Abstract:

While there is some evidence that exposure to individual polycyclic aromatic hydrocarbons (PAHs) impairs development of the early stage kidney (i.e. pronephros), little is known regarding the effects on the pronephros following exposure to complex crude oil mixtures at environmentally relevant concentrations. Transcriptional changes in genes with various structural, functional and signaling roles specific to different regions of the developing pronephros were assessed by QPCR and whole mount in situ hybridizations, in zebrafish. Morphological changes in the pronephros were determined by immunohistochemistry using an antibody raised against Na/K ATPase (NKA) in zebrafish. Additionally, latent effects of ELS crude oil exposure on kidney function and overall osmoregulation were assessed by subjecting red drum to an acute salinity transfer challenge. And, edema size was assessed in zebrafish after an acute salinity stress.

Suggested Citation:

Bonatesta, Fabrizio and Edward Mager. 2020. Effects on the teleost kidney following early life-stages exposure to Deepwater Horizon crude oil. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/BFXBPAD8

Purpose:

Establishing effects of crude oil on the teleost kidney morphology and gene expression and establishing osmoregulatory capacity in oil exposed fish.

Data Parameters and Units:

Multiple tests have been performed for the “Effects on the teleost kidney development and function following early life-stages exposure to Deepwater Horizon crude oil” dataset. They were performed during different days. Each test has been uploaded in the corresponding folder. The folder titles are abbreviated with codes. Please refer to the following list to match the test to the corresponding assigned code. M203: Zebrafish dose response for kidney mRNA expression M204: Zebrafish static 96 h LC50 bioassay M205: Zebrafish time course for kidney mRNA expression and salinity transfer M207: Zebrafish time course for whole mount in situs and immunostaining M211: Zebrafish kidney in situ hybridizations, immunohistochemistry, and salinity transfer E206: Red drum salinity transfer test post crude oil exposure. There is an extensive readme describing the dataset. Some relevant headers are -Survival, pH, dissolved oxygen (mg/L), salinity (mmol/kg, and ppt), PAH concentrations (µg/L), Heart rate (beats/min), Pericardial area (μm²)

Methods:

Water accommodated fractions (WAFs) of OFS crude oil were prepared with a dilution of 1 mg/L of oil to reconstituted hard water (RHW) and dilutions were made accordingly. Zebrafish (Danio rerio) embryos/larvae were used for the test starting the WAF exposure at 0 hours post fertilization (hpf) and ending at 96 hpf. Mortality and water parameters (pH, dissolved oxygen, temperature) were measured daily during the duration of the test. Samples were collected for: qPCR analysis of kidney specific genes, whole-mount in situ hybridization for the same genes, and whole-mount immunohistochemistry at 72 hpf using an antibody raised against the sodium-potassium ATPase. At the end of the WAF exposure, pictures and videos of zebrafish larvae were taken to assess heart rate and pericardial/ yolk-sac area. 2 salinity challenge tests were performed: one starting at 48 hpf and ending at 72 hpf, the other starting at 96 hpf and ending at 120 hpf. 3 different waters were used for the salinity challenge: Milli-Q water, Embryo (E3) Medium, and E3 Medium enriched with 25x the concentration of NaCl. Zebrafish larvae were randomly transferred from the treatment test to salinity test. Prior to transfer, they were rinsed twice in respective salinity solution. Pictures and videos of zebrafish larvae were taken prior and after the salinity test to assess potential osmoregulatory disfunctions by inspecting heart rate and pericardial/ yolk-sac area. An additional test was performed using red drum larvae (Sciaenops ocellatus) to asses osmoregulatory disfunctions due to crude oil exposure. Red drum embryos were collected the morning following spawning and transferred either into control seawater (35 ppt) or 1% OFS WAF for 24 h. Embryos were transferred to 164 L conical mesocosms containing clean saltwater up until 14 days post fertilization (dpf). The fish had first feeding at 3 dpf and were fed enriched rotifers. They were fed only enriched rotifers until 10 dpf. On day 10 they were fed enriched rotifers and unenriched artemia. On day 11 they were fed enriched rotifers and enriched artemia. Starting on day 12 and throughout our experiment they were fed enriched artemia. Samples were collected for qPCR analysis at 48 hpf and 7 dpf, and at 72 hpf for immunohistochemistry using antibody raised against NK-ATPase. Starting at 14 dpf a 96-h salinity challenge was performed, and mortality was scored every day, as well as water parameters (ph, salinity, temperature, dissolved oxygen). The salinity used for the test were: 30, 18, 11, 7, 4 and 2 ppt.

Individual Files: