The forgotten variable? Does the euthanasia method and sample storage condition influence an organisms transcriptome - a gene expression analysis on multiple tissues in pigs.

BACKGROUND: Transcriptomic studies often require collection of fresh tissues post euthanasia. The chosen euthanasia method might have the potential to induce variations in gene expressions that are unlinked with the experimental design. The present study compared the suitability of 'nitrogen gas in foam' (ANOXIA) in comparison to a non-barbiturate anaesthetic, T-61® (T61), for euthanizing piglets used in transcriptome research. Further, the effect of common tissue storage conditions, RNAlater™ (RL) and snap freezing in liquid nitrogen (LN2), on gene expression profiles were also analysed. RESULTS: On comparison of the 3'mRNA-Seq data generated from pituitary, hypothalamus, liver and lung tissues, no significant differential expression in the protein coding genes were detected between the euthanasia methods. This implies that the nitrogen anoxia method could be a suitable alternative for euthanasia of piglets used in transcriptomic research. However, small nuclear RNAs (snRNAs) that constitute the eukaryotic spliceosomal machinery were found to be significantly higher (log2fold change ≥ 2.0, and adjusted p value ≤ 0.1) in pituitary samples collected using ANOXIA. Non-protein coding genes like snRNAs that play an important role in pre-mRNA splicing can subsequently modify gene expression. Storage in RL was found to be superior in preserving RNA compared to LN2 storage, as evidenced by the significantly higher RIN values in representative samples. However, storage in RL as opposed to LN2, also influenced differential gene expression in multiple tissues, perhaps as a result of its inability to inhibit biological activity during storage. Hence such external sources of variations should be carefully considered before arriving at research conclusions. CONCLUSIONS: Source of biological variations like euthanasia method and storage condition can confound research findings. Even if we are unable to prevent the effect of these external factors, it will be useful to identify the impact of these variables on the parameter under observation and thereby prevent misinterpretation of our results.

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.

Differential gene expression in narrow- and broad-headed European glass eels (Anguilla anguilla) points to a transcriptomic link of head shape dimorphism with growth rate and chemotaxis.

One of the major challenges in evolutionary biology is to understand the mechanisms underlying morphological dimorphism and plasticity, including the genomic basis of traits and links to ecology. At the yellow eel stage of the European eel (Anguilla anguilla), two morphotypes are found: broad- and narrow-heads. This dimorphism has been linked to dietary differences, with broad-heads feeding on harder, larger prey than narrow-heads. However, recent research showed that both morphotypes could be distinguished at the glass eel stage, the nonfeeding predecessor of the yellow eel stage, implying that nondietary factors play a role in the development of this head shape dimorphism. Here, we used transcriptome profiling (RNAseq) to identify differentially expressed genes between broad- and narrow-headed glass eels. We found 260 significantly differentially expressed genes between the morphotypes, of which most were related to defence and immune responses. Interestingly, two genes involved in growth (soma and igf2) were significantly upregulated in narrow-heads, while nine genes involved in chemotaxis showed significant differential expression. Thus, we found support for the observation that head shape is associated with somatic growth, with fast-growing eels developing a narrower head. Additionally, observations in the wild have shown that slow-growers prefer freshwater, while fast-growers prefer brackish water. The differential expression of genes involved in chemotaxis seems to indicate that glass eel growth rate and habitat choice are linked. We hypothesize that two levels of segregation could take place in the European eel: first according to habitat choice and second according to feeding preference.

Response of mixed methanotrophic consortia to different methane to oxygen ratios.

Methane (CH4) and oxygen (air) concentrations affect the CH4 oxidation capacity (MOC) and mixed methanotrophic community structures in compost (fresh) and landfill (age old) top cover soils. A change in the mixed methanotrophic community structure in response has implications for landfill CH4 bio-filter remediation and possible bio-product outcomes (i.e., fatty acid methyl esters (FAME) content and profiles and polyhydroxybutyrate (PHB) contents). Therefore the study aimed to evaluate the effect of variable CH4 to oxygen ratios (10-50% CH4 in air) on mixed methanotrophic community structures enriched from landfill top cover (LB) and compost soils (CB) and to quantify flow on impacts on MOC, total FAME contents and profiles, and PHB accumulation. A stable consortium developed achieving average MOCs of 3.0±0.12, 4.1±0.26, 6.9±0.7, 7.6±1.3 and 9.2±1.2mgCH4g-1DWbiomassh-1 in LB and 2.9±0.04, 5.05±0.32, 6.7±0.31, 7.9±0.61 and 8.6±0.48mgCH4g-1DWbiomassh-1 in CB for a 20day cultivation period at 10, 20, 30, 40 and 50% CH4, respectively. CB at 10% CH4 had a maximal FAME content of 40.5±0.8mgFAMEg-1DWbiomass, while maximal PHB contents (25mgg-1DWbiomass) was observed at 40% CH4 in LB. Despite variable CH4/O2 ratios, the mixed methanotrophic community structures in both LB and CB were relatively stable, dominated by Methylosarcina sp., and Chryseobacterium, suggesting that a resilient consortium had formed which can now be tested in bio-filter operations for CH4 mitigations in landfills.

