Characterization of a de novo assembled transcriptome of the Common Blackbird (Turdus merula).

BACKGROUND: In recent years, next generation high throughput sequencing technologies have proven to be useful tools for investigations concerning the genomics or transcriptomics also of non-model species. Consequently, ornithologists have adopted these technologies and the respective bioinformatics tools to survey the genomes and transcriptomes of a few avian non-model species. The Common Blackbird is one of the most common bird species living in European cities, which has successfully colonized urban areas and for which no reference genome or transcriptome is publicly available. However, to target questions like genome wide gene expression analysis, a reference genome or transcriptome is needed. METHODS: Therefore, in this study two Common Blackbirds were sacrificed, their mRNA was isolated and analyzed by RNA-Seq to de novo assemble a transcriptome and characterize it. Illumina reads (125 bp paired-end) and a Velvet/Oases pipeline led to 162,158 transcripts. For the annotation (using Blast+), an unfiltered protein database was used. SNPs were identified using SAMtools and BCFtools. Furthermore, mRNA from three single tissues (brain, heart and liver) of the same two Common Blackbirds were sequenced by Illumina (75 bp single-end reads). The draft transcriptome and the three single tissues were compared by their BLAST hits with the package VennDiagram in R. RESULTS: Following the annotation against protein databases, we found evidence for 15,580 genes in the transcriptome (all well characterized hits after annotation). On 18% of the assembled transcripts, 144,742 SNPs were identified which are, consequently, 0.09% of all nucleotides in the assembled transcriptome. In the transcriptome and in the single tissues (brain, heart and liver), 10,182 shared genes were found. DISCUSSION: Using a next-generation technology and bioinformatics tools, we made a first step towards the genomic investigation of the Common Blackbird. The de novo assembled transcriptome is usable for downstream analyses such as differential gene expression analysis and SNP identification. This study shows the importance of the approach to sequence single tissues to understand functions of tissues, proteins and the phenotype.

Characterisation of transcriptional responses to dioxins and dioxin-like contaminants in roach (Rutilus rutilus) using whole transcriptome analysis.

There is significant concern regarding the contamination of riverine sediments with dioxins and dioxin-like compounds (DLCs), including polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs) and some polycyclic aromatic hydrocarbons (PAHs). The majority of studies investigating the ecotoxicology of DLCs in fish have focused on a few standard model species. However, there is significant uncertainty as to whether these model species are representative of native river fish, particularly in Europe. In this study, the transcriptional responses following exposure to equipotent concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), PCB 156 or the dioxin-like PAH, benzo[k]fluoranthene (BkF), were investigated in juvenile roach (Rutilus rutilus), a fish species that constitutes a large proportion of the fish biomass in freshwater bodies throughout Europe. To this end, RNA sequencing analysis was used to comprehensively characterise the molecular mechanisms and pathways of toxicity of these DLCs. Whole transcriptome analyses using ClueGO software revealed that DLCs have the potential to disrupt a number of important processes, including energy metabolism, oogenesis, the immune system, apoptosis and the response to oxidative stress. However, despite using equipotent concentrations, there was very little conservation of the transcriptional responses observed in fish exposed to different DLCs. TCDD provoked significant specific changes in the levels of transcripts related to immunotoxicity and carbohydrate metabolism, while PCB 156 caused virtually no specific effects. Exposure to BkF affected the most diverse suite of molecular functions and biological processes, including blood coagulation, oxidative stress responses, unspecific responses to organic or inorganic substances/stimuli, cellular redox homeostasis and specific receptor pathways. To our knowledge, this is the first study of the transcriptome-wide effects of different classes of DLCs in fish. These findings represent an important step towards describing complete toxicity pathways of DLCs, which will be important in the context of informing risk assessments of DLC toxicity in native fish species.

Toward understanding the impacts of sediment contamination on a native fish species: transcriptional effects, EROD activity, and biliary PAH metabolites.

