Metal stress in free-living nematodes

Terrestrial invertebrates offer meaningful targets for assessing the potential adverse effects of chemicals on soil ecosystems. Invertebrates play a major role in the functioning of the soil ecosystem by enhancing the soil structure, mineralization and the decomposition of organic material, and because of their role in the foodweb. The most dominant group of terrestrial invertebrates, in fact of all multicellular organisms on earth, are nematodes, also called threadworms or roundworms. Nematodes are usually small (0.2-2 mm), transparent and present in almost every habitat on earth. In soil, they usually occur in high abundances and the nematode community comprises a considerable species diversity (Sohlenius, 1980). Nematodes belonging to the terrestrial bacterial feeders inhabit the interstitual water of soil particles (Houx and Aben, 1993). Therefore, they are subjected directly to the dissolved fraction of contaminants in soils, apart from being subjected indirectly via the foodsource. After extraction from the soil, many of these terrestrial bacterivorous nematodes can easily be reared in the laboratory in growth media or on agar plates with bacteria as foodsource.</p><p><em>Plectus acuminatus</em> (Nematoda, Torquentia, Plectidae) Bastian 1865 is an example of such a free-living terrestrial bacterivorous nematode species, easy to rear in the laboratory. This species has an egg-to-egg period of approximately 3 weeks and a life span of about 3 months at 20°C. <em>P. acuminatus</em> appeared to be a suitable species for toxicity tests (Kammenga <em>et al.</em> , 1996) and is ubiquitous in the moderate regions of the world (e.g. in soils in the Netherlands (Bongers, 1988) and the UK (Arts, unpublished)).</p><p><em>Caenorhabditis elegans</em> (Nematoda, Secernentea, Rhabditidae) Maupas 1899 is another example of a free-living terrestrial bacterivorous nematode species. It is the most investigated nematode species in laboratory experiments that exists. <em>C. elegans</em> strain N2 has originally been extracted from soil from the area of Bristol, UK, but has not been found in Dutch soils yet. It has been reared and maintained in the laboratory for decades. <em>C. elegans</em> is homozygous and doesn't suffer from any inbreed depression. Its life-cycle is very short with an egg-to-egg period of nearly 3 days and a total life span of about 20 days at 20<FONT FACE="Symbol">°</font>C (Wood, 1988). Therefore, this nematode species is very suitable to study life-cycle traits and to perform multi-generation experiments.</p><p>The purpose of this thesis is to evaluate metal stress in free-living terrestrial bacterivorous nematodes by measuring the response on the one hand at a very low organisational level (biomarker response) and on the other hand at a high organisational level (fitness consequences at the population level after consecutive generations). The biomarker response can predict effects of toxicants on cellular function, which might lead to changes in the physiology and/or histology of an organism. These changes possibly exert effects on life-cycle traits, which could lead to changes in fitness, which might eventually lead to extinction of the population.</p><p>Biomarkers are changes at the molecular, biochemical or cellular level in organisms following exposure to pollutants (Peakall and Shugart, 1992; Deplegde and Fossi, 1994) and are usually the first detectable responses to environmental perturbation. Because these alterations underlie all effects at higher organisational levels, they can be helpful tools in ecotoxicological risk assessment.</p><p>If we select as biomarker cellular and biochemical events which are intimately involved in protecting and defending the cell from environmental insults, we have ideal candidates for biomarkers of exposure and possibly of effect (Sanders, 1990). Cells dramatically alter their gene expression in response to environmental stress, attempting to protect themselves from damage and to repair existing damage (Schlesinger <em>et al.</em> , 1982). This response is called the cellular stress response. Changes in gene expression associated with the stress response are extremely rapid and result in the induced synthesis and accumulation of stress proteins. One group of stress proteins are the heat shock proteins (hsps), first discovered upon heat exposure but later found to be induced by a wide variety of chemical, physical and biological stressors (e.g. listed in Nover, 1991 and Sanders, 1993).Hsps possibly all function as molecular chaperones (Ellis, 1987), for one, facilitating the synthesis, folding, assembly and intracellular transport of many proteins, reducing protein denaturation and aggregation and aiding in protein renaturation (e.g. Ellis and van der Vies, 1991; Parsell and Lindquist, 1993). The common signal elicited by all hsp-inducing stressors involves an abnormally high concentration of damaged/aggregated proteins within cells, a phenomenon generally referred to as 'proteotoxicity' (Hightower, 1993). Hsp biomarkers give an integrated response summarizing the total proteotoxic damage caused within the target organism or organism tissue.