The Gut Microbiota of Farmed and Wild Brook Trout (Salvelinus fontinalis): Evaluation of Feed-Related Differences Using 16S rRNA Gene Metabarcoding.

The gut microbiota has become a topic of increasing importance in various fields, including aquaculture. Several fish species have been the subject of investigations concerning the intestinal microbiota, which have compared different variables, including the intestinal portions, the environment, and diet. In this study, the microbiota of farmed and wild brook trout (Salvelinus fontinalis) were analyzed, in which the wall and content of the medial portion of the intestine were considered separately. A total of 66 fish (age class 2+) were sampled, of which 46 were wild and 20 were farmed brook trout, in two different years. Microbiota data were obtained using a 16S metabarcoding approach by analyzing the V3-V4 hypervariable regions of the corresponding 16S rRNA. The data showed that the core microbiota of these species consist of Proteobacteria (Alpha- and Gammaproteobacteria), Actinobacteria, Firmicutes (Bacilli and Clostridia), and, only for farmed animals, Fusobacteria. The latter taxon's presence is likely related to the fishmeal-based diet administered to farmed brook trout. Indeed, alpha and beta diversity analysis showed differences between wild and farmed fish. Finally, statistically significant differences in the microbiota composition were observed between the intestinal walls and contents of wild fish, while no differences were detected in reared animals. Our work represents the first study on the intestinal microbiota of brook trout with respect to both farmed and wild specimens. Future studies might focus on the comparison of our data with those pertaining to other fish species and on the study of other portions of the brook trout intestine.

Metabolic rate and climate change across latitudes: evidence of mass-dependent responses in aquatic amphipods.

Predictions of individual responses to climate change are often based on the assumption that temperature affects the metabolism of individuals independently of their body mass. However, empirical evidence indicates that interactive effects exist. Here, we investigated the response of individual standard metabolic rate (SMR) to annual temperature range and forecasted temperature rises of 0.6-1.2°C above the current maxima, under the conservative climate change scenario IPCC RCP2.6. As a model organism, we used the amphipod Gammarus insensibilis, collected across latitudes along the western coast of the Adriatic Sea down to the southernmost limit of the species' distributional range, with individuals varying in body mass (0.4-13.57 mg). Overall, we found that the effect of temperature on SMR is mass dependent. Within the annual temperature range, the mass-specific SMR of small/young individuals increased with temperature at a greater rate (activation energy: E=0.48 eV) than large/old individuals (E=0.29 eV), with a higher metabolic level for high-latitude than low-latitude populations. However, under the forecasted climate conditions, the mass-specific SMR of large individuals responded differently across latitudes. Unlike the higher-latitude population, whose mass-specific SMR increased in response to the forecasted climate change across all size classes, in the lower-latitude populations, this increase was not seen in large individuals. The larger/older conspecifics at lower latitudes could therefore be the first to experience the negative impacts of warming on metabolism-related processes. Although the ecological collapse of such a basic trophic level (aquatic amphipods) owing to climate change would have profound consequences for population ecology, the risk is significantly mitigated by phenotypic and genotypic adaptation.

Microplastics accumulation in functional feeding guilds and functional habit groups of freshwater macrobenthic invertebrates: Novel insights in a riverine ecosystem.

Microplastics pose a major threat for aquatic ecosystems, but the contamination dynamics in organisms inhabiting freshwater ecosystems is still little studied. Largely used for biomonitoring, macrobenthic invertebrates provide a pivotal trophic resource for many fish and bird species. In this study, we investigated the microplastics contamination in a macrobenthic invertebrate community (2772 individuals belonging to 33 taxa identified) in a high-plain riverine ecosystem (Vipacco River, northeast Italy) and compared the amount of microplastics accumulated in functional feeding guilds/functional habit groups. Microplastics (cellulosic fibers associated with polyester) were found in 48.5% of the taxa, with the highest amount detected in the collector-gatherers, followed by predators. The collector-gatherers showed a significantly higher microplastic accumulation than the other functional feeding guilds, whereas there was no difference among the functional habit groups. The main source of microplastics pollution was most likely urban wastewater discharge points located along the river. Our study reports a novel approach about microplastic pollution assessment in lotic environments, as it focuses into the microplastic contamination dynamics in an entire macrobenthic invertebrate community perspective and underlines the need for further study.

