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Palaeolimnological techniques were applied to spring habitats in the Trentino Region (NE Italy) as a test in order to reconstruct past environmental changes in these crucial and fragile milieus. Three different sites were selected from a database of more than one hundred on the basis of morphological, geological, and biological factors, including human impact evaluation: Nambrone, Paul and Madonnina Val Lomasona. Sampling was performed by adapting standard lake-sediment coring methods, and the retrieved sediment was described and subsampled following standard "lake" procedures. Field work and sedimentological analyses revealed clear influence from nearby fluvial environment for the Nambrone site, whereas Paul and Madonnina Val Lomasona were selected for further analyses (e.g., dating, diatoms, chironomids). Lead-210 and Caesium-137 dating methods were used to obtain a chronological framework, in addition to historical information on land use and other events in the area. The upper part of the Paul sequence presented organic rich sediment compatible with spring environments, but the lower and essentially detrital part appeared to record alluvial deposition, probably linked to extraordinary flood events (1882, 1966). This interpretation is indirectly confirmed by the records of the fallout nuclides Caesium-137 and Lead-210, that were both unfortunately concentrated in the topmost 1 cm, making it impossible to construct a normal age-depth profile. Madonnina Val Lomasona sedimentological and biological indicators point to a marked charge around ca 5 cm, dividing the sequence in two units, corresponding to different environmental conditions. Although the interpretation of the dating results is not completely straightforward, the assembled data suggest that the upper 5 cm of the record represents ca the last 50 years. During this period we found typical limnocrenic, clear-water spring conditions, while before 1960s the coexistence of lotic and lentic chironomid species including several opportunistic ones and the low richness, emphasized the presence of a disturbance (e.g., highly variable flow and/or trophic conditions), as confirmed also by diatom assemblages and,sedimentological features. The recorded disturbance is probably linked to a water-level change due to the local land use, as also testified by historical data. The results of this study support the idea that carefully selected spring sites might be approached with adapted palaeolimnological techniques to extract valuable palaeoenvironmental information. However, it also highlighted the low percentage of "good sites" (1%) and considerable difficulties in coring and sampling without disturbing the sediment.

The diversity and distribution of chironomids (Diptera, Chironotnidae) were studied in relation to environmental factors in 81 springs under pristine conditions in the Italian Prealps and Alps (Trentino and Veneto, NE-Italy, 46 degrees N, 10-11 degrees E). Each spring was surveyed once, between May and November, in 2005 or in 2007-2008, within 50 m of the spring's source (eucrenal). A total of 173 macroinvertebrate samples were collected, in which 26,871 chironomids (including larvae, pupae, pupal exuviae and adults) were counted. Five subfamilies (Tanypodinae, Diamesinae, Prodiamesinae, Orthocladiinae and Chironominae), 54 genera and 104 species/groups of species were identified. As expected, Orthocladiinae accounted for a large part of specimens (82%), followed by Diamesinae (10%), Chironominae Tanytarsini (6%) and Tanypodinae (2%). Together the Chironominae Chironomini and Prodiamesinae contributed less than 0.05% of the fauna. Larvae represented 97.5% of specimens, mostly juveniles (62.6%). Maximum richness and diversity occurred at intermediate altitudes (ca 900-2100 m a.s.l.). Most taxa were found in a small proportion of sites, and frequencies declined gradually for more widely distributed species. A high number (67%) of rare (= present in less than 10% of sites) taxa were found. Three to 27 taxa were identified per spring. The rheocrene/rheo-helocrene springs were richest in taxa (generally > 15 taxa), the mineral spring was poorest, with only three taxa. Most taxa were crenophilous, including lentic, rheobiontic and blyophilous taxa. A Principal Component Analysis (PCA) was performed including 98 taxa. Axes were interpreted calculating the correlation coefficients between site scores and 24 environmental factors. The species with the highest scores were Pseudokiefferiella parva, Corynoneura sp. A, Metriocnemus eurynotus gr., Paratrichocladius skirwithensis and Tvetenia calvescens. Five clusters of sites were identified with K-means analysis on the basis of the first and second PCA axes and a Discriminant Analysis was used to detect environmental factors discriminating the clusters: altitude, canopy cover, hydrological regime, pH, and granulometty as percentage of cobbles and stones. The highly individual nature of springs was highlighted; within the same river basin, between springs and within a single spring. These results suggest that prudent and conservative land management should assume that all springs sheltering such unique faunal assemblages need protection.

