Fig 1.
(A) Evidence of bloody lesions seen in flounder exposed to oil. Red arrows indicate observed lesions. Fish were not tagged; therefore, lesions were not enumerated. (B) Survival rates in exposure treatments on Day 2 post-exposure to pathogenic bacteria Vibrio anguillarum. Bars represent average survival for three tanks per treatment and error bars represent standard error.
Fig 2.
Effect of oil exposure on relative gene expression for A) CYP1A in upper and lower gill tissue and B) Immunoglobulin M and C) Beta hemoglobin in spleen and kidney tissue of fish collected Day 1 post-pathogen exposure. Bars represent average gene expression (log2) relative to control fish for 3 fish per treatment and error bars represent minimum and maximum values of relative quantification based on standard deviation of Ct values from qPCR analyses.
Fig 3.
Microbial communities significantly affected by oil exposure.
(A) Comparison of the percent of Alcanivorax with (B) the experimental pathogen Vibrio anguillarum in microbiome found in the upper gill, lower gill and intestine from flounder tissue collected Day 1 post-pathogen challenge (3 fish/treatment). Mean percentage of microbiome (+/- 1 SEM) for Alcanivorax sp. and V. anguillarum. (C) Comparison of the relative abundance of PAH degrading bacterial groups such as Alcanivorax, Halomonas and Oceanospirillales in the gut, lower gill, upper gill, sediment and water collected Day 1 post-pathogen challenge after flounder were returned to experimental tanks and (D) a closer investigation comparing the relative abundance (%) of the bacterial challenge species Vibrio anguillarum and other Vibrio spp between water, sediment and experimental treatments sampled Day 1 post-pathogen challenge.
Fig 4.
Effect of oil exposure on bacterial diversity and function.
(A) Principal Coordinates Analysis (PCoA) plot of data at the Order level from fish collected on Day 1 post-pathogen exposure calculated with a Bray-Curtis dissimilarity in R (v. 3.0.2 [2013-09-25]–“Frisbee Sailing”). Water and sediment samples were collected on Day 0 of the experiment. Lower case letters indicate locations of various bacterial orders (a-Vibrio, b-Alcanivorax sp, c-Vibrio, dRoseobacter, e-Thalassolitus, f-Oceanobacter kriegii, g-Alcanivorax sp, h-Halolactibacillus miurensis, i-Cytophaga fermentans, j-Oceanobacter kriegii). Symbols represent different tissue types (● Intestine, ▲Lower Gill, ■ Upper Gill). Ellipses represent non-oil exposed vs. oilexposed each defined by treatment labels in their centers and indicate 95% Confidence Intervals, with orange ellipses representing both oiled treatments and gray ellipses representing both nonoiled treatments. (B) Predictive metagenomics analysis of the intestinal bacterial taxa in Oil/Pathogen Challenged fish (compared with control fish) indicated enrichment in degradation pathways. Pathway abundance was normalized via log2 fold change.
Fig 5.
Differentially expressed transcripts in livers of flounder exposed to oil.
(A) The Venn diagram illustrates the comparison of each treatment group with the control group based on the significantly differentially expressed genes (n = 13184 response genes). (B) The hierarchically clustered transcript heatmap was generated using average linking distance metric of the log2 transformed (FPKM) values. Green represents lower expression, red represents high expression compared to the controls, columns represent individual experiments (No Oil/No Pathogen (I), Pathogen/No Oil (II), Oil/No Pathogen (III), Oil/Pathogen (IV)), and rows represent transcriptional units.