Effect of emerging pollutants on the gut microbiota of freshwater animals: Focusing on microplastics and pesticides.

In recent years, emerging environmental pollutants have increasingly endangered the health of freshwater organisms. The gut microbiota exhibits sensitivity to medications, dietary factors and environmental pollutants, rendering it a novel target for toxicological studies. The gut microbiota can be a potential exposure route affecting the host's health. Herein, we review the current knowledge on two different but concurrent pollutants, microplastics and pesticides, regarding their impact on the gut microbiota, which includes alterations in microbial composition, gene expression, function, and health effects in the hosts. Moreover, synergetic interactions between microplastics and pesticides can exacerbate dysbiosis and health risks. We discuss health-related implications of gut microbial changes based on the consequences in metabolism, immunity, and physiology function. Further research is needed to discover the mechanisms underlying these effects and develop strategies for mitigating their harmful impacts on freshwater animals.

From Personal Care to Coastal Concerns: Investigating Polyethylene Glycol Impact on Mussel's Antioxidant, Physiological, and Cellular Responses.

Pharmaceutical and personal care products (PPCPs) containing persistent and potentially hazardous substances have garnered attention for their ubiquitous presence in natural environments. This study investigated the impact of polyethylene glycol (PEG), a common PPCP component, on Mytilus galloprovincialis. Mussels were subjected to two PEG concentrations (E1: 0.1 mg/L and E2: 10 mg/L) over 14 days. Oxidative stress markers in both gills and digestive glands were evaluated; cytotoxicity assays were performed on haemolymph and digestive gland cells. Additionally, cell volume regulation (RVD assay) was investigated to assess physiological PEG-induced alterations. In the gills, PEG reduced superoxide dismutase (SOD) activity and increased lipid peroxidation (LPO) at E1. In the digestive gland, only LPO was influenced, while SOD activity and oxidatively modified proteins (OMPs) were unaltered. A significant decrease in cell viability was observed, particularly at E2. Additionally, the RVD assay revealed disruptions in the cells subjected to E2. These findings underscore the effects of PEG exposure on M. galloprovincialis. They are open to further investigations to clarify the environmental implications of PPCPs and the possibility of exploring safer alternatives.

Fitness assessment of Mytilus galloprovincialis Lamarck, 1819 after exposure to herbicide metabolite propachlor ESA.

The lack of data on the chronic effects of chloroacetanilide herbicide metabolites on non-target aquatic organisms creates a gap in knowledge about the comprehensive impacts of excessive and repeated pesticide use. Therefore, this study evaluates the long-term effects of propachlor ethanolic sulfonic acid (PROP-ESA) after 10 (T1) and 20 (T2) days at the environmental level of 3.5 µg.L-1 (E1) and its 10x fold multiply 35 µg.L-1 (E2) on a model organism Mytilus galloprovincialis. To this end, the effects of PROP-ESA usually showed a time- and dose-dependent trend, especially in its amount in soft mussel tissue. The bioconcentration factor increased from T1 to T2 in both exposure groups - from 2.12 to 5.30 in E1 and 2.32 to 5.48 in E2. Biochemical haemolymph profile and haemocyte viability were not affected by PROP-ESA exposure. In addition, the viability of digestive gland (DG) cells decreased only in E2 compared to control and E1 after T1. Moreover, malondialdehyde levels increased in E2 after T1 in gills, and DG, superoxidase dismutase activity and oxidatively modified proteins were not affected by PROP-ESA. Histopathological observation showed several damages to gills (e.g., increased vacuolation, over-production of mucus, loss of cilia) and DG (e.g., growing haemocyte trend infiltrations, alterations of tubules). This study revealed a potential risk of chloroacetanilide herbicide, propachlor, via its primary metabolite in the Bivalve bioindicator species M. galloprovincialis. Furthermore, considering the possibility of the biomagnification effect, the most prominent threat poses the ability of PROP-ESA to be accumulated in edible mussel tissues. Therefore, future research about the toxicity of pesticide metabolites alone or their mixtures is needed to gain comprehensive results about their impacts on living non-target organisms.

