ECOLOGICAL IMPACTS OF THE INVASIVE AND CRYPTIC BARBU BARBUS (L.) (PISCES:CYPRINIDAE)

Invasive species are among the principal causes of community and ecosystem integrity loss worldwide and freshwaters fishes are among the most threatened and introduced species. The invasive riverine fish Barbus barbus was used in this thesis as a model to study the ecological consequences deriving by two key mechanisms: interspecific trophic interactions and introgressive hybridisation. B. barbus is a large bodied cyprinid native to central Europe that has been introduced outside its native range in western England and Italy. The consequences of interspecific competition with functionally analogous fishes were tested in a serious of experimental conditions at different scales (from tank aquaria to mesocosms) with impacts measured on trophic niches and fish growth rates. Trophic ecology of B. barbus was also investigated in 11 wild populations of the UK also in relation to the use of angler’s baits (pelletized meal) that can act as trophic subsidies and facilitate B. barbus integration into the invaded communities. Introgressive hybridization consequences on functional traits (i.e. trophic ecology, morphology and life traits) was instead tested in wild Italian populations where B. barbus readily hybridize with native co-generic analogous B. plebejus and B. tyberinus. Finally, a further aspect that was considered in this study was the cryptic diversity of Barbus fluvio-lacustrine species in Italy that can lead to an underestimation of the extinction risk faced by barbels also in relation to B. barbus invasion. The experimental approaches demonstrated that competitive interaction among B. barbus and other analogous cyprinids (i.e. Leuciscus idus and Squalius cephalus) can result in suppressed growth rate but trophic niche segregation and constriction (i.e. diet diversification and specialisation) allow fish to co-occur and avoid outcompetition. Compared to intra-specific competition, the effects on fish growth rate were similar (i.e. reduced in both cases), but contrastingly, intraspecific competition produces an increase in niche size (i.e. generalization of diets). This provided experimental evidence for the niche variation hypothesis and explains the strong niche partitioning observed in previous studies on invasive B. barbus populations in English rivers. Moreover, although B. barbus appeared as a weaker competitor than the invasive L. idus, its introduction can result in isotopic niche reorganizations that can scale out to other community members with this requiring further elucidations. In agreement with previous studies, we found that some adult individuals in 11 UK wild B. barbus populations specialized their diets on allochthonous anglers’ baits as shown by their carbon isotope ratio (δ13C) strongly differentiated from that of freshwater macroinvertebrates. However, this varied considerably over space also according to angling pressure and it is unlikely that it helped to ease the interspecific competition of the barbel with native species that is instead more likely to be driven by niche variation processes. Introgressive hybridization with Italian native barbel populations resulted in hybrid populations, with mitochondrial DNA skewed toward B. barbus genotype and only 23% to 4% purebred native genotypes remaining in nuclear DNA. Significant alterations in morphology, enhanced growth rate, different diet and trophic position were detected in one hybrid population highlighting as introgressive hybridization is not only eroding the genetic integrity of native barbel species, but it has the potential to alter the functional role of barbel with consequent impacts that may influence also non-barbel members of the receiving community. Conversely, the detection of hybrid vigour underlined the adaptive role of introgression with hybrids that may be able to persist in areas where native barbel are disfavoured thus raising contrasting conservation perspectives. Purebred native species are likely to be confined to locations where barriers prevent B. barbus expansion and therefore there is a need to reconcile conservation needs to restore fluvial connectivity with the important role of isolated river stretches in offering refuge to native species. Geometric morphometrics and molecular analyses revealed the presence of two previously undetected barbel lineages in southern Italian basins for which a new description (B. samniticus sp. nov.) and a re-establishment (B. fucini Costa 1853) are proposed. Evolutionary history of these lineages may reveal some new insights into the evolution of the southern Italian basins and are therefore of great conservation interest. However, like B. plebejus and B. tyberinus species, the southern Italian lineages are already threatened especially by fish translocations and B. barbus and other exotic species invasions and they urgently require adequate protection. In conclusion, this thesis enhanced our understanding of the complex mechanisms governing the ecological and evolutionary consequences associated with biological invasions and brought new insights into Barbus genus diversity in Italy with important conservation implication

Predicting the ecological impacts of an alien invader: experimental approaches reveal the trophic consequences of competition

