Immunofluorescence detection and localization of B[a]P and TCDD in earthworm tissues

An immunohistochemical method using antibodies against polycyclic aromatic hydrocarbons (PAHs) and dioxins was developed on frozen tissue sections of the earthworm Eisenia andrei exposed to environmentally relevant concentrations of benzo[a]pyrene (B[a]P) (0.1, 10, 50 ppm) and 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) (0.01, 0.1, 2 ppb) in spiked standard soils. The concentrations of B[a]P and TCDD in E. andrei exposed to the same conditions were also measured using analytical chemical procedures. The results demonstrated that tissues of worms exposed to even minimal amount of B[a]P and TCDD reacted positively and specifically to anti-PAHs and -dioxins antibody. Immunofluorescence revealed a much more intense staining for the gut compared to the body wall; moreover, positively immunoreactive amoeboid coelomocytes were also observed, i.e. cells in which we have previously demonstrated the occurrence of genotoxic damage. The double immunolabelling with antibodies against B[a]P/TCDD and the lysosomal enzyme cathepsin D demonstrated the lysosomal accumulation of the organic xenobiotic compounds, in particular in the cells of the chloragogenous tissue as well as in coelomocytes, involved into detoxification and protection of animals against toxic chemicals. The method described is timesaving, not expensive and easily applicable.

Validation of the detection of Pseudo-nitzschia spp. using specific RNA probes tested in a microarray format: Calibration of signal based on variability of RNA content with environmental conditions.

Harmful algal blooms (HAB) occur worldwide and cause health problems and economic damage to fisheries and tourism. Monitoring for toxic algae is therefore essential but is based primarily on light microscopy, which is time consuming and can be limited by insufficient morphological characters such that more time is needed to examine critical features with electron microscopy. Monitoring with molecular tools is done in only a few places world-wide. EU FP7 MIDTAL (Microarray Detection of Toxic Algae) used SSU and LSU rRNA genes as targets on microarrays to identify toxic species. In order to comply with current monitoring requirements to report cell numbers as the relevant threshold measurement to trigger closure of fisheries, it was necessary to calibrate our microarray to convert the hybridisation signal obtained to cell numbers. Calibration curves for two species of Pseudo-nitzschia for use with the MIDTAL microarray are presented to obtain cell numbers following hybridisation. It complements work presented by Barra et al. (2012b. Environ. Sci. Pollut. Res. doi: 10.1007/s11356-012-1330-1v) for two other Pseudo-nitzschia spp., Dittami and Edvardsen (2012a. J. Phycol. 48, 1050) for Pseudochatonella, Blanco et al. (2013. Harmful Algae 24, 80) for Heterosigma, McCoy et al. (2013. FEMS. doi: 10.1111/1574-6941.12277) for Prymnesium spp., Karlodinium veneficum, and cf. Chatonella spp. and Taylor et al. (2014. Harmful Algae, in press) for Alexandrium.