Unraveling the molecular mechanisms of fish physiological response to freshwater salinization: A comparative multi-tissue transcriptomic study in a river polluted by potash mining.

Freshwater salinization is an escalating global environmental issue that threatens freshwater biodiversity, including fish populations. This study aims to uncover the molecular basis of salinity physiological responses in a non-native minnow species (Phoxinus septimaniae x P. dragarum) exposed to saline effluents from potash mines in the Llobregat River, Barcelona, Spain. Employing high-throughput mRNA sequencing and differential gene expression analyses, brain, gills, and liver tissues collected from fish at two stations (upstream and downstream of saline effluent discharge) were examined. Salinization markedly influenced global gene expression profiles, with the brain exhibiting the most differentially expressed genes, emphasizing its unique sensitivity to salinity fluctuations. Pathway analyses revealed the expected enrichment of ion transport and osmoregulation pathways across all tissues. Furthermore, tissue-specific pathways associated with stress, reproduction, growth, immune responses, methylation, and neurological development were identified in the context of salinization. Rigorous validation of RNA-seq data through quantitative PCR (qPCR) underscored the robustness and consistency of our findings across platforms. This investigation unveils intricate molecular mechanisms steering salinity physiological response in non-native minnows confronting diverse environmental stressors. This comprehensive analysis sheds light on the underlying genetic and physiological mechanisms governing fish physiological response in salinity-stressed environments, offering essential knowledge for the conservation and management of freshwater ecosystems facing salinization.

High rates of antibodies against Toscana and Sicilian phleboviruses in common quail Coturnix coturnix birds.

Introduction: Birds are involved natural cycle of a number of vector-borne viruses in both rural and urban areas. Toscana (TOSV) and Sicilian (SFSV) phleboviruses are sandfly-borne viruses in the genus Phlebovirus that can cause diseases in human. However, there is limited information on the role of the birds in sandfly-borne phleboviruses natural cycle and reservoirs ofthese viruses remain unknown. Methods: In this study, we analyzed Common Quail (Coturnix coturnix) sera from Spain to identify the seroprevalence of these two phleboviruses. We tested respectively, 106 and 110 quail serum against TOSV and SFSV from 2018, 2019, and 2021 from two locations in northern Spain with using virus neutralization test. Results: We identified high neutralizing antibody rates for SFSV (45.45%) and TOSV (42.45%) with yearly fluctuation. Discussion: This is the first identification of SFSV and TOSV neutralizing antibodies in wild birds. High seroprevalence rates of TOSV and SFSV in quail birds raises the question whether birds have a role as amplifying hosts in the natural cycle of phleboviruses.

Improving water quality does not guarantee fish health: Effects of ammonia pollution on the behaviour of wild-caught pre-exposed fish.

Ammonia is a pollutant frequently found in aquatic ecosystems. In fish, ammonia can cause physical damage, alter its behaviour, and even cause death. Exposure to ammonia also increases fish physiological stress, which can be measured through biomarkers. In this study, we analysed the effect of sublethal ammonia concentrations on the behaviour and the oxidative stress of Barbus meridionalis that had been pre-exposed to this compound in the wild. Wild-caught fish from a polluted site (pre-exposed fish) and from an unpolluted site (non-pre-exposed fish) were exposed, under experimental conditions, to total ammonia concentrations (TAN) of 0, 1, 5, and 8 mg/L. Swimming activity, feeding behaviour, and oxidative stress response based on biomarkers were analysed. Pre-exposed fish showed both an altered behaviour and an altered oxidative stress response in the control treatment (0 mg/L). Differences in swimming activity were also found as pre-exposed fish swam less. Lower feeding activity (voracity and satiety) and altered response to oxidative stress were also observed at ≥ 1 mg/L TAN. Biomarker results confirmed pre-exposed fish suffer from a reduction in their antioxidant defences and, hence, showed increased oxidative tissue damage. In summary, pre-exposed fish showed more sensitivity to ammonia exposure than fish from a pristine site.

Effect of different rice farming practices on the bioavailability of mercury: A mesocosm experiment with common goldfish (Carassius auratus).

