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Animal populations are increasingly forced to live in small residual natural or semi-natural areas due to habitat loss and fragmentation. Here, the viability of populations is often compromised by intrinsic threat factors typical of small and isolated populations, such as inbreeding depression, genetic drift and environmental and demographic stochasticity. Under these circumstances, organisms may have low fitness due to inadequate physiological responses needed to face environmental challenges. However, few studies have investigated the relationship between habitat fragmentation and stress defences. In this study, we aimed to test whether an increase in the level of individual inbreeding produced an increase in the antioxidant system response. To this purpose, we genotyped 151 individuals of fire salamander Salamandra salamandra (Amphibia: Urodela) within five sampling populations, located in forest landscapes with different degree of fragmentation in northern Italy. For 113 individuals we also measured the glutathione-S-transferase (GST) and catalase (CAT) enzyme activity. Results showed a significant increase in individual GST activity for increasing levels of inbreeding, whereas no relationship was found for CAT. We also measured acetylcholinesterase to test the possible confounding effects of pesticides that might have occurred in fragmented landscapes with forests interspersed with agricultural areas. However, no difference in this enzyme activity was found among sampling populations. We argue that high levels of GST activity may be symptomatic of oxidative stress derived from inbreeding. An increased frequency of homozygous deleterious alleles due to inbreeding may cause homeostatic alterations and trigger the expression of GST for protection against hydrogen peroxide reactive oxygen species. We suggest using GST as a biomarker for environmental stressors with great caution and not to underestimate that the sources of stress deriving from habitat fragmentation could lead to an unbalance in the oxidative status, possibly increasing population susceptibility to infectious diseases and, potentially, spillover events and zoonoses.

Honeybee and general pollinator decline is extensively reported in many countries, adding new concern to the general biodiversity loss. Many studies were addressed to assess the causes of pollinator decline, concluding that in most cases multi-stress effects were the most probable ones. In this research, the combined effects of two possible stress sources for bees, pesticides and electromagnetic fields (multi-stress conditions), were analyzed in the field. Three experimental sites were chosen: a control one far from direct anthropogenic stress sources, a pesticide-stress site and multi-stress one, adding to the same exposure to pesticides the presence of an electromagnetic field, coming from a high-voltage electric line. Experimental apiaries were monitored weekly for one year (from April 2017 to April 2018) by means of colony survival, queen activity, storage and brood amount, parasites and pathogens, and several biomarkers in young workers and pupae. Both exposure and effect biomarkers were analysed: among the first, acetylcholinesterase (AChE), catalase (CAT), glutathione S-transferase (GST) and alkaline phosphatase (ALP) and Reactive Oxygen Species (ROS), and among the last, DNA fragmentation (DNAFRAGM) and lipid peroxidation (LPO). Results showed that bee health conditions were the worst in the multi-stress site with only one colony alive out of the four ones present at the beginning. In this site, a complex picture of adverse effects was observed, such as disease appearance (American foulbrood), higher mortality in the underbaskets (common to pesticide-stress site), behavioral alterations (queen changes, excess of honey storage) and biochemical anomalies (higher ALP activity at the end of the season). The overall results clearly indicate that the multi-stress conditions were able to induce biochemical, physiological and behavioral alterations which severely threatened bee colony survival.

The use of plant protection products (PPPs) in agricultural areas implies potential chemical loadings to surface waters, which can pose a risk to aquatic ecosystems and human health. Due to the spatio-temporal variability of PPP applications and of the processes regulating their transport to surface waters, aquatic organisms are typically exposed to pulses of contaminants. In small mountain watersheds, where runoff fluxes are more rapid due to the steep slopes, such exposure peaks are particularly likely to occur. In this work, a spatially explicit, dynamic model for predicting pesticide exposure in surface waters of cultivated mountain basins (DynAPlus) has been developed. The model has been applied to a small mountain watershed (133 km2) located in the Italian Eastern Alps and characterized by intensive agriculture (apple orchards) around the main river and its tributaries. DynAPlus performance was evaluated for chlorpyrifos through experimental monitoring, using samples collected during the 2011 and 2012 productive seasons. The comparison between predictions and measurements resulted in a good agreement (R2 = 0.49, efficiency factor 0.60), although a more accurate spatial information in the input scenario (e.g., field-specific applications, rainfall amount, soil properties) would dramatically improve model performance. A set of illustrative simulations performed for three PPPs highlighted the potential role of DynAPlus in improving exposure predictions for ecological risk assessment and pesticide management practices (e.g., for active ingredient and application rate selection), as well as for planning efficient monitoring campaigns and/or interpreting monitoring data. However, some model improvements (e.g., solid erosion and transport) and a more thorough model validation are desirable to enlarge the applicability domain.

The effect of pesticide mixtures and electromagnetic fields were evaluated on honeybees in three experimental sites located in northern Italy: a control site far from anthropogenic stress sources, a semi-natural site close to a high voltage electric line and an agricultural site with intensive pesticide treatments. From each experimental site, young workers and foraging bees were taken monthly from May to October and analysed for four enzymatic biomarkers: acetylcholinesterase (AChE), catalase (CAT), glutathione S-transferase (GST) and alkaline phosphatase (AP). Results revealed timeand site-specific effects in respect to control site, confirming the role of biomarkers as diagnostic and early-warning tools for multi-stress sources on honeybees. In the electromagnetic-stress site an effect of an over-activation of all analyzed biomarkers was observed at the end of the season. According to other literature findings, this event was related to a behavioral over-activation in a period in which bees should prepare themselves to overwintering. This finding poses potential problems to winter survival. In the pesticide-stress site, different pesticide-induced responses were identified. We demonstrated in the field that pesticide mixtures, currently used in agriculture, were able to greatly affect biochemical parameters of bees (with both enzymatic under- and over-activations).

Biomarkers have been widely employed in ecotoxicology as early warning indicators of exposure to toxicants. Very often, they are used to compare reference and polluted sites, or to analyse time trends. However, very few studies focus on the natural variability range of biomarkers in the environment, which is pivotal to understand if the detected differences are actually determined by any adverse effects due to pollution. This work assesses the natural spatio-temporal variability of some enzymatic levels, frequently used as biomarkers, in freshwater benthic invertebrates. The influence of some environmental parameters on the enzymatic levels was also evaluated. Three families of insect larvae (Perlidae, Baetidae, and Heptageniidae) were sampled in three pristine streams and in eight different dates. Four enzymes (acetylcholinesterase, glutathione-S-transferase, alkaline phosphatase, and catalase) were measured. The natural variability of enzymatic levels was often significant in all considered species across both space and time. The observed pattern was poorly explained by the monitored environmental parameters. The results of this work show that great care should be paid when interpreting monitoring data in which biomarker levels are measured and compared among sites or dates. Presuming that measured differences are due to anthropogenic factors can be misleading, when other potentially influencing factors have not been accounted for.

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.

The relationship between benthic macroinvertebrates and environmental variables from 42 Italian lakes were analysed with the aim of developing a biotic index. In the investigated lakes, 570 species were found of which 373 belonged to the family of Chironomidae and 85 to the class of oligochaeta. Rare species, those present in less than 10 samples, were excluded from the analysis, leaving 57 species for data analysis. Multivariate analysis: canonical correlation analysis (CANON) and multivariate analysis of variance and covariance (MANCOVA) were carried out on a large database of 1060 sampling points, for which both environmental data (16 chemical and morphometric variables) and 57 species counts from soft bottom samples were available. In addition, a second dataset of 94 sites from small lakes sampled in 2005 in Northern Italy (Lombardy), was analysed for comparison. The data analysis (CANON) from the large database gave the following results: (1) the first canonical variate was related to conductivity, pH and alkalinity and accounted for 17% of the total variation; (2) the second canonical variate was related to total phosphorus, N-NH4 and dissolved oxygen, and accounted for 15% of the total variation; (3) lake maximum depth, volume and water temperature were related to the third canonical variate, which accounted for 14% of the source of variation. The analysis of the database from small lakes revealed that in the plane of the first two canonical variates dissolved oxygen was inversely correlated with the depth of sampling and that the second canonical variate showed an inverse relationship between transparency and nutrients. MANCOVA found evidence for differences between lake types and sampling years, confirmed the CANON results and allowed the quantification of the contribution of different target variables on species composition. A trophic status index was calculated ranking percentage oxygen saturation (O2 %sat), transparency (transp) and total phosphorus (TP). The means of O2 %sat, transp and TP weighted by the species abundances were calculated to have optimum values for each species; a benthic quality index was then obtained by multiplying the weighted means of O2 %sat, transp and TP by species abundances and dividing the product by the total number of specimens present at each station. A significant correlation was observed between trophic status and the benthic quality index. A good agreement was also observed between the indexes calculated using the large database and the second dataset on small lakes.

The effect of sublethal concentrations of potassium dichromate and fenitrothion on sodium-leucine cotransport in brush border membrane vesicles from Chironomus riparius larvae has been investigated. Exposure to potassium dichromate and fenitrothion caused a dose- and time-dependent inhibition of leucine uptake. Transport inhibition is easily detectable at doses 100-fold lower than LD50. Kinetic experiments showed that inhibition was mainly caused by a decrease of the Vmax (680 ± 53 vs. 382 ± 23 and 555 ± 27 nmol/15s/mg protein in control and exposed larvae to K2Cr2O7 and fenitrothion, respectively). Inhibition is possibly related to a variation of sodium ions permeability as evidenced by increased membrane lipid peroxidation. Appropriate control experiments ruled out that the observed differences could be due to changes in general features of membrane preparations. Transport inhibition observed in larvae exposed to potassium dichromate was accompanied by changes in ascorbate peroxidase and dehydroascorbate reductase activities, whereas those exposed to fenitrothion displayed an increase in transaminase activity. The possible value of leucine uptake as biochemical biomarker is briefly discussed. Arch. Insect Biochem. Physiol. 55:90–101, 2004. © 2004 Wiley-Liss, Inc.

Macroinvertebrates are one of the key components of lake ecosystems and are required to be monitored alongside other biological groups to define ecological status according to European Union legislation. Macroinvertebrate communities are highly variable and complex and respond to a diverse series of environmental conditions. The purpose of this study was to examine the relative importance of environmental variables in explaining macroinvertebrate abundance. A total of 45 sub-alpine lakes were sampled for macroinvertebrates in the shallow sublittoral. Environmental variables were grouped into four types: (1) aquatic physical and chemical parameters, (2) littoral and riparian habitat, (3) lake morphometric parameters and (4) sediment chemical characteristics. Nonparametric multiplicative regression (NPMR) was used to model the abundance of individual macroinvertebrate taxa. Significant models were produced for nine out of the 24 taxa examined. Sediment characteristics were the group most frequently included in models and also the factors to which taxa abundance was the most sensitive. Aquatic physical and chemical variables were the next group most frequently included in models although chlorophyll a was not included in any of the models and total phosphorus in only one. This indicates that many taxa may not show a direct easily interpretable response to eutrophication pressure. Lake morphometric factors were included in several of the models although the sensitivity of macroinvertebrate abundance tended to be lower than for sediment and aquatic physical and chemical factors. Habitat factors were only included in three models although riparian vegetation was found to have a significant influence on the abundance of Ephemera danica indicating that ecotone integrity is likely to play a role in its ecology. Overall, the models tended to be specific for species with limited commonality across taxa. Models produced by NPMR indicate that the response of macroinvertebrates to environmental variables can be successfully described but further research is required focussing in more detail on the response of key taxa to relevant environmental parameters and anthropogenic pressures.

Changes in enzyme activities, metabolite concentrations, and membrane transport activity underlying the Chironomus riparius larvae adaptive response to anoxia were investigated. Trehalose, malate, and aspartate degradation and alanine accumulation were recorded. During anoxia exposure, there was a boost of antioxidant defenses as shown by an increase of the specific activity of the enzymes catalase, glutathione-S-transferase, glutathione peroxidase, glutathione-synthase, malic enzyme, and NADP-dependent isocitrate dehydrogenase. The ratio, glutathione reduced over glutathione oxidized, decreased. Except for alanine and catalase, the parameters return to their basal value when larvae are transferred to normoxic conditions. To test whether antioxidant defenses had protective effects on membrane functionality, L-leucine uptake into brush border membrane vesicles and membrane lipid peroxidation was measured. No difference between membranes prepared from larvae exposed to anoxia and control larvae was found. The amino acid alanine, when present inside the vesicles, trans-stimulated leucine uptake. This effect could represent a mechanism to stimulate amino acid uptake and catabolism in vivo when free alanine concentration increases during hypoxic periods.

Leucine transport across brush border membrane vesicles prepared from four insect species common to European freshwater streams has been characterized. The species studied were: Ephemera danica (Ephemeroptera: Ephemeridae), Isoperla grammatica (Plecoptera: Perlodidae), Hydropsyche pellucidula (Trichoptera: Hydropsychidae), and Hybomitra bimaculata (Diptera: Tabanidae). The transport differed among the studied taxa for several features, including pH and sodium dependence, substrate affinity and specificity, and efficiency. In H. pellucidula and E. danica, leucine uptake was higher at pH 7.4 than at more alkaline or acidic pH values, whereas in I. grammatica and H. bimaculata, the uptake was rather constant when pH varied from 5.0 to 7.4, then strongly decreased at pH 8.8. All but E. danica displayed a transient intravescicular leucine accumulation in the presence of sodium, suggesting the existence of a cation-leucine symport mechanism. The sodium dependence ranged according to the following order: H. pellucidula > I. grammatica > H. bimaculata > E. danica. Moreover, in H. pellucidula and I. grammatica, the sodium-dependence was stronger at pH 8.8 than at pH 7.4. In E. danica, leucine uptake was sodium-independent at all pH values. The highest value of Vmax (45.3 pmol·s–1·mg proteins–1) was in E. danica, which, however, displayed the lowest affinity (Km 137 μM) when compared to the kinetic parameters of other taxa. The Vmax and Km values were: 40 and 52.5, 32.1 and 12.5, and 4.5 and 230 for H. bimaculata, H. pellucidula, and I. grammatica, respectively. The obtained results are discussed within our current knowledge of amino acid transport systems in insects. Arch. Insect Biochem. Physiol. 63:110–122, 2006. © 2006 Wiley-Liss, Inc.