1. Ecological theory on the trophic impacts of invasive fauna on native competitors is equivocal. While increased inter-specific competition can result in coexisting species having constricted and diverged trophic niches, the competing species might instead increase their niche sizes to maintain energy intakes. Empirical experiments can test invasion theory on competitive interactions and niche sizes across different spatial scales and complexity. 2. The consequences of increased inter-specific competition from a model alien fish Leuciscus idus were tested on two taxonomically and trophically similar native fishes, Squalius cephalus and Barbus barbus. Competitive interactions were tested in tank aquaria using comparative functional responses (CFRs) and cohabitation trials. The consequences of these competitive interactions for the trophic niche sizes and positions of the fishes were tested in pond mesocosms. 3. CFRs revealed that compared to B. barbus, L. idus had significantly higher attack and consumption rates; cohabitation trials revealed B. barbus growth rates were depressed in sympatry with L. idus. For L. idus and S. cephalus, differences in their functional response parameters and growth rates were not significant. 4. Pond mesocosms used stable isotope metrics to quantify shifts in the trophic niche sizes of the fishes between allopatry and sympatry using a substitutive experimental design. Isotopic niches were smaller and more divergent in sympatric paired species than predicted by their allopatric treatments, suggesting trophic impacts from inter-specific competition. However, an all-species sympatric treatment revealed similar niche sizes with allopatry. This maintenance of niche sizes in the presence of all species potentially resulted from the buffering of direct competitive effects of the species-pairs by indirect effects. 5. Experimental predictions from tank aquaria assisted the interpretation of the constricted and diverged trophic niches detected in the paired-species sympatric treatments of the pond mesocosms. However, the all-species sympatric treatment of this experiment revealed greater complexity in the outcomes of the competitive interactions within and between the species. These results have important implications for understanding how alien species integrate into food webs and influence the trophic relationships between native species

Immunophenotyping of leukocytes in bovine placenta

Lymphocytes and macrophages are the main white cells involved in fetal-maternal tolerance. Little is known about these leukocytes in bovine placenta, such as the quantity and location of these cells. Thus, the objective of this study was to identify lymphocyte and macrophage populations in bovine placenta using specific markers and flow cytometry. This study analyzed samples of placentomes and intercaruncular regions of cows in the three quarters of pregnancy. In the placentomes, during the first quarter of pregnancy, mean percentage of labeled CD3+ cells was 2.34%; CD8+, 1.28%; CD14+, 1.66%; and CD335+, 0.96%. For the intercaruncular region, percentage of CD3+ cells was 0.71%; CD8+, 1.63%; CD14+, 2.81%; and CD335+, 2.81%. In the second quarter, placentomes showed 0.94% CD3+ cells; 0.77% CD8+; 0.72% CD14+; and 0.51% CD335+. In the intercaruncular region, percentage of labeled CD3+ cells was 0.50%; CD8+, 1.81%; CD14+, 2.64%; and CD335+, 0.51%. In the third quarter, placentomes showed labeling of 0.88% CD3+; 0.66% CD8+; 1.06% CD14+; and 0.74% CD335+ cells. In the intercaruncular region, percentage of labeled CD3+ cells was 0.19%; CD8+, 2.23%; CD14+, 2.43%; and CD335+, 0.16%. The results showed that there was a greater immunomarking of leukocytes CD3+ and CD335+ in the placentome when compared to the intercaruncular region during the third trimester. It can be concluded that leukocytes populations in bovine placenta is reduced, probably because of the syndesmochorial characteristic of bovine placenta. This represents a significant barrier for the immunological system of the mother, sharply decreasing the exposure of the conceptus to the mother’s immune system.Linfócitos e macrófagos são os principais leucócitos envolvidos na tolerância materno-fetal. Pouco se sabe sobre esses leucócitos na placenta bovina, como por exemplo, a quantidade e localização dessas células. Assim, o objetivo desse estudo foi identificar populações de linfócitos e macrófagos na placenta bovina utilizando marcadores específicos e citometria de fluxo. Este estudo analisou amostras de placentônios da região intercaruncular de bovinos nos três trimestres da gestação. No primeiro trimestre, nos placentônios, a porcentagem média de células CD3+ foi 2,34%; CD8+, 1,28%; CD14+, 1,66%; e CD335+, 0,96%. Na região intercaruncular, a porcentagem de células CD3+ foi 0,71%; CD8+, 1,63%; CD14+, 2,81%; e CD335+, 2,81%. No segundo trimestre, os placentonios apresentaram 0,94% de células CD3+; 0,77% de CD8+; 0,72% de CD14+e 0,51% de CD335+. Na região intercaruncular, a porcentagem de células CD3+ foi 0,50%; CD8+, 1,81%; CD14+, 2,64%; e CD335+, 0,51%. No terceiro trimestre, os placentônios apresentaram 0,88% de células marcadas CD3+; 0,66% de CD8+; 1,06% de CD14+ e 0,74% de CD335+. Na região intercaruncular, a porcentagem de células CD3+ foi 0,19%; CD8+, 2,23%; CD14+, 2,43% e CD335+, 0,16%. Os resultados mostraram que a imunomarcação de leucócitos na região do placentônio foi maior do que na região intercaruncular no terceiro trimestre. Concluiu-se que a população de leucócitos CD3+ e CD335+ na placenta bovina está reduzida, provavelmente devido a sua característica sindesmocorial. Essa característica representa uma barreira significante para o sistema imunológico da mãe, o que diminui drasticamente a exposição do concepto ao sistema de defesa da mãe

