1. Freshwater amphipods Gammarus fossarum and Gammarus pulex are widespread in Europe, with some evidence of cryptic diversity in the former. We used DNA barcoding to assess genetic diversity within and among amphipod populations and examined mate discrimination and pre-copulatory pair formation between genetically divergent individuals. 2. Eight distinct G. fossarum and four distinct G. pulex molecular operational taxonomic units (MOTUs) were detected. Among the 33 amphipod populations sampled, 11 contained a single MOTU, 11 had two and 11 were composed of three sympatric MOTUs. Genetic divergences between sympatric MOTUs (G. fossarum and G. pulex MOTUs combined) ranged up to 28% (Kimura two parameter estimates). 3. In amphipod populations containing sympatric MOTUs, pre-copulatory pair formation was random between MOTUs diverging by <4%. However, pre-copulatory pairs involving amphipod individuals from MOTUs diverging by >4% were rare, suggesting mate discrimination between sympatric, highly divergent MOTUs. 4. Although the likelihood decreased with genetic distance between partners, pre-copulatory pair formation in the laboratory can occur between MOTUs diverging by c. 16% and led to successful mating: most female amphipod carried viable, fertilised eggs 48 h post-mating. 5. Data showed high cryptic diversity in the G. fossarum/G. pulex groups, even at a small spatial scale. Mate discrimination between genetically divergent amphipods in natural populations suggests that some of the MOTUs found in G. fossarum and G. pulex may be separated into (sub)species. However, amphipods from different MOTUs can still form pre-copulatory pairs and females produce viable eggs. Overall, data suggest that cryptic diversity is common in the G. fossarum/G. pulex groups and that pre-zygotic isolation through mate discrimination, rather than post-zygotic incompatibility, is likely to drive cryptic speciation.

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Biobanks correspond to different situations: research and technological development, medical diagnosis or therapeutic activities. Their status is not clearly defined. We aimed to investigate human biobanking in Europe, particularly in relation to organisational, economic and ethical issues in various national contexts. Data from a survey in six EU countries (France, Germany, the Netherlands, Portugal, Spain and the UK) were collected as part of a European Research Project examining human and non-human biobanking (EUROGENBANK, coordinated by Professor JC Galloux). A total of 147 institutions concerned with biobanking of human samples and data were investigated by questionnaires and interviews. Most institutions surveyed belong to the public or private non-profit-making sectors, which have a key role in biobanking. This activity is increasing in all countries because few samples are discarded and genetic research is proliferating. Collections vary in size, many being small and only a few very large. Their purpose is often research, or research and healthcare, mostly in the context of disease studies. A specific budget is very rarely allocated to biobanking and costs are not often evaluated. Samples are usually provided free of charge and gifts and exchanges are the common rule. Good practice guidelines are generally followed and quality controls are performed but quality procedures are not always clearly explained. Associated data are usually computerised (identified or identifiable samples). Biobankers generally favour centralisation of data rather than of samples. Legal and ethical harmonisation within Europe is considered likely to facilitate international collaboration. We propose a series of recommendations and suggestions arising from the EUROGENBANK project.

Biological rhythms are a fundamental property of life. The deep ocean covers 66% of our planet surface and is one of the largest biomes. The deep sea has long been considered as an arrhythmic environment because sunlight is totally absent below 1,000 m depth. In the present study, we have sequenced the temporal transcriptomes of a deep-sea species, the ecosystem-structuring vent mussel Bathymodiolus azoricus. We reveal that tidal cycles predominate in the transcriptome and physiology of mussels fixed directly at hydrothermal vents at 1,688 m depth at the Mid-Atlantic Ridge, whereas daily cycles prevail in mussels sampled after laboratory acclimation. We identify B. azoricus canonical circadian clock genes, and show that oscillations observed in deep-sea mussels could be either a direct response to environmental stimulus, or be driven endogenously by one or more biological clocks. This work generates in situ insights into temporal organisation in a deep-sea organism.