The freshwater scud Echinogammarus stammeri is a very abundant benthic crustacean in riverine systems of northern Italy. The molecular physiology of L-[3H]leucine transport across cell membranes of E. stammeri was investigated. At pH 7.4, the presence of a NaCl gradient stimulated the leucine transport rate, when compared with KCl gradient. By contrast, alkaline pH strongly inhibited transport activity. In NaCl medium at acidic pH, leucine uptake displayed a specific activity 2.1-fold lower than that measured at neutral pH. Analysis of the uptake as a function of external leucine concentration revealed saturation kinetics. Leucine uptake appeared broadly unspecific and it was much strongly inhibited by the non-polar amino acids phenylalanine, methionine, and BCH, as well as by hydrophobic hydroxyacids. These results revealed important differences with respect to amino acid transport systems described in marine crustaceans. Their relevance in the feeding adaptation of the scud is briefly discussed.

Background and aim. Large interferon-based therapeutic trials are still lacking in children with hepatitis C and the long-term safety and efficacy of interferon is unknown. This study describes the outcome of hepatitis C in 43 children enrolled in an open-label interferon trial, and were followed up to 66 months after stopping treatment. Patients and methods. All patients received interferon alfa2a (5 MU/m 2) thrice weekly for 6 months; children with genotype 1b received 3 MU/m 2 thrice weekly for 6 additional months. Results. Nine children discontinued interferon for adverse events and three were not compliant to treatment. Eight (19%, intention to treat analysis), including 2/20 (10%) with genotype 1b and 6/12 (50%) with genotypes 2 or 3, were sustained responders 12 months after stopping therapy. During further follow-up (mean ± S.D.: 44.7 ± 14.6 months), response was maintained; two non-responders cleared viremia, while a young boy progressed to cirrhosis. Conclusions. Small sample size and therapy withdrawal are the major limitations in the interpretation of our results. Nevertheless, our data, suggesting that response to interferon in children with hepatitis C is genotype-related and stable, agree with the results of large studies in adults. The outcome in non-responders was variable, including persistence of viremia and mild–moderate cytolysis (most cases), progression to cirrhosis, or eventual sustained viremia clearance.

Benthic macroinvertebrates are an essential component of freshwater environments. The ecotoxicological risk of benthic communities, estimated through the assay of biochemical markers, can be used as an early warning signal for environment alterations. In this work the activities of a number of enzymes, regarded as potential biomarker of exposure to pollutants (catalase, acetylcholinesterase, glutathione-S-transferase) was determined in homogenates of whole organisms. Specimens were collected in the rivers Taro and Ticino, northern Italy, in stony bottom reaches in five and three stations respectively. The orders of aquatic Insects Diptera, Plecoptera, Odonata, Ephemeroptera and Trichoptera, the Crustacea Amphipoda, and Annelida Oligochaeta were examined. Additional enzymes, such as NADP-dependent malate dehydrogenase, isocitric dehydrogenase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, and lactate dehydrogenase were considered in the study. Results emphasize significant differences among taxa concerning the specific activity of most enzymes.

Leucine uptake into membrane vesicles from larvae of the midge Chironomus riparius was studied. The membrane preparation was highly enriched in typical brush border membrane enzymes and depleted of other membrane contaminants. In the absence of cations, there was a stereospecific uptake of l-leucine, which exhibited saturation kinetics. Parameters were determined both at neutral (Km 33 +/- 5 microM and Vmax 22.6 +/- 6.8 pmol/7s/mg protein) and alkaline (Km 46 +/- 5 microM and Vmax 15.5 +/- 2.5 pmol/7s/mg protein) pH values. At alkaline pH, external sodium increased the affinity for leucine (Km 17 +/- 1 microM) and the maximal uptake rate (Vmax 74.0 +/- 12.5 pmol/7s/mg protein). Stimulation of leucine uptake by external alkaline pH agreed with lumen pH measurements in vivo. Competition experiments indicated that at alkaline pH, the transport system readily accepts most L-amino acids, including branched, unbranched, and alpha-methylated amino acids, histidine and lysine, but has a low affinity for phenylalanine, beta-amino acids, and N-methylated amino acids. At neutral pH, the transport has a decreased affinity for lysine, glycine, and alpha-methylleucine. Taken together, these data are consistent with the presence in midges of two distinct leucine transport systems, which combine characters of the lepidopteran amino acid transport system and of the sodium-dependent system from lower neopterans.