From a study of glacier-fed catchments along a European latitudinal and climatic gradient, the relationships between the distribution of mayfly (Ephemeroptera) and stonefly (Plecoptera) nymphs and environmental variables were explored. Canonical Correspondence Analysis (CCA) and Generalised Additive Models (GAMs) demonstrated clear responses of mayfly and stonefly generic composition and taxa richness to environmental gradients, notably maximum water temperature and channel stability as these change downstream of glacial sources. GAMs developed for aquatic insects are well suited to detect environmental impacts, including climate change.

1. A study on glacial stream ecosystems was carried out in six regions across Europe, from Svalbard to the French Pyrenees. The main aim was to test the validity of the conceptual model of Milner & Petts (1994) with regard to the zonation of chironomids of glacier-fed rivers along altitudinal and latitudinal gradient. 2. Channel stability varied considerably, both on the latitudinal and altitudinal scale, being lowest in the northern regions (Svalbard, Iceland and Norway) and the Swiss Alps. Water temperature at the upstream sites was always

Are the effluents of wastewater treatment plants in high mountains of concern for aquatic biodiversity? To answer this question, we carried out an experimental study testing the short-term toxicity of some Pharmaceutical Active Compounds (PhACs) in the effluents of a plant in a mountain valley of the Italian Alps sampled during the high tourist season (i.e., the ski season) when PhACs contamination is higher. We used different tools, taking as a model the bacterium Aliivibrio fischeri: the “whole-mixture approach” (Microtox test), “component-based approach”, predictive models “Concentration Addition (CA)”, “Independent Action (IA)”, and Combination Index (CI)”. We investigated the nature of interactions potentially occurring among seven selected PhACs (clarithromycin, naproxen, acetaminophen (paracetamol), ibuprofen, diclofenac, carbamazepine, and amoxicillin). This study showed that anti-inflammatory ibuprofen and diclofenac have higher short-term toxicity (IC50

Some pollutants can be transported through the atmosphere and travel medium–long distances to be deposited in glaciers at high altitude and latitude. The increase in the rate of glacier melting due to global warming can release these pollutants in alpine streams. This study investigated the combined effects of rising temperatures and chlorpyrifos (CPF) contamination on the swimming behaviour of alpine chironomids collected in a shrinking alpine glacier. We assessed the individual and interaction effects of rising temperatures (2–11 °C) and CPF concentrations (0–110 ng L−1) on the swimming behaviour of Diamesa zernyi (Chironomidae) larvae. Distance (mm) and speed (mm s−1) were recorded using a video-tracking system after 24–72 h of treatment. The two stressors caused different effects on distance and speed, with increasing temperature generally causing hyperactivity and CPF from hyperactivity to reduced mobility. Two interactions were detected between stressors when combined: (i) CPF superimposed the effect of temperature on both behavioural endpoints i.e., with 110 ng L−1 of CPF, at 11 °C, larvae moved less; (ii) warming (11 °C) magnified the negative effect of CPF: the smallest distance and slowest speed were recorded at the highest values of the two stressors after 72 h. Our results suggest that water contamination by CPF, even at sub-lethal concentrations, might increase the sensitivity of chironomids to warming, and vice versa, raising concerns about freshwater biodiversity conservation under climate change.

This study aimed to assess how different concentrations of the insecticide chlorpyrifos (1.1, 5.24, 11, 52.4, 110, 262, 524 and 1100 ng L-1) affect the swimming behaviour of Diamesa zernyi larvae following exposure. A video tracking system was employed to analyse two swimming traits (total distance moved and average speed) of the larvae simultaneously after 3 days of exposure to the pesticide at 2 °C. The behavioural results were also interpreted according to biochemical responses to oxidative stress (OS) induced by chlorpyrifos, based on malondialdehyde (MDA) and protein carbonyl (PCC) content. Both distance and speed significantly decreased after 72 h of exposure to chlorpyrifos concentrations of ≥ 110 ng L-1, under which significant OS was detected as lipid peroxidation (level of MDA) and protein carbonylation (level of carbonyl). Analysis of altered swimming behaviour, along with MDA and carbonyl content, indicated that ≥ 110 ng L-1 contamination levels of the insecticide cause the organism to reallocate energy normally used for locomotor activity to repair cell damage, which might explain the strong impairment to locomotor performance. Locomotor performance is an ecologically relevant trait for elucidating the population dynamics of key species, with disturbance to this trait having long-term negative impacts on population and community structure. Therefore, chlorpyrifos insecticides represent a serious ecological risk for mountain aquatic species based on the detrimental effects observed in the current study, as the tested concentrations were those at which the insecticide is found in many Alpine rivers of Italy.

Recently, the use of Daphnia magna has been proposed in on-line and real-time biomonitoring programmes as an early warning system for evaluating the effluent quality of sewage treatment plants (STPs). These systems are based on recording behavioural changes in the test organism resulting from the stress caused by the effluents. Indeed, altered behavioural signals could be induced at sublethal concentrations that are significantly lower than the corresponding EC50. However, at present, it is unknown whether the sensitivity of D. magna can be representative of that of other aquatic organisms, particularly benthic macroinvertebrates. An experiment was designed to verify whether D. magna can be employed in biomonitoring programmes for STPs located in alpine areas as a surrogate for cold freshwater best-adapted species. The responses of survival and behaviour alteration to exposure to the effluent of the Tonale Pass plant (Trentino, Italian Alps, 46°N, 10°E; 1799 m a.s.l.) were compared in a laboratory population of D. magna and a wild population of the chironomid Diamesa cinerella. These larvae were collected from the Vermigliana stream 50 metres upstream of the effluent input. Both organisms were exposed for 24 and 48 hrs to the effluent as it is and to three dilutions (/10, /100, /1000). The mortality rate and behavioural responses (using video tracking systems) were recorded. No significant mortality or change in behaviour was observed in the two species when exposed to the undiluted effluent. Exposure to serial dilutions of the treated effluent did not affect the survival of either species but notably altered their behaviour at both exposure times (e.g., the time spent in activity in D. magna and the average speed of movement and the cumulative distance travelled in both), especially when exposed to the ten-times-diluted effluent. Overall, the findings of this study emphasize that even though D. magna and D. cinerella use different behavioural strategies to cope with adverse environmental conditions, their overall sensitivity to treated effluents is similar. Accordingly, the use of D. magna in biological early warning systems protocols seems to also be sufficiently protective for local, cold-adapted species of alpine freshwater ecosystems.

Several studies have indicated the presence of contaminants in Alpine aquatic ecosystems. Even if measured concentrations are far below those that cause acute effects, continuous exposure to sub-lethal concentrations may have detrimental effects on the aquatic species present in these remote environments. This may lead to a cascade of indirect effects at higher levels of the ecological hierarchy (i.e., the community). To improve the determination of ecologically relevant risk endpoints, behavioural alterations in organisms due to pollutants are increasingly studied in ecotoxicology. In fact, behaviour links physiological function with ecological processes, and can be very sensitive to environmental stimuli and chemical exposure. This is the first study on behavioural alteration in a wild population of an Alpine species. In the present study, a video tracking system was standardized and subsequently used to identify contaminant-induced behavioural alterations in Diamesa zernyi larvae (Diptera, Chironomidae). Diamesa zernyi larvae, collected in an Italian Alpine stream (Rio Presena, Trentino Region), were acclimated for 24 h and successively exposed to several aquatic contaminants (pesticides: chlorpyrifos, metolachlor, boscalid, captan; pharmaceuticals: ibuprofen, furosemide, trimethoprim) at concentrations corresponding to their Lowest Observed Effect Concentration (LOEC). After 24, 48, 72, and 96 h of exposure, changes in the distance moved, the average speed, and the frequency of body bends were taken to reflect contaminant- and time-dependent effects on larval behaviour. In general, metolachlor, captan, and trimethoprim tended to reduce all the endpoints under consideration, whereas chlorpyrifos, boscalid, ibuprofen, and furosemide seemed to increase the distances moved by the larvae. This could be related to the different mechanisms of action of the investigated chemicals. Independently of the contaminant, after 72 h a general slowing down of all the behavioural activities occurred. Finally, we propose a behavioural stress indicator to compare the overall behavioural effects induced by the various contaminants. Sub-lethal concentrations of pharmaceuticals and pesticides induce significant behavioural changes on Diamesa zernyi.