Multi-characteristic toxicity of enantioselective chiral fungicide tebuconazole to a model organism Mediterranean mussel Mytilus galloprovincialis Lamarck, 1819 (Bivalve: Mytilidae).

The survey of available scientific literature shows a lack of data on the chronic effects of tebuconazole (TEB) on non-target aquatic organisms. Therefore, this study evaluates toxicity (10 and 20 days) of two considered concentrations 2 ng/L (E1) and 2 µg/L (E2) of TEB to bioindicator species Mytilus galloprovincialis. To this end, the TEB concentrations measured in soft mussel tissues showed a time-dependent increasing trend. The viability of haemocyte and digestive gland (DG) cells was higher than 95 % during the experiment. However, DG cells lost the ability to regulate their volume in both groups after 20-d. The E1 treatment increased Cl- and Na+ levels, and E2 decreased Na+ levels in the haemolymph. In addition, levels of superoxide dismutase (SOD) activity and oxidatively modified protein (OMP) increased after 10- and 20-d in both treatments. Histopathological findings showed abnormalities in the E2, e.g., haemocyte infiltration, hypertrophy, and hyperplasia in gills and DG. This study reveals the potential risks of TEB usage in the model organism M. galloprovincialis, primarily via bioaccumulation of TEB in food web links, and improves knowledge about its comprehensive toxicity.

Stress related blood values in Scyliorhinus canicula as live-indicators of physiological status after bottom trawling capture activity.

The quantification of capture-related physiological stress is an important factor when assessing the potential for post-release survival in sharks that are incidentally captured. In the absence of these biological data and when the post-release fate is unknown, effective management plans cannot be formulated and may lead to highly susceptible shark populations being overfished. Here, we measured the levels of lactate, glucose, alanine amino transferase (ALT), aspartate amino transferase (AST), Ca2+, Na+ K+,Cl - Mg 2+ and Pi in the plasma of mature and immature lesser spotted dogfish (Scyliorhinus canicula, herein dogfish) which were incidentally captured at two depths (shallow: 50-200 m, and deep: 201-500 m) by bottom trawl off the coast of southern Sicily. These values were used as biomarkers and physiological indicators of the secondary stress response associated with capture. This study found that dogfish captured in deeper waters (below 200 m) had elevated levels of glucose, Na+, Ca2+ and K+ compared to those inhabiting depths less than <200 m. We hypothesize that the elevated levels of physiological stress in dogfish captured at greater depths may be related to the prolonged duration of the interactions with the fishing gear in the area off southern Sicily. Our findings provide new data on the capture-related stress in dogfish and increase the understanding of the potential for post-release survival in sharks captured at two depths by bottom trawl, information that is important for improving the general management plans for the fishery. However, our PC Analysis results revealed that Maturity have a positive contribution from the sample weight, sample length, ALT, AST and a negative contribution from Pi.

Preliminary Study on the Host Response to Bivalent and Monovalent Autogenous Vaccines against Mycoplasma agalactiae in Dairy Sheep.

In Italy, dairy sheep farming represents a vital agro-industry sector, but it is still challenged by contagious agalactia (CA), which is endemic there, and vaccination is the most economical and sustainable tool for control. This study aimed to evaluate the combined Mycoplasma agalactiae (Ma)-Staphylococcus aureus (Sa) vaccine (Ma-Sa) against the Ma monovalent vaccine in ewes. Twelve primiparous Ma-free ewes were randomly grouped into three equal groups: first, the control group injected with placebo, second, the group vaccinated with the Ma monovalent vaccine, and third, the group vaccinated with Ma-Sa combined vaccine, with two S/C doses at 45-day intervals. The animals were examined for serological, hematological, and somatic cell count (SCC) changes for 17 successive weeks. A significant increase in anti-Ma antibody mean titers, leukocytes, and platelets was observed in the vaccinated animals, with the highest values in those who received the combined vaccine. Neutrophils were high only in the animals who received the combined vaccine. SCC was lower in the vaccinated animals during the first six weeks. This study concludes that the combined Ma-Sa vaccines enhance immune response and potentiate its efficacy against Ma. This improvement might be attributed to the sensitization/activation effect of S. aureus on platelets, which are recoded to act as a key regulator for the coordination of all components of the innate immune system. Even though this study included a small number of animals, its findings about the potentialities of this inactivated vaccine in the control of CA are strongly encouraging. Further confirmation might be needed through additional replicates and a challenge study is needed before proceeding with widespread use.

