Performance and precision of double digestion RAD (ddRAD) genotyping in large multiplexed datasets of marine fish species.

The development of Genotyping-By-Sequencing (GBS) technologies enables cost-effective analysis of large numbers of Single Nucleotide Polymorphisms (SNPs), especially in "non-model" species. Nevertheless, as such technologies enter a mature phase, biases and errors inherent to GBS are becoming evident. Here, we evaluated the performance of double digest Restriction enzyme Associated DNA (ddRAD) sequencing in SNP genotyping studies including high number of samples. Datasets of sequence data were generated from three marine teleost species (>5500 samples, >2.5 × 1012 bases in total), using a standardized protocol. A common bioinformatics pipeline based on STACKS was established, with and without the use of a reference genome. We performed analyses throughout the production and analysis of ddRAD data in order to explore (i) the loss of information due to heterogeneous raw read number across samples; (ii) the discrepancy between expected and observed tag length and coverage; (iii) the performances of reference based vs. de novo approaches; (iv) the sources of potential genotyping errors of the library preparation/bioinformatics protocol, by comparing technical replicates. Our results showed use of a reference genome and a posteriori genotype correction improved genotyping precision. Individual read coverage was a key variable for reproducibility; variance in sequencing depth between loci in the same individual was also identified as an important factor and found to correlate to tag length. A comparison of downstream analysis carried out with ddRAD vs single SNP allele specific assay genotypes provided information about the levels of genotyping imprecision that can have a significant impact on allele frequency estimations and population assignment. The results and insights presented here will help to select and improve approaches to the analysis of large datasets based on RAD-like methodologies.

Intermediate number of major histocompatibility complex class IIB length variants relates to enlarged perivisceral fat deposits in the blunt-head cichlid Tropheus moorii.

Studying the genetic basis of host-parasite interactions represents an outstanding opportunity to observe eco-evolutionary processes. Established candidates for such studies in vertebrates are immunogenes of the major histocompatibility complex (MHC). The MHC has been reported to reach high intra- and interindividual diversity, and a diverse MHC might be advantageous when facing infections from multiple parasites. However, other studies indicated that individuals with an intermediate number of MHC alleles are less infected with parasites or have other fitness advantages. In this study, we assessed the optimal number of MHC alleles in the blunt-head cichlid Tropheus moorii from Lake Tanganyika. We investigated the influence of the interindividual variation in number of MHC length variants on parasite infection and body condition, measured by the amount of perivisceral fat reserves. Surprisingly, there was no correlation between parasite infection and number of MHC length variants or perivisceral fat deposits. However, the individual number of MHC length variants significantly correlated with the amount of perivisceral fat deposits in males, suggesting that male individuals with an intermediate number of alleles might be able to use their fat reserves more efficiently.

Regional environmental pressure influences population differentiation in turbot (Scophthalmus maximus).

Unravelling the factors shaping the genetic structure of mobile marine species is challenging due to the high potential for gene flow. However, genetic inference can be greatly enhanced by increasing the genomic, geographical or environmental resolution of population genetic studies. Here, we investigated the population structure of turbot (Scophthalmus maximus) by screening 17 random and gene-linked markers in 999 individuals at 290 geographical locations throughout the northeast Atlantic Ocean. A seascape genetics approach with the inclusion of high-resolution oceanographical data was used to quantify the association of genetic variation with spatial, temporal and environmental parameters. Neutral loci identified three subgroups: an Atlantic group, a Baltic Sea group and one on the Irish Shelf. The inclusion of loci putatively under selection suggested an additional break in the North Sea, subdividing southern from northern Atlantic individuals. Environmental and spatial seascape variables correlated marginally with neutral genetic variation, but explained significant proportions (respectively, 8.7% and 10.3%) of adaptive genetic variation. Environmental variables associated with outlier allele frequencies included salinity, temperature, bottom shear stress, dissolved oxygen concentration and depth of the pycnocline. Furthermore, levels of explained adaptive genetic variation differed markedly between basins (3% vs. 12% in the North and Baltic Sea, respectively). We suggest that stable environmental selection pressure contributes to relatively strong local adaptation in the Baltic Sea. Our seascape genetic approach using a large number of sampling locations and associated oceanographical data proved useful for the identification of population units as the basis of management decisions.

