Diamesa species (Diptera, Chironomidae) are widely distributed in freshwater ecosystems, and their life cycles are closely linked to environmental variables such as temperature, water quality, and sediment composition. Their sensitivity to environmental changes, particularly in response to pollution and habitat alterations, makes them valuable indicators of ecosystem health. The challenges associated with the morphological identification of larvae invoke the use of DNA barcoding for species determination. The mitochondrial cytochrome oxidase subunit I (COI) gene is regularly used for species identification but faces limitations, such as similar sequences in closely related species. To overcome this, we explored the use of the internal transcribed spacers (ITS) region in addition to COI for Diamesa larvae identification. Therefore, this study employs a combination of molecular markers alongside traditional morphological identification to enhance species discrimination. In total, 129 specimens were analysed, of which 101 were sampled from a glacier-fed stream in Rotmoostal, and the remaining 28 from spring-fed streams in the neighbouring valleys of Königstal and Timmelstal. This study reveals the inadequacy of utilizing single COI or ITS genes for comprehensive species differentiation within the genus Diamesa. However, the combined application of COI and ITS markers significantly enhances species identification resolution, surpassing the limitations faced by traditional taxonomists. Notably, this is evident in cases involving morphologically indistinguishable species, such as Diamesa latitarsis and Diamesa modesta. It highlights the potential of employing a multi-marker approach for more accurate and reliable Diamesa species identification. This method can be a powerful tool for identifying Diamesa species, shedding light on their remarkable adaptations to extreme environments and the impacts of environmental changes on their populations.

Chironomids of the genus Diamesa (Meigen, 1835, Diptera: Chironomidae) inhabit cold, oxygen-rich running waters. We have investigated the presence of Diamesa and other freshwater macroinvertebrates at 22 stream sampling sites in 3 European high mountain regions (the Central Pyrenees, the Ötztal Alps, and the Tatra Mountains) to establish suitable temperature conditions for Diamesa dominance. It has been generally accepted that their high abundance was linked to the presence of glaciers; however, we have shown that in the Tatra Mountains, where there are no glaciers, the conditions for the dominance of Diamesa species are created due to permanent snowfields, the geographical orientation of the valley and shading by the surrounding high peaks. The historical connection of Diamesa to glaciers was investigated from the paleolimnological records of subfossil chironomid assemblages from the Bohemian Forest, where glaciers disappeared before or during the Late Glacial period. As expected, water temperature seems to be the main driver of Diamesa distribution, and we determined that the relative abundance of Diamesa species was significantly higher at the sites with a mean July water temperature below 6.5 °C. The Diamesa-dominated stream communities seems to be endangered due to ongoing climate warming and this assumption is supported by our paleolimnological results from the Bohemian Forest lakes, where Diamesa has disappeared due to warming of lake inflows at the beginning of the Holocene. These findings strengthen the former suggestions that some Diamesa species could be used as an indicator for tracking recent environmental changes in vulnerable ecosystems of cold mountain streams.

Chironomids (non-biting midges) inhabit almost every wet or semi-wet continental environment on Earth with probably 10,000 different species. Species occurrence and composition are undoubtedly limited by environ -mental harshness and food availability being reflected in their energy stores. Most animals store energy as glycogen and lipid. They enable the animals to survive adverse situations and to continue growth, development, and reproduction. This general statement is also true for insects and also particularly true for chironomid larvae. The rationale behind this research was, that probably any stress, any environmental burden, and any harmful influence increases the energy requirement of individual larvae depleting energy stores. We developed new methods to measure the glycogen and lipid content in small tissue samples. Here we show how to apply these methods to single chironomid larvae to demonstrate their energy stores. We compared different locations of the high Alpine rivers along harshness gradient densely populated and dominated by chironomid larvae. All samples show very low energy stores without any major differences. We found glycogen concentrations below 0.01% of dry weight (DW) and lipid concentrations below 5% of DW irrespective of the specific sampling point. These values are among the lowest ever observed in chironomid larvae. We demonstrate that individuals living in extreme environment are stressed leading to reduced energy stores in their bodies. This appears to be a general feature of high altitude regions. Our results provide new insights and a better understanding of population and ecological dynamics in harsh mountainous areas, also in view of a changing climate.