Serum endocan levels associated with hypertension and loss of renal function in pediatric patients after two years from renal transplant

Endocan is an important biomarker of inflammation and endothelial dysfunction that increases in association with several chronic diseases. Few published data have described the role of endocan in pediatric renal transplant (RT) patients. We evaluated the endocan concentrations in 62 children who underwent renal transplantation and assessed their relationships with the patients' blood pressure and loss of renal function. The endocan levels were significantly elevated in the pediatric RT patients who had hypertension and a loss of renal function. We determined positive correlations between the endocan concentrations and the hemodynamic variables (systolic blood pressure: r = 0.416P = 0.001pulse pressure: r = 0.412P = 0.003). The endocan levels were inversely correlated with the estimated glomerular filtration rate (r = -0.388P = 0.003). An endocan cutoff concentration of 7.0 ng/mL identified pediatric RT patients who had hypertension and a loss of renal function with 100% sensitivity and 75% specificity. In conclusion, the endocan concentrations were significantly elevated in pediatric RT patients who had both hypertension and a loss of renal function. The correlations between the endocan levels and the hemodynamic variables and the markers of renal function strengthen the hypothesis that it is an important marker of cardiorenal risk.FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, Brazil) [2013/03139-0]CNPq (Conselho-Nacional de Desenvolvimento Cientifico e Tecnologico) [443248/2014-1]Nephrology Division, School of Medicine, Federal University of São Paulo, São Paulo, SP, BrazilPediatrics Department, School of Medicine, Federal University of São Paulo, São Paulo, SP, BrazilNephrology Division, School of Medicine, Federal University of São Paulo, São Paulo, SP, BrazilPediatrics Department, School of Medicine, Federal University of São Paulo, São Paulo, SP, BrazilFAPESP: 2013/03139-0CNPq: 443248/2014-1Web of Scienc

Environmental DNA reveals the temporal and spatial extent of spawning migrations of European shad in a highly fragmented river basin

1. Anthropogenic barriers on lowland rivers impede the spawning migrations of anadromous fishes, preventing access to historical spawning areas. In the cryptic European shads Alosa alosa and Alosa fallax (‘shad’ hereafter), this has resulted in population declines across their range. Conservation programmes aim to facilitate the passage of migrators over these barriers and so require baseline knowledge on spatial and temporal extent of current migrations. 2. Here, a shad-specific environmental DNA (eDNA) assay was used to quantify the spatial extent of shad spawning migrations in the River Severn basin, Western England. This basin is characterised by the presence of multiple anthropogenic barriers in the lower catchment. In 2017, the eDNA assay was piloted in the River Teme, an important shad spawning tributary, and then applied in 2018 and 2019 across the lower Severn basin. 3. In all years, shad DNA was detected between mid-May and mid-June, with the maximum spatial extent of shad distribution being in early June when shad eDNA was detected upstream of weirs that were generally considered as impassable. In 2018, this included the detection of shad above the most upstream weir on the main River Severn that required individual fish to have passed six weirs. 4. Although anthropogenic barriers inhibit the spawning migrations of shad, this eDNA assay revealed some highly vagile individuals might be able to ascend these barriers and migrate considerable distances upstream. This suggests that efforts to increase the permeability of these barriers could result in relatively high numbers of migrating shad reaching upstream spawning areas. These results demonstrate that this eDNA assay could be also utilised across their range, outside the study system, to further quantify the spatial extent of their spawning, including in highly fragmented rivers and those where shad are believed to only spawn occasionally and are rarely observed

Secretory Leukoprotease Inhibitor (Slpi) Expression Is Required for Educating Murine Dendritic Cells Inflammatory Response Following Quercetin Exposure

