Heterogeneity of otolith chemical composition from two-dimensional mapping: Relationship with biomineralization mechanisms and implications for microchemistry analyses.

Although otoliths are widely used as archives to infer life-history traits and habitat use in fishes, their biomineralization process remains poorly understood. This lack of knowledge is problematic as it can lead to misinterpretation of the different types of signals (e.g., optical or chemical) that provide basic data for research in fish ecology, fisheries management, and species conservation. Otolith calcification relies on a complex system involving a pericrystalline fluid, the endolymph, whose organic and inorganic compositions are spatially heterogeneous for some constituents. This property stems from the particular structure of the calcifying saccular epithelium. In this study, we explored the spatial heterogeneity of elemental incorporation in otoliths for two species of high economic interest, European hake Merluccius merluccius (L. 1758) and European sea bass Dicentrarchus labrax (L. 1758). Two-dimensional mappings of chemical elements were obtained using UV high-repetition-rate femtosecond laser ablation (fs-LA) system coupled to a high-resolution inductively coupled plasma sector field mass spectrometer analyses on transverse sections of sagittae. Results highlighted a clear asymmetry between proximal (sulcus) and distal (antisulcus) concentrations for elements such as magnesium (Mg), phosphorus (P), manganese (Mn), and potassium (K) with concentration gradient directions that varied depending on the element. Strontium (Sr) and barium (Ba) did not show a proximo-distal gradient. These results are discussed in light of current knowledge on the endolymph composition and the mechanisms that lead to its compartmentalization, highlighting the need for further research on otolith biomineralization. Operational implications for studies based on otolith chemical composition are also discussed with emphasis on advice for sampling strategies to avoid analytical biases and the need for in-depth analyses of analytical settings before comparing otolith signatures between species or geographical areas.

Juvenile downstream migration patterns of an anadromous fish, allis shad (Alosa alosa), before and after the population collapse in the Gironde system, France.

Diadromous fish have exhibited a dramatic decline since the end of the 20th century. The allis shad (Alosa alosa) population in the Gironde-Garonne-Dordogne (GGD) system, once considered as a reference in Europe, remains low despite a fishing ban in 2008. One hypothesis to explain this decline is that the downstream migration and growth dynamics of young stages have changed due to environmental modifications in the rivers and estuary. We retrospectively analysed juvenile growth and migration patterns using otoliths from adults caught in the GGD system 30 years apart during their spawning migration, in 1987 and 2016. We coupled otolith daily growth increments and laser ablation inductively-coupled plasma mass spectrometry measurements of Sr:Ca, Ba:Ca, and Mn:Ca ratios along the longest growth axis from hatching to an age of 100 days (i.e., during the juvenile stage). A back-calculation allowed us to estimate the size of juveniles at the entrance into the brackish estuary. Based on the geochemistry data, we distinguished four different zones that juveniles encountered during their downstream migration: freshwater, fluvial estuary, brackish estuary, and lower estuary. We identified three migration patterns during the first 100 days of their life: (a) Individuals that reached the lower estuary zone, (b) individuals that reached the brackish estuary zone, and (c) individuals that reached the fluvial estuary zone. On average, juveniles from the 1987 subsample stayed slightly longer in freshwater than juveniles from the 2016 subsample. In addition, juveniles from the 2016 subsample entered the brackish estuary at a smaller size. This result suggests that juveniles from the 2016 subsample might have encountered more difficult conditions during their downstream migration, which we attribute to a longer exposure to the turbid maximum zone. This assumption is supported by the microchemical analyses of the otoliths, which suggests based on wider Mn:Ca peaks that juveniles in 2010s experienced a longer period of physiological stress during their downstream migration than juveniles in 1980s. Finally, juveniles from the 2016 subsample took longer than 100 days to exit the lower estuary than we would have expected from previous studies. Adding a new marker (i.e., Ba:Ca) helped us refine the interpretation of the downstream migration for each individual.

Growth potential can affect timing of maturity in a long-lived semelparous fish.

Many diadromous fishes such as salmon and eels that move between freshwater and the ocean have evolved semelparous reproductive strategies, but both groups display considerable plasticity in characteristics. Factors such as population density and growth, predation risk or reproduction cost have been found to influence timing of maturation. We investigated the relationship between female size at maturity and individual growth trajectories of the long-lived semelparous European eel, Anguilla anguilla A Bayesian model was applied to 338 individual growth trajectories of maturing migration-stage female silver eels from France, Ireland, the Netherlands and Hungary. The results clearly showed that when growth rates declined, the onset of maturation was triggered, and the eels left their growth habitats and migrated to the spawning area. Therefore, female eels tended to attain larger body size when the growth conditions were good enough to risk spending extra time in their growth habitats. This flexible maturation strategy is likely related to the ability to use diverse habitats with widely ranging growth and survival potentials in the catadromous life-history across its wide species range.

Differences in brain gene transcription profiles advocate for an important role of cognitive function in upstream migration and water obstacles crossing in European eel.

BACKGROUND: European eel is a panmictic species, whose decline has been recorded since the last 20 years. Among human-induced environmental factors of decline, the impact of water dams during species migration is questioned. The main issue of this study was to pinpoint phenotypic traits that predisposed glass eels to successful passage by water barriers. The approach of the study was individual-centred and without any a priori hypothesis on traits involved in the putative obstacles selective pressure. We analyzed the transcription level of 14,913 genes. RESULTS: Transcriptome analysis of three tissues (brain, liver and muscle) from individuals sampled on three successive forebays separated by water obstacles indicated different gene transcription profiles in brain between the two upstream forebays. No differences in gene transcription levels were observed in liver and muscle samples among segments. A total of 26 genes were differentially transcribed in brain. These genes encode for, among others, keratins, cytokeratins, calcium binding proteins (S100 family), cofilin, calmodulin, claudin and thy-1 membrane glycoprotein. The functional analysis of these genes highlighted a putative role of cytoskeletal dynamics and synaptic plasticity in fish upstream migration. CONCLUSION: Synaptic connections in brain are solicited while eels are climbing the obstacles with poorly designed fishways. Successful passage by such barriers can be related to spatial learning and spatial orientation abilities when fish is out of the water.

Quantifying larval dispersal portfolio in seabass nurseries using otolith chemical signatures.

The temporal asynchronies in larvae production from different spawning areas are fundamental components for ensuring stability and resilience of marine metapopulations. Such a concept, named portfolio effect, supposes that diversifying larval dispersal histories should minimize the risk of recruitment failure by increasing the probability that at least some larvae successfully settle in nursery. Here, we used a reconstructive approach based on otolith chemistry to quantify the larval dispersal portfolio of the European seabass, Dicentrarchus labrax, across six estuarine nursery areas of the northeast Atlantic Ocean. The analysis of natal and trajectory signatures indicated that larvae hatch in distinct environments and then dispersed in water masses featured by contrasting chemical signatures. While some trace elements appeared affected by temporal changes (Mn and Sr), others varied spatially during the larval stage but remained poorly affected by temporal fluctuation and fish physiology (Ba, Cu, Rb and Zn). We then proposed two diversity metrics based on richness and variations of chemical signatures among populations to reflect spatio-temporal diversity in natal origins and larval trajectories (i.e., estimates of dispersal portfolio). Along the French coast, the diversity estimates were maximum in nurseries located at proximity of offshore spawning sites and featured by complex offshore hydrodynamic contexts, such as the Mont St-Michel bay. Finally, our findings indicate that the dispersal portfolio was positively related with the local abundance of seabass juveniles, supporting the assumption that heterogeneity in dispersal history contributes to promote recruitment success in nurseries.

New insights into detecting alizarin from autofluorescence in marked glass eels.