Serum Cortisol and Its Correlation with Leucocyte Profile and Circulating Lipids in Donkeys (Equus asinus).

The values for basal serum cortisol concentrations of horses are available in many studies. However, there are limited data about serum cortisol in donkeys. The present study aimed to determine the baseline values for serum cortisol, to evaluate the influence of age and pregnancy on its levels, and to correlate its values with leucocyte profile, serum cholesterol, and triglycerides. Serum samples were collected from 97 healthy donkeys. Cortisol was analyzed by chemo-luminescent assay. The median and the 2.5th and 97.5th percentiles of serum cortisol measured and calculated in all donkeys were 5.64, 3.40, and 10.54 µg/dL, respectively. Females (n.91) were divided into three groups: Group A (young), Group B (adult), and Group C (pregnant at the 9th−11th months). The effect of age and physiological status was investigated by the Mann−Whitney test. Group C showed significantly higher levels than Group B (p < 0.05). Significant correlations were found in Group B with monocytes (r = 0.37, p < 0.01) and triglycerides (r = 0.30, p < 0.05), and in Group C with monocytes (r = 0.79, p < 0.01), basophils (r = 0.6, p < 0.05), and neutrophil/lymphocyte ratio (r = −0.63, p < 0.05). Higher cortisol values related to late pregnancy are also found in this species. These preliminary results provide evidence for a relationship between cortisol and the immune system as well as cortisol and lipid metabolism modulated by age and pregnancy when parameters are within normal values.

Cistus salviifolius Toxicity in Cattle.

Toxicity related to consumption of Cistus sp. pl. has been described in ruminants in some countries. This report describes the clinical and pathological findings of Cistus salviifolius toxicosis in 3 beef cattle herds located in 2 different areas of Sicily, Italy. Outbreaks were observed after grazing in poor winter pasture where C. salviifolius was abundant. Mean morbidity and mortality were 29% and 21%, respectively. Most of the affected animals (6 to 36 months old) showed anorexia, weight loss, and pollakiuria culminating in recumbency and death. Occasionally, abortion and neurological signs were observed. In animals with acute signs, there was a moderate decrease of sodium and chloride concentrations in serum. Animals with chronic signs showed an increase of serum urea, creatine phosphokinase (CPK), aspartate transaminase (AST), lactate dehydrogenase (LDH), and phosphorus and a decrease in total serum protein, calcium, chloride, and magnesium concentrations. Moderate anemia and slight neutropenia, lymphocytosis, and eosinophilia were detected in all groups. At necropsy, the main lesion was severe distention of the urinary bladder with turbid hemorrhagic urine and crystalluria. Histologically, chronic cystitis, interstitial nephritis, eosinophilic enteritis, and nonsuppurative necrotizing hepatitis were observed. To our knowledge, this is the first report of C. salviifolius toxicosis in cattle in Italy.

Assessing the effects of neonicotinoid insecticide on the bivalve mollusc Mytilus galloprovincialis.

