Input of organochlorine compounds by snow to European high mountain lakes

1.  Persistent organic pollutants (POPs) can be trapped by and accumulate in cold regions. To understand POP accumulation in temperate high mountain lakes, we collected samples of snow from the catchments of several high mountain lakes in Europe.
2.  Organochlorine compounds (OCs) are regularly found in snow collected in European high mountain sites. Polychlorobiphenyls (PCBs) were found in all samples examined. Hexachlorocyclohexanes (HCHs) are also common whereas DDTs and hexachlorobenzene were found less frequently.
3.  Comparison of the concentrations of these pollutants in snow with OC levels in the waters of these lakes or atmospheric deposition during cold periods shows that the snowpack constitutes a significant seasonal PCB reservoir in all catchments and that snow trapping is a major mechanism for the incorporation of HCHs in the lakes situated in the coldest sites, e.g. Gossenköllesee in the Alps.
4.  Correlation of the concentrations of the PCB congeners in snow versus mean annual winter temperature shows higher accumulation at lower temperatures. The less volatile PCBs exhibit higher temperature dependences than the more volatile congeners. This trend differs from altitudinal dependences observed in other sites such as Canada and may be related to the specific range of winter temperature in each area of study.     

Mitochondrial DNA polymorphism among alpine populations of Gammarus lacustris (Crustacea, Amphipoda)

1. Mitochondrial DNA sequence analysis was used to investigate the phylogeographic structure among populations of the amphipod Gammarus lacustris isolated in high altitude lakes from the Northern to the Southern Alps, and to investigate the historical and demographic events that may have accompanied this differentiation.
2. The comparison of a 376‐base segment of the mitochondrial cytochrome c oxidase I gene in fifty‐four specimens from nine different populations reveals no variation within populations. However, northern populations appear divergent from those from the Southern Alps. A particular population, located at the limit between the Northern and Southern Alps shows an intermediate haplotype related to the southern lineage.
3. Consideration of the different mitochondrial lineages, together with geographical differences among populations, suggests a scenario for the post‐glacial colonization of G. lacustris in the alpine lakes.     

The genetic structure of natural and reintroduced roe deer (Capreolus capreolus) populations in the Alps and central Italy,with reference to the mitochondrial DNA phylogeography of Europe

The first hypervariable fragment (HVI) of the mitochondrial DNA control region was sequenced in 90 individuals of the European roe deer (Capreolus capreolus) from the Alps, central Italy and Spain. Pooling these data with 70 published sequences from several European regions, we were able to identify patterns of divergence within the Italian peninsula, and in Europe in general. The results we obtained can be summarized as follows. First, the genetic structure of European roe deer populations is substantial (PhiST values around 0.6). Second, the divergence between some central Italian populations, the Alpine group (which is genetically close to the French, the Spanish and the Norwegian samples) and the Eastern European populations seems to reflect Upper Pleistocene subdivisions, possibly related to three southern European refugia. Third, a different group of central Italian individuals probably diverged more recently from the Alpine group, and their attribution to the subspecies C. c. italicus does not appear justified. Fourth, the analysis of mitochondrial DNA in the roe deer can be used to identify recently reintroduced animals in the western Alps which clearly cluster within the Eastern European group, thus providing an important tool for conservation and management strategies for this species.     

Littoral Chironomid Communities of Alpine Lakes in Relation to Environmental Factors