</p><p>Each hsp is the member of a multigene family, regulated by different promotors and coding for closely related protein isoforms (Lindquist, 1986). Based on their molecular weight, hsps can be classified into different families (Sanders, 1993). The family of 55-65 kDa is called chaperonin. The members of this family have thus far been found in eubacteria and in eukaryotic cells, almost exclusively in organelles which are probably of endosymbiotic origin (mitochondria, chloroplasts) (Hemmingsen <em>et al.</em> , 1988) designated hsp60, stress-60, cpn60, GroEL ( <em>E. coli</em> ) or RuSBP (Rubisco Subunit Binding Protein (chloroplast)). Hsp60 is a nucleus-encoded, constitutively expressed protein. Under stressfull conditions, the hsp60 expression can be dramatically increased. Together with the ubiquitous hsp70 family, which is the most highly conserved and the largest of all the hsp families, the hsp60 family has great potential as a biomarker for general stress (Sanders, 1990).</p><p>Therefore, both the hsp70 and hsp60 response were qualitatively analyzed in the nematode <em>P. acuminatus</em> (see chapter 2) in order to select the most sensitive hsp-biomarker to increasing metal concentrations. The hsp70 and hsp60 responses were studied following exposure to heat, to copper chloride and to cadmium chloride. Mini two-dimensional polyacrylamide gel electrophoresis was used for protein separation. Poly- and monoclonal antibodies raised against hsp70 or hsp60 in various organisms were used to detect the respective hsps by immunoblotting. Both hsp60 and hsp70 could be identified after exposure of the nematodes to heat, indicating the broad cross reactivity among species to the antibodies used. The induction of hsp60 in <em>P. acuminatus</em> was related to increased concentrations of cadmium and copper chloride. For copper chloride, the induction of hsp60 was 3 orders of magnitude more sensitive than was the EC20 for reproduction; for cadmium chloride, the hsp60 induction was 2 orders of magnitude more sensitive. The hsp70 response in <em>P. acuminatus</em> was also elevated after exposure of the nematodes to cadmium and copper chloride, but this response was relatively weak compared to the hsp60 response. Therefore, it was concluded that the hsp60 response may be suitable as a potential biomarker to metal stress in <em>P. acuminatus</em> .</p><p>The hsp60 response in <em>P. acuminatus</em> has been further investigated quantitatively in the laboratory, at the protein level as well as at the mRNA level after exposure to various metals (see chapter 3). The mRNA response may be more sensitive and reproducible compared to the protein response and was therefore worth considering. Both the hsp60 protein and mRNA response were measured after 24 hours of exposure to either zinc chloride (0-550<FONT FACE="Symbol">m</font>M) or copper chloride (0-59<FONT FACE="Symbol">m</font>M), the protein response also after 24 hours of exposure to cadmium chloride (0-109<FONT FACE="Symbol">m</font>M). Furthermore, we identified hsp60 in <em>P. acuminatus</em> by elucidating its full-length mRNA sequence and deduced amino acid translation and comparing this to other known sequences. After exposure of the nematodes to zinc chloride, a significant optimum curve was found for the hsp60 response at the protein level, with a maximum induction of over 8 fold the control response at a concentration of 291<FONT FACE="Symbol">m</font>M zinc chloride. Most likely, the hsp60 response increased until the ability of the heat shock system to react to increasing metal concentrations reached its climax, after which a further increase in metal concentrations resulted in a decline of the hsp60 level, which might be interpreted as a result of pathological tissue damage as described by Eckwert <em>et al</em> . (1997) concerning the hsp70 response in the isopod species <em>Oniscus asellus</em> . A significant hsp60 increase at the protein level was also detected with increasing copper chloride concentrations, but the maximum hsp60 induction was not reached within the investigated copper concentration range. When the nematodes were exposed to cadmium chloride, no significant trend was observed. At the mRNA level, in <em>P. acuminatus</em> no considerable hsp60 induction was obtained when compared to control levels and to the protein levels at the investigated metal concentration range after 24 hours of exposure. Though the variability at the hsp60 protein level in <em>P. acuminatus</em> was much higher compared to the hsp60 mRNA level, the increase upon metal exposure was much higher at the protein level and occurred at higher metal concentrations. Therefore, the hsp60 protein response in <em>P. acuminatus</em> may have more potential as a biomarker for metal stress than the hsp60 mRNA response.