Jason M. Weeks

Biomarkers in Earthworms

Earthworms are believed to be so-called key species within ecosystems and are often exposed to a wide range of anthropogenic compounds released to the terrestrial environment. As a consequence, they may suffer from the toxicity of these compounds. For these and other reasons, earthworms have been used extensively in ecotoxicological studies. In recent years the use of other biological responses (biomarkers) to estimate either exposure or resultant effects of chemicals has received increased attention. Biomarkers address the question of bioavailability by only responding to the bioactive fraction. They may incorporate effects following exposure to a mixture of chemicals. Biomarkers may also reduce extrapolation of results from the laboratory to the field, as they may be applicable under both conditions. The present review has drawn together current knowledge on potential biomarkers in earthworms and appraised them in relation to basic requirements needed for supplying information relevant to devising satisfactory risk assessment. A wide range of potential biomarkers have been measured in earthworms, including DNA alteration, induction of metal-binding proteins (MTs and MBP), depression of ChE activity and other enzymatic responses, energy reserve responses, responses in neural impulse conductivity, lysosomal membrane stability, immunological responses, changes in sperm numbers, histopathological changes, and behavioral changes. Both organic and inorganic compounds have been included; however, for each biomarker the main emphasis historically has been placed on only a few chemicals. Dose-response relationships were in some cases observed. Little information is available on the linkage of the biomarker response to effects at population or community levels. The influence of other factors, biotic and abiotic, on the biomarker responses and their temporal duration have been only sporadically reported.

An NMR‐based metabonomic approach to the investigation of coelomic fluid biochemistry in earthworms under toxic stress

The endogenous metabolites of the coelomic fluid of the earthworm Eisenia veneta were characterised using high‐resolution one‐dimensional and two‐dimensional 1H nuclear magnetic resonance spectroscopy. Signals from common organic acids, such as acetate, fumarate, malonate, malate, formate, and succinate, were identified together with adenosine and nicotinamide mononucleotide. The potential use of this information as a baseline dataset for future toxicological or physiological studies was demonstrated by a metabonomic analysis: a series of earthworms were dosed with the model compound 3‐fluoro‐4‐nitrophenol, and toxic effects followed by multivariate analysis of the spectral data of the coelomic fluid. Relative concentrations of acetate and malonate were decreased in the dosed worms compared to the controls.

Use of an earthworm lysosomal biomarker for the ecological assessment of pollution from an industrial plastics fire

Abstract A low-cost field technique employing retention of the dye neutral-red by lysosomes in coelomocyte cells taken from earthworms (Lumbricus castaneus), was used as a means of assessing the ecological effects (if any) of an industrial accident. Earthworms and soil samples were collected at the site of a large industrial plastics fire in Thetford, UK along a 200 m transect leading from the factory perimeter fence, over a layer of molten plastic impregnated soil and into the surrounding forest. Coelomic fluid extracted from the earthworms was dye-loaded with neutral-red and lysosomal leaking observed. Metal residues in soil and earthworms were found to be highly elevated close to the factory perimeter and to rapidly drop to background levels within the first 50 m of the transect. Coelomocyte cells taken from earthworms adjacent to the factory perimeter showed the shortest period of neutral-red retention (2 min); cells taken from worms further into the surrounding forest had a longer retention time (12 min), whilst cells taken from worms from a control site showed even greater retention times (25 min). Thus, the neutral-red retention times correlated negatively with measured residues of heavy metals in the earthworms, the higher the body metal concentration the shorter the retention time. This field trial has demonstrated the validity of using an in vitro cellular biomarker technique for use in biological impact assessment along gradients of contamination.

Toxicity of Nickel to the Earthworm and the Applicability of the Neutral Red Retention Assay

The toxic effects of nickel on survival, growth, and reproduction of Eisenia veneta were investigated following 4 weeks of exposure to a nickel-chloride spiked loamy sand soil. The ability of a simple earthworm biomarker, the lysosomal membrane stability of coelomocytes, to reflect nickel exposure was also studied. Nickel caused a significant toxic effect on E.veneta at soil concentrations above 85 mg Ni/kg. Reproduction (cocoon production) was the most sensitive parameter being reduced at soil concentrations above 85 mg Ni/kg (EC10 = 85 mg Ni/kg). Survival of adults was only reduced at concentrations above 245 mg Ni/kg, while adult and cocoon wet weight were not affected by soil nickel concentrations up to 700 mg Ni/kg. The lysosomal membrane stability, measured as neutral-red retention time, was reduced at soil nickel concentrations similar to those that reduced reproduction, and demonstrated a dose-response relationship. The neutral-red retention time showed large individual variation for the earthworms within each exposure concentration. It was concluded that the lysosomal membrane stability, measured as neutral red retention time, has a potential role in risk assessment, but care should be taken conducting this test.