The results of a study of littoral chironomid communities from 89 lakes in the Alps are presented. The lakes are located on the northern and southern sides of the Alps from the western Bernese to the eastern Julian Alps. Due to the different origins of the data set here considered, different sampling years and sampling methodologies were adopted. All of the lakes were sampled during the ice-free season, but 79/89 lakes were sampled mainly in autumn 2000, while for a few of them the sample dated back to 1996 or the early 90s. Samples were taken by kicking the different substrata or a stretch of lakeshore (10–30 m) for at least 2–5 min with a standard net and were then preserved in alcohol. Larvae and pupal exuviae were sampled by skimming the lake surface with the same net, to extend identification to species level. Notwithstanding the large altitudinal gradient, almost all of the lakes are situated above the timberline, and show a wide range of morphological (depth, watershed and lake area) and chemical characteristics. Chironomids were the most abundant insects represented in these lakes, with highest abundances at pH between 6.0 and 7.5. In particular, Orthocladiinae and Chironominae were the two subfamilies with the widest distribution. Altitude and temperature played an important role in determining their distribution, the higher altitudes were dominated by the former, whereas the latter were more numerous in lower lakes. The prevalence of Orthocladiinae and Tanytarsini at high altitudes was regarded as a general pattern of taxonomic composition in harsh climatic areas. A reverse condition occurred only in the Julian Alps, where Tanytarsini dominate, followed by Tanypodinae and rare Orthocladiinae. Although our results demonstrated that the composition of chironomid assemblages was also determined by pH, the pattern was unbalanced. Due to the fact that no cases of strong water acidification were present, taxa restricted to very low pH were poorly represented (only three taxa—Limnophyes, Endochironomus and Orthocladius (O.) spp.) compared with circumneutral (8) and alkaline (14) taxa, which occurred in slightly acid and non-acidified soft water lakes. As regard conductivity and altitude, we found that almost all the taxa present at higher pH were contemporary present at conductivity higher than 100 μeq l⁻¹ and altitude lower than 2100 m a.s.l. At species level, Pseudodiamesa branickii, Corynoneura arctica, Heterotrissocladius marcidus and Paratanytarsus austriacus resulted to be the most ubiquitous and abundant species in the Alps. When the west to east distribution of taxa was taken into account, Chironomini (particularly the genera Chironomus and Dicrotendipes) and Tanypodinae (Larsia and Paramerina) were more typical of the eastern lakes (JA). They may be better indicators of more alkaline waters. Assembling all the existing data and presenting a comprehensive panorama of lake-littoral chironomid taxa of the Alps was one of the aims of this paper. Further monitoring, but also harmonization of the sampling methodologies and taxonomical identification including inter-calibration exercises among laboratories are recommended, to provide reliable basis and more knowledge for future studies of comparative biogeography, and to preserve these environments where global climatic changes may cause abrupt shifts in the faunal assemblages with a loss of taxa typical of these mountainous areas.     

Remote European mountain lake ecosystems: regionalisation and ecological status

1. A survey of c. 350 remote high altitude and high latitude lakes from 12 different mountain regions across Europe was undertaken to explore ecosystem variability, climate forcing, environmental conditions and pollution threats at a scale not previously attempted.
2. Lakes were sampled for a range of contemporary and sub-fossil organisms including planktonic crustaceans, rotifers, littoral invertebrates, chironomids, diatoms and cladocerans. Survey and cartographic data were used to determine environmental characteristics at each site. Organic pollutants and trace metal concentrations were measured in the lake sediment.
3. A number of separate studies were undertaken which examined the environmental characteristics of the surveyed lakes (climate forcing and chemical composition), distribution of biota relative to local, regional and biogeographical factors and pollution threats (acidification, heavy metals and persistent organic pollutants) to these sensitive ecosystems.
4. There is a strong regional element to the way that environmental factors combine (including climate and pollution threats) and the biota responds in mountain lakes across Europe. From a management perspective it is clear that lake classification and the development of useful typologies and assessments of reference conditions should be undertaken at regional rather than pan-European scales.
5. There are some common features across lake districts related to the timing of industrialisation, but the studies carried out on metals, organic pollutants and nitrate deposition indicate that each lake district has distinct pollution threats. Climate warming already affects most of the lake districts and there are considerable uncertainties as to how this will modify conditions in remote European mountain systems.
6. The lake district concept goes beyond a geographical construct and merits further theoretical and experimental development as an ecological concept.     

Use of [125I]4'-iodoflavone as a tool to characterize ligand-dependent differences in Ah receptor behavior

We have synthesized [(125)I]4'-iodoflavone to study Ah receptor (AhR)-ligand interactions by a class of AhR ligands distinct from the prototypic ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). This radioligand allows the comparison of AhR-ligand interactions using a ligand that differs in AhR affinity, and yet has the same radiospecific activity as [(125)I]2-iodo-7,8-dibromodibenzo-p-dioxin. Specific binding of [(125)I]4'-iodoflavone with the AhR was detected as a single radioactive peak ( approximately 9.7 S) following density sucrose gradient analysis. Cytosolic extracts from both Hepa 1 and HeLa cells were used as the source of mouse and human AhR, respectively. A approximately 6.7 S form of radioligand-bound Ah receptor was detected in the high salt nuclear extracts of both cell lines. In HeLa cells approximately twofold more [(125)I]4'-iodoflavone-AhR 6 S complex, compared with [(125)I]2-iodo-7,8-dibromodibenzo-p-dioxin, was recovered in nuclear extracts. A comparison of the ability of 4'-iodoflavone and TCDD to cause time-dependent translocation of AhR-yellow fluorescent protein revealed that 4'-iodoflavone was more efficient at enhancing nuclear accumulation of the receptor. These results suggest that [(125)I]4'-iodoflavone is a particularly useful and easily synthesized ligand for studying the AhR.     