Hybridisation between the two major African schistosome species of humans.

It is generally accepted that Schistosoma mansoni and Schistosoma haematobium, causing intestinal and urinary schistosomiasis, respectively, are not able to hybridise, due to the high phylogenetic distance between them. Cloning of nuclear internal transcribed spacer rDNA and partial mitochondrial cytochrome c oxidase 1 fragments revealed two internal transcribed spacer rDNA genotypes within single eggs and miracidia, one identical to S. mansoni and the other identical to S. haematobium, suggesting hybrid ancestry. The cytochrome c oxidase 1 clones always belonged to only one of the parental species. This demonstrates that offspring of heterologous pairing between these two species is not (always) parthenogenetic.

Gene transcription reflects poor health status of resident European eel chronically exposed to environmental pollutants.

Understanding the effects of chronic exposure to pollutants on the genome and transcriptome of diadromous fish populations is crucial for their resilience under combined anthropogenic and environmental selective pressures. The catadromous European eel (Anguilla anguilla L.) has suffered a dramatic decline in recruitment for three decades, necessitating a thorough assessment of the transcriptional effects of environmental pollutants on resident and migrating eels in natural systems. We investigated the relationship between muscular bioaccumulation levels of metals (Hg, Cd, Pb, Cu, Zn, Ni, Cr, As and Se), PCBs and organochlorine pesticides (DDTs), the health status (condition factor and lipid reserves) and the associated transcriptional response in liver and gill tissues for genes involved in metal detoxification (metallothionein, MT) and oxidative metabolism (cytochrome P4501A, CYP1A) of xenobiotic compounds. In total 84 resident eels originating from three Belgian river basins (Scheldt, Meuse and Yzer) were analyzed along with five unpolluted aquaculture samples as control group. There was a large spatial variation in individual contaminant intensity and profile, while tissue pollution levels were strongly and negatively associated with condition indices, suggesting an important impact of pollution on the health of sub-adult resident eels. Gene transcription patterns revealed a complex response mechanism to a cocktail of pollutants, with a high variation at low pollution levels, but strongly down-regulated hepatic and gill gene transcription in highly polluted eels. Resident eels clearly experience a high pollution burden and seem to show a dysfunctional gene transcription regulation of detoxification genes at higher pollutant levels, correlated with low energy reserves and condition. To fully understand the evolutionary implications of pollutants on eel reproductive fitness, analyses of mature migrating eels and the characterization of their transcriptome-wide gene transcription response would be appropriate to unveil the complex responses associated with multiple interacting stressors and the long-term consequences at the entire species level. In the meanwhile, jointly monitoring environmental and tissue pollution levels at a European scale should be initiated, while preserving high quality habitats to increase the recovery chance of European eel in the future.

Permanent genetic resources added to Molecular Ecology Resources Database 1 December 2011-31 January 2012.

This article documents the addition of 473 microsatellite marker loci and 71 pairs of single-nucleotide polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Barteria fistulosa, Bombus morio, Galaxias platei, Hematodinium perezi, Macrocentrus cingulum Brischke (a.k.a. M. abdominalis Fab., M. grandii Goidanich or M. gifuensis Ashmead), Micropogonias furnieri, Nerita melanotragus, Nilaparvata lugens Stål, Sciaenops ocellatus, Scomber scombrus, Spodoptera frugiperda and Turdus lherminieri. These loci were cross-tested on the following species: Barteria dewevrei, Barteria nigritana, Barteria solida, Cynoscion acoupa, Cynoscion jamaicensis, Cynoscion leiarchus, Cynoscion nebulosus, Cynoscion striatus, Cynoscion virescens, Macrodon ancylodon, Menticirrhus americanus, Nilaparvata muiri and Umbrina canosai. This article also documents the addition of 116 sequencing primer pairs for Dicentrarchus labrax.

