by day 2 due to spraying, final treatment level being 2.8 x control. Life-stage composition indicated an increased juvenile proportion from 55% to 75% due to treatment. Spraying caused surfacing juveniles to increase significantly by day 2, reaching a final level 3.8 x control, whereas for mature worms a significant increase did not occur until day 4. Species rank-order was Aporrectodea longa>A. rosea>Lumbricus terrestris>A. caliginosa in control areas, but A. longa>L. terrestris>A. rosea>A. caliginosa in sprayed areas; spraying altered the rank-order such that the anecic A. longa and L. terrestris dominated, jointly increasing from 59% to 78%. At species level, L. terrestris and A. longa exhibited significant increases of 4.6 x and 3.6 x in final migration levels in treated areas, the endogeic A. rosea and A. caliginosa having trends for increase. Species-specific differences for reaction time occurred, with significantly elevated migration already by day 1 for L. terrestris, and day 2 for A. longa and A. caliginosa. For each species, juveniles consistently showed greater increases than mature worms due to spraying, significantly so for juvenile L. terrestris, A. longa, and A. caliginosa, the two anecics reaching as high as 5.3 x and 4.7 x. The response of mature worms differed: A. longa and A. rosea increased surfacing due to treatment, L. terrestris showed a delayed reaction, whereas A. caliginosa exhibited suppressed migration. Results are discussed relative to behavior, ecological category, and risk of toxic exposure.

Animals↗

Measurement of annetocin gene expression: a new reproductive biomarker in earthworm ecotoxicology.

The emergence of new technologies from the genomics revolution will transform the potential application of biomarkers to assess how pollutants impact people, animals, and ecosystems. Genetic databases provide a huge resource from which candidate molecular biomarkers can be identified and, subsequently, exploited to address these issues. However, a major challenge is to link these novel molecular indices to ecologically relevant whole-organism life-cycle traits (such as reproduction and growth). Such a functional link is provided by annetocin, previously characterized as a member of the vasopressin/oxytocin superfamily of neuropeptides. It is expressed in annelid worms within the neurons of the central nervous system and has been shown to be involved in the induction of egg-laying behavior. This paper outlines the validation of annetocin as a novel biomarker of reproductive fitness in the earthworm Eisenia fetida. The design of primer pairs targeted toward oligochaete annetocin has facilitated the isolation of a full-length annetocin cDNA from this species. Optimization of a real-time quantitative PCR procedure exploiting the fluorescent DNA-binding molecule, Sybr Green, has allowed the measurement of annetocin transcript levels over a range covering six orders of magnitude. Using this approach, gene expression was measured in earthworms exposed to soils polluted with high concentrations of zinc and lead. Traditional growth and reproductive indices, including cocoon production, were also recorded and related to the molecular parameter. The future use of annetocin as a molecular genetic biomarker in terrestrial ecotoxicology is discussed.

Amino Acid Sequence↗

The bioavailability of chemicals in soil for earthworms.

The bioavailability of chemicals to earthworms can be modified dramatically by soil physical/chemical characteristics, yet expressing exposure as total chemical concentrations does not address this problem. In order to understand the effects of modifying factors on bioavailability, one must measure and express chemical bioavailability to earthworms in a consistent, logical manner. This can be accomplished by direct biological measures of bioavailability (e.g., bioaccumulation, critical body residues), indirect biological measures of bioavailability (e.g., biomarkers, reproduction), or indirect chemical measures of bioavailability (e.g., chemical or solid-phase extracts of soil). If indirect chemical measures of bioavailability are to be used, they must be correlated with some biological response. Bioavailability can be incorporated into ecological risk assessment during risk analysis, primarily in the estimation of exposure. However, in order to be used in the site-specific ecological risk assessment of chemicals, effects concentrations must be developed from laboratory toxicity tests based on exposure estimates utilizing techniques that measure the bioavailable fraction of chemicals in soil, not total chemical concentrations.

Animals↗