The metabolic effects of low oxygen content on alcohol-dehydrogenase (ADH) activity and hemoglobin (Hb) concentration were investigated in IV-instar larvae of Chironomus riparius (Diptera: Chironomidae) from an Italian stream. Two series of short-term (48 h) experiments were carried out: exposure to (1) progressive hypoxia (95 to 5% of oxygen saturation) and (2) anoxia (at 50% and decreased significantly only after 48 h of treatment (= 2 gAFDW-1 h-1. ADH activity showed two phases of growth, within the first 14 h and over 18 h of exposure. Overall, we inferred that i) Hb might function as short-term oxygen storage, enabling animals to delay the on-set of anaerobiosis; and ii) alcoholic fermentation co-occurs for a short time with aerobic respiration, becoming the prevalent metabolic pathway below 5% of oxygen saturation (-1). These considerations were supported also by results from anoxia exposure (2). In such condition, larvae were visibly stressed, becoming immobile after few minutes of incubation, and ADH reached higher values than in the hypoxia treatment (2.03±0.15 UADH mg prot-1). Overall, this study showed a shift from aerobic to anaerobic activity in C. riparius larvae exposed to poorly oxygenated water with an associated alteration of ADH activity and the Hb amount. Such metabolites might be valid candidate biomarkers for the environmental monitoring of running waters.

1.A collection of approximately 100 000 chironomids (Diptera; Chironomidae) inhabiting glacial areas of the Southern Alps that were collected over a period of approximately four decades from 1977 to 2014 were analysed to evaluate the impact of environmental traits on the distribution of chironomid species. Although the list of species has not substantially changed over time, some rare species captured in the 1970s have not been collected in recent years, while other species have only been collected recently. 2.The recovered correlation between altitude of the sampling sites and the date of collection since the first sampling event emphasises the need, in recent years, to sample at higher altitudes to collect species living near the glacier snout. This result confirms the impact of glacial retreat on chironomid communities. 3.Temporal trends in diversity were not emphasised throughout the collecting period. 4.A canonical correspondence analysis (CCA) showed that the influence of glaciers is the ecological trait that is the most robust predictor of the spatial distribution of species. This trait was highly correlated with the first CCA axis. Diamesa steinboecki, Diamesa latitarsis, Diamesa modesta, Diamesa goetghebueri and Diamesa laticauda are species that are known to be restricted to glacial habitats and were correlated with the first axis. The second CCA axis separated Western from Central-Eastern glaciers, with Diamesa cinerella prevailing in the former. 5.The threshold indicator taxa analysis, which detects changes in the distribution of taxa along an environmental gradient, confirmed the results of CCA in selecting Diamesa species with the strongest preference for glacial habitats.

Rapid and efficient DNA-based tools are recommended for the evaluation of the insect biodiversity of high-altitude streams. In the present study, focused principally on larvae of the genus Diamesa Meigen 1835 (Diptera: Chironomidae), the congruence between morphological/ molecular delimitation of species as well as performances in taxonomic assignments were evaluated. A fragment of the mitochondrial cox1 gene was obtained from 112 larvae, pupae and adults (Diamesinae, Orthocladiinae and Tanypodinae) that were collected in different mountain regions of the Alps and Apennines. On the basis of morphological characters 102 specimens were attributed to 16 species, and the remaining ten specimens were identified to the genus level. Molecular species delimitation was performed using: i) distance-based Automatic Barcode Gap Discovery (ABGD), with no a priori assumptions on species identification; and ii) coalescent tree-based approaches as the Generalized Mixed Yule Coalescent model, its Bayesian implementation and Bayesian Poisson Tree Processes. The ABGD analysis, estimating an optimal intra/interspecific nucleotide distance threshold of 0.7%-1.4%, identified 23 putative species; the tree-based approaches, identified between 25–26 entities, provided nearly identical results. All species belonging to zernyi, steinboecki, latitarsis, bertrami, dampfi and incallida groups, as well as outgroup species, are recovered as separate entities, perfectly matching the identified morphospecies. In contrast, within the cinerella group, cases of discrepancy arose: i) the two morphologically separate species D. cinerella and D. tonsa are neither monophyletic nor diagnosable exhibiting low values of between-taxa nucleotide mean divergence (0.94%); ii) few cases of larvae morphological misidentification were observed. Head capsule color is confirmed to be a valid character able to discriminate larvae of D. zernyi, D. tonsa and D. cinerella, but it is here better defined as a color gradient between the setae submenti and genal setae. DNA barcodes performances were high: average accuracy was ~89% and precision of ~99%. On the basis of the present data, we can thus conclude that molecular identification represents a promising tool that could be effectively adopted in evaluating biodiversity of high-altitude streams. PLOS ONE