Cadmium tolerance in a soil arthropod a model of real-time microevolution Insects are suitable models for the study of micro- Nico M. van Straalen & Dick Roelofs evolution in real time. Springtails (Collembola) Institute of Ecological Science are common inhabitants of organic soils. When Vrije Universiteit the soil is polluted by heavy metals, some spring- De Boelelaan 1085 tails are still able to survive due to genetic adap- 1081 HV Amsterdam The Netherlands tation. We have shown that the production of a [email protected] metal-binding protein, metallothionein, is in- creased in strains of Orchesella cincta cultured from sites with heavy metal contamination. A higher production of metal-binding protein al- lows more metal to be retained in the gut cells and consequently more metal to be excreted when the gut epithelium is regenerated at moult. DNA analysis has shown that the promoter of the metallothionein gene shows a considerable degree of polymorphism in springtail popula- tions while some promoter alleles confer in- creased expression of the gene. In populations tion in the oxygen transporting protein haemocyanin, but in exposed to high metal concentrations the fre- general our knowledge of insect mineral nutrition is scanty quency of these alleles is increased compared to compared to that of vertebrates and plants. reference populations, which is most likely due Notwithstanding the long history of interactions between organisms and heavy metals, adaptation processes can still to a selective advantage of high expresser phe- be observed in present ecosystems. This is due to the fact notypes in polluted ecosystems. The data illus- that in a relatively short timespan, human activities have led trate a new mechanism of microevolution, se- to a large-scale redistribution of heavy metals over the pla- net. This redistribution basically takes the form of depletion lection on transcriptional regulation, rather than in a few places (ore deposits) and enrichments in many ot- on gene structure. her places (around mines and smelters, in industrial areas and around urban settlements). In this way, ecosystems are Entomologische Berichten 65(4): 105-111 loaded with metal levels that were not there before. The question addressed in this paper is whether exposures asso- ciated with these increased metal loadings of the environ- Keywords: cadmium, Collembola, evolution, heavy metals, ment lead to new adaptations and what would be the me- metallothionein, Orchesella cincta, soil pollution chanisms by which animals can develop such adaptations. Adaptation to heavy metals can be considered an examp- Introduction le of ‘evolution through pollution’. In fact, one of the finest examples of evolution in action is provided by the adapta- Like all animals, insects require heavy metals in their meta- tion of plants to metal-containing soils in areas that are bolism, but suffer from metal toxicity when they are exposed naturally enriched with heavy metals. These examples have to levels above their capacity for detoxification (box 1). Since found their way to textbooks and are cited as evidence that life began, organisms have used heavy metals to perform evolution is still going on around us (Futuyma 1998). The ve- certain functions in their metabolism. Some authors even getation of metal-enriched soils includes metal-tolerant argue that heavy metals played a crucial role in the origin of varieties of species that are often rare in other places and life itself (Russell & Hall 1997). Anyway, life has been very se- species that are only known from metal-enriched sites. lective in the choice of metals for biological functions, be- In insects, the issue of ‘evolution through pollution’ is cause there is no correlation whatsoever between the abun- mostly known from pesticide resistance (Oppenoorth 1987). dance of metals in the earth’s crust and their abundance in Metal tolerance is not often studied, partly because many in- biological tissues (Ernst & Joosse 1983). Also insects have sects are not exposed to high concentrations of heavy me- adopted heavy metals as essential elements in their metabo- tals, at least not during their adult life. The story is different lism. The role of copper is well-known because of its posi- for insects that live in close contact with soils or sediments Entomologische Berichten 65(4) 2005 105 and their associated dead organic matter, because these en- Evidence for metal tolerance is suggested for at least vironmental compartments are sinks for many pollutants eighteen species, most of them invertebrates, but including and heavy metals tend to accumulate there. Consequently, two fishes (table 1). Only in a few cases it was actually pro- tolerance development to heavy metals in insects is best do- ven that the adaptation was due to genetic change. The re- cumented in soil- and sediment-living species. In this paper view (table 1) reinforces the previous conclusion that many we document recent progress in the study of a soil-living animals can develop tolerance to metals, that invertebrates collembolan and its adaptation to cadmium. seem to be more prone to develop tolerance than vertebra- tes, and that some animals cannot develop tolerance at all. Adaptation to metals The strongest evidence for tolerance comes from studies at grossly polluted sites where selection is expected to be parti- Since the 1970s, many authors have reported cases of adap- cularly strong, e.g. ore mines and point sources of industrial tation to metal pollution in field populations of animals emission. It is interesting to note that no cases of tolerance (Joosse & Buker 1979, Van Straalen et al. 1987, Hopkin 1989, (at least not in animals) are reported from some common Posthuma et al. 1992, Baird & Barata 1999, Stürzenbaum et cases of environmental pollution, e.g. lead in roadside ver- al. 2001). The term ‘adaptation’ is used in variable senses ges and zinc below overhead electric power cables. Also, (see box 2). The aquatic studies published in the 1970s and there is no evidence for copper tolerance in earthworms fr- most of the 1980s were summarized by Klerks & Weis (1987). om vineyeard soils. Their review showed that metal tolerance had been reported Whereas table 1 illustrates cases of metal tolerance in more often in algae and invertebrates than in vertebrates field populations, the phenomenon of tolerance is also known (fish and amphibians). The authors also stated that if the pu- from laboratory test species. Extensive work has been done blished literature accurately represents the situation in pol- on the variation between clones in parthenogenetic or g a- luted areas, it must be concluded that most, but not all, pop- nisms, such as Daphnia magna Straus for the aquatic environ - ulations can develop tolerance. However, they warned ment and Folsomia candida (Willem) for soil. A study by against relaxing water quality criteria on this basis, because Baird & Barata (1999) has shown that substantial variability many populations failed to survive in polluted environments. in the susceptibility to cadmium is present between clones How the tolerances were acquired, by physiological or gene- of Daphnia. This has important implications for the use of tic adaptation, was unclear in most of the studies. Posthuma these cladocerans as standard test animals in ecotoxicology, & Van Straalen (1993) conducted a review with a scope simi- because the same level of water pollution may be evaluated lar to Klerks & Weis, but aimed to cover the terrestrial en- differently depending on the clone used. The issue of inter- vironment. These authors concluded that there was good clonal differences was also investigated by Crommentuijn et evidence for metal tolerance in seven species of terrestrial al. (1995) in four strains of the parthenogenetic collembolan invertebrate. F. candida, exposed to three toxicants, including cadmium. Differences between clones amounted to a factor 4. The evidence reviewed here (see table 1) suggests that Box 1. What is a heavy metal? adaptation to metals accompanied by genetic change is not an All elements which have a density greater than 5 g/cm3 fall in the uncommon phenomenon in the wild. The following sections category ‘heavy metals’. Heavy metals are a normal part of na- deals with one of the mechanisms underlying adaptation, in- ture: they cycle through all compartments including living bio- duction of the metal-binding protein, metallothionein. mass, although their concentrations are usually very low (hence ‘trace metals’). Some metals perform specific functions in the Metallothionein metabolism, for example: copper is an essential constituent of the oxygen-transporting Since its discovery in 1957, metallothionein (MT) has been haemolymph protein of arthropods and molluscs, haemocya- subject to extensive research in a wide variety of organisms. nin, The protein was called metallothionein because of its very zinc is a crucial element of enzymes such as carbonic anhydrase high metal and sulphur content. The high sulphur content is and many DNA-binding peptides which regulate the expression due to an exceptional number of cysteine residues which re- of genes (‘zinc fungers’), sults in a high affinity for heavy metals such as cadmium iron is an essential part of cytochromes such as cytochrome and copper. The cysteines are usually arranged in a Cys- P450, a family of enzymes associated with the metabolism of XaaCys (Xaa being any amino acid) configuration. steroid hormones and biotransformation of plant secondary Metallothio- nein lacks aromatic amino acids. Notwithstan- compounds. ding these properties shared by all metallothioneins, the size Some metals such as lead and cadmium have no known bio- of protein and amino acid composition can vary enormously logical function, but it cannot be excluded that they may have a between MTs of different organisms or even between iso- function in very low concentrations. For example, recently a bio- forms with-in an organism. logical role was suggested for cadmium in a specific form of car- Studies on vertebrates have shown that each molecule of bonic anhydrase in a marine diatom.