The structural gene for the Escherichia coli enzyme amylomaltase, malQ, is the second gene in the malPQ operon. The nucleotide sequence of malQ shows that the gene encodes an Mr 78360 protein close to the experimentally determined Mr of purified amylomaltase (72000-74000). The malQ initiation codon was identified by sequence analysis of clustered deletions around the 5' end of the gene. One of these deletions removed the first 5 bases from the malQ coding sequence. Strains carrying a plasmid with this truncated malQ gene under lacZ promoter control and out-of-frame with the first four codons of lacZ were Mal-. The Mal+ phenotype could be restored by inserting small, random fragments of E. coli chromosomal DNA into the unique EcoRI site. Nucleotide sequencing showed that the inserts either joined the lacZ and malQ sequences in frame, or contained a new translation start signal and coding sequence in frame with malQ. These results indicate that amylomaltase could be useful as a reporter protein in gene fusion studies.

Aquaculture including shellfish production is an important food resource worldwide which is particularly vulnerable to infectious diseases. Marteilia refringens, Bonamia ostreae and Bonamia exitiosa are regulated protozoan parasites infecting flat oysters Ostrea edulis that are endemic in Europe. Although some PCR assays have been already developed for their detection, a formal validation to assess the performances of those tools is often lacking. In order to facilitate the diagnosis of flat oyster regulated diseases, we have developed and evaluated a new multiplex Taqman® PCR allowing the detection of both M. refringens and Bonamia sp. parasites in one step. Highlights► M. refringens, B. ostreae and B. exitiosa are regulated parasites of flat oysters. ► First validated PCR detecting all regulated pathogens of flat oysters in one step. ► Multiplex PCR performances similar or higher than currently recommended PCR assays. ► Represents a substantial improvement in time, resources and accuracy. ► Useful to monitors parasites prevalence or for demonstration of freedom.

Body condition is an important determinant of fitness in many natural populations. However, as for many fitness traits, the underlying genes that regulate body condition remain elusive. The dopamine receptor D4 gene (DRD4) is a promising candidate as dopamine is known to play an important role in the regulation of food intake and the metabolism of both glucose and lipids in vertebrates. In this study, we take advantage of a large data set of greater flamingos, Phoenicopterus roseus, to test whether DRD4 polymorphism predicts early body condition (EBC) while controlling for whole-genome effects of inbreeding and outbreeding using microsatellite multilocus heterozygosity (MLH). We typed 670 of these individuals for exon 3 of the homologue of the human DRD4 gene and 10 microsatellite markers. When controlling for the effects of yearly environmental variations and differences between sexes, we found strong evidence of an association between exon 3 DRD4 polymorphisms and EBC, with 2.2-2.3% of the variation being explained by DRD4 polymorphism, whereas there was only weak evidence that MLH predicts EBC. Because EBC is most likely a polygenic trait, this is a considerable amount of variation explained by a single gene. This is to our knowledge, the first study to show an association between exon 3 DRD4 polymorphism and body condition in non-human animals. We anticipate that the DRD4 gene as well as other genes coding for neurotransmitters and their receptors may play an important role in explaining variation in traits that affect fitness.

Understanding how fitness is related to genetic variation is of crucial importance in both evolutionary ecology and conservation biology. We report a study of heterozygosity-fitness correlations in a wild, noninbred population of Zenaida Doves, Zenaida aurita, based on a sample comprising 489 individuals (382 adults and 107 juveniles) typed at 13 microsatellite loci, resulting in a data set comprising 5793 genotypes. In both adults and juveniles, and irrespective of sex, no evidence was found for an effect of either multilocus or single-locus heterozygosity on traits potentially related to fitness such as foraging tactic, competitive ability, and fluctuating asymmetry. In contrast, a significant negative correlation between body condition and multilocus heterozygosity, indicative of outbreeding depression, was found in juveniles, whereas no such trend was observed in adults. However, the frequency distribution of heterozygosity did not differ between the two age classes, suggesting compensatory growth by heterozygous juveniles. We discuss our results in relation to some practical limitations associated with studies of heterozygosity-fitness correlations, and suggest that tropical bird species with allopatric divergence between island populations may provide a good biological model for the detection of outbreeding depression.