Metal detoxification is crucial for animals to cope with environmental exposure. In snails, a pivotal role in protection against cadmium (Cd) is attributed to metallothioneins (MTs). Some gastropod species express, in a lineage-specific manner, Cd-selective MTs devoted exclusively to the binding and detoxification of this single metal, whereas other species of snails possess non-selective MTs, but still show a high tolerance against Cd. An explanation for this may be that invertebrates and in particular snails may also synthetize phytochelatins (PCs), originally known to be produced by plants, to provide protection against metal or metalloid toxicity. Here we demonstrate that despite the fact that similar mechanisms for Cd inactivation exist in snail species through binding of the metal to MTs, the actual detoxification pathways for this metal may follow different traits in a species-specific manner. In particular, this depends on the detoxification capacity of MTs due to their Cd-selective or non-specific binding features. In the terrestrial slug Arion vulgaris, for example, Cd is solely detoxified by a Cd-selective MT isoform (AvMT1). In contrast, the freshwater snail Biomphalaria glabrata activates an additional pathway for metal inactivation by synthesizing phytochelatins, which compensate for the insufficient capacity of its non-selective MT system to detoxify Cd. We hypothesize that in other snails and invertebrate species, too, an alternative inactivation of the metal by PCs may occur, if their MT system is not Cd-selective enough, or its Cd loading capacity is exhausted.

The tiny contribution of cadmium (Cd) to the composition of the earth's crust contrasts with its high biological significance, owing mainly to the competition of Cd with the essential zinc (Zn) for suitable metal binding sites in proteins. In this context it was speculated that in several animal lineages, the protein family of metallothioneins (MTs) has evolved to specifically detoxify Cd. Although the multi-functionality and heterometallic composition of MTs in most animal species does not support such an assumption, there are some exceptions to this role, particularly in animal lineages at the roots of animal evolution. In order to substantiate this hypothesis and to further understand MT evolution, we have studied MTs of different snails that exhibit clear Cd-binding preferences in a lineage-specific manner. By applying a metallomics approach including 74 MT sequences from 47 gastropod species, and by combining phylogenomic methods with molecular, biochemical, and spectroscopic techniques, we show that Cd selectivity of snail MTs has resulted from convergent evolution of metal-binding domains that significantly differ in their primary structure. We also demonstrate how their Cd selectivity and specificity has been optimized by the persistent impact of Cd through 430 million years of MT evolution, modifying them upon lineage-specific adaptation of snails to different habitats. Overall, our results support the role of Cd for MT evolution in snails, and provide an interesting example of a vestigial abiotic factor directly driving gene evolution. Finally, we discuss the potential implications of our findings for studies devoted to the understanding of mechanisms leading to metal specificity in proteins, which is important when designing metal-selective peptides.

In most organisms, the concentration of free Zn2+ is controlled by metallothioneins (MTs). In contrast, no significant proportions of Zn2+ are bound to MTs in the slug, Arion vulgaris. Instead, this species possesses cytoplasmic low-molecular-weight Zn2+ (LMW Zn) binding compound that divert these metal ions into pathways uncoupled from MT metabolism. Zn2+ is accumulated in the midgut gland calcium cells of Arion vulgaris, where they associate with a low-molecular-weight ligand with an apparent molecular mass of ~ 2,000 Da. Mass spectrometry of the semi-purified LMW Zn binding compound combining an electrospray ion source with a differential mobility analyser coupled to a time-of-flight mass spectrometer revealed the presence of four Zn2+-containing ion signals, which arise from disintegration of one higher MW complex resulting in an ion-mobility diameter of 1.62 nm and a molecular mass of 837 Da. We expect that the novel Zn2+ ion storage pathway may be shared by many other gastropods, and particularly species that possess Cd-selective MT isoforms or variants with only very low affinity to Zn2+.

Arion vulgaris is a land-living European slug belonging to the gastropod clade of Stylommatophora. The species is known as an efficient pest organism in vegetable gardening and horticulture, which may in part be the consequence of its genetically based innate immunity, along with its high ability to withstand toxic metal stress by intracellular detoxification. Like many species of terrestrial snails, slugs possess a distinct capacity for Cd accumulation in their midgut gland, where the metal is stored and inactivated, conferring to these animals an increased metal tolerance. Although midgut gland Cd fractions in slugs have been shown to be variably allocated between different metal-binding protein pools, depending on the level of environmental metal contamination, a true metallothionein (MT) was so far never characterized from slugs. Instead, the Cd binding proteins identified so far were described as Metallothionein-like proteins (MTLPs). In the present study, the slug A. vulgaris was used as a model organism, in order to verify the presence of true MTs in experimentally metal-exposed slugs. We wanted to find out if these suggested slug MTs have similar metal binding properties and metal-selective features like those previously reported from helicid snails. To this aim, two MT isoform genes (AvMT1 and AvMT2) were characterized from midgut gland extracts and localized in the cells of this tissue. The AvMT1 and AvMT2 proteins were purified and partially sequenced, and their metal-binding features analysed after recombinant expression. Eventually, we wanted to understand if and by how much the metal binding features of the two MT isoforms of A. vulgaris may be related, owing to their reciprocal amino acid sequence similarities, to the binding properties of metal-specific MTs from terrestrial snails.

Human activity has spread trace amounts of chemically stable endocrine-disrupting pollutants throughout the biosphere. These compounds have generated a background level of estrogenic activity that needs to be assessed. Fish are adequate sentinels for feminization effects as male specimens are more sensitive than humans to exogenous estrogenic compounds. High mountain lakes, the most distant environments of continental areas, only receive semi-volatile compounds from atmospheric deposition. We analyzed the expression levels of estrogen-regulated genes in male fish from these mountain lakes in Europe. Incipient feminization involving expression of estrogen receptor and zona radiata genes revealed a widespread diffuse estrogenic impact. This effect was correlated with the concentrations of some organochlorine compounds in fish and was consistent with the persistent occurrence of these tropospheric pollutants in the most remote planet regions. These results should be of general concern given the increasing endocrine disruption effects in human populations.

The brown trout (Salmo trutta L.) represents one of the most differentiated vertebrate species. However, human activities such as habitat alteration, overexploitation and introgression of non­ native hatchery fish led to an alarming reduction of population variability. This is particularly evident for vast parts of the highly fragmented alpine area. Because of these facts the Interrreg IIIA-project "Trout-Examinvest" was initiated in order to achieve the following goals: ♦ Genetic characterisation of local trout populations ♦ Identification of potential autochthonous populations ♦ Establishm ent o f indigenous hatchery strains for Conservation management In the framework of our project two molecular techniques were applied: (i) sequence analysis of the complete mitochondrial DNA control region and (ii) analyses of a number of variable microsatellite DNA loci. As was shown in previous studies, mitochondrial DNA revealed to be a useful tool in the screening of frequencies and distribution patterns ofthe major trout lineages. On the other hand, microsatellite DNA data delivered more detailed information about withinpopulation genetic diversity and population structure as well as about hybridisation between native and introduced trout lineages. Based on these findings we point to the necessity of using a combined approach of molecular analyses to select and establish indigenous trout breeding strains for future stocking and repopulation measures.

1. The watercourses of Austria are mainly part of the Danube drainage system. Nonetheless, only few brown trout (Salmo trutta) populations of the Danubian phylogenetic lineage have been found in this region, not being introgressed by hatchery-reared fish of the allochthonous Atlantic lineage. The present study was aimed at identifying further waterbodies that might harbour the non-admixed gene pool of the Danubian clade trout. 2. In a first step, the complete mitochondrial DNA control region of 447 specimens from 20 sampling sites was sequenced to screen for the presence of specific haplotypes. In a second step, 332 individuals from 15 populations were analysed at 10 microsatellite DNA loci. 3. Although sampling concentrated on remote Alpine headwater streams, introgression of hatchery strains was more common than expected from existing stocking records. For seven populations, however, no evidence of recent hatchery trout introgression was found and it is suggested that they might represent the indigenous gene pool. 4. Populations showing no evidence of hatchery introgression displayed lower levels of intrapopulation variability and higher degrees of differentiation compared to purported admixed populations. 5. Our study demonstrates the necessity of combining different molecular markers to reveal the impact of introgression into wild populations, since at some sampling sites admixture would have remained undetected if only a single molecular technique was applied. 6. The identification and characterisation of non-introgressed populations provides important information for setting conservation priorities and preventing further loss of genetic integrity of a unique element of the native fish fauna.

Cytochrome p450 1A (CYP1A) gene expression in fish liver increases upon exposure to a variety of chemical compounds, including organochlorine compounds (OCs) and polycyclic aromatic hydrocarbons (PAHs). To use this physiological response as a marker of environmental impact, we developed and validated a set of primers to quantify CYP1A expression by qRT-PCR in the brown trout, Salmo trutta. These primers were used to explore the natural variability of CYP1A expression in 8 isolated populations (65 samples) from European remote lakes, in a geographical distribution encompassing the Tyrolean Alps, Pyrenees, Rila, Tatras, and Norwegian and Scottish mountains. CYP1A expression values varied more than 2 orders of magnitude among samples, with strong variations within each population. CYP1A expression values were significantly elevated in Tatras and Pyrenees fish populations, whereas the lowest median values were found in populations from the Tyrolean Alps and Rila. These values correlated with the content of different environmentally relevant pollutants in the sediments of the lakes harboring each fish population, particularly with HCB and 4,4'-DDE contents. To our knowledge, this works represents a first report of a physiological response linked to persistent organic pollutants in fish from mountain lakes.

Southern populations of the European minnow fromremote oligotrophic mountain lakes along an Alpinenorth-south transect accumulated more p,p′ DDE andPCBs than northern populations. As these semi-volatileorganochlorines predominantly evaporate in warmcountries, higher rates of condensation (deposition)are assumed to occur in the southern slopes of theAlps. The higher accumulation of lead and cadmium insouthern population is rather attributed to geogenicand specific environmental impacts than to atmosphericdeposition. Increasing liver ratios of [glutathionedisulfide]/[glutathione], a potential indicator foroxidative stress, from north to south reflect thegenerally higher toxic load at remote sites at thesouthern edge of the Alps. However, histopathologicalchanges in the liver did not correlate withaccumulated toxicants indicating that deposition doesnot lead to severe lesions but induces specificmechanisms for detoxification.

Total protein, nitrogen and lipid as well as calorific content, ash content, inorganic ions and amino acids were measured in various types of zooplankton, Artemia salina nauplii and Moina sp, including summer and winter samples from Austrian lakes. The activities of three enzyme systems, L-lactate dehydrogenase (LDH), proteases and NADH2-dependent dehydrogenases, in zooplankton are not affected by freezing or freeze-drying, or by storage below −18°C. The half-life time of LDH in freeze-dried material is 280 days at room temperature, and 3 h at 80°C. When frozen or freeze-dried zooplankton material is introduced into water, enzyme activity is rapidly lost. After 10 min at 9°C, only 27% of the original LDH activity remain in the plankton material, whereas the rest can be recovered in the incubation medium. Nearly all the free and about one-third of free plus protein-bound amino acids are lost from plankton samples incubated for one hour. The leaching experiments suggest the presence of unknown, non-protein sources of “bound” citrulline and histidine in the plankton. During the process of leaching nearly all of the bound lysine and arginine are liberated from plankton proteins by the action of the still viable proteases. Pelleting freeze-dried plankton reduces the rate of leaching considerably. After 10 min in water at 9°C, about 85% of the original LDH activity can still be found in the potential food material.