Abstract:

This study was performed to profile the ultrastructure of control, environmental sponge individuals not exposed to oil or dispersants. Sectioned, stained sponges were examined in a Transmission Electron Microscope (TEM). Scanning Electron Microscope micrographs were taken with a Gatan digital camera mounted on the TEM.

Suggested Citation:

Lopez, Jose and Blackwelder, Patricia. 2015. Ultrastructure of Cinachyrella sponges before and after treatment with oil and dispersants. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N7VX0DGQ

Purpose:

This study was performed to profile the ultrastructure of control, environmental sponge individuals not exposed to oil or dispersants.

Data Parameters and Units:

TEM and SEM micrographs - naming convention of images in folder [Experiment (X1, X2, X3)) Type of Dosing (C = control, O = oil at 0.5 ppm, D = dispersant at 0.5 ppm, OD = oil/dispersant mixture at 0.5 ppm) Time of Exposure (0 time, 8 hours, 24 hours, 48 hours) Date of Experiment. file 4.26.14 Joe BP Spreadsheet.xlsx : Cinachyrella Sample (sample name), EM micrograph file (name of image file), Oil dosing experiment - X1 = experiment 1, X2 = experiment 2, X3 = experiment 3,

Methods:

We performed ex situ dosing experiments on wild Cinachyrella sp. sponges acclimated to laboratory aquaculture conditions on three different occasions ( = three replicate experiments each labeled X1 – X3) at FIU laboratories. The experiments were conducted in closed seawater tanks. For each experiment, one whole individual was exposed to sub-lethal concentrations (0.5 ppm) of Macondo crude oil, Corexit 9500 dispersant, or oil-Corexit 9500 dispersant mixtures (in the form of chemically enhanced water accommodated fractions or CE-WAFs). Each sponge individual and tank water were tracked and collected at four time points – 0, 1, 8, 24 and 48 hours. This yielded a 4 x 5 treatment matrix, or a minimum number of 16 sponges per experiment. Preparation of WAFs and CE-WAFs, and dosing followed standard ecotoxicological methods (“Chemical Response to Oil Spills: Ecological Effects Research Forum” or CROSERF) for the dosing of Cinachyrella individuals . To begin each experiment, at least 20 sponges were collected from local reefs in Dania Beach FL, and brought back to the laboratory alive. At least one immediately sacrificed as a “wild” type control (labeled “Fresh Cina”) for microscopy characterizations to determine the baseline microbial communities and tissue status. All sponges were placed in tanks for 1-2 weeks prior to the dosing experiments for acclimation. These are labeled “tank Cina”. Once experiments started, coding for each sample followed the protocol: Experiment, Type of Dosing, Time of Exposure, Date of Experiment. For example, sample “X1C8S10Aug11” was a control sample, not dosed, but sacrificed after 8 hours in Experiment X1. Each treated sponge was subsampled for electron microscopy (current data set). Designations of “inner” vs “outer” refer to the section of the sponge. Following the experiments, used seawater with oil and dispersant mixtures were treated as hazardous waste and disposed according to FIU standard waste removal protocols. Transmission Electron Microscopy (TEM) Sponge samples were placed in a gluteraldehyde fixative (2% gluteraldehyde in 0.05M Na cacodylate buffered 0.22 micron filtered seawater) for a minimum of 2-3 h. They were then rinsed in three changes of 0.0 5M Na cacodylate buffer for 10 min each. Samples were then postfixed in 1% osmium tetroxide in buffer for 45 min. and rinsed in buffer for three changes at 10 min each. Samples were dehydrated in a graded series of ethanol (20, 40, 60, 70, 90 and 100%). Following dehydration, samples were then embedded in Spurr embedding resin in flat embedding molds for 24 h at 600C. Blocks were sectioned using a Diatome 35 degree angle diamond knife. Sections were then stained with lead citrate (Reynolds 1963) and examined in a JEOL JEM 1200 Transmission Electron Microscope (TEM). Micrographs were taken with a Gatan digital camera mounted on the TEM.

Individual Files: