Selection of insensitive phenotypes
It is indisputable that isopods and diplopods can inherently tolerate exposure to metals to some extent. This ability is mainly based on the intracellular precipitation of metal ions as phosphates or organic complexes in membrane-surrounded granules (spherites), which withdraw them for the most part from the animal’s metabolism (summarized in Hopkin, 1989). Nevertheless, high concentrations of cadmium, lead or zinc have been shown to surpass this detoxification system and to result in significantly reduced growth or moult rates in P. scaber ( Drobne & Štrus, 1996; Donker et al., 1998 ; Witzel, 1998) and may therefore exert a strong selection pressure toward the evolution of mechanisms to overcome the effect of these ions. It was an aim of this study to reveal the existence of adapted phenotypes in metal-contaminated habitats, and several of our results indicate selection of stress-insensitive phenotypes in some of the investigated populations. Although the stress response level covaried with certain abiotic parameters (soil pH, soluble metal fractions), models using these variables alone were only able to explain a few per cent of the intersite variability in the hsp70 level.
In contrast, the importance of the site-specific pollution history suggests that time is required for the formation of populations indicative of the evolution of stress insensitivity. Statistical models incorporating time explained much variability in hsp70 and, furthermore, suggested additional parameters, metal ions, which comprised the selection of stress insensitivity. Further support for the selection of insensitive phenotypes in the long-term polluted mining area and its surrounding was given by the laboratory and, particularly, the microcosm experiments. In the laboratory, isopods of different origin displayed a divergent behaviour and, concomitantly, different biochemical responses to stress situations posed by metal contamination. Although the differences in food uptake and stress response may result from pre-exposure and may represent after-effects of different acclimation, the long-term microcosm experiment clearly showed that any acclimation of control isopods to the conditions at the mine site was impossible within 3 months of exposure. This finding suggests the involvement of a genetic component to tolerance at least for the tested Oniscus populations. The observed trend towards a decrease in hsp70 variability (relative standard deviations) also supports the notion of the evolution of tolerance ( Posthuma & van Straalen, 1993).
Metal insensitivity was associated with a low stress protein level. However, a low hsp70 level in metal-exposed animals does not necessarily indicate stress insensitivity. Although heavy metals were shown usually to induce stress proteins in isopods ( Eckwert et al., 1997 ) and diplopods ( Zanger et al., 1996 ), the hsp70 level follows a curve with a maximum at rather high but not extreme concentrations. In response to very high metal concentrations, the stress protein level decreases ( Guven et al., 1994 ; Eckwert et al., 1997 ; Pyza et al., 1997 ) due to severe pathological impact upon target organs, such as the hepatopancreas of isopods ( Köhler et al., 1996 ). It is doubtful that a population of pathologically injured animals can maintain itself under field conditions. Indeed, Gräff (1997) and Schill (1999) did not find pathologically damaged organs in free-living animals, even if they derived from heavily metal-polluted stands. That low hsp70 levels of the populations at the long-term contaminated sites arise through histopathological disintegration of tissues can hence be refuted.
Parameters of importance
Interestingly, the main factors besides pollution history explaining both high hsp70 levels in nontolerant populations and low but increasing hsp70 levels with increasing metal pollution in stress-insensitive populations differed between the investigated species. For O. asellus and J. scandinavius, cadmium and lead were of greater importance, while zinc was associated with P. scaber response to metal pollution. Zinc appears responsible for the absence of P. scaber in a number of sites in the surrounding of a metal smelting works close to Avonmouth, south-west England ( Hopkin, 1990). Hopkin & Hames (1994) argued that zinc amongst a metal ‘cocktail’ will be the limiting factor for P. scaber, if the zinc to cadmium ratio is >10:1 and the zinc to lead ratio is >20:1. The latter conjecture was not confirmed by our investigations since in none of our sites was the zinc concentration 20 or more times higher than the lead concentration. Nevertheless, zinc was found to be significantly associated with stress insensitivity. Therefore, we conclude that under natural conditions, regardless of the actual total concentration of heavy metals in soil, zinc will either limit the survival of nontolerant populations of P. scaber (as shown by Donker, 1992; Jones & Hopkin, 1998) and/or will act as a main component of selection for metal stress tolerance.
The reason for the species-specificity of selection pressure components may lie in the kinetics of uptake, accumulation and loss of metals, and in the specific cellular, subcellular and physiological responses to these metals which most likely form the material upon which selection acts. The amount of accumulated zinc, lead and cadmium during artificial exposure is higher in O. asellus than in P. scaber ( Hopkin, 1989; Hopkin, 1990; Drobne & Hopkin, 1995). Though apparently contradictory to the view that zinc has a profound effect on P. scaber, Hopkin’s (1989) experiments were conducted with nontolerant specimens. Secondly, zinc was retained by contaminated P. scaber fed on uncontaminated leaf litter while it was rapidly lost from the hepatopancreas of O. asellus ( Hopkin, 1990). It therefore seems crucial for the survival of O. asellus in polluted stands that it selects food carefully from less contaminated patches and, consequently, allows zinc to be excreted from the midgut gland. This is all the more true since Hopkin (1990) estimated the ‘critical’ concentrations of zinc in the midgut gland (the maximum storage capacity) beyond which survival is impossible, to about 15 000 μg per g dry weight in O. asellus but to about 25 000 μg per g in P. scaber. The observations on feeding selectivity from the present study concur with this suggestion and indicate, furthermore, that this selectivity is not a general behavioural criterion of O. asellus, but seems to be selected for only in few populations. Our observations of selective feeding by O. asellus may mean that the external zinc concentrations are less important for its stress status than other metals.