Alizarin detection in fish fins is extensively employed because it is easy to use. However, in eels, the eelGFP fluorescent protein may impede the detection of the fluorescent markers in the eel tissues. The study tests the effectiveness of three of the most up-to-date alizarin-detecting technologies on the living body and fins of European glass eels (Anguilla anguilla L.). The findings demonstrated that the control group had a high autofluorescence at alizarin and eelGFP maxima bands. With fluorescence reflectance imaging (FRI), the eel living body autofluorescence impeded the detection of the marked eels. In contrast with experimental excitation-emission-matrix (EEM) fluorescence analyses, 99% of the marked eels were correctly assigned to their group from fluorescence analyses of their fin cellular contents. With epifluorometry (EPI), 100% of the marked eels were detected with the caudal fin tips when excited at 450-490 nm wavelengths due to a weaker autofluorescence signal. EEM and FRI assays unveiled an average fluorescence quenching 60% and 44% of the marked group respectively, in the alizarin and eelGFP maxima bands. The fluorescence quenching observed is discussed. Results will benefit experimental design by examining autofluorescence effects on mark detection and the development of non-invasive detection methods in this critically endangered species.

Impact of environmental micropollutants and diet composition on the gut microbiota of wild european eels (Anguilla anguilla).

In fish, the gut microbiome plays a crucial role in homeostasis and health and is affected by several organic and inorganic environmental contaminants. Amphidromous fish are sentinel species, particularly exposed to these stressors. We used whole metagenome sequencing to characterize the gut microbiome of wild European eels (Anguilla anguilla) at a juvenile stage captured from three sites with contrasted pollution levels in term of heavy metals and persistent organic pollutants. The objectives were to identify what parameters could alter the gut microbiome of this catadromous fish and to explore the potential use of microbiota as bioindicators of environment quality. We identified a total of 1079 microbial genera. Overall, gut microbiome was dominated by Proteobacteria, Firmicutes and Actinobacteria. Alpha and beta diversity were different amongst sites and could be explained by a reduced number of environmental and biological factors, specifically the relative abundance of fish preys in eels' diet, PCB101, γHCH (lindane), transnonachlor and arsenic. Furthermore, we identified a series of indicator taxa with differential abundance between the three sites. Changes in the microbial communities in the gut caused by environmental pollutants were previously undocumented in European eels. Our results indicate that microbiota might represent another route by which pollutants affect the health of these aquatic sentinel organisms.

Variable outcomes of hybridization between declining Alosa alosa and Alosa fallax.

Hybridization dynamics between co-occurring species in environments where human-mediated changes take place are important to quantify for furthering our understanding of human impacts on species evolution and for informing management. The allis shad Alosa alosa (Linnaeus, 1758) and twaite shad Alosa fallax (Lacépède, 1803), two clupeids sister species, have been severely impacted by human activities across Europe. The shrinkage of A. alosa distribution range along with the decline of the remaining populations' abundance threatens its persistence. The main objective was to evaluate the extent of hybridization and introgression between those interacting species. We developed a set of 77 species-specific SNP loci that allowed a better resolution than morphological traits as they enabled the detection of hybrids up to the third generation. Variable rates of contemporary hybridization and introgression patterns were detected in 12 studied sites across the French Atlantic coast. Mitochondrial markers revealed a cyto-nuclear discordance almost invariably involving A. alosa individuals with an A. fallax mitochondrial DNA and provided evidence of historical asymmetric introgression. Overall, contemporary and historical introgression revealed by nuclear and mitochondrial markers strongly suggests that a transfer of genes occurs from A. fallax toward A. alosa genome since at least four generations. Moreover, the outcomes of introgression greatly depend on the catchments where local processes are thought to occur. Undoubtedly, interspecific interaction and gene flow should not be overlooked when considering the management of those species.

Fast sequence-based microsatellite genotyping development workflow.

