Additional file 1: of Zebrafish sexual behavior: role of sex steroid hormones and prostaglandins

Table S1. Primers used for qRT-PCR analysis

Additional file 2: of Zebrafish sexual behavior: role of sex steroid hormones and prostaglandins

Figure S1. Male and female zebrafish were exposed separately to 25 nM E2 for 24 h and the brain tissue was isolated and dissected into three regions. RNA was isolated from individual samples followed by cDNA synthesis and qRT-PCR analysis. Statistical significance was determined as outlined in the materials and methods section. Statistically significant differences were determined using Student’s t test (*p < 0.05; **p < 0.01; *** p < 0.001). n = 4. Figure S2. Male and female zebrafish were exposed separately to 25 nM E2 for 24 h and the brain tissue was isolated followed by cDNA synthesis and qRT-PCR analysis. Statistical significance was determined as outlined in the materials and methods section. Statistically significant differences were determined using Student’s t test (*p < 0.05; *** p < 0.001). n = 4. Figure S3. Male and female zebrafish were exposed separately to 25 nM E2 for 24 h and the brain tissue was isolated followed by cDNA synthesis and qRT-PCR analysis. Statistical significance was determined as outlined in the materials and methods section. Statistically significant differences were determined using Student’s t test (*p < 0.05; **p < 0.01; *** p < 0.001). n = 4

Chemical analysis of water samples from Lake Hornträsket and the Vormbäcken stream.

<p>^aThe following substances were below detection limits: aliphatics >C10-C12, >C12-C16, >C16-C35; chlorobenzine; total PCB; naphthalene; acenaphthylen; acenaphthene; fluorene; phenanthrene; anthracene; fluoranthene; pyrene; benzo(a)anthracene; chrysene; benzo(b)fluoranthene; benzo(k)fluoranthene; benzo(a)pyrene; benzo(ah)anthracene; benzo(ghi)perylene; indeno(123cd)pyrene; total PAH.</p><p>Chemical analysis of water samples from Lake Hornträsket and the Vormbäcken stream.</p

Effect of metal exposure on <i>C</i>. <i>elegans</i> genes involved in apoptosis and innate immunity.

<p>Apoptosis marker genes <i>ape-1</i> and <i>hus-1</i> and innate immunity modulators <i>tir-1</i>, <i>clec-60</i>, and <i>bar-1</i> were analysed in <i>C</i>. <i>elegans</i> treated with the metal cocktail (MC) or waters from Lake Hornträsket (H) and Vombäcken stream at Björnås (B) and the exit to Vindelälven (V). One way analysis of variance (ANOVA) followed by Tukey’s post-test was used for multiple group comparison where * refers to <i>p</i><0.01 as compared to control and # represents significant differences in between the sites H, B and V. The dotted line represents the expression level normalized to that of the control (K-medium prepared with Milli-Q water).</p

Principal component analysis.

<p>The PCA analysis was based on the gene expression pattern in <i>C</i>. <i>elegans</i> exposed to water samples from lake Hornträsket (H), Vormbäcken stream at Björkås (B), Vormbäcken stream at the outlet to Vindelälven (V), K-medium prepared with Milli-Q water (C) and metal cocktail (MC). A. Score plot showing the distribution following exposure to environmental waters and control water based on gene expression. The ellipse shows Hotellings T² (0.05). B. PCA loading plot showing the distribution of the measured variables following exposure to the environmental waters and the K-medium control. C. Score plot showing the distribution following exposure to the MC, environmental waters and control water based on gene expression. The ellipse shows Hotellings T² (0.05). D. PCA loading plot shows the distribution of the measured variables following exposure to the MC, environmental waters and the control (K-medium prepared with Milli-Q water).</p

Metal exposure alters the expression of heat shock response genes in <i>C</i>. <i>elegans</i>.