In the present work, the marine invertebrate Mytilus galloprovincialis was used as model organism to evaluate the toxic effects of the neonicotinoid Calypso 480 SC (CAL) following 20 days of exposure to sub-lethal concentrations of 7.77 mg L-1 (0.1% 96 h-LC50) and 77.70 mg L-1 (1% 96 h-LC50), and a recovery period of 10 days in uncontaminated seawater. Results revealed that exposure to both concentrations of CAL increased significantly mortality rate in the cells of haemolymph and digestive gland, while digestive gland cells were no longer able to regulate cell volume. Exposure significantly reduced haemolymph parameters (Cl-, Na+), affected the enzymatic activities of superoxide dismutase of digestive gland and catalase of gill, and caused also histopathological alterations in digestive gland and gills. Main histological damages detected in mussels were lipofuscin accumulation, focal points of necrosis, mucous overproduction and infiltrative inflammations. Interestingly, alterations persisted after the recovery period in CAL-free water, especially for haemocyte parameters (K+, Na+, Ca2+, lactate dehydrogenase, glucose). A slight recovery of histological conditions was detected. These findings suggested that sub-chronic exposure to the neonicotinoid insecticide caused significant alterations in both cell and tissue parameters of M. galloprovincialis. Considering the ecologically and commercially important role of mussels in coastal waters, a potential risk posed by neonicotinoids to this essential aquatic resource can be highlighted.

Co-inertia multivariate approach for the evaluation of anthropogenic impact on two commercial fish along Tyrrhenian coasts.

Aliphatic hydrocarbon levels were determined by the GC/MS technique in fish livers of Engraulis encrasicolus (Ee) and Trachurus trachurus (Tt), collected from a particular area of the Mediterranean Sea, called GSA 10, which is located exactly in Tyrrhenian Sea between Campania coast and North Sicily coast. The aim was to evaluate their potential use as specific bioindicators towards this class of contaminants. Both Tt and Ee are considered to be pollution monitoring bioindicators, due to their dominance in marine communities and economic fishing interest. Ee showed a higher tendency to bioaccumulate TAHs, due to the lower quantity of fatty acids in liver tissues with respect to Tt. The area under study has been characterised a) chemically with the acquisition of temperature, oxygen and salinity profiles along the water column, and b) ecologically with the determination of amino acid contents in fish eyes, in order to gain information on the adaptation to environmental changes. Moreover, specific activities of two hydrolytic enzymes, such as alkaline phosphatase and peroxidase in fish epidermal mucus, together with lactate in blood plasma and cortisol levels, have been investigated for the first time, in order to obtain insights into the effects of hydrocarbons on animal welfare. A multiple co-inertia analysis was also applied to chemical and environmental parameters, in order to explore any possible correlation between different variables. The multivariate approach showed a clear spatial distribution between environmental and chemical variables in Ee, whilst there was an absence of a spatial trend in Tt. Moreover, the chemometric analysis showed a very high correlation between amino acid profiles and environmental variables for both species, confirming the possibility of being used as ecological welfare indices for short-term environmental variations.

Assessment of Electrolytes and Metals Profile of the Faro Lake (Capo Peloro Lagoon, Sicily, Italy) and Its Impact on Mytilus galloprovincialis.

Faro Lake is a coastal meromictic lagoon with singular characteristics in the Mediterranean (Messina, Sicily - Italy). It is part of the Natural Oriented Reserve of Capo Peloro (38° 15' 57″ N; 15° 37' 50″ E). In this area, traditional mollusc farming activity persists, producing 'autochthonous' mussels. This study reports of the Mytilus galloprovincialis haemolymph chemical profile and water variables determination of 1 year-lasted survey (April 2016 - March 2017). The determinations of electrolytes (Na+ , Cl- , K+ , Ca2+ , Mg2+ , P inorganic) and heavy metals in both Faro lake water and haemolymph have been carried out. Heavy metals are elements with high density and are quite toxic in low concentrations, such as Aluminum (Al), Arsenic (As), Cobalt (Co), Chrome (Cr), Copper (Cu), Iron (Fe), Magnesium (Mg), Manganese (Mn), Lead (Pb), Tin (Sn), Zinc (Zn). Heavy metals toxicity depends, principally, on bioaccumulation processes. M. galloprovincialis is a good bio-indicator, ideal for assess levels of environmental pollution thanks to its biological, ecological and physiological characteristics. The results of this study showed a typical fluctuation range in haemolymph and water parameters, related to the water ones; chemical-physical parameters affected the ions (electrolytes and metals) levels in some period of the year. The study reports the interactions between biotic (Mytilus galloprovincialis) and abiotic (water parameters) components of Faro Lake, and creates reference data for further future study on the same area or on similar ones.

