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.

Biochemical and proteomic effects in Procambarus clarkii after chlorpyrifos or carbaryl exposure under sublethal conditions.

In vivo effects of two sublethal doses of chlorpyrifos and carbaryl were studied in Procambarus clarkii after 2 and 7 days of exposure, and after pesticide removal. Chlorpyrifos inhibited carboxylesterase activity in a concentration-dependent manner, but acetylcholinesterase was less sensitive. Compared with chlorpyrifos, carbaryl had a less marked effect on esterase activity. The effects of selected pesticides on biotransformation or oxidative stress biomarkers were contradictory. Chlorpyrifos lowered ethoxyresorufin-O-deethylase (EROD), catalase and oxidized glutathione (GSSG) levels but raised glutathione-S-transferase activity, while carbaryl raised EROD, catalase and glutathione-S-transferase, but lowered glutathione peroxidase and reduced glutathione (GSH) levels. The effects on protein expression patterns depending on pesticide type and the tissue used for analysis were studied in parallel by 2-DE. In gill and nervous tissue about 2000 spots (pI 4-7) were resolved, with quite different expression patterns. Chlorpyrifos altered 72 proteins, mostly in nervous tissue, and carbaryl 35, distributed evenly between organs. Several specific spots were selected as specific protein expression signatures for chlorpyrifos or carbaryl exposure in gills and nervous tissue, respectively.

Assessment of Doñana National Park contamination in Procambarus clarkii: integration of conventional biomarkers and proteomic approaches.

Aquatic ecosystems of Doñana National Park (DNP) were monitored using Procambarus clarkii as bioindicator in four campaigns carried out between 2003 and 2004 to assess environmental quality possibly threatened by agrochemicals used in nearby areas. An integrated approach was carried out, by combining the responses of well-established biomarkers and the massive analysis of biological effects at the proteomic level. In sites potentially polluted, lower catalase, glucose-6-P dehydrogenase, and esterase activities, and higher malondialdehyde, metallothionein and glutathione levels were found. Two-dimensional gel electrophoresis resolved >2500 gill spots, and image analysis detected that 35 showed significant intensity differences between the reference site and the other seven sites studied. The superiority of proteomic approaches was clearly recognized in our study since four different protein expression patterns were established based in the fold-number of up-/down-regulation of the 35 differentially expressed proteins. Sites located within Doñana Biological Reserve were essentially free of contaminants and those near the DNP limits were only slightly polluted. The higher proteomic responses found at the upper "Rocina" and "Partido" courses indicate that non-persistent agrochemicals are mainly used in Doñana surroundings. The highest responses corresponded to rice growing areas placed between the Guadiamar stream and the Guadalquivir River, according to the extended and intensive use of agrochemicals in such areas.