The use of a lysosome assay for the rapid assessment of cellular stress from copper to the freshwater snail Viviparus contectus (Millet)

To enable efficient monitoring and the early detection of toxic metal effects a rapid low cost test has been developed for use with freshwater gastropods. A cellular assay formerly developed for marine organisms has been modified for use with freshwater snails. A modified neutral-red retention assay makes use of the fact that lysosomes in unstressed cells retain the neutral-red dye for a long duration after uptake. In contrast, following uptake in stressed cells, the dye will leak from the lysosomes into the cytoplasm more rapidly, an indication of cell-membrane damage. The higher the stress level, the shorter the period of dye retention. In this study freshwater snails Viviparus contectus were exposed to dissolved copper. Blood cells extracted from snails exposed to the highest concentrations exhibited the shortest lysosomal neutral-red retention times. This early detectable change may be useful for routine environmental monitoring, identifying low level contamination, the moment it occurs.

Effects of phenotypic plasticity on pathogen transmission in the field in a Lepidoptera-NPV system

Abstract In models of insect–pathogen interactions, the transmission parameter (ν) is the term that describes the efficiency with which pathogens are transmitted between hosts. There are two components to the transmission parameter, namely the rate at which the host encounters pathogens (contact rate) and the rate at which contact between host and pathogen results in infection (host susceptibility). Here it is shown that in larvae of Spodoptera exempta (Lepidoptera: Noctuidae), in which rearing density triggers the expression of one of two alternative phenotypes, the high-density morph is associated with an increase in larval activity. This response is likely to result in an increase in the contact rate between hosts and pathogens. Rearing density is also known to affect susceptibility of S. exempta to pathogens, with the high-density morph showing increased resistance to a baculovirus. In order to determine whether density-dependent differences observed in the laboratory might affect transmission in the wild, a field trial was carried out to estimate the transmission parameter for S. exempta and its nuclear polyhedrosis virus (NPV). The transmission parameter was found to be significantly higher among larvae reared in isolation than among those reared in crowds. Models of insect–pathogen interactions, in which the transmission parameter is assumed to be constant, will therefore not fully describe the S. exempta-NPV system. The finding that crowding can influence transmission in this way has major implications for both the long-term population dynamics and the invasion dynamics of insect–pathogen systems.

Developing a new method for soil pollution monitoring using molecular genetic biomarkers.

Physiological responses to environmental stressors may induce changes in gene expression as part of an organisms homeostatic mechanisms. Thus molecular genetic biomarkers have the potential to be used for monitoring sublethal chemical exposure in ecosystems. This paper describes a methodological assessment of the suitability of a protocol to monitor selected biomarkers. The TaqMan® real-time quantitative polymerase chain reaction was used to measure gene transcription in earthworms (Lumbricus rubellus) maintained on control or cadmium- or copper-spiked soil. Changes in the expression of two target genes, that encoding metallothionein isoform 2 (MT-2) and that encoding the mitochondrial large ribosomal subunit (MLRS), were quantified against the internal control gene β-actin. The protocol used produced reliable and reproducible results. Transcript levels displayed qualitative and quantitative differences in the responses to the two metal ions. MLRS gene levels were unaffected by exposure to cadmium but displayed a response to high levels of copper. Conversely, cadmium greatly induced MT-2 gene expression, but copper only altered transcription of this gene at high exposure concentrations. This study demonstrates that it is now technically feasible to use gene expression as an index of pollution exposure in environmentally relevant organisms.

1H NMR spectroscopic investigations of tissue metabolite biomarker response to Cu II exposure in terrestrial invertebrates: identification of free histidine as a novel biomarker of exposure to copper in earthworms

High resolution 1H NMR spectroscopy of biofluids, cells and tissue extracts allows rapid, non destructive analysis for a wide range of metabolites and organic compounds with minimal sample pre treatment. We have applied high resolution 1H NMR spectroscopy to investigate the biochemical effects of Cu II in two earthworm species Eisenia andrei n =78 and Lumbricus rubellus n =45 exposed under laboratory and semi field conditions respectively. The most marked metabolic response was the elevation of endogenous whole body free histidine in animals which positively correlated with increasing copper exposure and total copper burden in the semi field experiment. Histidine forms thermodynamically stable copper complexes under a wide range of physico chemical conditions and we proposed that the elevation of free histidine in response to copper challenge provides an energetically low cost detoxification mechanism. Histidine elevation may also provide a novel molecular biomarker of Cu II exposure in environmental situations.High resolution 1H NMR spectroscopy of biofluids, cells and tissue extracts allows rapid, non destructive analysis for a wide range of metabolites and organic compounds with minimal sample pre treatment. We have applied high resolution 1H NMR spectroscopy to investigate the biochemical effects of Cu II in two earthworm species Eisenia andrei n =78 and Lumbricus rubellus n =45 exposed under laboratory and semi field conditions respectively. The most marked metabolic response was the elevation of endogenous whole body free histidine in animals which positively correlated with increasing copper exposure and total copper burden in the semi field experiment. Histidine forms thermodynamically stable copper complexes under a wide range of physico chemical conditions and we proposed that the elevation of free histidine in response to copper challenge provides an energetically low cost detoxification mechanism. Histidine elevation may also provide a novel molecular biomarker of Cu II exposure in environmental situ...

Influence of dimethoate on acetylcholinesterase activity and locomotor function in terrestrial isopods

Locomotor behavior in terrestrial organisms is crucial for burrowing, avoiding predators, food seeking, migration, and reproduction; therefore, it is a parameter with ecological relevance. Acetylcholinesterase (AChE) is a nervous system enzyme inhibited by several compounds and widely used as an exposure biomarker in several organisms. Moreover, changes in energy reserves also may indicate an exposure to a stress situation. The aim of this study is to link biomarkers of different levels of biological organization in isopods exposed to increasing doses of dimethoate in semifield conditions. Locomotor parameters, AChE activity, and energy reserves (lipid, glycogen, and protein contents) were evaluated in the isopod Porcellio dilatatus after 48-h and 10-d exposure to dimethoate-contaminated soil. Results showed a clear impairment of both locomotor and AChE activity during the entire study, although effects were more pronounced after 48 h. Most locomotor parameters and AChE activity showed a clear dose-response relationship. By contrast, no clear trend was observed on energetic components. A positive and significant relationship was found between AChE activity and those locomotor parameters indicating activity, and the opposite was observed with those locomotor parameters indicating confusion and disorientation. The results obtained in this study enhance the importance of linking biochemical responses to parameters with ecological relevance at individual level, the value of locomotor behavior as an important marker to assess effects of toxicants, and also the usefulness and the acquisition of ecological relevance by AChE as a biomarker, by linking it with ecologically relevant behavioral parameters.

An NMR-based metabonomic investigation of the toxic effects of 3-trifluoromethyl-aniline on the earthworm Eisenia veneta

(1)H NMR spectra of earthworms Eisenia veneta treated with 3-trifluoromethyl-aniline in a 72-h contact filter paper test have been analysed using pattern recognition techniques to determine the biochemical response. Various strategies for data reduction of the metabolite profile, and illustration by principal components analysis are applied and discussed. The use of mean principal components plots in simplifying group data representation and highlighting the dose-response function is demonstrated. Hierarchical cluster analysis, and cluster significance analysis of the principal components were also used to examine the relative distribution of dose groups. Identification and assignment of metabolite responses to toxicity were found via correlation coefficient-shift plots. As measured by the correlation coefficients alanine was the most significant metabolite, but increased levels of other amino acids such as glycine and asparagine were also observed. Further, elevated levels of glucose, and the citric acid cycle intermediates citrate and succinate were noted as potential biomarkers of toxicity. This work provides a basis for examining the biochemical response of invertebrates to toxins. This should provide a framework to examine toxicity effects of other halogenated aromatic pollutants to earthworms used as environmental monitors.1H NMR spectra of earthworms Eisenia veneta treated with 3-trifluoromethyl-aniline in a 72-h contact filter paper test have been analysed using pattern recognition techniques to determine the biochemical response. Various strategies for data reduction of the metabolite profile, and illustration by principal components analysis are applied and discussed. The use of mean principal components plots in simplifying group data representation and highlighting the dose-response function is demonstrated. Hierarchical cluster analysis, and cluster significance analysis of the principal components were also used to examine the relative distribution of dose groups. Identification and assignment of metabolite responses to toxicity were found via correlation coefficient-shift plots. As measured by the correlation coefficients alanine was the most significant metabolite, but increased levels of other amino acids such as glycine and asparagine were also observed. Further, elevated levels of glucose, and the citric acid cycle intermediates citrate and succinate were noted as potential biomarkers of toxicity. This work provides a basis for examining the biochemical response of invertebrates to toxins. This should provide a framework to examine toxicity effects of other halogenated aromatic pollutants to earthworms used as environmental monitors.