The impact of captivity on some haematological parameters of griffon vultures (Gyps fulvus).

Haematological analysis is an essential field of veterinary medicine that provides inexpensive and reliable support to determinate animal health. The knowledge of how different factors affect the normal mean values of blood parameters is key to understand and improve animal health. In order to investigate how captivity can affect the haematological profile of birds of prey, the erythrocyte count, haemoglobin concentration, haematocrit, mean corpuscular volume, total leukocytes count of 123 griffon vultures (Gyps fulvus) were analysed. The birds were divided into 4 groups according to their life conditions: a control group of free-living griffons, 2 semi-captive groups held in an aviary for 15 and 30 days respectively, representing short-term captivity, and a captive group that had lived in cage for about 2 years. Our results showed that long-term captivity could influence haematocrit value and haemoglobin concentration. Furthermore, discriminant analysis highlighted significant separation between the captive birds on one hand and the control group and the semi-captive birds on the other. Instead, short-term captivity did not seem to affect prominently haemocytometric profile.

Changes in some blood parameters, milk composition and yield of buffaloes (Bubalus bubalis) during the transition period.

In this study changes in hematochemical parameters, milk composition and yield were investigated in buffaloes during the transition period. A total of 93 buffaloes 113.9 ± 8.03 months old and 535 ± 50 kg average body weight were used. Parity was recorded, blood samples were collected from 80 days pre-partum until 70 days post-partum; milk samples were collected from 5 days to 70 days post-partum. On serum samples, the values of non-esterified fatty acids, ß-hydroxybutyrate, glucose, cholesterol, triglycerides, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, urea, total proteins were evaluated. On milk, percentage of fat, protein and lactose, along with the somatic cell count (SCC), milk yield and daily milk production (DMP) were assessed. The peripartum period significantly (P ≤ 0.01) influenced all studied parameters with the exception of glucose. Milk fat percentage showed decreasing trend from 10 until 40 days post-partum; DMP significantly (P ≤ 0.01) increased from 1 day post-partum until 40 days post-partum. Milk yield significantly (P ≤ 0.01) decreased in animals over the sixth lactation. Our results confirmed the importance of transit period in buffaloes. Blood parameters and milk composition alterations are crucial to predict the energy balance status of buffaloes in order to improve their management and feed intake during the transition period.

Cytotoxicity, haemolymphatic parameters, and oxidative stress following exposure to sub-lethal concentrations of quaternium-15 in Mytilus galloprovincialis.

The presence of a xenobiotic in the environment can often represent a risk for living organisms. Quaternium-15, a preservative, is one of the most used substances and is added to several cosmetics and other industrial products. For this reason,kwowing the bio-indicator of the marine environment, the toxicological effects potentially elicited by this preservative on the marine invertebrate Mytilus galloprovincialis were studied. The results of this work confirm that quaternium-15, used at 0.1 and 1mg/l concentrations, while metabolized in M. galloprovincialis, causes a decrease in cellular viability, and remarkable changes to the defense and antioxidant system. In fact, haemocyte viability is dramatically reduced, and haemolymphatic parameter measurements indicate a stress on the animal. Moreover, an increase in radical species production, in Thiobarbituric Acid Reactive Species (TBARS) concentration, and in the Heat Shock Protein 70 amount, were observed in hepatopancreas. These changes suggest that the antioxidant systems are activated to overwhelm the oxidative damage induced by quaternium-15. Quaternium-15 jeopardizes both the defense and antioxidant systems. These results provide essential information with the biological fate of quaternium-15 in aquatic organisms, and confirm that biomarkers represent an important tool for modern environmental assessments as they can help with the prediction of pollutants involved in the monitoring program.