Dendritic cells’ (DCs) ability to present antigens and initiate the adaptive immune response confers them a pivotal role in immunological defense against hostile infection and, at the same time, immunological tolerance towards harmless components of the microbiota. Food products can modulate the inflammatory status of intestinal DCs. Among nutritionally-derived products, we investigated the ability of quercetin to suppress inflammatory cytokines secretion, antigen presentation, and DCs migration towards the draining lymph nodes. We recently identified the Slpi expression as a crucial checkpoint required for the quercetin-induced inflammatory suppression. Here we demonstrate that Slpi-KO DCs secrete a unique panel of cytokines and chemokines following quercetin exposure. In vivo, quercetin-enriched food is able to induce Slpi expression in the ileum, while little effects are detectable in the duodenum. Furthermore, Slpi expressing cells are more frequent at the tip compared to the base of the intestinal villi, suggesting that quercetin exposure could be more efficient for DCs projecting periscopes in the intestinal lumen. These data suggest that quercetin-enriched nutritional regimes may be efficient for suppressing inflammatory syndromes affecting the ileo-colonic tract

Secretory leukoprotease inhibitor is required for efficient quercetin-mediated suppression of TNFα secretion

Dendritic cells (DCs) are professional antigen presenting cells (APCs) that in response to microbial infections generate long-lasting adaptive immune response. Following microbial uptake, DCs undergo a cascade of cellular differentiation that ultimately leads to “mature” DCs. Mature DCs produce a variety of inflammatory cytokines, including tumor necrosis factor-α (TNFα) a key cytokine for the inflammatory cascade. In numerous studies, polyphenols, including quercetin, demonstrated their ability to suppress TNFα secretion and protect from the onset of chronic inflammatory disorders. We show that murine bone marrow derived DCs express Slpi following quercetin exposure. Slpi is known to suppress LPS mediated NFκB activation, thus, it was hypothesized that its expression could be the key step for polyphenol induced inflammatory suppression. Slpi-KO DCs poorly respond to quercetin administration failing to reduce TNFα secretion in response to quercetin exposure. Supernatant from quercetin exposed DCs could also reduce LPS-mediated TNFα secretion by unrelated DCs, but this property is lost using an anti-Slpi antibody. In vivo, oral administration of quercetin is able to induce Slpi expression. Human biopsies from inflamed tract of the intestine reveal the presence of numerous SLPI(+) cells and the expression level could be further increased by quercetin administration. We propose that quercetin induces Slpi expression that in turn reduces the inflammatory response. Our data encourages the development of nutritional strategies to improve the efficiency of current therapies for intestinal chronic inflammatory syndrome and reduce the risks of colorectal cancer development

A Bronze-Tomato Enriched Diet Affects the Intestinal Microbiome under Homeostatic and Inflammatory Conditions

Inflammatory bowel diseases (IBD) are debilitating chronic inflammatory disorders that develop as a result of a defective immune response toward intestinal bacteria. Intestinal dysbiosis is associated with the onset of IBD and has been reported to persist even in patients in deep remission. We investigated the possibility of a dietary-induced switch to the gut microbiota composition using Winnie mice as a model of spontaneous ulcerative colitis and chow enriched with 1% Bronze tomato. We used the near isogenic tomato line strategy to investigate the effects of a diet enriched in polyphenols administered to mild but established chronic intestinal inflammation. The Bronze-enriched chow administered for two weeks was not able to produce any macroscopic effect on the IBD symptoms, although, at molecular level there was a significant induction of anti-inflammatory genes and intracellular staining of T cells revealed a mild decrease in IL17A and IFNγ production. Analysis of the microbial composition revealed that two weeks of Bronze enriched diet was sufficient to perturb the microbial composition of Winnie and control mice, suggesting that polyphenol-enriched diets may create unfavorable conditions for distinct bacterial species. In conclusion, dietary regimes enriched in polyphenols may efficiently support IBD remission affecting the intestinal dysbiosis

Aquaporin-9 Contributes to the Maturation Process and Inflammatory Cytokine Secretion of Murine Dendritic Cells

Dendritic cells (DCs) are the most potent antigen-presenting cells able to trigger the adaptive immune response to specific antigens. When non-self-antigens are captured, DCs switch from an “immature” to a “mature” state to fulfill their function. Among the several surface proteins involved in DCs maturation, the role of aquaporins (AQPs) is still poorly understood. Here we investigated the expression profile of Aqps in murine bone marrow derived dendritic cells (BMDCs). Among the Aqps analyzed, Aqp9 was the most expressed by DCs. Its expression level was significantly upregulated 6 h following LPS exposure. Chemical inhibition of Aqp9 led to a decreased inflammatory cytokines secretion. BMDCs from AQP9-KO mice release lower amount of inflammatory cytokines and chemokines and increased release of IL-10. Despite the reduced release of inflammatory cytokines, Aqp9-KO mice were not protected from DSS induced colitis. All together, our data indicate that AQP9 blockade can be an efficient strategy to reduce DCs inflammatory response but it is not sufficient to protect from acute inflammatory insults such as DSS induced colitis