Assessment of phytostabilization of Pb/Zn mine soils with organic amendments and a native metallicolous ecotype of Festuca Rubra l.: implications for amendment selection and recovery of soil health

La contaminación del suelo es una de las principales amenazas para los ecosistemas y la salud humana. Actualmente, desde un punto de vista tanto económico como ambiental, la fitoestabilización es la mejor tecnología para remediar suelos contaminados con elevadas concentraciones de metales como son los suelos mineros. La fitoestabilización asistida consiste en el empleo de plantas y enmiendas orgánicas y/o inorgánicas con el fin de reducir la movilidad y la biodisponibilidad de los contaminantes y recuperar la salud de suelo. En este trabajo se han realizado ensayos en microcosmos y en campo centrándonos en la salud del suelo minero contaminado con Pb y Zn durante un proceso de fitoestabilización empleando enmiendas orgánicas (purines vacunos, gallinaza, estiércol de oveja y lodos de papelera mezclados con gallinaza) y/o la especie metalífera Festuca rubra con el objetivo de (i) estudiar las interacciones suelo-enmienda responsables de los cambios inducidos por el proceso de quimioestabilización en las propiedades físicoquímicas y biológicas del suelo, (ii) evaluar la efectividad del proceso de fitoestabilización sobre suelos vegetados y de la revegetación sobre suelos desnudos (iii) valorar la idoneidad de distintos indicadores químicos y biológicos (parámetros microbianos y de la vegetación) para monitorizar la efectividad de la fitoestabilización asistida en términos de reducción de la biodisponibilidad de metales en el suelo, mejora de la vegetación y de la recuperación de la salud del suelo. La aplicación de enmiendas al suelo minero supone una entrada de materia orgánica y nutrientes que conduce a una disminución de la biodisponibilidad de metales, facilitando la colonización de las plantas y el crecimiento de la vegetación nativa, además de estimular la actividad microbiana del suelo. El pH del suelo es un factor crítico que condiciona la movilidad de los metales y la toxicidad del suelo. Las poblaciones microbianas de las enmiendas no modificaron la diversidad funcional de las comunidades microbianas nativas de la mina. Los purines vacunos y los lodos de papelera mezclados con gallinaza son los tratamientos más efectivos en el proceso de fitoestabilización asistida bajo condiciones de campo. La gallinaza fue el tratamiento que más estimuló el crecimiento de la vegetación nativa y la colonización en los suelos desnudos. El bioensayo de elongación radical de lechuga es un test sensible, sencillo y barato para evaluar la biodisponibilidad de metal y la ecotoxicidad del suelo. Los tocoferoles son biomarcadores de exposición a metales con potencial para su implementación en bioensayos de toxicidad. Este trabajo permite concluir que la población metalífera de F. rubra, combinada con enmiendas orgánicas, es una excelente candidata para los proyectos de fitoestabilización asistida. Además, la monitorización simultánea de los parámetros fisicoquímicos y microbiológicos del suelo y de su ecotoxicidad permite una evaluación adecuada de la salud del suelo, así como la selección de enmiendas apropiadas para el desarrollo de un proceso fitoestabilizador.

Fishing Long-Fingered Bats (Myotis capaccinii) Prey Regularly upon Exotic Fish

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Language distance and non-native syntactic processing: Evidence from event-related potentials

[EN] In this study, we explore native and non-native syntactic processing, paying special attention to the language distance factor. To this end, we compared how native speakers of Basque and highly proficient non-native speakers of Basque who are native speakers of Spanish process certain core aspects of Basque syntax. Our results suggest that differences in native versus non-native language processing strongly correlate with language distance: native/non-native processing differences obtain if a syntactic parameter of the non-native grammar diverges from the native grammar. Otherwise, non-native processing will approximate native processing as levels of proficiency increase. We focus on three syntactic parameters: (i) the head parameter, (ii) argument alignment (ergative/accusative), and (iii) verb agreement. The first two diverge in Basque and Spanish, but the third is the same in both languages. Our results reveal that native and non-native processing differs for the diverging syntactic parameters, but not for the convergent one. These findings indicate that language distance has a significant impact in non-native language processing.

Application of Entropy and Fractal Dimension Analyses to the Pattern Recognition of Contaminated Fish Responses in Aquaculture

The objective of the work was to develop a non-invasive methodology for image acquisition, processing and nonlinear trajectory analysis of the collective fish response to a stochastic event. Object detection and motion estimation were performed by an optical flow algorithm in order to detect moving fish and simultaneously eliminate background, noise and artifacts. The Entropy and the Fractal Dimension (FD) of the trajectory followed by the centroids of the groups of fish were calculated using Shannon and permutation Entropy and the Katz, Higuchi and Katz-Castiglioni's FD algorithms respectively. The methodology was tested on three case groups of European sea bass (Dicentrarchus labrax), two of which were similar (C1 control and C2 tagged fish) and very different from the third (C3, tagged fish submerged in methylmercury contaminated water). The results indicate that Shannon entropy and Katz-Castiglioni were the most sensitive algorithms and proved to be promising tools for the non-invasive identification and quantification of differences in fish responses. In conclusion, we believe that this methodology has the potential to be embedded in online/real time architecture for contaminant monitoring programs in the aquaculture industry.

Mugilid Fish Are Sentinels of Exposure to Endocrine Disrupting Compounds in Coastal and Estuarine Environments

Effects on fish reproduction can result from a variety of toxicity mechanisms first operating at the molecular level. Notably, the presence in the environment of some compounds termed endocrine disrupting chemicals (EDCs) can cause adverse effects on reproduction by interfering with the endocrine system. In some cases, exposure to EDCs leads to the animal feminization and male fish may develop oocytes in testis (intersex condition). Mugilid fish are well suited sentinel organisms to study the effects of reproductive EDCs in the monitoring of estuarine/marine environments. Up-regulation of aromatases and vitellogenins in males and juveniles and the presence of intersex individuals have been described in a wide array of mullet species worldwide. There is a need to develop new molecular markers to identify early feminization responses and intersex condition in fish populations, studying mechanisms that regulate gonad differentiation under exposure to xenoestrogens. Interestingly, an electrophoresis of gonad RNA, shows a strong expression of 5S rRNA in oocytes, indicating the potential of 5S rRNA and its regulating proteins to become useful molecular makers of oocyte presence in testis. Therefore, the use of these oocyte markers to sex and identify intersex mullets could constitute powerful molecular biomarkers to assess xenoestrogenicity in field conditions.

Identification of Sex and Female’s Reproductive Stage in Commercial Fish Species through the Quantification of Ribosomal Transcripts in Gonads

The estimation of maturity and sex of fish stocks in European waters is a requirement of the EU Data Collection Framework as part of the policy to improve fisheries management. On the other hand, research on fish biology is increasingly focused in molecular approaches, researchers needing correct identification of fish sex and reproductive stage without necessarily having in house the histological know-how necessary for the task. Taking advantage of the differential gene transcription occurring during fish sex differentiation and gametogenesis, the utility of 5S ribosomal RNA (5S rRNA) and General transcription factor IIIA (gtf3a) in the molecular identification of sex and gametogenic stage was tested in different economically-relevant fish species from the Bay of Biscay. Gonads of 9 fish species (, Atlantic, Atlantic-chub and horse mackerel, blue whiting, bogue, European anchovy, hake and pilchard and megrim), collected from local commercial fishing vessels were histologically sexed and 5S and 18S rRNA concentrations were quantified by capillary electrophoresis to calculate a 5S/18S rRNA index. Degenerate primers permitted cloning and sequencing of gtf3a fragments in 7 of the studied species. 5S rRNA and gtf3a transcript levels, together with 5S/18S rRNA index, distinguished clearly ovaries from testis in all of the studied species. The values were always higher in females than in males. 5S/18S rRNA index values in females were always highest when fish were captured in early phases of ovary development whilst, in later vitellogenic stages, the values decreased significantly. In megrim and European anchovy, where gonads in different oogenesis stages were obtained, the 5S/18S rRNA index identified clearly gametogenic stage. This approach, to the sexing and the quantitative non-subjective identification of the maturity stage of female fish, could have multiple applications in the study of fish stock dynamics, fish reproduction and fecundity and fish biology in general.

18S rRNA V9 metabarcoding for diet characterization: a critical evaluation with two sympatric zooplanktivorous fish species

The potential of the 18S rRNA V9 metabarcoding approach for diet assessment was explored using MiSeq paired-end (PE; 2 9 150 bp) technology. To critically evaluate the method's performance with degraded/digested DNA, the diets of two zooplanktivorous fish species from the Bay of Biscay, European sardine (Sardina pilchardus) and European sprat (Sprattus sprattus), were analysed. The taxonomic resolution and quantitative potential of the 18S V9 metabarcoding was first assessed both in silico and with mock and field plankton samples. Our method was capable of discriminating species within the reference database in a reliable way providing there was at least one variable position in the 18S V9 region. Furthermore, it successfully discriminated diet between both fish species, including habitat and diel differences among sardines, overcoming some of the limitations of traditional visual-based diet analysis methods. The high sensitivity and semi-quantitative nature of the 18S V9 metabarcoding approach was supported by both visual microscopy and qPCR-based results. This molecular approach provides an alternative cost and time effective tool for food-web analysis.