Lethal and Sublethal Responses of Hydropsyche pellucidula (Insecta, Trichoptera) to Commercial Polypropylene Microplastics after Different Preconditioning Treatments.

Microplastics (MPs) pose biological and chemical hazards in aquatic and terrestrial food webs across the globe. Research on microplastic contamination has long focused on marine ecosystems, whereas the toxicological impact on freshwater organisms is still little explored. In this study, the lethal and sublethal response of the freshwater macroinvertebrate Hydropsyche pellucidula exposed to polypropylene MPs after different pre-conditioning treatments was assessed. Field samples were collected in a riverine system (Vipacco river; northeast Italy) to assess the characteristics of the MPs in the aquatic environment Both water and sediment were contaminated by MPs (3.73 ± 2.11 items m-3 per min and 3.33 ± 4.16 items dm-3, respectively). The chemical MPs composition included polystyrene, polyethylene terephthalate, polyurethane, polyamide, polypropylene, and polyethylene. Polypropylene (PP), although not the most abundant polymer recorded in the study area, was preferred over the other types according to its abundance in freshwater and H. pellucidula feeding behavior. A housing test was performed to recreate the natural conditions of larvae sampled for a reliable response to the ecotoxicological tests. The microplastics underwent either preconditioning with Vipacco River water (PP-river) and surfactant Triton X-100 (PP-sf) or no pre-treatment (PP). Submersion of microplastics in 10 µg L-1 of surfactant solution for 24 h was sufficient to induce consistent spectral changes and modify the chemical profile of the plastic surface. Mortality rate differed according to treatment: PP and PP-river > positive control > PP-sf > negative control. Integrated biomarker response (IBRv2) and analysis of oxidative stress biomarker levels showed a greater response of superoxide dismutase and lipid peroxidation (malondialdehyde) in larvae treated with PP conditioned in surfactant. Our findings enhance knowledge on the toxicity of PP and conditioning phases on H. pellucidula larvae.

Relationship between the prevalence of Dibothriocephalus latus (Cestoda: Diphyllobothriidea) and the load of Escherichia coli: New findings in a neglected fish-borne parasitic zoonosis.

The sub-Alpine lakes of Switzerland, Italy and France have long been reported as an endemic area of diphyllobothriosis, a parasitic zoonosis caused by Dibothriocephalus latus. With this study, we explored the hypothesis for a relationship between the prevalence of D. latus in Perca fluviatilis and the Escherichia coli load in lake water. To do this, we identified eleven sampling sites in three areas (north, centre and south) of Lake Iseo (north Italy) to determine E. coli load and the prevalence of D. latus in P. fluviatilis. Prevalence and 95% confidence interval (CI) of D. latus infestation ranged from 0% (95% CI: 0.71-0.0) in Sarnico (southern area) to 20% (95% CI: 33.0-11.2) in Pisogne (northern area). There were significant differences in prevalence between the sites (χ2  = 31.12; p-value = .0006) and in E. coli load (Kruskal-Wallis test; p-value = .0005). There was decreasing gradient of E. coli load and prevalence of D. latus infestation from north to south. A significant positive correlation (r = .881; p-value = .003) was found between E. coli load and prevalence of D. latus. Also, linear regression showed a significant relationship between E. coli load and prevalence of infestation (R2  = .775). Our findings offer an explanation for the link between E. coli load in water and D. latus prevalence. The potential factors in this link are the efficiency of the local wastewater treatment plant, the bathymetric profile of the lake and the life cycle of D. latus, which is mainly affected by light and water temperature.

Ecology of oxidative stress in the Danube barbel (Barbus balcanicus) from a winegrowing district: Effects of water parameters, trace and rare earth elements on biochemical biomarkers.