The aryl hydrocarbon receptor activates the retinoic acid receptoralpha through SMRT antagonism

Aryl hydrocarbon receptor (AhR) ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benzo(a)pyrene interfere with hormonal regulatory pathways, leading to endocrine disruption. Notably, the activated AhR exerts complex effects on estrogens and retinoids at both levels of their metabolism and regulation of cognate genes. Our current investigation of these AhR effects revealed the TCDD-dependent activation of a subset of retinoid-dependent genes (tissue-transglutaminase, IGF binding protein-3, AhR) in MCF-7 breast cancer cells. A collection of in vitro hormone-dependent reporter gene models showed that AhR activation by TCDD stimulated transactivation by several class I heteromeric receptors (retinoic and thyroid hormone receptors) while it antagonized homodimeric nuclear receptors (estrogen and progesterone receptors, ER and PR). TCDD exerted a dose-dependent effect on a retinoic acid-dependent reporter gene expressed in MCF-7 cells. AhR was shown to be involved in a mutual antagonism with RARalpha corepressor SMRT (silencing mediator of retinoid and thyroid receptors). This, and the documented physical interaction between AhR and SMRT suggested that SMRT sequestration by AhR might activate RARalpha in the absence of ligand. Immunocytochemical studies of AhR and SMRT strongly suggested they colocalized in nuclear bodies during this sequestration. Concurring with this interpretation, we observed an interaction in vitro between AhR and the PML protein, the core component of nuclear bodies. This ability of AhR to elicit spurious activation of retinoid receptors expands the scope of AhR ligands influence beyond ER antagonism and specific Dioxin-responsive genes. Unknown AhR endogenous ligands may also elicit gene transactivation by class I receptors, while being inactive on classic xenobiotic-responsive genes.     

European common frog (Rana temporaria) recolonized Switzerland from multiple glacial refugia in northern Italy via trans‐ and circum‐Alpine routes

The high mountain ranges of Western Europe had a profound effect on the biotic recolonization of Europe from glacial refugia. The Alps present a particularly interesting case because they form an absolute barrier to dispersal for most taxa, obstructing recolonization from multiple refugia in northern Italy. Here, we investigate the effect of the European Alps on the phylogeographic history of the European common frog Rana temporaria. Based on partial cytochrome b and COXI sequences from Switzerland, we find two mitochondrial lineages roughly north and south of the Alpine ridge, with contact zones between them in eastern and western Switzerland. The northern haplogroup falls within the previously identified Western European haplogroup, while the southern haplogroup is unique to Switzerland. We find that the lineages diverged ~110 kya, at approximately the onset of the last glacial glaciation; this indicates that they are from different glacial refugia. Phylogenetic analyses suggest that the northern and southern haplogroups colonized Switzerland via trans‐ and circum‐Alpine routes from at least two separate refugia in northern Italy. Our results illustrate how a complex recolonization history of the central European Alps can arise from the semi‐permeable barrier created by high mountains.     

Ligand displaces heat shock protein 90 from overlapping binding sites within the aryl hydrocarbon receptor ligand-binding domain

Hsp90 (heat shock protein of 90 kDa) is often found associated with functional domains of client proteins, including those for ligand binding, dimerization, DNA binding, and enzymatic activity. Although Hsp90 can maintain the conformation of functionally important domains prior to activation of the client protein, its specific binding site and the mechanism(s) of Hsp90 dissociation during activation are unknown. Here, we have identified and characterized residues involved in Hsp90 binding within the aryl hydrocarbon receptor (AhR) ligand-binding domain and demonstrate that they overlap with those involved in ligand binding. In agreement with this spatial model, ligand binding results in Hsp90 dissociation from the AhR Per-ARNT-Sim B fragment. Interestingly, whereas Hsp90-binding residues within the ligand-binding domain were not involved in Hsp90-dependent AhR protein stability, several of these residues are important for ligand-dependent AhR activation, and their mutation resulted in conversion of two AhR antagonists/partial agonists into full AhR agonists. These studies reveal co-localization of a tentative Hsp90-binding site with that for AhR ligand binding and provide the first molecular mechanism for Hsp90 dissociation in the activation of a client protein.