Wetlands such as rice paddies are important ecosystems that provide habitat for a numerous range of species but are considered as a major source of mercury in the most toxic form of methylmercury. An in situ mesocosm experiment was conducted during the rice cultivation season of 2018 in rice paddies from the Ebro Delta (NE Spain) to investigate the bioaccumulation of mercury in fish. Common goldfish (Carassius auratus) were exposed to three types of rice-fields subject to different agricultural management (conventional or ecological agriculture) and different hydric practices (wet, dry) and were sampled monthly. Total mercury concentration in fish increased over time for almost all the fields under study, but the mercury increase was particularly higher for fish exposed to ecological fields. We conducted δ15N and δ13C measurements to follow up fish diet changes within or among fields. The results have shown there were no variations in the diet of the fish over time within fields, although differences among management practices were observed, which could explain to some extent the THg variation in fish exposed to each of the agricultural practices. Results indicate that wetting and drying rice fields might be a greater source of mercury for fish and a potential hotspot for MeHg production enhanced by physicochemical parameters. Thus, mercury bioaccumulation in fish inhabiting rice paddies depend on several factors what, in turn, could lead to biomagnification across the food webs.

Play Behavior in Two Captive Bottlenose Dolphin Calves (Tursiops Truncatus): Ethogram, Ontogeny, and Individual Differences.

This research focused on different aspects of play behavior including ethogram, ontogeny, and individual differences, in one male and one female captive bottlenose dolphin calves (Tursiops truncatus) from November 2003 to June 2004. We presented the first peer-reviewed description of a play ethogram in bottlenose dolphin calves whose behaviors were grouped into three hierarchical levels: two categories: solitary and social play; four subcategories: locomotor, object, bubble play, and in the presence of humans, and 35 entries. It was conducted in two phases: the descriptive phase - from 3 to 5 months old - with 29 entries and the quantitative phase - from 6 to 10 months old -with six entries. All social and solitary locomotor behaviors appeared when animals were 3 months old meanwhile more complex behaviors concerning playing with objects, bubbles, and in the presence of humans were observed for 6 months old. There were no statistically significant intra-individual and inter-individual differences in the play behavioral diversity, in the time invested in play and in the Shannon's evenness index. However, we observed statistically significant inter-individual but not intra-individual differences for the Shannon's diversity index.

Inter-individual Distance in Different Captive Packs of Iberian Wolf (Canis lupus signatus): Management Applications.

There is a tendency for the wolves of a pack to stay together assuming that there is a social bond between the pack members. A rough estimate of the strength of this bond can be obtained by measuring the distances between pack members. The aim of this study was to determine the inter-individual distance by calculating the Euclidian distance to determine the Iberian wolf cohesion in different pack compositions. The study subjects were seven captive Iberian wolves (Canis lupus signatus) housed at the Barcelona Zoo. Multifocal sampling methods were used for data collection, and instantaneous scans were made at 15-min during different sessions. Proximity relationships among these wolves in different pack composition were visualized using two-dimensional scaling analysis. The pair leader without offspring and the breeding pair with their offspring were significantly closer than the rest of the dyads which combinations were equidistant. During the pregnancy period, the breeding pair was significantly further than during the juvenile period. More studies in field and captivity by using Euclidian distance are necessary to improve social husbandry techniques, animal welfare and conservation programs.

Biochemical aspects of susceptibility to stressors in two small cyprinids Squalius laietanus and Barbus meridionalis from the NW Mediterranean.

