Abstract:

Sheepshead minnows, Cyprinodon variegatus, were exposed to control, low oil (150 µg/L tPAH), or high oil (300 µg/L tPAH) throughout the embryonic stage. Exposure solutions were refreshed every 24 hours. At four days post-fertilization (just before hatch), embryos were imaged and scored for evidence of cardiotoxic phenotypes (pericardial edema), as well as calculation of cardiac output through heart rate observation and quantification of ventricular stroke volume. qPCR analysis was performed following imaging for expression of cyp1a to verify exposures to PAHs.

Suggested Citation:

Sepulveda, Maria S. and Elizabeth Allmon. 2021. Cardiac development and function in embryonic sheepshead minnows (Cyprinodon variegatus) following oil exposure. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/ZCZFTXT3

Purpose:

Identify cardiotoxic phenotypes and functional responses in the cardiac system of sheepshead minnows (Cyprinodon variegatus) exposed to oil during embryonic development.

Data Parameters and Units:

Readme file contains details on file naming. Time: hours (h), seconds (s), hours post fertilization (hpf), days post fertilization (dpf) Volume: milliliters (mL), liters (L) HEWAF concentrations: micrograms per liter total PAH (µg/L tPAH) Fluorescence: Fluorescent Signal Units (FSU) Stroke Volume (SV): nanoliter (nL) Heart Rate (HR): beats per minute (bpm) Cardiac output (CO): nanoliters per minute (nL/min)

Methods:

Sheepshead minnow (Cyprinodon variegatus) embryos collected <12 hpf and exposed to control (0 µg/L tPAH), low oil (150 µg/L tPAH), or high oil (300 µg/L tPAH) 100% HEWAF stocks made daily and diluted to 150 and 300 µg/L tPAH nominal concentrations. Exposure solutions are refreshed every 24 hrs. tPAH analysis of four 100% HEWAF stocks conducted by ALS Environmental, Kelso, WA, USA. Fluorescence measurements conducted on 100% HEWAF, 0h exposures, and 24h exposures for each concentration (0, 150, or 300 µg/L tPAH) Images and videos collected on a Nikon SMZ1500 stereomicroscope affixed with a Nikon Ds-Ri2 camera with Nikon NIS-Elements D software. Image and video analysis through ImageJ software. RNA extraction was done with Qiagen RNEsay Kits. cDNA synthesized from 100 ng total RNA starting material and SuperScript III reverse transcriptase following DNase-1 incubation. qPCR performed on a Bio-Rad CFX Connect Real-Time PCR System with iQ SYBR Green Supermix for b-actin and cyp1a with primers designed in-house and supplied through IDT.

Instruments:

Fluorometer: Turner Designs AU-10 Microscope: Nikon SMZ1500 stereomicroscope Camera: Nikon Ds-Ri2 Image capture: Nikon NIS-Elements D software Image analysis: ImageJ software qPCR: Bio-Rad CFX Connect Real-Time PCR System

Individual Files: