The environmental quality of Doñana surrounding areas affects the immune transcriptional profile of inhabitant crayfish Procambarus clarkii.

This study aimed to identify differentially expressed genes in Procambarus clarkii crayfish collected from locations of different environmental qualities in the Doñana National Park surrounding areas. The pollution sustained by the crayfish was confirmed by their hepatopancreatic metal concentration. We generated forward and reverse libraries by suppression subtractive hybridization (SSH) to analyze the transcriptional profiles of crayfish from moderately and highly polluted zones in comparison with the control site within the Doñana Biological Reserve. Forty-three differentially expressed genes were detected, and most of them were identified as genes involved in a variety of biological functions, particularly in the innate immune response. To verify the SSH results and assess interindividual variability nine transcripts (ALP, AST, BTF3, CHIT, CTS, ferritin, HC, HC2, and SPINK4) were selected for absolute quantification by real-time qRT-PCR. The qRT-PCR data revealed substantial differences in the absolute amounts of the nine transcripts and confirmed their up- or down-regulation in the polluted sites. Additionally, a positive and significant linear correlation was found between the hepatopancreatic copper concentration and the levels of the transcripts encoding hemocyanins. Finally, the transcriptomic study was complemented with a detailed analysis of SNP profiles of the selected transcripts that revealed point mutations that might underlie adaptive response to environmental stress in P. clarkii. Overall, this work provides novel insights into the molecular pathways that could mediate the response to environmental pollutants in P. clarkii emphasizing the central role of the immune function and thus, should clearly benefit further immunotoxicological research in this organism.

Omic approaches in environmental issues.

Biomonitoring requires the application of batteries of different biomarkers, as environmental contaminants induce multiple responses in organisms that are not necessarily correlated. Omic technologies were proposed as an alternative to conventional biomarkers since these techniques quantitatively monitor many biological molecules in a high-throughput manner and thus provide a general appraisal of biological responses altered by exposure to contaminants. As the studies using omic technologies increase, it is becoming clear that any single omic approach may not be sufficient to characterize the complexity of ecosystems. This work aims to provide a preliminary working scheme for the use of combined transcriptomic and proteomic methodologies in environmental biomonitoring. There are difficulties in working with nonmodel organisms as bioindicators when combining several omic approaches. As a whole, our results with heterologous microarrays in M. spretus and suppressive subtractive hybridization (SSH) in P. clarkii indicated that animals sustaining a heavy pollution burden exhibited an enhanced immune response and/or cell apoptosis. The proteomic studies, although preliminary, provide a holistic insight regarding the manner by which pollution shifts protein intensity in two-dimensional gel electrophoresis (2-DE), completing the transcriptomic approach. In our study, the sediment element concentration was in agreement with the intensity of protein expression changes in C. maenas crabs. In conclusion, omics are useful technologies in addressing environmental issues and the determination of contamination threats.

Immune- and stress-related transcriptomic responses of Solea senegalensis stimulated with lipopolysaccharide and copper sulphate using heterologous cDNA microarrays.

The sole, Solea senegalensis, is a common flatfish of Atlantic and Mediterranean waters with a high potential for aquaculture. However, its cultivation is hampered by high sensitivity to different stresses and several infectious diseases. Improving protection from pathogens and stressors is thus a key step in reaching a standardized production. Fish were exposed to lipopolysaccharide (LPS), a mimetic of bacterial infections, and copper sulphate (CuSO(4)), used in aquaculture to control algae and outbreaks of infectious diseases. We employed a European flounder cDNA microarray to determine the transcriptomic responses of Senegalese sole to these exposures. Microarray analyses showed that many genes were altered in expression following both LPS and copper treatments in comparison to vehicle controls. Gene ontology analysis highlighted copper-specific induction of genes related to cellular adhesion and cell signalling, LPS-specific induction of genes related to the immune response, and a common induction of genes related to unfolded protein binding, intracellular transport/secretion and proteasome. Additionally transcripts for glutathione-S-transferases were down-regulated by LPS, and those for digestive enzymes were down-regulated by both treatments. We selected nine changing genes for absolute quantification of transcript copy numbers by real-time RT-PCR to validate microarray differential expression and to assess inter-individual variability in individual fishes. The quantitative RT-PCR data correlated highly with the microarray results. Overall, data reported provide novel insights into the molecular pathways that could mediate the immune and heavy metal stress responses in Senegalese sole and thus might have biotechnological applications in the culture of this important fish species.

Solea senegalensis genes responding to lipopolysaccharide and copper sulphate challenges: large-scale identification by suppression subtractive hybridization and absolute quantification of transcriptional profiles by real-time RT-PCR.

Solea senegalensis is a commercially relevant aquaculture species that remains largely unexplored at the genomic level. The aim of this study was to identify novel genomic responses to lipopolysaccharide and copper sulphate challenges using suppression subtractive hybridization (SSH) and real-time RT-PCR. Forward- and reverse-subtractive libraries were generated for the identification of genes whose transcription is altered in response to lipopolysaccharide (LPS) (immunomodulator) in head kidney (immunologically important organ) and to CuSO(4) (common algacide) in liver (central metabolic organ and important source of immune transcripts). A total of 156 genes involved in major physiological functions were identified by SSH, the identified sequences representing a significant increase in the number of sole ESTs in public databases. Fifteen genes represented in the subtracted libraries were selected for further tissue, temporal and inducible transcriptional profiling by real-time RT-PCR. A rigorous quantification of transcript copy numbers was performed for this purpose in both pooled and individual samples from two independent experiments. More than half of the investigated mRNAs encode proteins that deal with different aspects of the immune response, like NCCRP1 (non-specific cytotoxic cell receptor), C3 and C7 (complement components), and ferritin M, HP and TF (iron homeostasis), or play a crucial role in its regulation, like TRAF3. Other mRNAs studied encode proteins involved in metabolism, like TKT and NDUFA4, the response to stimulus, like CEBPB (transcription factor) and CIRBP (RNA-binding protein), and other cell processes. Highly abundant (>500 molecules/pg total RNA) and rare (< or =1 molecules/pg) mRNA species were quantified in each sole organ examined, and outstanding differences were also recorded in the comparison between the two organs, e.g. C3 and TF mRNAs were largely overexpressed in liver (>5000 molecules/pg) as compared to head kidney (<5 molecules/pg). Most investigated mRNAs displayed significant alterations in their steady-state copy number following LPS and/or CuSO(4) stimulation, i.e. they were (i) up-regulated in response to both treatments in at least one of the two organs (NCCRP1, CEBPB, SQSTM1, NDUFA4, C7 and HP), (ii) up-regulated (TF, CIRBP, TRFA, C3) or down-regulated (TKT) by LPS, their levels remaining essentially unchanged upon CuSO(4) challenge, or (iii) down-regulated by LPS, though up-regulated by CuSO(4) (ferritin M). Quantifications in individual fish were consistent with those in pooled samples with respect to both the direction and the absolute changes in transcript abundance.