Permanent genetic resources added to Molecular Ecology Resources Database 1 August 2011-30 September 2011.

This article documents the addition of 299 microsatellite marker loci and nine pairs of single-nucleotide polymorphism (SNP) EPIC primers to the Molecular Ecology Resources (MER) Database. Loci were developed for the following species: Alosa pseudoharengus, Alosa aestivalis, Aphis spiraecola, Argopecten purpuratus, Coreoleuciscus splendidus, Garra gotyla, Hippodamia convergens, Linnaea borealis, Menippe mercenaria, Menippe adina, Parus major, Pinus densiflora, Portunus trituberculatus, Procontarinia mangiferae, Rhynchophorus ferrugineus, Schizothorax richardsonii, Scophthalmus rhombus, Tetraponera aethiops, Thaumetopoea pityocampa, Tuta absoluta and Ugni molinae. These loci were cross-tested on the following species: Barilius bendelisis, Chiromantes haematocheir, Eriocheir sinensis, Eucalyptus camaldulensis, Eucalyptus cladocalix, Eucalyptus globulus, Garra litaninsis vishwanath, Garra para lissorhynchus, Guindilla trinervis, Hemigrapsus sanguineus, Luma chequen. Guayaba, Myrceugenia colchagüensis, Myrceugenia correifolia, Myrceugenia exsucca, Parasesarma plicatum, Parus major, Portunus pelagicus, Psidium guayaba, Schizothorax richardsonii, Scophthalmus maximus, Tetraponera latifrons, Thaumetopoea bonjeani, Thaumetopoea ispartensis, Thaumetopoea libanotica, Thaumetopoea pinivora, Thaumetopoea pityocampa ena clade, Thaumetopoea solitaria, Thaumetopoea wilkinsoni and Tor putitora. This article also documents the addition of nine EPIC primer pairs for Euphaea decorata, Euphaea formosa, Euphaea ornata and Euphaea yayeyamana.

Mito-nuclear discordance in the degree of population differentiation in a marine goby.

An increasing number of phylogeographic studies on marine species shows discordant patterns in the degree of population differentiation between nuclear and mitochondrial markers. To understand better which factors have the potential to cause these patterns of discordance in marine organisms, a population genetic study was realized on the sand goby Pomatoschistus minutus (Pallas 1770; Gobiidae, Teleostei). Sand gobies from eight European locations were genotyped at eight microsatellite markers. Microsatellites confirmed the global phylogeographical pattern of P. minutus observed with mitochondrial DNA (mtDNA) markers and nuclear allozyme markers. Three groups consistent with the mitochondrial lineages were defined (the Mediterranean, Iberian and North Atlantic groups) and indications of a recent founder event in the northern Baltic Sea were found. Nevertheless, differences in the degree of population differentiation between the nuclear and mitochondrial markers were large (global F(ST)-values for microsatellites=0.0121; for allozymes=0.00831; for mtDNA=0.4293). Selection, sex-biased dispersal, homoplasy and a high effective population size are generally accepted as explanations for this mitonuclear discrepancy in the degree of population differentiation. In this study, selection on mtDNA and microsatellites, male-biased dispersal and homoplasy on microsatellite markers are unlikely to be a main cause for this discrepancy. The most likely reason for the discordant pattern is a recent demographical expansion of the sand goby, resulting in high effective population sizes slowing down the differentiation of nuclear DNA.

QTL for body weight, morphometric traits and stress response in European sea bass Dicentrarchus labrax.

