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.

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

7 p.

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.

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.

Quantification of key biological processes determining the dynamics and the assessment of the anchovy population in the Bay of Biscay: growth, reproduction, demography and natural mortality.

333 p.

Combined Fluorescent-Chromogenic In Situ Hybridization for Identification and Laser Microdissection of Interphase Chromosomes

Chromosome territories constitute the most conspicuous feature of nuclear architecture, and they exhibit non-random distribution patterns in the interphase nucleus. We observed that in cell nuclei from humans with Down Syndrome two chromosomes 21 frequently localize proximal to one another and distant from the third chromosome. To systematically investigate whether the proximally positioned chromosomes were always the same in all cells, we developed an approach consisting of sequential FISH and CISH combined with laser-microdissection of chromosomes from the interphase nucleus and followed by subsequent chromosome identification by microsatellite allele genotyping. This approach identified proximally positioned chromosomes from cultured cells, and the analysis showed that the identity of the chromosomes proximally positioned varies. However, the data suggest that there may be a tendency of the same chromosomes to be positioned close to each other in the interphase nucleus of trisomic cells. The protocol described here represents a powerful new method for genome analysis

SNP Discovery in European Anchovy (Engraulis encrasicolus, L) by High-Throughput Transcriptome and Genome Sequencing

12 p.

New Nuclear SNP Markers Unravel the Genetic Structure and Effective Population Size of Albacore Tuna (Thunnus alalunga)

In the present study we have investigated the population genetic structure of albacore (Thunnus alalunga, Bonnaterre 1788) and assessed the loss of genetic diversity, likely due to overfishing, of albacore population in the North Atlantic Ocean. For this purpose, 1,331 individuals from 26 worldwide locations were analyzed by genotyping 75 novel nuclear SNPs. Our results indicated the existence of four genetically homogeneous populations delimited within the Mediterranean Sea, the Atlantic Ocean, the Indian Ocean and the Pacific Ocean. Current definition of stocks allows the sustainable management of albacore since no stock includes more than one genetic entity. In addition, short-and long-term effective population sizes were estimated for the North Atlantic Ocean albacore population, and results showed no historical decline for this population. Therefore, the genetic diversity and, consequently, the adaptive potential of this population have not been significantly affected by overfishing.

Effects of Spring Temperatures on the Strength of Selection on Timing of Reproduction in a Long-Distance Migratory Bird

Climate change has differentially affected the timing of seasonal events for interacting trophic levels, and this has often led to increased selection on seasonal timing. Yet, the environmental variables driving this selection have rarely been identified, limiting our ability to predict future ecological impacts of climate change. Using a dataset spanning 31 years from a natural population of pied flycatchers (Ficedula hypoleuca), we show that directional selection on timing of reproduction intensified in the first two decades (1980-2000) but weakened during the last decade (2001-2010). Against expectation, this pattern could not be explained by the temporal variation in the phenological mismatch with food abundance. We therefore explored an alternative hypothesis that selection on timing was affected by conditions individuals experience when arriving in spring at the breeding grounds: arriving early in cold conditions may reduce survival. First, we show that in female recruits, spring arrival date in the first breeding year correlates positively with hatch date; hence, early-hatched individuals experience colder conditions at arrival than late-hatched individuals. Second, we show that when temperatures at arrival in the recruitment year were high, early-hatched young had a higher recruitment probability than when temperatures were low. We interpret this as a potential cost of arriving early in colder years, and climate warming may have reduced this cost. We thus show that higher temperatures in the arrival year of recruits were associated with stronger selection for early reproduction in the years these birds were born. As arrival temperatures in the beginning of the study increased, but recently declined again, directional selection on timing of reproduction showed a nonlinear change. We demonstrate that environmental conditions with a lag of up to two years can alter selection on phenological traits in natural populations, something that has important implications for our understanding of how climate can alter patterns of selection in natural populations.