Application of high-throughput sequencing technologies to microsatellite genotyping (SSRseq) has been shown to remove many of the limitations of electrophoresis-based methods and to refine inference of population genetic diversity and structure. We present here a streamlined SSRseq development workflow that includes microsatellite development, multiplexed marker amplification and sequencing, and automated bioinformatics data analysis. We illustrate its application to five groups of species across phyla (fungi, plant, insect and fish) with different levels of genomic resource availability. We found that relying on previously developed microsatellite assay is not optimal and leads to a resulting low number of reliable locus being genotyped. In contrast, de novo ad hoc primer designs gives highly multiplexed microsatellite assays that can be sequenced to produce high quality genotypes for 20-40 loci. We highlight critical upfront development factors to consider for effective SSRseq setup in a wide range of situations. Sequence analysis accounting for all linked polymorphisms along the sequence quickly generates a powerful multi-allelic haplotype-based genotypic dataset, calling to new theoretical and analytical frameworks to extract more information from multi-nucleotide polymorphism marker systems.

Whether European eel leptocephali use the Earth's magnetic field to guide their migration remains an open question.

European eels (Anguilla anguilla) migrate between the southwestern Sargasso Sea and the European and Mediterranean coasts. In a recent paper in Current Biology, Naisbett-Jones et al.[1] claim to "provide the first evidence that they [eels] derive positional information from the Earth's magnetic field" and that this information guides their migration. The evidence reported by Naisbett-Jones et al.[1] in support of this conclusion was derived from eels collected in the Severn River (UK), approximately 50 km upstream of the estuary (i.e. not "in the Severn Estuary" as stated by the authors). Eels collected this far into rivers are benthic and fully adapted to freshwater; that is, they are late-stage glass eels (∼ 2 years old), not the pelagic leptocephalus (larval) life stage that actually undertakes the trans-Atlantic migration. The entire interpretive framework for the Naisbett-Jones et al.[1] study rests on the assumption that the behaviour of these late-stage freshwater glass eels, and their responses to magnetic fields, can be used as a proxy for the responses of eel leptocephali. The authors present no evidence in support of this key assumption.

Can analysis of Platichthys flesus otoliths provide relevant data on historical metal pollution in estuaries? Experimental and in situ approaches.

Despite recent efforts to manage them more efficiently, estuaries are natural sinks for a wide range of metal contaminants, many of which accumulate at potentially toxic concentrations for fish populations, posing a threat to recruitment and stocks. While analysis of metal concentrations in soft tissue and water samples calls for continuous and long-term sampling operations, the use of otoliths to study metal pollution may be one way of providing a historical record of pollutant exposure. In this study, we examine the potential use of otoliths as natural tracers of metal contamination. A "cocktail" of different metals (Cd, Pb and Ni) was used to test bio-accumulation in otoliths and tissue (liver, kidney, muscle and gills) extracted from juvenile flounder (Platichthys flesus). Assessment took place under controlled conditions over a three month period, with water exposure concentrations increasing every 3weeks. The concentrations used were natural (T1), X5 (T2), X10 (T3), and null (T4). Chemical analyses were carried out using an inductively coupled plasma optical emission spectrometer ICP-OES and atomic absorption spectrometer AAS for water and tissue, while otolith microchemistry analyses were performed using a femtosecond laser ablation-high resolution inductively coupled plasma mass spectrometer (fsLA-ICP-MS-HR). Significant differences between control and exposed individuals, as well as an increase in metal concentrations according to exposure level, were observed in all tissues except in muscle tissue. Significant increases in Pb were also detected in contaminated fish otoliths compared with control specimens, with the highest concentrations occurring in T3. Cartographies of Pb distribution in otoliths of both control and contaminated fish only showed high concentrations of Pb at the edge of contaminated fish otoliths, indicating an accumulation of metal during the experiment. Although the relationships between exposure level and Pb concentration in otoliths were complex, the concentrations were correlated with those in the water. Analysis of flounder specimens collected from 2007 to 2014 in the Gironde estuary (SW France) showed interannual variability in Pb concentrations, with higher values for fish otoliths from 2007 to 2010 than those from 2012 to 2014. This trend indicated a decrease in Pb in the Gironde estuary over the last decade, which is consistent with the results of other surveys on bivalves. Our study demonstrates that it is possible to use otolith microchemistry as a tool in assessing and retracing long-term metal pollution in estuarine systems.