Genome-wide single-generation signatures of local selection in the panmictic European eel.

Next-generation sequencing and the collection of genome-wide data allow identifying adaptive variation and footprints of directional selection. Using a large SNP data set from 259 RAD-sequenced European eel individuals (glass eels) from eight locations between 34 and 64(o) N, we examined the patterns of genome-wide genetic diversity across locations. We tested for local selection by searching for increased population differentiation using F(ST) -based outlier tests and by testing for significant associations between allele frequencies and environmental variables. The overall low genetic differentiation found (F(ST) = 0.0007) indicates that most of the genome is homogenized by gene flow, providing further evidence for genomic panmixia in the European eel. The lack of genetic substructuring was consistent at both nuclear and mitochondrial SNPs. Using an extensive number of diagnostic SNPs, results showed a low occurrence of hybrids between European and American eel, mainly limited to Iceland (5.9%), although individuals with signatures of introgression several generations back in time were found in mainland Europe. Despite panmixia, a small set of SNPs showed high genetic differentiation consistent with single-generation signatures of spatially varying selection acting on glass eels. After screening 50 354 SNPs, a total of 754 potentially locally selected SNPs were identified. Candidate genes for local selection constituted a wide array of functions, including calcium signalling, neuroactive ligand-receptor interaction and circadian rhythm. Remarkably, one of the candidate genes identified is PERIOD, possibly related to differences in local photoperiod associated with the >30° difference in latitude between locations. Genes under selection were spread across the genome, and there were no large regions of increased differentiation as expected when selection occurs within just a single generation due to panmixia. This supports the conclusion that most of the genome is homogenized by gene flow that removes any effects of diversifying selection from each new generation.

Assessing patterns of hybridization between North Atlantic eels using diagnostic single-nucleotide polymorphisms.

The two North Atlantic eel species, the European eel (Anguilla anguilla) and the American eel (Anguilla rostrata), spawn in partial sympatry in the Sargasso Sea, providing ample opportunity to interbreed. In this study, we used a RAD (Restriction site Associated DNA) sequencing approach to identify species-specific diagnostic single-nucleotide polymorphisms (SNPs) and design a low-density array that combined with screening of a diagnostic mitochondrial DNA marker. Eels from Iceland (N=159) and from the neighboring Faroe Islands (N=29) were genotyped, along with 94 larvae (49 European and 45 American eel) collected in the Sargasso Sea. Our SNP survey showed that the majority of Icelandic eels are pure European eels but there is also an important contribution of individuals of admixed ancestry (10.7%). Although most of the hybrids were identified as F1 hybrids from European eel female × American eel male crosses, backcrosses were also detected, including a first-generation backcross (F1 hybrid × pure European eel) and three individuals identified as second-generation backcrosses originating from American eel × F1 hybrid backcrosses interbreeding with pure European eels. In comparison, no hybrids were observed in the Faroe Islands, the closest bodies of land to Iceland. It is possible that hybrids show an intermediate migratory behaviour between the two parental species that ultimately brings hybrid larvae to the shores of Iceland, situated roughly halfway between the Sargasso Sea and Europe. Only two hybrids were observed among Sargasso Sea larvae, both backcrosses, but no F1 hybrids, that points to temporal variation in the occurrence of hybridization.

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.

A resource of genome-wide single-nucleotide polymorphisms generated by RAD tag sequencing in the critically endangered European eel.

Reduced representation genome sequencing such as restriction-site-associated DNA (RAD) sequencing is finding increased use to identify and genotype large numbers of single-nucleotide polymorphisms (SNPs) in model and nonmodel species. We generated a unique resource of novel SNP markers for the European eel using the RAD sequencing approach that was simultaneously identified and scored in a genome-wide scan of 30 individuals. Whereas genomic resources are increasingly becoming available for this species, including the recent release of a draft genome, no genome-wide set of SNP markers was available until now. The generated SNPs were widely distributed across the eel genome, aligning to 4779 different contigs and 19,703 different scaffolds. Significant variation was identified, with an average nucleotide diversity of 0.00529 across individuals. Results varied widely across the genome, ranging from 0.00048 to 0.00737 per locus. Based on the average nucleotide diversity across all loci, long-term effective population size was estimated to range between 132,000 and 1,320,000, which is much higher than previous estimates based on microsatellite loci. The generated SNP resource consisting of 82,425 loci and 376,918 associated SNPs provides a valuable tool for future population genetics and genomics studies and allows for targeting specific genes and particularly interesting regions of the eel genome.

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.

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 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.

Temporal genetic stability and high effective population size despite fisheries-induced life-history trait evolution in the North Sea sole.

Heavy fishing and other anthropogenic influences can have profound impact on a species' resilience to harvesting. Besides the decrease in the census and effective population size, strong declines in mature adults and recruiting individuals may lead to almost irreversible genetic changes in life-history traits. Here, we investigated the evolution of genetic diversity and effective population size in the heavily exploited sole (Solea solea), through the analysis of historical DNA from a collection of 1379 sole otoliths dating back from 1957. Despite documented shifts in life-history traits, neutral genetic diversity inferred from 11 microsatellite markers showed a remarkable stability over a period of 50 years of heavy fishing. Using simulations and corrections for fisheries induced demographic variation, both single-sample estimates and temporal estimates of effective population size (N(e) ) were always higher than 1000, suggesting that despite the severe census size decrease over a 50-year period of harvesting, genetic drift is probably not strong enough to significantly decrease the neutral diversity of this species in the North Sea. However, the inferred ratio of effective population size to the census size (N(e) /N(c) ) appears very small (10(-5) ), suggesting that overall only a low proportion of adults contribute to the next generation. The high N(e) level together with the low N(e) /N(c) ratio is probably caused by a combination of an equalized reproductive output of younger cohorts, a decrease in generation time and a large variance in reproductive success typical for marine species. Because strong evolutionary changes in age and size at first maturation have been observed for sole, changes in adaptive genetic variation should be further monitored to detect the evolutionary consequences of human-induced selection.

Application of SNPs for population genetics of nonmodel organisms: new opportunities and challenges.

Recent improvements in the speed, cost and accuracy of next generation sequencing are revolutionizing the discovery of single nucleotide polymorphisms (SNPs). SNPs are increasingly being used as an addition to the molecular ecology toolkit in nonmodel organisms, but their efficient use remains challenging. Here, we discuss common issues when employing SNP markers, including the high numbers of markers typically employed, the effects of ascertainment bias and the inclusion of nonneutral loci in a marker panel. We provide a critique of considerations specifically associated with the application and population genetic analysis of SNPs in nonmodel taxa, focusing specifically on some of the most commonly applied methods.

Shape-controlled synthesis of NIR absorbing branched gold nanoparticles and morphology stabilization with alkanethiols.

Gold nanoparticles are ideal candidates for clinical applications if their plasmon absorption band is situated in the near infrared region (NIR) of the electromagnetic spectrum. Various parameters, including the nanoparticle shape, strongly influence the position of this absorption band. The aim of this study is to produce stabilized NIR absorbing branched gold nanoparticles with potential for biomedical applications. Hereto, the synthesis procedure for branched gold nanoparticles is optimized varying the different synthesis parameters. By subsequent electroless gold plating the plasmon absorption band is shifted to 747.2 nm. The intrinsic unstable nature of the nanoparticles' morphology can be clearly observed by a spectral shift and limits their use in real applications. However, in this article we show how the stabilization of the branched structure can be successfully achieved by exchanging the initial capping agent for different alkanethiols and disulfides. Furthermore, when using alkanethiols/disulfides with poly(ethylene oxide) units incorporated, an increased stability of the gold nanoparticles is achieved in high salt concentrations up to 1 M and in a cell culture medium. These achievements open a plethora of opportunities for these stabilized branched gold nanoparticles in nanomedicine.

Impact of pre-concentration to covalently biofunctionalize suspended nanoparticles.

The effective biofunctionalization of nanoparticles is crucial for biomedical applications. In this study we investigated the covalent biofunctionalization of magnetic nanoparticles based on carbodiimide activation. An important aspect in the covalent biofunctionalization of nanoparticles has been neglected, namely pre-concentration. Exploiting the electrostatic attraction forces between a protein and the nanoparticle surface will favor the covalent immobilization. We showed that low ionic strength buffers with a pH slightly lower than the pI of the selected biomolecules is needed to increase the yield of covalent immobilization. Additionally, it is demonstrated that the covalently immobilized proteins are bioactive, relying on a sandwich assay using gold nanoparticles as reporter labels.

Increased stability of mercapto alkane functionalized Au nanoparticles towards DNA sensing.