Natural mating and mass spawning in the European sea bass (Dicentrarchus labrax L., Moronidae, Teleostei) complicate genetic studies and the implementation of selective breeding schemes. We utilized a two-step experimental design for detecting QTL in mass-spawning species: 2122 offspring from natural mating between 57 parents (22 males, 34 females and one missing) phenotyped for body weight, eight morphometric traits and cortisol levels, had been previously assigned to parents based on genotypes of 31 DNA microsatellite markers. Five large full-sib families (five sires and two dams) were selected from the offspring (570 animals), which were genotyped with 67 additional markers. A new genetic map was compiled, specific to our population, but based on the previously published map. QTL mapping was performed with two methods: half-sib regression analysis (paternal and maternal) and variance component analysis accounting for all family relationships. Two significant QTL were found for body weight on linkage group 4 and 6, six significant QTL for morphometric traits on linkage groups 1B, 4, 6, 7, 15 and 23 and three suggestive QTL for stress response on linkage groups 3, 14 and 23. The QTL explained between 8% and 38% of phenotypic variance. The results are the first step towards identifying genes involved in economically important traits like body weight and stress response in European sea bass.

A combined AFLP and microsatellite linkage map and pilot comparative genomic analysis of European sea bass Dicentrarchus labrax L.

European sea bass (Dicentrarchus labrax L., Moronidae, Teleostei) sustains a regional fishery and is commonly farmed in the Mediterranean basin, but has not undergone much long-term genetic improvement. An updated genetic linkage map of the European sea bass was constructed using 190 microsatellites, 176 amplified fragment length polymorphisms and two single nucleotide polymorphisms. From the 45 new microsatellite markers (including 31 type I markers) reported in this study, 28 were mapped. A total of 368 markers were assembled into 35 linkage groups. Among these markers, 28 represented type I (coding) markers, including those located within the peptide Y, SOX10, PXN1, ERA and TCRB genes (linkage groups 1, 7, 16, 17 and 27 respectively). The sex-averaged map spanned 1373.1 centimorgans (cM) of the genome. The female map measured 1380.0 cM, whereas the male map measured 1046.9 cM, leading to a female-to-male (F:M) recombination rate ratio of 1.32:1. The intermarker spacing of the second-generation linkage map of the European sea bass was 3.67 cM, which is smaller than that of the first-generation linkage map (5.03 cM). Comparative mapping of microsatellite flanking regions was performed with five model teleosts and this revealed a high percentage (33.6%) of evolutionarily conserved regions with the three-spined stickleback.

Review on the immunology of European sea bass Dicentrarchus labrax.

European sea bass (Dicentrarchus labrax L.) is a marine species of great economic importance, particularly in Mediterranean aquaculture. However, numerous pathogenic viruses, bacteria, fungi and parasites affect the species, causing various infectious diseases and thereby leading to the most heavy losses in aquaculture production of sea bass. In this respect, knowledge on molecular and genetic mechanisms of resistance to pathogens and specific features of immune response against various infectious agents should greatly benefit the development of effective vaccines and proper vaccination strategies in marker-assisted selection of fish resistant to a range of infections. To date, genetic knowledge on sea bass immune regulatory genes responsible for resistance to pathogens is relatively poor but tends to accumulate rapidly. In this review, we summarize and update current knowledge on the immune system and immune regulatory genes of the sea bass.

Evidence for isolation by time in the European eel (Anguilla anguilla L.).

Life history traits of highly vagile marine species, such as adult reproductive success and larval dispersal, are strongly determined by oceanographic and climatic forces. Nevertheless, marine organisms may show restricted dispersal in time and space. Patterns of isolation by distance (IBD) have been repeatedly observed in marine species. If spawning time is a function of geographical location, temporal and spatial isolation, can easily be confounded or misinterpreted. In this study, we aimed at discriminating between various forces shaping the genetic composition of recruiting juveniles of the European eel (Anguilla anguilla L.). By controlling for geographical variation, we assessed temporal variation and tested for possible isolation by time (IBT) between recruitment waves within and between years. Using 12 polymorphic allozyme and six variable microsatellite loci, we show that genetic differentiation was low (F(ST) = 0.01-0.002) and significant between temporal samples. Regression analysis between genetic and temporal distance, was consistent with a subtle interannual pattern of IBT. Our data suggest that the population dynamics of the European eel may be governed by a double pattern of temporal variance in genetic composition: (i) a broad-scale IBT of spawning cohorts, possibly as a consequence of the large migration loop in anguillids and strong variance in annual adult reproductive contribution; and (ii) a smaller-scale variance in reproductive success (genetic patchiness) within cohorts among seasonally separated spawning groups, most likely originating from fluctuating oceanic and climatic forces. The consistency of both mechanisms remains to be verified with fine-scale analyses of both spawning/migrating aged adults and their offspring to confirm the stochastic/deterministic nature of the IBT pattern in eel.

Evidence for fine-scale genetic structure and estuarine colonisation in a potential high gene flow marine goby (Pomatoschistus minutus).

Marine fish seem to experience evolutionary processes that are expected to produce genetically homogeneous populations. We have assessed genetic diversity and differentiation in 15 samples of the sand goby Pomatoschistus minutus (Pallas, 1770) (Gobiidae, Teleostei) from four major habitats within the Southern Bight of the North Sea, using seven microsatellite and 13 allozyme loci. Despite its high dispersal potential, microsatellite loci revealed a moderate level of differentiation (overall F(ST)=0.026; overall R(ST)=0.058). Both hierarchical analysis of molecular variance and multivariate analysis revealed significant differentiation (P<0.01) between estuarine, coastal and marine samples with microsatellites, but not with allozymes. Comparison among the different estimators of differentiation (F(ST) and R(ST)) pointed to possible historical events and contemporary habitat fragmentation. Samples were assigned to two breeding units in the estuary and coastal region. Despite this classification, there were indications of a complex and dynamic spatiotemporal structure, which is, most likely, determined by historical events and local oceanic currents.

Phylogeography of the common goby, Pomatoschistus microps, with particular emphasis on the colonization of the Mediterranean and the North Sea.

The phylogeographical patterns of a small marine fish, the common goby, Pomatoschistus microps, were assessed at 12 sites along the northeastern Atlantic coasts and the western Mediterranean Sea. A combination of two genetic markers was employed: cellulose acetate allozyme electrophoresis (CAGE) and sequence analysis of a 289 bp fragment of the mitochondrial locus cytochrome b. Both markers were congruent in revealing significant differences between samples (global FST = 0.247 for the allozymes and PhiST = 0.437 for the mitochondrial DNA data) and a pattern of isolation-by-distance. Phylogeographical analyses yielded a shallow branching structure with four groups. Three of those were confined to the Atlantic basin and showed a star-like pattern. The fourth group contained a central haplotype occurring at the edges of the species' distribution, accompanied by a few more rare variants, which were restricted to the Mediterranean Sea. A genetic break was observed around the British Isles, with distinct haplotypes dominating at either side of the English Channel. A significantly negative correlation between the degree of genetic diversity and latitude was recorded both for mitochondrial DNA (mtDNA) and allozymes in the Atlantic basin. Gene flow analysis suggested that recolonization of the North Sea and the coasts of western Scotland and Ireland may have taken place from a glacial refugium in the Southern Bight of the North Sea. These results are discussed in the perspective of possible postglacial migration routes of marine fish along the northeastern Atlantic coasts.

Development and linkage relationships for new microsatellite markers of the sea bass (Dicentrarchus labrax L.).

Twenty-eight polymorphic microsatellites were isolated from the sea bass, Dicentrarchus labrax, using a microsatellite enrichment protocol and selective hybridization with oligonucleotide probes. Analysis for these markers and 11 recently described microsatellites of D. labrax found linkage between 26 loci and revealed eight linkage groups.

Phylogenetic relationships among Palearctic and Nearctic burbot (Lota lota): Pleistocene extinctions and recolonization.

