Heinz-R. Köhler

Trehalose and anhydrobiosis in tardigrades – evidence for divergence in responses to dehydration

To withstand desiccation, many invertebrates such as rotifers, nematodes and tardigrades enter a state known as anhydrobiosis, which is thought to require accumulation of compatible osmolytes, such as the non‐reducing disaccharide trehalose to protect against dehydration damage. The trehalose levels of eight tardigrade species comprising Heterotardigrada and Eutardigrada were observed in five different states of hydration and dehydration. Although many species accumulate trehalose during dehydration, the data revealed significant differences between the species. Although trehalose accumulation was found in species of the order Parachela (Eutardigrada), it was not possible to detect any trehalose in the species Milnesium tardigradum and no change in the trehalose level has been observed in any species of Heterotardigrada so far investigated. These results expand our current understanding of anhydrobiosis in tardigrades and, for the first time, demonstrate the accumulation of trehalose in developing tardigrade embryos, which have been shown to have a high level of desiccation tolerance.

Drought acclimation confers cold tolerance in the soil collembolan Folsomia candida.

It has been noted that both summer drought and sub-zero winter temperatures induce the synthesis of sugars and polyols in invertebrate tissues. This has led several authors to suggest that many of the adaptations, previously viewed as a response to cold, might be part of a more universal desiccation tolerance mechanism. Here we show that acclimation of the soil dwelling collembolan Folsomia candida to a sublethal desiccation stress confers tolerance to cold shock and a significant increase in the molar percent of membrane fatty acids with a mid-chain double bond. These changes in membrane fatty acids are interpreted as conferring a significant reduction in the transition temperature of cell membranes, as would be expected in acclimation to cold, and these changes are therefore interpreted as contributing to the cross-tolerance. Drought acclimation was also shown to trigger the synthesis of the 70kDa family of heat-shock proteins (Hsp70). This group of heat shock proteins is implicated in the reestablishment of the normal three-dimensional structure of partially unfolded proteins and therefore are also likely to contribute to the observed cross-tolerance. This study provides evidence that the stresses exerted by desiccation and cold at the cellular level have sufficient similarities to induce overlapping adaptations.

Induction of heat shock proteins, changes in liver ultrastructure, and alterations of fish behavior: are these biomarkers related and are they useful to reflect the state of pollution in the field?

Heat shock protein (hsp70) expression, changes inliver ultrastructure, and alterations of swimmingbehavior were simultaneously investigated in browntrout (Salmo trutta f. fario) exposed towater from test streams in southern Germany undersemi-field conditions during 1995 and 1996. Chemicalanalyses revealed different levels of contamination ofthe two streams by pesticides, PAHs, PCBs, and heavymetals, whereas stream pollution was only partlyreflected by body burdens of contaminants in fish.Biomarker responses were compared after differenttimes of exposure and with respect to differenttemperature conditions. Differences in pollutionstates of the streams were reflected by differentbiomarker responses of the trout. In the highlycontaminated stream, the biomarker responses were morecoincident with one another than in the moderatelypolluted stream in which the biomarkers showed littlerelation to one another. This is primarily due to thedifferential capacities of the biomarkers to integratestress responses over time. Understanding the dynamicsof the stress response curves and the effects oftemperature on biomarker responses is essential forevaluating the effects of environmental stressors suchas contaminants on the health of organisms.

The induction of stress proteins (hsp) in Oniscus asellus (Isopoda) as a molecular marker of multiple heavy metal exposure: I. Principles and toxicological assessment

The induction of stress-70 (hsp70) and stress-60 (hsp60, chaperonin 60) in the terrestrial woodlouse, Oniscus asellus, after exposure to various heavy metals and heavy metal combinations was investigated by means of standard Western-blot using monoclonal antibodies. The expression of stress-70 was found to react much more sensitively to metal contamination than that of stress-60. The general course of the stress-70 response to increasing metal concentration was found to be similar for all treatments. Based on the obtained results, this course could be divided into three zones: in the first zone, the expression of stress-70 proteins was only marginally increased by comparatively low metal concentrations; in the second zone, strong induction of stress-70 occurred and the ability of the heat shock system to react to increased metal concentrations reached its climax; and in the third, non-compensation zone, a further increase in heavy metal concentration resulted in a decline of stress-70 levels, which can be interpreted as a result of pathological tissue damage. Combinations of metals were found to increase the stress- 70 level more intensely than related concentrations of singularly applied metals. Based on these studies, a ranking scheme of increasing toxicological impact of a variety of heavy metal combinations was composed

Stress gene (hsp70) sequences and quantitative expression in Milnesium tardigradum (Tardigrada) during active and cryptobiotic stages

SUMMARY The eutardigrade Milnesium tardigradum can undergo cryptobiosis, i.e. entry into a reversible ametabolic stage induced by dehydration, cooling and, probably, osmotic and anoxic stress. For the first time in tardigrades, we described partial sequences of three heat-shock protein (hsp70 family) genes and examined gene expression on the way from an active to a cryptobiotic and back to an active stage again. Results showed different patterns of gene expression in the hsp70 isoforms. All three isoforms seem to be true heat-shock proteins since transcription could be clearly enhanced by temperature elevation. Isoform 1 and, at a lower level, isoform 3 do not seem to have a specific function for cryptobiosis. By contrast, transcription of isoform 2 is significantly induced in the transitional stage between the active and the cryptobiotic stage, resulting in a comparatively high mRNA copy number also during cryptobiosis. This pattern of induction implies that isoform 2 is the most relevant hsp70 gene for M. tardigradum individuals entering the cryptobiotic stage.

Behavioural and developmental toxicity of chlorpyrifos and nickel chloride to zebrafish (Danio rerio) embryos and larvae.

In order to assess the combined toxicity of environmental chemicals with different modes of action in acute (2 h) and subchronic (11 d) exposures, embryos and larvae of Danio rerio were exposed to a heavy metal salt, nickel chloride (NiCl2), the insecticide chlorpyrifos (CHP) and their binary mixtures. Chlorpyrifos is an acetylcholine esterase inhibitor, which is likely to affect behaviour of the organism. NiCl2 targets the active sites of enzymes and is regarded as an unspecific toxicant for aquatic organisms. Several endpoints, such as locomotor activity, morphological abnormalities, and mortality of D. rerio embryos and larvae were studied. During acute exposures to > or =0.25 mg/L of chlorpyrifos, locomotor activity tended to increase. However, this activity decreased significantly at > or =7.5 mg Ni/L. Subchronic exposures to CHP resulted in behavioural changes at much lower concentrations (> or =0.01 mg/L) and considerably earlier than the observed increase in morphological abnormalities and mortality (LC(50) (10 d): 0.43 mg/L). Combined CHP and NiCl2 mixtures led to an antagonistic deviation from the concept of independent action, in the case of locomotor activity. Compared to developmental or survival parameters, behaviour was the most sensitive endpoint for CHP exposure in this study; therefore we recommend this parameter to complement already established endpoints.

Acute effects of diclofenac and DMSO to Daphnia magna: Immobilisation and hsp70-induction

To determine the toxicity of the anti-rheumatic drug diclofenac to Daphnia magna, acute toxicity tests according to the OECD guideline 202 were combined with biochemical investigations of the hsp70 level as a biomarker for proteotoxicity. Particular attention was paid to the impact of the solvent DMSO as a confounding factor to diclofenac toxicity by means of testing different variations of producing stock solutions. In the acute immobilisation tests, diclofenac was most toxic as a singular test substance, with indication of a slight antagonistic interaction between the two substances. The highest EC50 values were obtained in those approaches using diclofenac pre-dissolved in DMSO. Thus, the observed antagonism seems to be intensified by pre-dissolution. Hsp70 levels of 12- to 19-days-old D. magna were determined after 48h exposure using a highly reproducible immunological protocol. Hsp70 induction occurred at a LOEC of 30mgl(-1) diclofenac plus 0.6mll(-1) DMSO, and at a LOEC of 40mgl(-1) for diclofenac alone. In summary, DMSO showed only slight confounding effects on diclofenac action in the applied range of concentrations.

Temperature-dependent effects of the pesticides thiacloprid and diazinon on the embryonic development of zebrafish (Danio rerio)