Relationship between serum cortisol levels and some physiological parameters following reining training session in horse.

The changes of cortisol, red blood cells (RBC), hemoglobin (Hb), hematocrit (Hct), heart rate (HR), respiratory rate (RR) and rectal temperature (RT) were evaluated after a reining training session in eight Quarter Horses. All parameters were measured before exercise (T0), immediately after exercise (T1), 1 h after exercise (T2), 2 h after exercise (T3) and 24 h after exercise (T4). One-way analysis of variance (ANOVA) for repeated measures, followed Bonferroni's post hoc test, showed a significant effect of the reining training session (P < 0.0001) on cortisol, RBC, Hb, Hct, HR, RR and RT. Simple linear regression analysis showed the positive correlation (P < 0.05) between cortisol changes and variations of studied parameters in T1, T3 and T4. Exercise-induced cortisol concentrations reflect the physiological response of reining training, suggesting that the changes observed are useful to assess the performance in reining horses and their reining training adaptability.

Evaluation of seasonal changes of serum and plasma estradiol-17ß, progesterone and testosterone in dolphins (Tursiops truncatus) by chemiluminescence.

AIM: The purpose of the research was to test and validate an innovative and safe chemiluminescence method to evaluate sexual hormones in serum and plasma samples of Tursiops truncatus. MATERIALS AND METHODS: The research was performed on 9 bottlenose dolphins entertained in Oltremare and in Zoomarine aquatic parks, sampled by the tail vein or from the ventral one and an ultrasound monitoring, throughout a 6 months period. Blood samples were analyzed using a chemiluminescence method. Data obtained were compared to radioimmunoassay and enzyme immuno assay reference data, with the purpose to test and validate this method, through the calculation of the coefficient of variability, and its reliability on serum and plasma samples. A one-way analysis of variance was applied to test the effect of time on serum and plasma hormonal changes. RESULTS: Mean concentrations of estradiol-17ß in serum were equal to 149.07±6.82 pmol/L, and in plasma equal to 159.14±12.99 pmol/L; mean values of progesterone in serum were equal to 0.69±0.05 pmol/L, and in plasma equal to 0.64±0.05 pmol/L; mean values of testosterone in serum were equal to 44.43±14.42 nmol/L, and in plasma equal to 48.99±11.20 nmol/L. CONCLUSION: It would be interesting to widen the investigations on a larger number of subjects, in which the relationship between the concentrations of free and binding steroid hormones, with the dosing of binding proteins, would define the physiological ranges of reference in the T. truncatus.

Reference intervals for total protein concentration, serum protein fractions, and albumin/globulin ratios in clinically healthy dairy cows.

The aim of the current study was to evaluate total serum protein concentration measured by the biuret reaction as well as albumin and globulin protein fractions determined by agarose gel electrophoresis. These data were used to establish reference intervals in dairy cows of different ages. Blood was collected from 111 clinically healthy Modicana dairy cows by means of jugular venipuncture. Reference intervals (mean ± standard deviation) were determined for total protein (67.54 ± 11.53 g/l), albumin (31.86 ± 4.60 g/l), α(1)-globulin (5.77 ± 2.20 g/l), α(2)-globulin (5.84 ± 1.90 g/l), ß-globulin (7.46 ± 1.94 g/l), and γ-globulin (16.73 ± 4.54 g/l) concentrations as well as for albumin/globulin (A/G) ratio (0.88 ± 0.43). Values from 2-, 3-, 4-, 5-, and 6-year-old cows were compared statistically. One-way analysis of variance showed age-related differences for α-globulin and ß-globulin fractions only. The results of the current study provide reference intervals for total protein concentration as well as albumin and globulin protein fractions in 2- to 6-year-old dairy cows.