Jose Martin Pujolar

Sequencing, de novo annotation and analysis of the first Anguilla anguilla transcriptome: EeelBase opens new perspectives for the study of the critically endangered european eel

BackgroundOnce highly abundant, the European eel (Anguilla anguilla L.; Anguillidae; Teleostei) is considered to be critically endangered and on the verge of extinction, as the stock has declined by 90-99% since the 1980s. Yet, the species is poorly characterized at molecular level with little sequence information available in public databases.ResultsThe first European eel transcriptome was obtained by 454 FLX Titanium sequencing of a normalized cDNA library, produced from a pool of 18 glass eels (juveniles) from the French Atlantic coast and two sites in the Mediterranean coast. Over 310,000 reads were assembled in a total of 19,631 transcribed contigs, with an average length of 531 nucleotides. Overall 36% of the contigs were annotated to known protein/nucleotide sequences and 35 putative miRNA identified.ConclusionsThis study represents the first transcriptome analysis for a critically endangered species. EeelBase, a dedicated database of annotated transcriptome sequences of the European eel is freely available at http://compgen.bio.unipd.it/eeelbase. Considering the multiple factors potentially involved in the decline of the European eel, including anthropogenic factors such as pollution and human-introduced diseases, our results will provide a rich source of data to discover and identify new genes, characterize gene expression, as well as for identification of genetic markers scattered across the genome to be used in various applications.

Surviving in a toxic world: transcriptomics and gene expression profiling in response to environmental pollution in the critically endangered European eel.

BackgroundGenomic and transcriptomic approaches have the potential for unveiling the genome-wide response to environmental perturbations. The abundance of the catadromous European eel (Anguilla anguilla) stock has been declining since the 1980s probably due to a combination of anthropogenic and climatic factors. In this paper, we explore the transcriptomic dynamics between individuals from high (river Tiber, Italy) and low pollution (lake Bolsena, Italy) environments, which were measured for 36 PCBs, several organochlorine pesticides and brominated flame retardants and nine metals.ResultsTo this end, we first (i) updated the European eel transcriptome using deep sequencing data with a total of 640,040 reads assembled into 44,896 contigs (Eeelbase release 2.0), and (ii) developed a transcriptomic platform for global gene expression profiling in the critically endangered European eel of about 15,000 annotated contigs, which was applied to detect differentially expressed genes between polluted sites. Several detoxification genes related to metabolism of pollutants were upregulated in the highly polluted site, including genes that take part in phase I of the xenobiotic metabolism (CYP3A), phase II (glutathione-S-transferase) and oxidative stress (glutathione peroxidase). In addition, key genes in the mitochondrial respiratory chain and oxidative phosphorylation were down-regulated at the Tiber site relative to the Bolsena site.ConclusionsTogether with the induced high expression of detoxification genes, the suggested lowered expression of genes supposedly involved in metabolism suggests that pollution may also be associated with decreased respiratory and energy production.

Genetic patchiness in European eel adults evidenced by molecular genetics and population dynamics modelling.

Disentangling the demographic processes that determine the genetic structure of a given species is a fundamental question in conservation and management. In the present study, the population structure of the European eel was examined with a multidisciplinary approach combining the fields of molecular genetics and population dynamics modelling. First, we analyzed a total of 346 adult specimens of known age collected in three separate sample sites using a large panel of 22 EST-linked microsatellite loci. Second, we developed a European eel-specific model to unravel the demographic mechanisms that can produce the level of genetic differentiation estimated by molecular markers. This is the first study that reveals a pattern of genetic patchiness in maturing adults of the European eel. A highly significant genetic differentiation was observed among samples that did not follow an Isolation-by-Distance or Isolation-by-Time pattern. The observation of genetic patchiness in adults is likely to result from a limited parental contribution to each spawning event as suggested by our modelling approach. The value of genetic differentiation found is predicted by the model when reproduction occurs in a limited number of spawning events isolated from each other in time or space, with an average of 130-375 breeders in each spawning event. Unpredictability in spawning success may have important consequences for the life-history evolution of the European eel, including a bet-hedging strategy (distributing reproductive efforts over time) which could in turn guarantee successful reproduction of some adults.