Specimens of two endemic cyprinids, Squalius laietanus (Catalan chub) and Barbus meridionalis (Mediterranean barbel), were sampled from a reference site in a small stream of the Ripoll River (NW Mediterranean) outside of their reproductive season. Biomarkers involved in xenobiotic-mediated responses were individually contrasted in fish of both species and 17 perfluoroalkyl substances (PFASs) analysed in muscle to reveal bioaccumulation trends. The parameters were in muscle: cholinesterases, metabolic lactate dehydrogenase (LDH) and citrate synthase (CS); and in liver: cytochrome P450 dependent activities (EROD and BFCOD), carboxylesterase (CE), glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPX) and catalase (CAT). All markers are considered adaptive defence mechanism to face stress. Sensitivity to a model pesticide: dichlorvos was also contrasted in vitro in muscular acetylcholinesterase (AChE) and hepatic CE to reveal species sensitivity to neurotoxic chemicals. Enzymatic activities related to protective mechanisms such as butyrylcholinesterase (BuChE), CE and CAT were higher in chub whereas the antioxidant defences GR and GPX were higher in barbel. Aerobic CS was also higher in barbel while anaerobic LDH was so in chub. EROD activity did not differ between the two species but BFCOD activity was higher in barbel. Levels of PFAS were higher in barbel likely due to its benthic habitat. The in vitro tests revealed higher sensitivity to dichlorvos of muscular AChE in chub (lower IC50) which was probably compensated by a higher catalytic efficiency of CE. All these former biochemical particularities are discussed in terms of fish ecological performance in front of anthropogenic stressors.

Effects of industrial pollution on the reproductive biology of Squalius laietanus (Actinopterygii, Cyprinidae) in a Mediterranean stream (NE Iberian Peninsula).

Mediterranean rivers are severely affected by pollutants from industry, agriculture and urban activities. In this study, we examined how industrial pollutants, many of them known to act as endocrine disruptors (EDCs), could disturb the reproduction of the Catalan chub (Squalius laietanus). The survey was conducted throughout the reproductive period of S. laietanus (from March to July 2014) downstream an industrial WWTP located in the River Ripoll (NE Iberian Peninsula). Eighty fish (28 females and 52 males) were caught by electrofishing upstream and 77 fish (33 females and 44 males) downstream a WWTP. For both sexes, the gonadosomatic index (GSI) and gonadal histology were examined and related to water chemical analysis and fish biomarkers. Female fecundity was assessed using the gravimetric method. Fish from the polluted site showed enhanced biomarker responses involved in detoxification. Also, in the polluted site, lower GSI values were attained in both sexes and females displayed lower numbers of vitellogenic oocytes. Gonadal histology showed that all maturation stages of testicles and ovaries were present at the two study sites but fish males from the polluted site had smaller diameter seminiferous tubules. Water chemical analysis confirmed greater presence of EDCs in the river downstream the industrial WWTP. The chemicals benzotriazole and benzothiazole could be partially responsible for the observed alterations in the reproductive biology of S. laietanus.

Towards Non-Invasive Methods in Measuring Fish Welfare: The Measurement of Cortisol Concentrations in Fish Skin Mucus as a Biomarker of Habitat Quality.

Cortisol levels in fish skin mucus have shown to be good stress indicators in farm fish exposed to different stressors. Its applicability in free-ranging animals subject to long-term environmental stressors though remains to be explored. The present study was therefore designed to examine whether skin mucus cortisol levels from a wild freshwater fish (Catalan chub, Squalius laietanus) are affected by the habitat quality. Several well-established hematological parameters and cortisol concentrations were measured in blood and compared to variations in skin mucus cortisol values across three habitats with different pollution gradient. Fluctuations of cortisol in skin mucus varied across the streams of differing habitat quality, following a similar pattern of response to that detected by the assessment of cortisol levels in blood and the hematological parameters. Furthermore, there was a close relationship between cortisol concentrations in skin mucus and several of the erythrocytic alterations and the relative proportion of neutrophils to lymphocytes. Taken together, results of this study provide the first evidence that skin mucus cortisol levels could be influenced by habitat quality. Although results should be interpreted with caution, because a small sample size was collected in one studied habitat, the measurement of cortisol in skin mucus could be potentially used as a biomarker in freshwater fish.

Patterns of animal-enrichment interaction in captive brown bears.