</p><p>In chapter 4, the application of the hsp60 protein response in <em>P. acuminatus</em> as a biomarker for metal pollution is evaluated in an in situ bio-assay in a field experiment along a metal gradient near Avonmouth, UK. Because it is impossible to determine nematodes to the species level without killing or at least heavily stressing them, <em>P. acuminatus</em> specimens were transplanted into six field sites along the metal gradient and the hsp60 protein response was measured. The response appeared to be significantly higher in the nematodes transplanted into the field site with the lowest metal concentrations compared to the other field sites. The responses of the nematodes in the other field sites did not significantly differ from each other. It can be concluded that the hsp60 response in <em>P. acuminatus</em> alone was not a suitable biomarker for heavily contaminated soils. However, this biomarker had indicative value when related to other biomarker responses measured simultaneously in the same field sites (e.g. the hsp70 response in the isopod species <em>O. asellus</em> and <em>Porcellio scaber</em> ). Furthermore, it might be a suitable biomarker for less heavily contaminated soils. This would have to be investigated in field experiments, because laboratory experiments provide no alternative.</p><p>From this thesis it can be concluded that the hsp60 response in <em>P. acuminatus</em> could be used as a biomarker for metal exposure, but with the following limitations:</p><OL><LI>An increase of the hsp60 response in the nematode indicates the presence of a proteotoxic stress factor. To identify this stress factor, additional measurements would have to be performed, e.g. chemical analyses, the application of biomarkers which identify specific stressors.</LI><LI>The hsp60 response in <em>P. acuminatus</em> is only elevated within a restricted and relatively low concentration range of the metal(s). No elevation of the response means that the stressor level is either too low or too high. The hsp60 response should always be related to other biomarker responses in order to interpret the response. This is not only important in case no elevation is measured, but also to determine where the hsp60 response should be located compared to the maximum induction value, indicating whether the response is increasing or already quenching.</LI></OL><p>The outcome of short-term toxicity studies, such as the hsp responses described in this thesis, may not be used for predicting long-term demographic effects. Because effects at the population level are mediated through effects on fitness, the change in fitness under metal stress in a multi-generation experiment is studied using the nematode <em>C. elegans</em> (chapter 5). The strong advantage of testing multiple generations instead of one generation is the detection of possible trade-off mechanisms among life-history traits and fitness consequences, thus eliciting the probable course of the final consequences of chronic metal stress on the existence of the population. A life-history model of <em>C. elegans</em> was developed to calculate fitness maximisation in populations exposed to cadmium chloride during multiple generations. It was shown that the maximum fitness of <em>C. elegans</em> depended strongly on the trade-off between sperm maturation time and juvenile development. Once <em>C. elegans</em> was exposed to cadmium chloride, fitness decreased during the first generation. After exposure of consecutive generations, fitness increased slightly but significantly compared to the first exposed generation, while various life-cycle traits were strongly affected. The life-history modelling of <em>C. elegans</em> showed that cadmium chloride decreased fitness by impairment of juvenile development. The sperm maturation time remained constant. After long-term exposure of multiple generations, <em>C. elegans</em> counteracted the effect on juveniles by growing faster and increasing reproduction and fitness. This chapter illustrates that the combination of detailed knowledge of the life-cycle and life-history modelling provides insight into the underlying mechanisms of toxicant induced life-cycle changes and fitness consequences.</p&gt