In contrast to P. scaber, cadmium and, to a lesser degree, lead seems to be the limiting factor for the diplopod, J. scandinavius. In an ultrastructural study on cytopathological effects of metals on the main target organs of a variety of soil animals, the midgut epithelium of J. scandinavius was particularly sensitive to cadmium and displayed cytopathological alterations at cadmium concentrations which were about one order of magnitude lower than those which induced comparable effects in the midgut gland of P. scaber. For other metals, differences between species were not detected ( Köhler & Triebskorn, 1998). This particular sensitivity to cadmium is probably mirrored by the present finding that cadmium, in combination with lead and long-term pollution, seems to have acted as the main component of selection for tolerance in J. scandinavius.
Survival strategies and trade-offs
Since either excessive accumulation of metals or their pathological impact limits survival of isopods and diplopods under chronic exposure, selection should favour those response mechanisms and life-cycle traits which led to the avoidance of the stressor or its impact with little cost in terms of survival and reproduction. Avoidance of metal uptake can never be complete and thus both isopods and diplopods exhibit increasing metal concentrations with increasing age due to storage-excretion ( Hopkin, 1989). Also, altered behaviour may limit the ingestion of excessively polluted food and, consequently, the damage exerted by it. It is most likely that adaptation of the O. asellus populations from the polluted sites around the Wiesloch mine (site 14) is based on selective feeding. This, rather than physiological acclimation, enables O. asellus to survive under high metal ion concentrations, since a control population from an unpolluted site could not acclimatize to the same conditions within 3 months. Reduction of food uptake, however, is also a general response of nontolerant isopods to extreme metal concentrations (e.g. Joosse et al., 1981 ; Donker, 1992; Drobne & Hopkin, 1995) and does not necessarily have a genetic basis. Yet tolerance of long-term exposed soil invertebrate populations may at least partly be based on the evolution of behavioural patterns that reduce contact with or consumption of contaminated material.
If stress insensitivity in our study populations involved selection of phenotypes which are able to choose their food more carefully than others and restrict their metal absorption and proteotoxicity, this would explain the low hsp70 levels recorded in populations exposed to long-term pollution. Is then a low hsp70 level only a by-product of altered behaviour or might it be subject itself to evolution? At least for O. asellus the latter possibility cannot be excluded. After ingestion of contaminated food, it is essential for O. asellus to feed selectively on less contaminated litter to excrete zinc from its midgut gland ( Hopkin, 1990). Accumulation is quicker and the critical zinc level lower in O. asellus than in P. scaber. One therefore should expect zinc to be the most important ion involved in the selection of behavioural adapted phenotypes of O. asellus. If the stress protein level was just a side-effect of altered behaviour, zinc must also be assumed as being most relevant for the hsp70 level. However, in our study, zinc has been shown to be of only marginal importance to explain selection of insensitive populations in O. asellus and, thus, the hsp70 level itself (or any other factor determining it) must be regarded as being the subject to evolution in the investigated populations.
Even though a transiently elevated hsp70 level undoubtedly helps organisms to restrict the effects of a stress factor, our insensitive populations have been seemingly selected for a low stress protein level. If the stress protein level is subject to evolution, and a low hsp70 level is favoured instead of a constitutively elevated level, the latter likely trades off against other fitness consequences across the entire life cycle. The constitutive formation of stress proteins at a high rate would be an energy-intense strategy to cure the symptoms of proteotoxicity and, surely, would alter processes of energy allocation. In this context, it has been shown that the higher stress tolerance of individuals with an elevated hsp70 level or extra copies of hsp70 genes in the genome ( Welte et al., 1993 ; Feder et al., 1996 ; Krebs et al., 1998 ) was associated with reduced juvenile-to-adult survival and lengthened development in Drosophila melanogaster ( Krebs & Feder, 1997a, b; Krebset al., 1998 ).
The evolution of greater protein integrity in long-term stressed populations to mitigate the cost of hsp70 chaperoning, however, may result in other costs, such as, for example, increased energy expenditure due to selective feeding or increased detoxification mechanisms, reduced heterozygosity, reduced food uptake or nutrient absorption. Interestingly, Donker et al. (1993b ) found that P. scaber from around a smelter at Budel, The Netherlands, showed increased mortality and slower growth rates than control populations, but persist through earlier reproduction and increased reproductive allocation, suggestive of trade-offs ( Southwood, 1988; Sibly & Calow, 1989). Further studies indicate that P. scaber from the Budel population had a greater capacity to detoxify zinc ( Donker et al., 1996 ) and cadmium ( Donker & Bogert, 1991) relative to those from other sites. The energy costs of detoxifying high concentrations of metals in the diet most likely were responsible for the smaller average size of P. scaber in the contaminated areas ( Donker, 1992; Donker et al., 1993a ). Both P. scaber and O. asellus from a metal gradient at Avonmouth, UK, were also smaller than typical individuals from noncontaminated sites ( Jones & Hopkin, 1998), but there was no change in their reproductive allocation ( Jones & Hopkin, 1996).
Although the molecular mechanisms of metal detoxification which may have been induced in insensitive populations of isopods and diplopods remain unclear, they may represent variables that selection has also acted upon in the polluted southern German sites. One may argue that an induction of metal-scavenging proteins could be responsible for tolerance but, as yet, such proteins are unknown for both isopods and diplopods.