The burbot (Lota lota Linnaeus, 1758) is the only freshwater species from the cod family. Various taxonomic hypotheses were tested against molecular data by sequencing the mitochondrial cytochrome b locus of 120 burbot from 41 populations together with the related species Molva molva (ling) and Brosme brosme (tusk), which represented the other Lotinae genera. Within the genus Lota two distinct phylogroups were observed: one in North America south of the Great Slave Lakes (Lota lota maculosa) and one in Eurasia and the remainder of the Nearctic region (Lota lota lota). The burbot lineage separated 10 Myr BP from the other Lotinae, while the genetic variation within burbot appeared to be approximately 1 Myr old. However, fossil evidence suggested that burbot already existed in the Early Pliocene in Europe, from were it probably colonized North America in the Early Pleistocene. While Nearctic burbot survived climatic oscillations and diverged in several refugia, the Eurasian form became extinct or was reduced to a very small population. In the Late Pleistocene the species recolonized the Palearctic region to establish its present distribution range.

Late glacial history of the cold-adapted freshwater fish Cottus gobio, revealed by microsatellites.

The distribution of genetic diversity at 10 highly polymorphic microsatellite loci within the European freshwater fish, Cottus gobio, L. was examined. The sampling range comprised a large geographical scale including lineages known to be highly divergent at both mitochondrial DNA (mtDNA) and allozymes. An analysis of genetic variability within populations showed that expected heterozygosity and allelic richness could be explained largely by current effective population sizes. Evidence was found, however, that historical processes predating the last major glaciation affected allelic richness. In addition to confirming the large-scale patterns from earlier studies, the microsatellite data revealed new insights into recent processes by analysing genetic structure within ancient lineages defined by mtDNA data. Stepwise mutation model (SMM) and nonSMM-based methods demonstrated a clear genetic structuring within the Northwestern European lineage comprising populations from Britain and Belgium, and within the Central European lineage populations from the rivers Danube, Elbe and Main. Supported by an analysis of genetic variability within populations these results showed that the bullhead populations most probably persisted throughout the last major glaciation within the British Isles and within the drainages of the rivers Elbe and Main. Such observations provide the first genetic evidence for a glacial refugium in such close proximity to the European glacial margins.

The nuclear rDNA region of Gyrodactylus arcuatus and G. branchicus (Monogenea: Gyrodactylidae).

The primary structure of the ribosomal DNA internal transcribed spacers (ITS-1 and ITS-2) and 5.8S rRNA gene were used to characterize and identify 2 monogenean species of Gyrodacrylus living externally on the threespine stickleback (Gasterosteus aculeatus). The ITS region was amplified by PCR from freshwater, brackish, and marine isolates of Gyrodactylus arcuatus and G. branchicus, and the ends of the coding regions were identified by comparative alignment. No intraspecific and very low interspecific variation were observed in the 5.8S rRNA gene; high inter- and low intraspecific variation were revealed in the ITS-1 and ITS-2 regions. The morphological species identification was in all cases confirmed by the molecular identification. Intraspecifically, samples from 2 locations in the North Sea could be differentiated, but the Baltic sample resembled North Sea genotypes. Our approach offers perspectives for a multimetric genetical, morphometrical, and ecological taxonomy of the genus Gyrodactylus.

Replication, expression, and fate of foreign DNA during embryonic and larval development of the African catfish (Clarias gariepinus).

The transfer of exogenous DNA in fish represents a powerful strategy to study the regulation of gene expression in vivo. The African catfish (Clarias gariepinus) was chosen for this study because of its scientific and economic importance due to its easy husbandry, its short developmental period, and its value as a protein source in Africa and Asia. Fertilized eggs (1- and 2-cell stage) were cytoplasmatically injected with either supercoiled or linearized plasmids harboring the fusion genes encoding beta-galactosidase (lacZ) or luciferase (Luc) without a promoter or fused to the promoter/enhancer of human cytomegalovirus (CMV). Replication of the exogenous DNA peaked at 4 hours (early gastrula) and again at 2 days (which corresponds to the developmental stage of yolksac resorption). Foreign DNA persisted during embryogenesis, and it was still detectable 8 months after injection. In vivo transient expression of both CMV fusion genes was mosaic and peaked within 24 hours after DNA injection. Transient expression of the luciferase reporter gene could be detected with a much higher sensitivity than the lacZ gene. These data establish African catfish as a suitable in vivo assay system, and they confirm the luciferase reporter gene as a high quality reporter gene in fish.