M.H. Donker

Early reproduction and increased reproductive allocation in metal-adapted populations of the terrestrial isopod Porcellio scaber

Organisms inhabiting metal-contaminated areas can be stressed by metal exposure and are possibly subject to selection, resulting in increased metal tolerance and changes in growth and/or reproduction characteristics. In a previous study it was found that in the terrestrial isopod Porcellio scaber, sampled from the vicinity of a zine smelter, the body size was small and the brood size was large compared to isopods from a reference area. To assess whether these differences were due to genetic differentiation between strains, isopods collected from a reference wood, a zinc smelter area and a lead mine were cultured on non-polluted food, while growth, reproduction and metal concentrations were studied in first and second laboratory generations. The isopods from the three populations differed in age and weight at first reproduction, although there were hardly any differences in growth. The females of the mine and the smelter population started to reproduce earlier, at a lower weight, which resulted in fewer young per female. However, reproductive allocation (=wight of young relative to the weight of the mother) was higher in mine and smelter isopods. We conclude that the isopods at the metal-contaminated sites have been selected for early reproduction and increased reproductive allocation. The results indicate that populations inhabiting metal-polluted sites have probably undergone evolutionary changes. This study showed that growth and reproduction characteristics of different populations under laboratory conditions may provide information on selection processes in the field.

Metallothionein-bound cadmium in the gut of the insect Orchesella cincta (Collembola) in relation to dietary cadmium exposure

Metallothionein is considered to be a potential biomarker for heavy metal exposure in the terrestrial environment. However, limited information is available on metallothioneins from insects, a major class of terrestrial invertebrates. In this study we have quantified metallothioneins in the springtail Orchesella cincta by determining metallothionein-bound cadmium after separation of these proteins using gel filtration and reversed phase chromatography from total body homogenates of animals dietary exposed to different concentrations of cadmium. Furthermore, we have studied in more detail where cadmium and metallothionein-bound cadmium is located within this animal. The concentration of metallothionein-bound cadmium increases with the exposure concentration in the same way as the total internal concentration. Both reach a plateau at an exposure concentration of approximately 1.0 micromol Cd/dry food. Cadmium is primarily located within the gut of O. cincta and isolation of metallothionein from this organ gives results identical to isolations from total bodies. Based on this results an estimation of the metallothionein level at the highest exposure concentration results in a concentration of about 115 microg metallothionein/g fresh gut. The O. cincta metallothionein gives the possibility of using this protein as a biomarker for heavy metal exposure in soil insects.

The Role of Zinc Regulation in the Zinc Tolerance Mechanism of the Terrestrial Isopod Porcellio scaber

1. To elucidate mechanisms underlying adaptation to metals in the terrestrial isopod Porcellio scaber (Isopoda) responses to zinc were compared in field animals and laboratory-reared generations of three populations, one from a contaminated smelter site and the others from reference sites. 2. Isopods were exposed to a range of zinc concentrations in the food and their consumption, food and zinc assimilation, zinc concentration and internal zinc distribution were determined. 3. Isopods refused to eat food contaminated with 54 μmol Zn g - 1 4. The zinc concentration of field isopods from the smelter site was three times higher than from the reference isopods. The mean fraction of zinc assimilated, determined as the difference between consumption and defecation (AE Zn ), was 25.3% for the reference isopods and 20.1% in the smelter isopods. When zinc assimilation was determined as the increase of the zinc body burden, the reference field isopods accumulated 3% of the zinc consumed: the increase in the smelter isopods was not significant. Isopods from both populations translocated zinc into the hepatopancreas. 5. In the laboratory-reared generations, food assimilation was reduced by zinc, this effect was more pronounced in the smelter isopods. In the smelter isopods, reduced food assimilation did not, however, result in less growth in comparison to the reference isopods. The AE Zn did not differ significantly between the populations, but the increase in zinc body burden was again lower in the smelter isopods. 6. The higher growth efficiency of smelter isopods and their lower increase in zinc body burden may be regarded as an adaptation to zinc and may be one of the mechanisms contributing to survival of this population at a site severely polluted with zinc.

Metal binding compounds in hepatopancreas and haemolymph of porcellio scaber (isopoda) from contaminated and reference areas

Abstract 1. In order to evaluate the role of metal-binding proteins in the tolerance mechanism of Porcellio scaber to heavy metals, a comparative study was made using isopods from three locations: a zinc-lead mine (Plombieres), a zinc smelter (Budel) and a reference wood (Spanderswoud). The Cu, Zn and Cd concentrations and the protein composition were determined in the haemolymph and hepatopancreas from the isopods. 2. A constant Cu/Zn molar ratio of about 5 was found in the haemolymph of all populations and no correlation was found between hepatopancreas and haemolymph Cu and Zn content. 3. Using fast-performance liquid chromatography (FPLC), most of the haemolymph Cu and Zn appeared to be associated with a single UV absorbing peak corresponding with an apparent molecular weight of ± 70 kD; this peak is probably the monomer of hemocyanin. 4. The hepatopancreas Zn and Cd concentration were elevated compared to the hepatopancreas of the smelter and mine isopods; after homogenization and centrifugation 70–80% of the metals were found in the supernatant. 5. In all populations the hepatopancreas Cu-, Zn- and Cd-binding compounds eluted in separate peaks of low molecular weight, suggesting the absence of an MT-like compound in Porcellio scaber. 6. The similarity of the protein profiles in haemolymph, and the similar distribution of the metals over the fractions in haemolymph and hepatopancreas suggests that inducible metal binding compounds are not involved in metal tolerance differences between populations.

Two metal binding peptides from the insect Orchesella cincta (Collembola) as a result of metallothionein cleavage.

Metallothionein (MT) is an ubiquitous heavy metal-binding protein which has been identified in animals, plants, protists, fungi and bacteria. In insects, primary structures of MTs are known only for Drosophila and the collembolan, Orchesella cincta. The MT cDNA from O. cincta encodes a 77 amino acid protein with 19 cysteines. Isolations of the protein itself have demonstrated the presence of two smaller metal-binding peptides, whose amino acid sequences correspond to parts of the cDNA, and which apparently result from cleavage of the native protein. The present study was undertaken to complete the picture of cleavage sites within the MT protein by applying protein isolation techniques in combination with mass spectrometry and N-terminal sequence analysis. Further, recombinant expression allowed us to study the intrinsic stability of the MT and to perform in vitro cleavage studies. The results show that the MT from O. cincta is specifically cleaved at two sites, both after the amino acid sequence Thr-Gln (TQ). One of these sites is located in the N-terminal region and the other in the linker region between two putative metal-binding clusters. When expressed in Escherichia coli, the recombinant O. cincta MT can be isolated in an uncleaved form; however, this protein can be cleaved in vitro by the proteolytic activity of O. cincta. In combination with other studies, the results suggest that the length of the linker region is important for the stability of MT as a two domain metal-binding protein.

From theory to practice: intervention fidelity in a randomized controlled trial aiming to optimize weight development during pregnancy.

Background. Intervention fidelity is an increasingly important methodological concept in process evaluations. In this article, the authors investigated the intervention fidelity in a randomized controlled trial on excessive weight gain prevention in pregnancy. Method. A sample of 109 audiotaped counseling sessions, linked to 65 women in the intervention group of the New Life(style) trial, was drawn. The following criteria were quantitatively evaluated using a fidelity checklist: (a) reach, (b) dose, (c) adherence to study objectives, (d) adherence to underlying problem-solving treatment (PST) theory, and (e) counselor competence. Results. A total of 60.4% received all counseling sessions. The dose of intervention components was generally moderate (50.9% to 60.4%), and the dose of PST components was low (17.3%). Adherence to study objectives was moderate (64.2%) and adherence to PST theory was low (43.2%). The counselors sufficiently stimulated the participant to optimize lifestyle (54.2% of the sessions), provided positive feedback (50.5%), and left the initiative regarding problem solving to the participant (71%). One of the two counselors performed significantly better on all measured criteria (p < .001). Conclusions. Intervention fidelity in the New Life(style) trial was generally low to moderate. In future interventions, it is recommended to put more emphasis on counselor recruitment, training, and intervention protocol contents. Fellow researchers are encouraged to embed a process evaluation into all study stages, taking into account all essential process elements, and to link process outcomes to more distal, health outcomes.