The present study examined combined effects of the insecticides thiacloprid and diazinon and a physical stressor, elevated temperature (28, 30 and 33.5 degrees C, control at 26 degrees C), on embryonic development of zebrafish (Danio rerio). Early life stages were exposed to five different concentrations (thiacloprid: 1, 5, 10, 15 and 20mg/L; diazinon: 100, 500, 1000, 2000 and 3000 microg/L) for 72 or 96 h, respectively, depending on the temperature. Thiacloprid was shown to have no effects on early life stage parameters, except on the heart rate. This effect was probably due to an increased metabolism upon exposure to the pollutant. Exposure to diazinon resulted in mortality which strongly increased with elevated temperature. Within the tested temperature range, increased mortality occurred particularly at concentrations of 2000 and 3000 microg/L diazinon. Observed sublethal effects were a decrease of the heart rate with increasing substance concentration, yolk sac edema, heart sac edema and spine deformations, particularly at concentrations of 2000 and 3000 microg/L diazinon. The hatching date was shown to be preponed with increasing temperature, most strikingly in combination with 2000 or 3000 microg/L diazinon. Mixtures of temperature and substance concentration as independent variables were shown to act synergistically in a dose-level-dependent manner on hatching rate, whereas the magnitude of synergism depended on effect levels.

The Impact of Environmental Chemicals on Wildlife Vertebrates

A plethora of papers have been published that address the affects of chemicals on wildlife vertebrates. Collectively, they support a connection between environmental pollution and effects on wildlife vertebrate populations; however, causal relationships between exposure, and reproduction or population structure effects have been established for only a few species. In a vast number of fish species, particularly in teleosts, it is accepted that EDCs affect the endocrine system of individuals and may alter sexual development and fertility. However, only few studies have demonstrated population-level consequences as a result of exposure to EDCs. The same applies to fish populations exposed to contaminants or contaminant mixtures with non-endocrine modes of action; few studies link EDCs directly to population affects. Amphibian populations are declining in many parts of the world. Although environmental chemicals have been shown to affect reproduction and development in single organism tests, the degree to which chemicals contribute to the decline of amphibians, either alone, or in concert with other factors (habitat loss, climate change, introduction of neozoa, UV-B irradiation, and direct exploitation) is still uncertain. Because reptilian endocrinology is so variable among species, EDC effects reported for individual species cannot easily be extrapolated to others. Nevertheless, for some species and locations (e.g., the Lake Popka alligators), there is considerable evidence that population declines are caused or triggered by chemical pollution. In birds, there is ample evidence for EDC effects on the reproductive system. In some bird species, effects can be linked to population declines (e.g., based on egg-shell thinning induced by DDT/DDE). In contrast, other bird species were shown to be rather insensitive to endocrine disruption. Oil spills, which also may exert endocrine effects, are usually regarded to cause only transient bird population effects, although long-term data are largely missing. Mammal population declines have been correlated with organochlorine pollution. Moreover, numerous studies have attributed reproductive and non-reproductive dysfunctions in mammals to EDC exposure. However, in the majority of cases, it is uncertain if effects at the population level can be attributed to chemical-induced reproductive effects. Evidence shows that selected species from all vertebrate classes were negatively affected by certain anthropogenic chemicals. Affects on some species are well characterized at the organismal level. However, the proof of a direct link between chemical exposure and population decline was not given for the vast majority of studied species. This review clearly shows the gaps in knowledge that must be filled for the topic area addressed. We, herewith, make a plea for long-term studies to monitor effects of various environmental chemicals on wildlife vertebrate populations. Such studies may be augmented or combined with mechanistically-oriented histological, cytological and biochemical parallel investigations, to fill knowledge gaps.

B-type esterases in the snail Xeropicta derbentina : An enzymological analysis to evaluate their use as biomarkers of pesticide exposure

The study was prompted to characterize the B-type esterase activities in the terrestrial snail Xeropicta derbentina and to evaluate its sensitivity to organophosphorus and carbamate pesticides. Specific cholinesterase and carboxylesterase activities were mainly obtained with acetylthiocholine (K(m)=77.2 mM; V(max)=38.2 mU/mg protein) and 1-naphthyl acetate (K(m)=222 mM, V(max)=1095 mU/mg protein) substrates, respectively. Acetylcholinesterase activity was concentration-dependently inhibited by chlorpyrifos-oxon, dichlorvos, carbaryl and carbofuran (IC50=1.35x10(-5)-3.80x10(-8) M). The organophosphate-inhibited acetylcholinesterase activity was reactivated in the presence of pyridine-2-aldoxime methochloride. Carboxylesterase activity was inhibited by organophosphorus insecticides (IC50=1.20x10(-5)-2.98x10(-8) M) but not by carbamates. B-esterase-specific differences in the inhibition by organophosphates and carbamates are discussed with respect to the buffering capacity of the carboxylesterase to reduce pesticide toxicity. These results suggest that B-type esterases in X. derbentina are suitable biomarkers of pesticide exposure and that this snail could be used as sentinel species in field monitoring of Mediterranean climate regions.