Fish can be highly vulnerable to environmental pressures because they are exposed to oxidative stressors in the aquatic environment. Such stressors can affect the levels of antioxidant biomarkers against reactive oxygen species (ROS). With this study we investigated the oxidative stress ecology in Danube barbel (Barbus balcanicus) from the Barbucina creek (northeast Italy), a watercourse in the Collio winegrowing district. To do this, superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) activity was measured in gills, liver, and muscle, while metallothioneins (MT) and trace and rare earth elements (REEs) levels were determined in muscle. The effect of environmental factors (physicochemical parameters of water, trace elements and REEs) on oxidative stress biomarkers was thus assessed. High concentrations were determined for cerium (Ce), scandium (Sc), neodymium (Nd), lanthanum (La), yttrium (Y), and praseodymium (Pr) among the REEs. Among the trace elements, arsenic (As), copper (Cu), and mercury (Hg) levels were higher compared to published data, suggesting their role as stressors. The multiple linear regression (MLR) model showed a statistically significant association (R2 = 0.858; F = 10.07; p = 0.015) between As, Cu, Hg, and Pr and SOD activity in the gills, indicating a functional relationship between them. Differently, CAT activity was significantly higher in the liver, probably in response to long-term Cu contamination of the watercourse. This was confirmed by the MLR model that showed a significant association (R2 = 0.638; F = 8.152; p = 0.02) between the concentration of MT and of Cu. Our data show a biochemical defensive response by Danube barbel to the disturbances in the aquatic ecosystem of the Barbucina creek. These insights advance our understanding of the role and the effects of environmental factors as trace elements and REEs on oxidative stress in fish.

The unusual isolation of carnobacteria in eyes of healthy salmonids in high-mountain lakes.

Carnobacteria are common bacteria in cold and temperate environments; they are also reported during fish mortality events. In a previous study, carnobacteria were isolated from the eyes of healthy wild salmonids from a high-mountain lake. To better understand these findings, salmonids were captured from three high-mountain lakes (Lower and Upper Balma Lake, Rouen Lake; northwest Italy) during August 2019 and subjected to bacteriological and histological examination. Although all were healthy, 8.7% (Lower Balma Lake), 24% (Upper Balma Lake), and 32.6% (Rouen Lake) were positive for carnobacteria colonization of the eyes. A Trojan-horse effect was hypothesized to explain carnobacteria isolation in the eye. This immune-escaping macrophage-mediated mechanism has been identified in other Gram-positive bacteria. Biochemical, molecular, and phylogenetic analysis were carried out on isolated bacteria (Carnobacterium maltaromaticum and C. divergens). Based on previous references for carnobacteria isolated from fish, C. maltaromaticum strains were tested for the pisA precursor gene of the bacteriocin piscicolin 126. Carnobacterium maltaromaticum strains were found to display genotypic heterogeneity and a low percentage of pisA positive amplification. Features of geomorphology, geographic isolation, and microbiota common to the three lakes are thought to be possibly related to our findings. Moreover, terrestrial insects collected from the lake shoreline and the stomach contents were screened for the presence of carnobacteria. The salmonids in these high-mountain environments feed mainly on terrestrial insects, which are considered possible vectors for carnobacteria that might catabolize the exoskeleton chitin. All insects tested negative for carnobacteria, but as a small number of samples were analyzed, their role as possible vectors of infection cannot be excluded. Further studies are needed to corroborate our research hypothesis.

First Insights Into Trace Element Accumulation by Philoscia affinis (Crustacea, Isopoda): a Novel Tracer to Assess Soil Contamination in Lowland Plains?

Isopods are terrestrial invertebrates that accumulate trace elements in large quantities, thus providing information on levels of soil contamination. However, the accumulation pattern seems to be species dependent. For this study, specimens of Philoscia affinis (Isopoda, Oniscidea) and soil samples were collected from both a protected area (site 1) and urban roadside (site 2) in the low plain of Friuli-Venezia Giulia (northeast Italy) to determine whether P. affinis could serve as a potential candidate for monitoring soil contamination. To do this, the following objectives were achieved: a) the level of trace elements (Al, Cd, Cu, Fe, Hg, Mn, Pb, Zn) were detected in soils and isopods; 2) the difference in trace elements accumulation was compared in the two sampling sites; 3) the bioaccumulation factor (BAF) was calculated for each element. With some exceptions, trace element concentrations were higher in both isopods and soil samples from the urban roadside compared to the protected area. Furthermore, except for Cd, Cu, and Zn, trace element levels were higher in the soil than in the isopod samples. The higher mean BAF values were recorded for Cd (6.169 and 6.974 for site 1 and 2, respectively), Cu (10.324 and 11.452 for site 1 and 2, respectively), and Zn (1.836 and 2: 1.943 for site 1 and 2, respectively), whereas BAF values <1 were recorded for the other elements. Philoscia affinis was found to be a potential candidate to monitor soil contamination as a macro-concentrator of Cu and Cd and a micro-concentrator of Zn.

First insights into plastic and microplastic occurrence in biotic and abiotic compartments, and snow from a high-mountain lake (Carnic Alps).

Plastic pollution has become a pervasive environmental problem on a global scale, from the ocean depths to the aquatic ecosystems of the Tibetan Plateau. To date, data on plastic and microplastic occurrence in pristine ecosystems like high-mountain lakes are lacking. In this study, plastic (>5000 µm) and microplastic (10-5000 µm) levels were measured in snow at the end of the winter season (April 2020), and in water, sediment, and biological samples collected monthly (June-October 2019) during the ice-free season from the Dimon Lake, a high-mountain lake in the Carnic Alps, northeast Italy. Biological samples consisted of chironomids (Diptera, Chironomidae; n = 150) and stomach contents of Cottus gobio (n = 40). Analysis of the water, sediment, and biological samples revealed the absence of plastic and microplastics larger than 10 µm, whereas the snow samples contained microplastics of polyethylene terephthalate (PET) albeit at very low levels (0.11 ± 0.19 L-1). These results show that while the lake ecosystem could be considered unpolluted by microplastics, abundant snow precipitation in winter can trap microplastic particles that deposit on the ground. The very low levels of PET microparticles recorded in the snow samples suggest the need for further research to better understand the source of microplastic pollution in this environmental matrix.

Long-Term Assessment of Baseline Blood Biochemistry Parameters in Rainbow Trout (Oncorhynchus mykiss) Maintained under Controlled Conditions.

Twelve biochemical parameters were measured in serum blood samples from rainbow trout (Oncorhynchus mykiss) maintained under controlled conditions. Forty-five samples were taken every 3 months (T1-T4) over the course of one year to define baseline values. The effect of fish total weight (TW) was also evaluated. Principal component analysis showed a clear separation between T4 and T1, and T2 and T3, indicating an increase in certain biochemical parameters with weight. Linear regression analysis showed how TW significantly explained 11-67% of the variability observed for nine parameters out of 12. Pearson's correlation matrix showed a significant positive correlation (p < 0.05) between TW and albumin, alkaline phosphatase, alanine-aminotransferase, creatinine, gamma-glutamyl transferase, magnesium, phosphorus and total protein. Furthermore, significant correlations (p < 0.05) between the majority of the biochemical parameters were found, indicating that growth can influence several parameters at the same time. The present study provides several useful baseline values for assessing the health of O. mykiss, indicating that fish weight is an important factor for interpreting the blood biochemical profile.

Occurrence and Spatial Distribution of Dibothriocephalus Latus (Cestoda: Diphyllobothriidea) in Lake Iseo (Northern Italy): An Update.

Dibothriocephalus latus (Linnaeus, 1758) (Cestoda: Diphyllobothriidea; syn. Diphyllobothrium latum), is a fish-borne zoonotic parasite responsible for diphyllobothriasis in humans. Although D. latus has long been studied, many aspects of its epidemiology and distribution remain unknown. The aim of this study was to investigate the prevalence, mean intensity of infestation, and mean abundance of plerocercoid larvae of D. latus in European perch (Perca fluviatilis) and its spatial distribution in three commercial fishing areas in Lake Iseo (Northern Italy). A total of 598 specimens of P. fluviatilis were caught in 2019. The total prevalence of D. latus was 6.5%. However, there were significant differences between areas (10.2% North; 7.3% Center; 1.5% South) (Chi-square test, p = 0.0018). The mean intensity of infestation ranged from 1 larva in southern area to 1.2 larvae in both the central and northern (Pisogne) areas. In addition, the mean abundance ranged from 0.02 in the southern area to 0.26 in the northern area (Pisogne). The total number of larvae (anterior dorsal-AD = 21; anterior ventral-AV = 1; posterior dorsal-PD = 15; posterior ventral-PV = 5) differed significantly between the four anatomical quadrants (Kruskal-Wallis test; p = 0.0001). The prevalence of D. latus plerocercoid larvae in European perch from Lake Iseo has long been investigated, but without an appropriate sampling design. With the present study, a broader analysis in spatial distribution has been added to the existing literature, revealing new information about D. latus distribution and occurrence in Lake Iseo, with new data that will be useful for health authorities and future studies.

First Occurrence of Eustrongylides spp. (Nematoda: Dioctophymatidae) in a Subalpine Lake in Northwest Italy: New Data on Distribution and Host Range.

The genus Eustrongylides includes nematodes that infect fish species and fish-eating birds inhabiting freshwater ecosystems. Nematodes belonging to the genus Eustrongylides are potentially pathogenic for humans; infection occurs after the consumption of raw or undercooked fish. In the two-year period 2019-2020, a total of 292 fish belonging to eight species were examined for the occurrence of Eustrongylides spp. from Lake San Michele, a small subalpine lake in northwest Italy. The prevalence of infestation was 18.3% in Lepomis gibbosus, 16.7% in Micropterus salmoides, and 10% in Perca fluviatilis. The other five fish species (Ameiurus melas, Ictalurus punctatus, Squalius cephalus, Carassius carassius, and Scardinius erythrophthalmus) were all negative for parasite presence. There were no significant differences in prevalence between the three fish species (Fisher's exact test; p = 0.744). The mean intensity of infestation ranged from 1 (M. salmoides and P. fluviatilis) to 1.15 (L. gibbosus), and the mean abundance ranged from 0.1 (P. fluviatilis) to 0.28 (L. gibbosus). There were significant differences in the infestation site between the four muscle quadrants (anterior ventral, anterior dorsal, posterior ventral, and posterior dorsal) and the visceral cavity (Kruskal-Wallis test; p = 0.0008). The study findings advance our knowledge about the distribution and host range of this parasite in Italy.

Accumulation of As, Cd, Pb, and Zn in sediment, chironomids and fish from a high-mountain lake: First insights from the Carnic Alps.

Though mountain lakes are generally much less influenced by human activities than other habitats, anthropogenic threats can still alter their natural condition. A major source of global environmental pollution in mountain ecosystems is trace element contamination. For this study we investigated for the first time the accumulation of As, Cd, Pb, and Zn in sediment, Diptera Chironomidae (prey), and bullhead Cottus gobio (predator) in a typical high-mountain lake (Dimon Lake) in the Carnic Alps. Significant differences in trace element levels were observed between sediment, Diptera Chironomidae, and C. gobio liver and muscle samples (Kruskal-Wallis test; p < .03 for all elements). As and Pb levels were highest in sediment, Cd and Zn levels were highest in Diptera Chironomidae, and the lowest values for all elements were measured in C. gobio muscle and liver. Bioaccumulation factor values were much higher in Diptera Chironomidae than fish muscle and liver, with the highest values recorded for Cd (5.16) and Zn (4.37). Trophic transfer factor values were very low for all elements in fish muscle and liver, suggesting a biodilution effect along the food chain. Further studies are needed to expand on these first findings that provide useful insights to inform environmental monitoring and policy in remote high-mountain lakes.

First Report of Clinostomum complanatum (Trematoda: Digenea) in European Perch (Perca fluviatilis) from an Italian Subalpine Lake: A Risk for Public Health?

Clinostomum complanatum, a digenean trematode of the Clinostomidae family, is a fish-borne zoonotic parasite responsible for Halzoun syndrome in humans and is transmitted through the consumption of raw or undercooked freshwater fish. Of the total of 112 specimens of European perch (Perca fluviatilis) sampled from a subalpine lake (Lake Endine) in North Italy in 2019, 21 (18.75%) tested positive for encysted metacercariae in the fillet. This study reports the first isolation of C. complanatum in P. fluviatilis and highlights the possible zoonotic risk for consumers, since P. fluviatilis is a food fish used in the traditional local cuisine.

Oxidative stress ecology in brook trout (Salvelinus fontinalis) from a high-mountain lake (Cottian Alps).