BACKGROUND: Both frequency and intensity of flood events are expected to increase as a result of global climate change in the upcoming decades, potentially resulting in increased re-suspension of sediments in fluvial systems. Contamination of these re-suspended sediments with legacy contaminants, including dioxins and dioxin-like compounds (DLCs), as well as polycyclic aromatic hydrocarbons (PAHs) is of great ecotoxicological concern. DLCs, and to some extent also PAHs, exhibit their toxicity through activation of the aryl hydrocarbon receptor (AhR). However, interactions of DLCs with pathways other than those known to be mediated through the AhR are not fully understood to date. METHODS: This study aimed to investigate molecular and biochemical effects in roach (Rutilus rutilus) during a 10 days exposure to suspensions of three natural sediments that differed in the level of DLC contamination. Concentrations of biliary PAH metabolites and hepatic 7-ethoxyresorufin-O-deethylase activity were quantified in exposed fish. Furthermore, the abundance of transcripts of several genes related to energy metabolism, response to oxidative stress, and apoptosis, as well as cytochrome P450 1A (cyp1a) was quantified. RESULTS: Biliary PAH metabolites and activation of the AhR were confirmed as suitable early warning biomarkers of exposure to suspended sediments containing DLCs and PAHs that corresponded well with analytically determined concentrations of those contaminants. Although the abundances of transcripts of superoxide dismutase (sod), protein kinase c delta (pkcd), and ATP-binding cassette transporter c9 (abcc9) were altered by the treatment compared with unexposed control fish, none of these showed a time- or concentration-dependent response. The abundance of transcripts of pyruvate carboxylase (pc) and transferrin variant d (tfd) remained unaltered by the treatments. CONCLUSIONS: We have shown that contaminated sediments can become a risk for fish during re-suspension events (e.g., flooding and dredging). We have also demonstrated that roach, which are native to most European freshwater systems, are suitable sentinel species due to their great sensitivity and ecological relevance. Roach may be particularly suitable in future field studies to assess the toxicological concerns associated with the release of DLCs and PAHs during sediment re-suspension.

Towards science-based sediment quality standards-Effects of field-collected sediments in rainbow trout (Oncorhynchus mykiss).

Sediments can act as long-term sinks for environmental pollutants. Within the past decades, dioxin-like compounds (DLCs) such as polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) have attracted significant attention in the scientific community. To investigate the time- and concentration-dependent uptake of DLCs and PAHs in rainbow trout (Oncorhynchus mykiss) and their associated toxicological effects, we conducted exposure experiments using suspensions of three field-collected sediments from the rivers Rhine and Elbe, which were chosen to represent different contamination levels. Five serial dilutions of contaminated sediments were tested; these originated from the Prossen and Zollelbe sampling sites (both in the river Elbe, Germany) and from Ehrenbreitstein (Rhine, Germany), with lower levels of contamination. Fish were exposed to suspensions of these dilutions under semi-static conditions for 90 days. Analysis of muscle tissue by high resolution gas chromatography and mass spectrometry and of bile liquid by high-performance liquid chromatography showed that particle-bound PCDD/Fs, PCBs and PAHs were readily bioavailable from re-suspended sediments. Uptake of these contaminants and the associated toxicological effects in fish were largely proportional to their sediment concentrations. The changes in the investigated biomarkers closely reflected the different sediment contamination levels: cytochrome P450 1A mRNA expression and 7-ethoxyresorufin-O-deethylase activity in fish livers responded immediately and with high sensitivity, while increased frequencies of micronuclei and other nuclear aberrations, as well as histopathological and gross pathological lesions, were strong indicators of the potential long-term effects of re-suspension events. Our study clearly demonstrates that sediment re-suspension can lead to accumulation of PCDD/Fs and PCBs in fish, resulting in potentially adverse toxicological effects. For a sound risk assessment within the implementation of the European Water Framework Directive and related legislation, we propose a strong emphasis on sediment-bound contaminants in the context of integrated river basin management plans.