MOESM2 of Influence of different wastewater treatment technologies on genotoxicity and dioxin-like toxicity in effluent-exposed fish

Additional file 2.  All data

MOESM1 of Influence of different wastewater treatment technologies on genotoxicity and dioxin-like toxicity in effluent-exposed fish

Additional file 1: Semi-quantitative histolopathological assessment of liver, kidney and gill samples of fish exposed up- and downstream of WWTP B. Samples were classified into five categories described in detail by Wilhelm et al. [17] according to the symptoms displayed. Shortly, class 1 includes samples in control state, class 2 has been assigned to tissue samples displaying slight reactions, class 3 to samples with pronounced reactions, class 4 describes samples expressing beginning destructive alterations and class 5 has been assigned to liver samples with severe cellular destruction. Asterisks and horizontal lines indicate significant differences between two datasets according to likelihood ratio chi-square tests with subsequent Holm correction

Intraspecific Variation in Cellular and Biochemical Heat Response Strategies of Mediterranean <i>Xeropicta derbentina</i> [Pulmonata, Hygromiidae]

<div><p>Dry and hot environments challenge the survival of terrestrial snails. To minimize overheating and desiccation, physiological and biochemical adaptations are of high importance for these animals. In the present study, seven populations of the Mediterranean land snail species <i>Xeropicta derbentina</i> were sampled from their natural habitat in order to investigate the intraspecific variation of cellular and biochemical mechanisms, which are assigned to contribute to heat resistance. Furthermore, we tested whether genetic parameters are correlated with these physiological heat stress response patterns. Specimens of each population were individually exposed to elevated temperatures (25 to 52°C) for 8 h in the laboratory. After exposure, the health condition of the snails' hepatopancreas was examined by means of qualitative description and semi-quantitative assessment of histopathological effects. In addition, the heat-shock protein 70 level (Hsp70) was determined. Generally, calcium cells of the hepatopancreas were more heat resistant than digestive cells - this phenomenon was associated with elevated Hsp70 levels at 40°C.We observed considerable variation in the snails' heat response strategy: Individuals from three populations invested much energy in producing a highly elevated Hsp70 level, whereas three other populations invested energy in moderate stress protein levels - both strategies were in association with cellular functionality. Furthermore, one population kept cellular condition stable despite a low Hsp70 level until 40°C exposure, whereas prominent cellular reactions were observed above this thermal limit. Genetic diversity (mitochondrial cytochrome c oxidase subunit I gene) within populations was low. Nevertheless, when using genetic indices as explanatory variables in a multivariate regression tree (MRT) analysis, population structure explained mean differences in cellular and biochemical heat stress responses, especially in the group exposed to 40°C. Our study showed that, even in similar habitats within a close range, populations of the same species use different stress response strategies that all rendered survival possible.</p></div

Correlation of relative Hsp70 level vs. histopathological mean assessment values.

<p>Data obtained for the populations of the respective exposure groups (25, 33, 40, 43 and 48°C) are framed, respectively. <b>A.</b> Relative Hsp70 level vs. condition of the tubules. <b>B.</b> Relative Hsp70 level vs. condition of the digestive cells. <b>C.</b> Relative Hsp70 level vs. condition of the calcium cells.</p

Relative Hsp70 level of different populations after exposure to elevated temperature for 8 h.

<p>Shown are means and SD; <i>n</i> = 10. Asterisks show significant differences of the respective exposure groups compared to the control at 25°C after Bonferroni correction: 0.0017<<i>P</i>≤0.0083: (*) and 0.00017<<i>P</i>≤0.0017 (**).</p

Results of the MRT analyses of PCoA transformed physiological heat stress response data (Hsp70 and histology) constrained with population structure information of <i>Xeropicta derbentina</i> under four temperature conditions.

<p>R²: cross-validated proportion of variance explained by the primary grouping (i.e., first split of the tree); P1–P7: populations studied; π: nucleotide diversity; H_MH3: axis 3 of transformed haplotype diversity; F_ST1, F_ST2: axes 1 and 2 of transformed pairwise fixation index; (+): positive correlation; (−): negative correlation; improved histopathology (i); deteriorated histopathology (d).</p

The condition of the digestive cells of the hepatopancreas.

<p>Mean assessment values for each population at elevated temperature. Shown are means and SD; <i>n</i> = 8. Asterisks show significant differences of the respective exposure groups compared to the control at 25°C after Bonferroni correction: 0.0025<<i>P</i>≤0.0125: (*) and 0.00025<<i>P</i>≤0.0025 (**).</p

Within- and between-site genetic differentiation calculated for <i>Xeropicta derbentina</i> populations (1–7) from Southern France based on the COI gene.

<p>On diagonal line: nucleotide diversity (π); above diagonal: haplotype divergence (H_MH) based on the Morisita-Horn index; below diagonal: pairwise fixation index (F_ST).</p

The condition of the calcium cells of the hepatopancreas.

<p>Mean assessment values for each population at elevated temperature. Shown are means and SD; <i>n</i> = 8. Asterisks show significant differences of the respective exposure groups compared to the control at 25°C after Bonferroni correction: 0.0025<<i>P</i>≤0.0125: (*) and 0.00025<<i>P</i>≤0.0025 (**).</p