<p>Synchronized young adult <i>C</i>. <i>elegans</i> were exposed for six hours to the metal cocktail (MC) or waters from Lake Hornträsket (H) and Vormbäcken stream at Björkås (B) and the exit to Vindelälven (V). qRT-PCR was performed for genes <i>hsp-16</i>.<i>1</i>, <i>hsp-16</i>.<i>2</i>, <i>hsp-16</i>.<i>48</i> and <i>hsp-70</i>. One way analysis of variance (ANOVA) followed by Tukey’s post-test was used for multiple group comparison where * refers to <i>p</i><0.01 as compared to control, and # represents significant differences between the environmental sites H, B and V. The dotted line represents the expression level normalized to that of the control (K-medium prepared with Milli-Q water).</p

Comparative Analysis of Stress Induced Gene Expression in <i>Caenorhabditis elegans</i> following Exposure to Environmental and Lab Reconstituted Complex Metal Mixture

<div><p>Metals are essential for many physiological processes and are ubiquitously present in the environment. However, high metal concentrations can be harmful to organisms and lead to physiological stress and diseases. The accumulation of transition metals in the environment due to either natural processes or anthropogenic activities such as mining results in the contamination of water and soil environments. The present study used <i>Caenorhabditis elegans</i> to evaluate gene expression as an indicator of physiological response, following exposure to water collected from three different locations downstream of a Swedish mining site and a lab reconstituted metal mixture. Our results indicated that the reconstituted metal mixture exerted a direct stress response in <i>C</i>. <i>elegans</i> whereas the environmental waters elicited either a diminished or abrogated response. This suggests that it is not sufficient to use the biological effects observed from laboratory mixtures to extrapolate the effects observed in complex aquatic environments and apply this to risk assessment and intervention.</p></div

Exposure of <i>C</i>. <i>elegans</i> to metals affects the expression of oxidative stress response genes.

<p>Synchronized <i>C</i>. <i>elegans</i> treated with the metal cocktail (MC) or waters from Lake Hornträsket (H) and Vormbäcken stream at Björkås (B) and the exit to Vindelälven (V) were analyzed for the expression of <i>gst-4</i>, <i>sod-1</i>, <i>cat-2</i>, <i>cyp-35A2</i>, <i>daf-2</i>, <i>nnt-1</i>, <i>kel-8</i> and <i>gcs-1</i> using qRT-PCR. One way analysis of variance (ANOVA) followed by Tukey’s post-test was used for multiple group comparison where * refers to <i>p</i><0.01 as compared to control and # represents significant differences in between sites H, B and V. The dotted line represents the expression level normalized to that of the control (K-medium prepared with Milli-Q water).</p

Effect of metals exposure on nuclear hormone receptors and developmental genes in <i>C</i>. <i>elegans</i>.

<p><i>C</i>. <i>elegans</i> treated with the metal cocktail (MC) or waters from Lake Hornträsket (H) and Vombäcken stream at Björnås (B) and the exit to Vindelälven (V) were analyzed for the expression of genes associated with hormone receptors (<i>fshr-1</i>, <i>nhr-8</i> and <i>nhr-14)</i> and development (<i>vit-2</i>, <i>daf-12</i>, and <i>apl-1)</i> were tested using qRT-PCR. One way analysis of variance (ANOVA) followed by Tukey’s post-test was used for multiple group comparison where * refers to <i>p</i><0.01 as compared to control whereas # represents significant differences in between the sites H, B and V. The dotted line represents the expression level normalized to that of the control (K-medium prepared with Milli-Q water).</p

Effect of <i>C</i>. <i>elegans</i> exposure on the metal response genes.

<p>Adult <i>C</i>. <i>elegans</i> treated with the metal cocktail (MC) or waters from Lake Hornträsket (H) and Vombäcken stream at Björnås (B) and the exit to Vindelälven (V) were analyzed for the expression of ten metal response genes; <i>mtl-1</i>, <i>mtl-2</i>, <i>ftn-1</i>, <i>pgp-5</i>, <i>cdf-2</i>, <i>hif-1</i>, <i>numr-1</i>, <i>aip-1</i>, <i>cdr-1</i>, and <i>hmt-1</i>. One way analysis of variance (ANOVA) followed by Tukey’s post-test was used for multiple group comparison where * refers to <i>p</i>< 0.01 as compared to control and # represents significant differences in between the sites H, B and V. The dotted line represents the expression level normalized to that of the control (K-medium prepared with Milli-Q water).</p