High-mountain lakes are pristine ecosystems characterized by extreme environmental conditions. The atmospheric transport of pollutants from lowlands may add further stress to organisms inhabiting these environments. We investigated the environmental stress pressure on brook trout (Salvelinus fontinalis) from a high-mountain lake in the Cottian Alps (Piedmont, northwest Italy). To do this, males and females of brook trout were sampled from Balma Lake in summer (August) and autumn (October) 2017 in order to assess the influence of trace elements accumulation and environmental parameters (physicochemical parameters and nutrient characteristics of water) on oxidative stress biomarkers. Bioaccumulation of Al, As, Cd, Cr, Cu, Fe, Hg, Pb, Ni, Se, and Zn and metallothionein levels were measured in muscle tissue of males and females. Liver, gills, kidney, and spleen tissue samples were analyzed for superoxide dismutase, catalase, total glutathione peroxidase, selenium-dependent glutathione peroxidase, glutathione reductase, and glutathione S-transferase activity. Analysis of environmental parameters showed changes in biomarker levels with seasonal variations. Water temperature was significantly higher in summer than autumn (Wilcoxon test; p = .0078), while pH was significantly higher in autumn than in summer (Wilcoxon test; p = .0078). Sex-related differences in oxidative stress biomarkers in tissues were unremarkable, whereas seasonal variability of oxidative stress biomarkers was observed, with major differences occurred for liver in summer and for gills, kidney, spleen and muscle in autumn. Positive correlations between environmental parameters and biomarkers were noted. Major fluctuations in water temperature, pH, Cu, Pb and Hg produced changes in biomarker levels; however, increased food intake during the ice-free season was probably the main factor that influenced changes in oxidative stress biomarker levels in brook trout in this extreme ecosystem.

Macrobenthic invertebrates as tracers of rare earth elements in freshwater watercourses.

Rare earth elements (REEs) are emergent contaminants in aquatic ecosystems in parallel with their growing use in science, technology, and industry. In this study we measured the concentration of 16 REEs in freshwater marcobenthic invertebrates from 6 watercourses in northeast Italy to determine their potential use as ecological tracers of REEs in aquatic ecosystems. The total REE concentration at the sampling sites followed this order: site 6 (7.05 mg Kg-1) > site 3 (5.76 mg Kg-1) > site 4 (3.58 mg Kg-1) > site 1 (3.0 mg Kg-1) > site 5 (2.36 mg Kg-1) > site 2 (1.95 mg Kg-1). There were no significant differences in REE concentrations across the six samplings sites (Kruskal Wallis test, p = 0.1773), but two (site 3 and 6) had higher amount of REEs and were classified with the ecological status "Moderate" sensu Water Framework Directive since affected by anthropogenic activities. Light REE were always greater than heavy REE concentrations at all six sites. A positive correlation was observed between certain REEs (La, Ce, Gd) and the density of genera Caenis and Baetis (Ehemeroptera, collector-gatherers) (ρS range 0.795-0.812), suggesting that non-predatory macrobenthic invertebrates accumulate more REEs than predatory organisms and that the intake of sediment is the most effective route of assimilation.

Macrobenthic invertebrates as bioindicators of trace elements in high-mountain lakes.

Alpine lakes are extreme ecosystems located in remote areas and populated by few but well-adapted species. Because of their remote location, they are often considered pristine, unpolluted ecosystems. Since the 1980s, however, they have been affected by global anthropogenic impacts. Macrobenthic invertebrates play a pivotal role in these ecosystems and can be used as bioindicators also for monitoring the accumulation of trace elements. We characterized the macrobenthic invertebrates community of Balma Lake (Cottian Alps, Northwest Italy) and Dimon Lake (Carnic Alps, Northeast Italy) in summer and autumn and measured the levels of nine trace elements (As, Cd, Cr, Cu, Fe, Ni, Pb, Se, Zn) in the most abundant taxa (Chironomidae and Oligochaeta in both lakes and Hirudinea in Dimon Lake) in both seasons. The highest levels of trace elements were recorded for Fe, Cu, and Zn according to their environmental availability and their function as essential elements. The total amount of trace elements was highest for the Chironomidae from both lakes compared to the other two taxa. As, Cd, Pb, and Zn were measured in sediment to calculate bioaccumulation factor (BAF) values. The amount of elements in sediment and macrobenthic invertebrates was higher for Dimon Lake, suggesting a greater flux via precipitation of contaminants from the lowland. The BAF values were decreased with increasing trace elements concentration in sediment, indicating mechanisms of elements excretion in biota where the environment is contaminated. This study is the first to report on the use of macrobenthic invertebrates to monitor trace elements in Alpine lakes.