We studied the behavioral responses of three brown bears (Ursus arctos) to different types of enrichment devices to assess the predominant pattern of animal-enrichment interaction (PAI) to each type of enrichment. We assessed the bears' responses to feeding, sensory, and occupational enrichments over nine sessions. Using Pearson's correlation (r) and the coefficient of variation (CV)-we describe four models of PAIs: habituation, increasing, continuous, and fluctuating. The habituation model (r < 0 and p < 0.05; CV (%) > 0) consists of a loss of interest in the device over time and it occurred with the occupational device. The increasing model (r > 0 and p < 0.05; CV (%) > 0) consists of a sustained interest in the device over time and it was not observed for any device of this study. The continuous model (0 > r > 0 and p > 0.05; CV (%) < 100) consists of a consistent and unchanging interaction over time and it occurred with the feeding enrichments used in this study. The fluctuating PAI (0 > r > 0 and p > 0.05; CV (%) ≥ 100) consists of wide oscillations in the degree of interaction over time and we observed this pattern with sensory devices. Review of previous literature supports these classes of PAI, and suggests similar relationships between PAIs and the type of enrichment as we observed in this study.

The ecology of sexual dimorphism in size and shape of the freshwater blenny Salaria fluviatilis.

Sexual selection is considered the major cause of sexual dimorphism, but recent observations suggest that natural selection may play a more important role in the evolution of sex differentiation than previously recognized. Therefore, studying the trade-offs between natural selection and sexual selection is crucial to a better understanding of the ecology underlying the evolution of sexual dimorphism. The freshwater blenny Salaria fluviatilis, a fish inhabiting lakes and rivers around the Mediterranean Sea, displays strong sexual dimorphism in size, shape, and behavior (i.e., larger body and head size for males and higher swimming requirements for females during the reproductive period). We tested for differences in sexual dimorphism in size and shape between the populations from lake and river habitats with the goal of identifying the trade-offs between natural and sexual selection that underlie variations in sexual dimorphism in this species. Our results show i) differences in sexual size dimorphism (SSizeD) in accordance to Rensch's rule (i.e., larger individuals in rivers associated with higher SSizeD), and ii) a decrease in shape differentiation between males and females in lake populations. Together, this suggests that the different environmental conditions between lake and river habitats (e.g., resource limitations, predation pressure, water velocity) affect the relative importance of sexual selection in the display of sexual dimorphism within the species. This study highlights the importance of considering the environmental conditions to which populations are exposed to better understand the ecology underlying the evolution of sexual dimorphism.

Improving the Welfare of a Zoo-Housed Male Drill (Mandrillus leucophaeus poensis) Aggressive Toward Visitors.

Improving the welfare of nonhuman animals in captivity and maintaining behavioral competence for future conservation purposes is of the highest priority for zoos. The behavior of an aggressive male drill (Mandrillus leucophaeus poensis) was assessed in Barcelona Zoo. The 2-year study presented in this article examined the effects of introducing changes in the exhibit of the drill to improve his welfare by analyzing scan behaviors. First, a partial visual barrier was applied and proved to be insufficient to decrease the long-term stress indicators assessed. Next, a feeding enrichment program was implemented. The results supported the hypothesis that feeding and explorative activities would increase, whereas apathetic and stereotypic behaviors would decrease. However, visitor-directed aggression did not vary, indicating that more profound structural modifications were needed to reduce the negative impact of the agonistic interactions between the drill and the public. The study emphasized the usefulness of environmental enrichment evaluations in assessing captive animal welfare.

Long-Term Macroevaluation of Environmental Enrichment in Three Brown Bears (Ursus arctos) at Barcelona Zoo.

The evaluation of enrichment programs is important to determine their effect on nonhuman animal welfare. The daily activity pattern and use of space of 3 brown bears (Ursus arctos) were used for long-term macroevaluation of enrichment to compare the baseline and enrichment phases. Focal sampling methods were used for data collection, and instantaneous scans were made at 2-min intervals during 15 sessions of 1 hr for each animal during the 2 study periods. The enrichment devices were categorized as feeding, occupational, and sensorial. The long-term macroevaluation in 3 bears showed statistically significant differences in some types of activity but not in others. There were also statistically significant differences for the use of space in 4 of the 8 zones in which the enclosures were divided. A more homogenous pattern in the use of space was only observed during the enrichment phase in the old female. The 3 brown bears followed different patterns concerning the enrichment program.