No apparent genetic bottleneck in the demographically declining European eel using molecular genetics and forward-time simulations

The stock of the European eel is considered to be outside safe biological limits, following a dramatic demographic decline in recent decades (90–99% drop) that involves a large number of factors including overfishing, contaminants and environmental fluctuations. The aim of the present study is to estimate the effective population size of the European eel and the possible existence of a genetic bottleneck, which is expected during or after a severe demographic crash. Using a panel of 22 EST-derived microsatellite loci, we found no evidence for a genetic bottleneck in the European eel as our data showed moderate to high levels of genetic diversity, no loss of allele size range or rare alleles, and a stationary population with growth values not statistically different from zero, which is confirmed by finding comparable value of short-term and long-term effective population size. Our results suggest that the observed demographic decline in the European eel did not entail a genetic decline of the same magnitude. Forward-time simulations confirmed that large exploited marine fish populations can undergo genetic bottleneck episodes and experience a loss of genetic variability. Simulations indicated that the failure to pick up the signal of a genetic bottleneck in the European eel is not due to lack of power. Although anthropogenic factors lowered the continental stock biomass, the observation of a stable genetic effective population size suggests that the eel crash was not due to a reduction in spawning stock abundance. Alternatively, we propose that overfishing, pollution and/or parasites might have affected individual fitness and fecundity, leading to an impoverished spawning stock that may fail to produce enough good quality eggs. A reduced reproduction success due to poor quality of the spawners may be exacerbated by oceanic processes inducing changes in primary production in the Sargasso Sea and/or pathway of transport across the Atlantic Ocean leading to a higher larval mortality.

Positive Darwinian selection in gamete recognition proteins of Strongylocentrotus sea urchins

Gamete recognition proteins commonly experience positive Darwinian selection and evolve more rapidly than nonreproductive proteins, but the selective forces responsible for their adaptive diversification remain unclear. We examined the patterns of positive selection in the cognate interacting pair of proteins formed by sperm bindin and its egg receptor (EBR1) and in two regions of the sea urchin sperm receptor for egg jelly suREJ3 gene (exons 22 and 26) among four species of Strongylocentrotus sea urchins (S. purpuratus, S. droebachiensis, S. pallidus and S. franciscanus). The signatures of selection differed at each reproductive protein. A strong signal of positive selection was detected at bindin in all lineages even though the species compared had highly variable gamete traits and experience different intensities and forms of sexual selection and sexual conflict in nature. Weaker selection was observed at EBR1 but the small region studied precluded a clear understanding of the extent of sexual conflict between bindin and the EBR1 protein. At the suREJ3 locus, diversifying selection was observed in exon 22 but not exon 26, suggesting that these regions experience different selective pressures and evolutionary constraints. Positive selection was also detected within S. pallidus at suREJ‐22 because of the presence of 12 amino acid replacement mutations segregating at frequencies >0.10. Our results suggest that sexual conflict may be the predominant evolutionary mechanism driving the rapid diversification of reproductive proteins between, and polymorphism within, strongylocentrotid sea urchins.

Genetic composition of Atlantic and Mediterranean recruits of European eel Anguilla anguilla based on EST-linked microsatellite loci.

Anguilla anguilla glass eels arriving at two Mediterranean and two Atlantic sites were tested for differences in genetic composition between regions using a total of 23 microsatellite loci developed from an expressed sequence tag (EST) library. Hierarchical analysis of molecular variance indicated a non-significant difference between regions (Mediterranean v. Atlantic), which contrasted with the significant differences observed between samples within regions. The existence of a single spawning site for all A. anguilla individuals and extensive migration loop with great opportunity for mixing of individuals might explain the homogeneity in genetic composition found between regions. The observation of a (small-scale) pattern of genetic patchiness among intra-annual samples (arrival waves) within geographic regions does not conflict with the lack of (large-scale) geographic sub-structuring found between the Mediterranean and Atlantic regions, but most likely is a consequence of the strong dependence of A. anguilla on oceanic conditions in the Sargasso Sea that might result in a limited parental contribution to each spawning event. The comparison of Atlantic and Mediterranean A. anguilla glass eel recruits based on EST-linked microsatellite loci provides evidence supporting the hypothesis of panmixia A. anguilla across Europe.

Detecting genome-wide gene transcription profiles associated with high pollution burden in the critically endangered European eel

The European eel illustrates an example of a critically endangered fish species strongly affected by human stressors throughout its life cycle, in which pollution is considered to be one of the factors responsible for the decline of the stock. The objective of our study was to better understand the transcriptional response of European eels chronically exposed to pollutants in their natural environment. A total of 42 pre-migrating (silver) female eels from lowly, highly and extremely polluted environments in Belgium and, for comparative purposes, a lowly polluted habitat in Italy were measured for polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs) and brominated flame retardants (BFRs). Multipollutant level of bioaccumulation was linked to their genome-wide gene transcription using an eel-specific array of 14,913 annotated cDNAs. Shared responses to pollutant exposure were observed when comparing the highly polluted site in Belgium with the relatively clean sites in Belgium and Italy. First, an altered pattern of transcription of genes was associated with detoxification, with a novel European eel CYP3A gene and gluthatione S-transferase transcriptionally up-regulated. Second, an altered pattern of transcription of genes associated with the oxidative phosphorylation pathway, with the following genes involved in the generation of ATP being transcriptionally down-regulated in individuals from the highly polluted site: NADH dehydrogenase, succinate dehydrogenase, ubiquinol-cytochrome c reductase, cytochrome c oxidase and ATP synthase. Although we did not measure metabolism directly, seeing that the transcription level of many genes encoding enzymes involved in the mitochondrial respiratory chain and oxidative phosphorylation were down-regulated in the highly polluted site suggests that pollutants may have a significant effect on energy metabolism in these fish.

The Effect of Recurrent Floods on Genetic Composition of Marble Trout Populations

A changing global climate can threaten the diversity of species and ecosystems. We explore the consequences of catastrophic disturbances in determining the evolutionary and demographic histories of secluded marble trout populations in Slovenian streams subjected to weather extremes, in particular recurrent flash floods and debris flows causing massive mortalities. Using microsatellite data, a pattern of extreme genetic differentiation was found among populations (global F ST of 0.716), which exceeds the highest values reported in freshwater fish. All locations showed low levels of genetic diversity as evidenced by low heterozygosities and a mean of only 2 alleles per locus, with few or no rare alleles. Many loci showed a discontinuous allele distribution, with missing alleles across the allele size range, suggestive of a population contraction. Accordingly, bottleneck episodes were inferred for all samples with a reduction in population size of 3–4 orders of magnitude. The reduced level of genetic diversity observed in all populations implies a strong impact of genetic drift, and suggests that along with limited gene flow, genetic differentiation might have been exacerbated by recurrent mortalities likely caused by flash flood and debris flows. Due to its low evolutionary potential the species might fail to cope with an intensification and altered frequency of flash flood events predicted to occur with climate change.

Managing Polyploidy in Ex Situ Conservation Genetics: The Case of the Critically Endangered Adriatic Sturgeon (Acipenser naccarii)

While the current expansion of conservation genetics enables to address more efficiently the management of threatened species, alternative methods for genetic relatedness data analysis in polyploid species are necessary. Within this framework, we present a standardized and simple protocol specifically designed for polyploid species that can facilitate management of genetic diversity, as exemplified by the ex situ conservation program for the tetraploid Adriatic sturgeon Acipenser naccarii. A critically endangered endemic species of the Adriatic Sea tributaries, its persistence is strictly linked to the ex situ conservation of a single captive broodstock currently decimated to about 25 individuals, which represents the last remaining population of Adriatic sturgeon of certain wild origin. The genetic variability of three F1 broodstocks available as future breeders was estimated based on mitochondrial and microsatellite information and compared with the variability of the parental generation. Genetic data showed that the F1 stocks have only retained part of the genetic variation present in the original stock due to the few parent pairs used as founders. This prompts for the urgent improvement of the current F1 stocks by incorporating new founders that better represent the genetic diversity available. Following parental allocation based on band sharing values, we set up a user-friendly tool for selection of candidate breeders according to relatedness between all possible parent-pairs that secures the use of non-related individuals. The approach developed here could also be applied to other endangered tetraploid sturgeon species overexploited for caviar production, particularly in regions lacking proper infrastructure and/or expertise.

Reconciling Deep Calibration and Demographic History: Bayesian Inference of Post Glacial Colonization Patterns in Carcinus aestuarii (Nardo, 1847) and C. maenas (Linnaeus, 1758)

A precise inference of past demographic histories including dating of demographic events using Bayesian methods can only be achieved with the use of appropriate molecular rates and evolutionary models. Using a set of 596 mitochondrial cytochrome c oxidase I (COI) sequences of two sister species of European green crabs of the genus Carcinus (C. maenas and C. aestuarii), our study shows how chronologies of past evolutionary events change significantly with the application of revised molecular rates that incorporate biogeographic events for calibration and appropriate demographic priors. A clear signal of demographic expansion was found for both species, dated between 10,000 and 20,000 years ago, which places the expansions events in a time frame following the Last Glacial Maximum (LGM). In the case of C. aestuarii, a population expansion was only inferred for the Adriatic-Ionian, suggestive of a colonization event following the flooding of the Adriatic Sea (18,000 years ago). For C. maenas, the demographic expansion inferred for the continental populations of West and North Europe might result from a northward recolonization from a southern refugium when the ice sheet retreated after the LGM. Collectively, our results highlight the importance of using adequate calibrations and demographic priors in order to avoid considerable overestimates of evolutionary time scales.