The transcriptome assembly of the European freshwater mussel Unio elongatulus C. Pfeiffer, 1825.

Freshwater mussels of the order Unionida are a global conservation concern. Species of this group are strictly freshwater, sessile, slow-growing animals and, extremely sensitive to environmental changes. Human-mediated changes in freshwater habitats are imposing enormous pressure on the survival of freshwater mussels. Although a few flagship species are protected in Europe, other highly imperilled species receive much less attention. Moreover, knowledge about biology, ecology, and evolution and proper conservation assessments of many European species are still sparse. This knowledge gap is further aggravated by the lack of genomic resources available, which are key tools for conservation. Here we present the transcriptome assembly of Unio elongatulus C. Pfeiffer, 1825, one of the least studied European freshwater mussels. Using the individual sequencing outputs from eight physiologically representative mussel tissues, we provide an annotated panel of tissue-specific Relative Gene Expression profiles. These resources are pivotal to studying the species' biological and ecological features, as well as helping to understand its vulnerability to current and future threats.

Integrative phylogenetic, phylogeographic and morphological characterisation of the Unio crassus species complex reveals cryptic diversity with important conservation implications.

The global decline of freshwater mussels and their crucial ecological services highlight the need to understand their phylogeny, phylogeography and patterns of genetic diversity to guide conservation efforts. Such knowledge is urgently needed for Unio crassus, a highly imperilled species originally widespread throughout Europe and southwest Asia. Recent studies have resurrected several species from synonymy based on mitochondrial data, revealing U. crassus to be a complex of cryptic species. To address long-standing taxonomic uncertainties hindering effective conservation, we integrate morphometric, phylogenetic, and phylogeographic analyses to examine species diversity within the U. crassus complex across its entire range. Phylogenetic analyses were performed using cytochrome c oxidase subunit I (815 specimens from 182 populations) and, for selected specimens, whole mitogenome sequences and Anchored Hybrid Enrichment (AHE) data on âˆ¼ 600 nuclear loci. Mito-nuclear discordance was detected, consistent with mitochondrial DNA gene flow between some species during the Pliocene and Pleistocene. Fossil-calibrated phylogenies based on AHE data support a Mediterranean origin for the U. crassus complex in the Early Miocene. The results of our integrative approach support 12 species in the group: the previously recognised Unio bruguierianus, Unio carneus, Unio crassus, Unio damascensis, Unio ionicus, Unio sesirmensis, and Unio tumidiformis, and the reinstatement of five nominal taxa: Unio desectusstat. rev., Unio gontieriistat. rev., Unio mardinensisstat. rev., Unio nanusstat. rev., and Unio vicariusstat. rev. Morphometric analyses of shell contours reveal important morphospace overlaps among these species, highlighting cryptic, but geographically structured, diversity. The distribution, taxonomy, phylogeography, and conservation of each species are succinctly described.

The complete maternal and paternal mitochondrial genomes of Unio crassus: Mitochondrial molecular clock and the overconfidence of molecular dating.

The availability of a rapidly growing number of complete mitochondrial genome sequences provokes high confidence dating approaches. However, even if the congruence between mitochondrial and nuclear markers is reasonable, the resulting topologies are frequently questionable. The unique opportunity to study the evolutionary history of two independent mitochondrial genomes in one phylogenetic context exists in the freshwater mussels family Unionidae. The two lineages function under doubly uniparental inheritance since well before the emergence of the family. Despite the relatively high number of available complete sequences of maternally inherited genomes, comparative analyses are limited by the small number of sequences of counterpart paternally inherited genomes. We have sequenced for the first time the representative set of five sequences (two maternal and three paternal) from the species Unio crassus. Comparative analysis of the phylogenies reconstructed using relevant mitogenomic data available in GenBank (13 species in total) reveal that single - genome inferences are congruent only if the relaxed clock is assumed.

The impact of in vivo and in vitro exposure to base analogue 5-FU on the level of DNA damage in haemocytes of freshwater mussels Unio pictorum and Unio tumidus.

The impact of in vivo and in vitro exposure to anticancer drug 5-Fluorouracil (5-FU) on the level of DNA damage was evaluated using comet assay on haemocytes of freshwater mussels Unio pictorum and Unio tumidus. For the in vivo experiment, the groups of 5 mussels per concentration were exposed for 72 h to 5-FU (0.04, 0.4, 4, 40 and 100 µM) with 0.4 µM being the lowest concentration to induce significant DNA damage. For the in vitro experiment, the primary cultures of haemocytes were treated with 0.04, 0.4, 4 and 40 µM 5-FU for 22 h and the treatment with CdCl2 was used as a positive control. In contrast to in vivo exposure, 5-FU did not induce significant increase of DNA damage in vitro, possibly because of the absence of haemocytes proliferation in primary cultures.

Specific proteomic response of Unio pictorum mussel to a mixture of glyphosate and microcystin-LR.

Cyanobacterial toxins and pesticides regularly impact freshwaters. Microcystin-LR is one of the most toxic and common cyanobacterial toxins whereas glyphosate is the active ingredient of a widely use herbicide. As filter feeders, freshwater mussels are particularly exposed. Like many native bivalve species, Unio pictorum suffers from a continuous decline in Europe. In order to get a deeper insight of its response to contaminants, U. pictorum was exposed to either 10 µg L(-1) of microcystin-LR or 10 µg L(-1) of glyphosate or a mixture of both. Proteins of the digestive glands were extracted and analyzed by DIGE. Gel analysis revealed 103 spots with statistical variations, and the response seems to be less toward glyphosate than to microcystin-LR. Specific spots have variations only when exposed to the mixture, showing that there is an interaction of both contaminants in the responses triggered. The proteins of 30 spots have been identified. They belong mostly to the cytoskeleton family, but proteins of the oxidative pathway, detoxification, and energetic metabolism were affected either by glyphosate or microcystin-LR or by the mixture. These results demonstrate the importance to study contaminants at low concentrations representative of those found in the field and that multicontaminations can lead to different response pathways.

Identification and characterization of the first microsatellite loci for the thick-shelled river mussel Unio crassus (Bivalvia: Unionidae).

The thick-shelled river mussel Unio crassus is critically endangered throughout its range as a result of increasing human activity and habitat loss. Next generation DNA sequencing was used to develop a set of microsatellite markers that can be used for future ecological and population genetics studies of U. crassus. A total of 11 polymorphic loci were identified and characterized using 57 individuals from two Polish populations. Numbers of alleles ranged from 2 to 15 and expected heterozygosity levels ranged from 0.069 to 0.899. Deficiency of heterozygous genotypes was observed in four loci. Marker independence was confirmed with tests for linkage disequilibrium, however, analyses indicated evidence of null alleles in four loci. The microsatellite markers developed specifically for U. crassus provide a valuable tool for future ecological, population genetic assessments, and conservation management of this species.

Complete sequences of maternally inherited mitochondrial genomes in mussels Unio pictorum (Bivalvia, Unionidae).

Mitochondrial genomes are frequently used to infer phylogenetic relationships. Some taxa are, however, poorly represented. To facilitate better understanding of the potential of mitochondrial genome data in freshwater mussels, we present here, for the first time, the mitochondrial sequences of 4 complete F-type mitochondrial genomes from the European freshwater bivalve Unio pictorum (Unionidae). These genomes are very compact (15,761 bp) but have a typical gene complement for bilaterian mitochondrial genomes and a very similar organization to other unionid genomes available in databases. Very low nucleotide diversity within the species suggests a small effective population size of Polish U. pictorum, a phenomenon of potential importance for environmental management policies.

SOD and CAT cDNA cloning, and expression pattern of detoxification genes in the freshwater bivalve Unio tumidus transplanted into the Moselle river.

The cDNA sequences encoding manganese superoxide dismutase (Mn-SOD) and catalase (CAT) were isolated in the freshwater bivalve Unio tumidus by reverse-transcription polymerase chain reaction (RT-PCR) using degenerate primers. Quantitative real-time PCR approach was used to evaluate the mRNA expression patterns of SOD, CAT, selenium-dependent glutathione peroxidase (Se-GPx), pi class glutathione S-transferase (pi-GST) and metallothionein (MT), in the digestive gland of Unio tumidus transplanted from a control site to four stations in the Moselle River (M1-M4), for periods of 8 and 21 days. These sites were chosen upstream and downstream of populated areas. Chemical analysis performed on sediments from the Moselle river sites did not show high levels of pollutants. Decrease of SOD, CAT, Se-GPx and MT mRNA levels were observed at M3 site after a 21-day exposure compared to control site. These results suggest inefficiency of antioxidant systems affected by cytotoxic mechanisms and confirm an environmental perturbation. Organisms transplanted at M4 site showed a strong increase of biomarkers transcription levels after 21 days of exposure. These inductions could correspond to an adaptive response to an altered environment. Our results showed that biological approaches using multibiomarkers appear as essential tools complementary to measurement of contaminants, to detect environmental degradations.

Identification, sequencing and expression of selenium-dependent glutathione peroxidase transcript in the freshwater bivalve Unio tumidus exposed to Aroclor 1254.

Glutathione peroxidases (GPx) and glutathione S-transferases (GST) are essential enzymes of the cellular defense system. The aim of this work was the identification of GPx transcript in a freshwater bivalve, Unio tumidus, and the effects of Aroclor 1254 on GPx and pi-class GST (pi-GST) expression pattern. The GPx full-length coding sequence was obtained by reverse transcription PCR using degenerated primers followed by 5' and 3' rapid amplification of cDNA ends. The GPx cDNA encodes a protein of 232 amino acids. The 72nd amino acid corresponds to a selenocysteine encoded by a TGA codon. Residues essential to the enzymatic function are conserved in GPx of U. tumidus. Specific amplifications of the Se-GPx mRNA from U. tumidus were performed on the digestive gland, the excretory system and the gills. Se-GPx expression level is highest in the digestive gland. No induction of the Se-GPx was observed at the transcriptional level in the digestive gland and the excretory system of Aroclor-treated mussels, while an increase of the pi-GST mRNA level was observed in the excretory system.