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.

Sasoiaren menpeko erreferentzia geneen transkripzio-profilen ikerketa Chelon labrosus lazunean

Chelon labrosus lazuna oso erabilia da kutsaduraren eragina aztertzeko organismo zentinela bezala, oso kutsatuta dauden itsaso zabaleko zein itsasadarretako uretan bizirauteko gai baita. Kutsatzaileek zelulen, ehunen eta organismoen transkripzio-profilen aldaketa eragin dezaketela ondo deskribatuta dago. Aldaketa hauek neurtzeko, gene-espresioaren normalizazioa burutu behar da. Eskuarki, normalizazio prozesua burutzeko, metabolismo orokorreko geneak erabiltzen dira, hau da, erreferentzia geneak. Gene hauek, zelularen oinarrizko funtzioak garatzeko beharrezkoak diren geneak dira eta zelula guztietan konstitutiboki adierazi ohi dira. Aurretik egin diren zenbait ikerketetan ordea, gene hauen transkripzio maila egoera esperimentalen, ehun motaren, garapen fasearen edota zelularen zikloaren arabera aldatu egiten dela behatu da. Hortaz, erreferentzia geneen transkripzio mailen aldaketak, aztergai den itu genearen interpretazio okerra eman dezake. Hori dela eta, azken urte hauetan, beste metodo batzuk garatzen ari dira geneen transkripzio maila normalizatzeko, esaterako, QuanT-it OliGreen metodoa. Testuinguru honetan, Chelon labrosus lazunean toxikologia-analisiak egiteko gehien erabiltzen diren erreferentzia geneen transkripzio-profilak aztertu dira indibiduo ar zein emeetan ugalketa zikloaren zeharreko fase gametogeniko desberdinetan (bost guztira). Horretarako, erreferentzia gene bat, itu gene kontsideratuz, normalizazioa beste erreferentzia geneekiko zein QuanT-it OliGreen metodotik lortutako emaitzekiko burutu da. Azterturiko geneak β-aktina (BA), 18S RNA erribosomikoa (18S rRNA), 1-α elongazio faktorea (EF-1-α) eta glizeraldehido-3-fosfato deshidrogenasa (GAPDH) izan dira. Aztergai zeuden erreferentzia geneen transkripzio maila qRT-PCR bidez kuantifikatu ondoren, aurretik aipatutako bi metodoekiko normalizatu ziren. Normalizazioa erreferentzia geneekiko egin zenean, itu gene berak transkripzio-profil desberdinak aurkeztu zituen normalizaziorako erabili zen erreferentzia genearen arabera. QuanT-it OliGreen metodoaren bidezko normalizazioa egin zenean berriz, aztertutako erreferentzia gene guztien transkripzio-profila, fase gametogenikoen arabera aldatzen zela ondorioztatu zen. Beraz, itu gene baten transkripzio-profilaren azterketa egin nahi denean QuanT-it OliGreen metodoa erreferentzia geneak erabiltzea baino fidagarriagoa da.

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.

Amplicon metagenomics for elucidating microbial community changes associated to conventional and organic farming of Beta vulgaris and Solanum lycopersicum

Soil microbial community changes associated to conventional and organic farming of two relevant crops (Beta vulgaris and Solanum lycopersicum) were analysed through 16s rRNA amplicon sequencing. This study revealed microbial communities in the agricultural soils studied to be similar to other reported nutrient-rich microbiomes, and some significant differences between the microbial communities associated to the two farming practices were found. Some phyla (Chloroflexi and Thermi) were found to be present in different abundances according to soil treatment. As chloroplast interference can be a stumbling block in plant-associated 16s rRNA amplicon metagenomics analysis of aerial plant tissues, two protocols for bacterial cell detachment (orbital shaking and ultrasound treatment) and two protocols for microbial biomass recovery (centrifugation and filtration) were tested regarding their efficiency at excluding plant-DNA. An alternative method to the one proposed by Rastogi et al (2010) for evaluating the chloroplast-amplicon content in post-PCR samples was tested, and the method revealed that filtration was the most efficient protocol in minimising chloroplast interference.

Number of Players and Relative Pitch Area per Player: Comparing Their Influence on Heart Rate and Physical Demands in Under-12 and Under-13 Football Players

The aim of the present study is to analyse the influence of different large-sided games (LSGs) on the physical and physiological variables in under-12s (U12) and -13s (U13) soccer players. The effects of the combination of different number of players per team, 7, 9, and 11 (P7, P9, and P11, respectively) with three relative pitch areas, 100, 200, and 300 m(2) (A100, A200, and A300, respectively), were analysed in this study. The variables analysed were: 1) global indicator such as total distance (TD); work:rest ratio (W:R); player-load (PL) and maximal speed (V-max); 2) heart rate (HR) mean and time spent in different intensity zones of HR (<75%, 75-84%, 84-90% and >90%), and; 3) five absolute (<8, 8-13, 13-16 and >16 Km h(-1)) and three relative speed categories (<40%, 40-60% and >60% V-max). The results support the theory that a change in format (player number and pitch dimensions) affects no similarly in the two players categories. Although it can seem that U13 players are more demanded in this kind of LSG, when the work load is assessed from a relative point of view, great pitch dimensions and/or high number of player per team are involved in the training task to the U12 players. The results of this study could alert to the coaches to avoid some types of LSGs for the U12 players such as:P11 played in A100, A200 or A300, P9 played in A200 or A300 and P7 played in A300 due to that U13>U12 in several physical and physiological variables (W:R, time spent in 84-90% HRmax, distance in 8-13 and 13-16 Km h(-1) and time spent in 40-60% V-max). These results may help youth soccer coaches to plan the progressive introduction of LSGs so that task demands are adapted to the physiological and physical development of participants.

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.