The use of gold nanoparticles (GNPs) in bioassays is often hampered by their colloidal stability. In this study, gold nanoparticles coated with different mercapto alkanes were investigated towards their stability. Hereto, the effects of the alkane chain length (5-11 methylene groups), the type of functional end-group (-OH or -COOH) and the amount of incorporated poly-ethylene oxide units (none, 3 or 6) on the GNP stabilization was evaluated. Based on these results, an optimal mercapto alkane (HS(CH(2))(11)PEO(6)COOH) was selected to increase the colloidal stability up to 2 M NaCl. Furthermore, it was proved that this mercapto alkane is ideally suited to enhance the stability of DNA functionalized GNPs in high electrolytic hybridization buffers. The effectiveness of these DNA functionalized GNPs was demonstrated in a sandwich assay using a surface plasmon resonance biosensor. The superior stability of these nanoparticles during hybridization may lead to enhanced biosensor technologies.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 December 2009-31 January 2010.

This article documents the addition of 220 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Allanblackia floribunda, Amblyraja radiata, Bactrocera cucurbitae, Brachycaudus helichrysi, Calopogonium mucunoides, Dissodactylus primitivus, Elodea canadensis, Ephydatia fluviatilis, Galapaganus howdenae howdenae, Hoplostethus atlanticus, Ischnura elegans, Larimichthys polyactis, Opheodrys vernalis, Pelteobagrus fulvidraco, Phragmidium violaceum, Pistacia vera, and Thunnus thynnus. These loci were cross-tested on the following species: Allanblackia gabonensis, Allanblackia stanerana, Neoceratitis cyanescens, Dacus ciliatus, Dacus demmerezi, Bactrocera zonata, Ceratitis capitata, Ceratitis rosa, Ceratits catoirii, Dacus punctatifrons, Ephydatia mülleri, Spongilla lacustris, Geodia cydonium, Axinella sp., Ischnura graellsii, Ischnura ramburii, Ischnura pumilio, Pistacia integerrima and Pistacia terebinthus.

Chemical and biological characterization of thiol SAMs for neuronal cell attachment.

Cellular adhesion and growth on solid-state surfaces is the central theme in the development of cell-based biosensors and implantable medical devices. Suitable interface techniques must be applied to construct stable and well-organized thin films of biologically active molecules that would control the development of neuronal cells on chips. Peptides such as RGD fragments, poly-L-lysine (PLL), or basal lamina proteins, such as laminin or fibronectin, are often used in order to promote cellular adhesion on surfaces. In this paper we describe the characterization of several self-assembled monolayers (SAMs) for their ability to anchor a laminin-derived synthetic peptide, PA22-2, a peptide known to promote neuronal attachment and stimulate neurite outgrowth. We have evaluated the immobilization of PA22-2 onto 16-mercaptohexadecanoic acid, 4-maleimide-N-(11-undecyldithio)butanamide, and 2-(maleimide)ethyl-N-(11-hexaethylene oxide-undecyldithio)acetamide SAM functionalized Au substrates. The neuronal attachment and outgrowth have been evaluated in embryonic mouse hippocampal neuron cultures up to 14 days in vitro. Our results show that differences in the cell morphologies were observed on the surfaces modified with various SAMs, despite the minor differences in chemical composition identified using standard characterization tools. These different cell morphologies can most probably be explained when investigating the effect of a given SAM layer on the adsorption of proteins present in the culture medium. More likely, it is the ratio between the specific PA22-2 adsorption and nonspecific medium protein adsorption that controls the cellular morphology. Large amounts of adsorbed medium proteins could screen the PA22-2 sites required for cellular attachment.

Conservation of the introgressed European water frog complex using molecular tools.

In Belgium, the Pelophylax esculentus complex has recently been subjected to multiple introductions of non-native water frogs, increasing the occurrence of hybridisation events. In the present study, we tested the reliability of morphometric and recently developed microsatellite tools to identify introgression and to determine the origin of exotic Belgian water frogs. By analysing 150 individuals of each taxon of the P. esculentus complex and an additional 60 specimens of the introduced P. cf. bedriagae, we show that neither of the currently available tools appears to have sufficient power to reliably distinguish all Belgian water frog species. We therefore aimed at increasing the discriminatory power of a microsatellite identification tool by developing a new marker panel with additional microsatellite loci. By adding only two new microsatellite loci (RlCA5 and RlCA1b20), all taxa of the P. esculentus complex could be distinguished from each other with high confidence. Three more loci (Res3, Res5 and Res17) provided a powerful discrimination of the exotic species.

Isolation and characterization of expressed sequence tag-linked microsatellite loci for the European eel (Anguilla anguilla).

A European eel (Anguilla anguilla) expressed sequence tag database consisting of 795 contigs and 4008 singletons was screened for microsatellites sequences. Primers were designed to amplify 96 repeats, of which 86 gave good quality amplification products. Twenty-eight microsatellites were selected for further microsatellite genotyping. Only two loci were found to be monomorphic; out of the 26 polymorphic loci, number of alleles per locus ranged from two to 14, while the observed and expected heterozygosities ranged from 0.05 to 0.93, and from 0.05 to 0.95, respectively. All 28 primer sets tested revealed positive amplification in American eel (Anguilla rostrata).