Exposure to Oil and Hypoxia Results in Alterations of Immune Transcriptional Patterns in Developing Sheepshead Minnows (Cyprinodon variegatus)

Abstract

The area and timing of the Deepwater Horizon oil spill highlight the need to study oil and hypoxia exposure in early life stage fishes. Though critical to health, little research has targeted the effect of oil and hypoxia exposure on developing immune systems. To this end, we exposed sheepshead minnows (Cyprinodon variegatus) at three early life stages: embryonic; post-hatch; and post-larval, to a high energy water accommodated fraction (HEWAF) of oil, hypoxia, or both for 48 hours. We performed RNAseq to understand how exposures alter expression of immune transcripts and pathways. Under control conditions, the embryonic to post-hatch comparison (first transition) had a greater number of significantly regulated immune pathways than the second transition (post-hatch to post-larval). The addition of oil had little effect in the first transition, however, hypoxia elicited changes in cellular and humoral immune responses. In the second transition, oil exposure significantly altered many immune pathways (43), and while hypoxia altered few pathways, it did induce a unique signature of generally suppressing immune pathways. These data suggest that timing of exposure to oil and/or hypoxia matters, and underscores the need to further investigate the impacts of multiple stressors on immune system development in early life stage fishes.

Figure 1

Representation of comparisons examined in the first life stage transition (A) and second life stage transition (B). Photographs taken by Danielle Simning.

Figure 2

Hierarchical clustering for all genes represented in any immune-related pathway (A), and for the top 25 most common immune-related genes from all pathways (B) generated in Java TreeView software version 1.1.6r4 (http://jtreeview.sourceforge.net/). Principal component analysis for all samples (C) generated in CLC Genomics Workbench 11 (https://www.qiagenbioinformatics.com/products/clc-genomics-workbench/).

Figure 3

Hierarchical clustering of immune-related canonical pathways of all treatment groups in the first and second life stage transitions,* indicates a pathway shared between all treatment groups in the first life stage transition, +indicates a pathway unique to the normoxic oil group in the second life stage transition, O indicates a pathway shared between normoxic oil and hypoxic oil groups in the second life stage transition, C indicates a cellular immune response pathway, and H indicates a humoral immune response pathway (A). (A) was generated in IPA (no version number) (https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis/). Comparison of number of significantly regulated immune-related pathways in the first (B) and second (C) life stage transitions for each treatment. (B,C) were generated in Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/).

Figure 4

Cellular and humoral immune responses in the first (A) and second (B) life stage transitions. Note that humoral immune response values are not shown in part B as there were no significant data. Both figures were generated in IPA (no version number) (https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis/).