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Fri Sep 13, 2019, 12:10 AM

Genetic Sequencing of Mahi Mahi to Determine the Exposure Level From the Deep Horizon Oil Disaster.

The paper I'll discuss in this relatively brief post is this one: Whole-Transcriptome Sequencing of Epidermal Mucus as a Novel Method for Oil Exposure Assessment in Juvenile Mahi-Mahi (Coryphaena hippurus) (Justin B. Greer,*,† Nicolette E. Andrzejczyk,*,† Edward M. Mager,‡ John D. Stieglitz,§ Daniel Benetti,§ Martin Grosell,∥ and Daniel Schlenk†,⊥, Environ. Sci. Technol. Lett. 2019, 6, 538−544)

One of the joys of writing posts in this "sleepy little DU science forum" - as I've heard it described - is the privilege of learning things as I write. Over the last few weeks, I've been writing a somewhat involved post comparing two recently published scientific papers about two radioactive contamination events, one of which is everyone's favorite with the possible exception of Chernobyl, Fukushima, and the other involving radioactive contamination of a San Joaquin oil field in California. Writing this rather long post has stimulated me to do some interesting reading on the human physiology of certain radioactive nuclides, including two that surely killed a rather famous scientist.

One of the side notes I found myself going down is the case of the famous scientific paper about the "Fukushima Tuna Fish" which received vast international attention, much to the chagrin of the paper's authors, and believe me, when a scientist gets a paper with an international attention all over the news media, "chagrin" is not usually the word associated with the attention.

Anyway, this is not that post. I'm still working on it and it isn't done.

It proves to timely: The recent Fukushima attention concerns the proposal to dump "radioactive" seawater into the sea - it happens that there is no such thing, and never has been such a thing as seawater that isn't radioactive - the dumping is perfectly OK with me. If one supports nuclear energy as I do, one has to greet such ignorance with a mixture of amusement and despair. If the Fukushima "radioactive seawater" is dumped, we can expect the morons at Greenpeace to have a festival of clownish trivializing stupidity featuring dressing up and cruising around in diesel powered boats in their ongoing efforts to be sure the planet commits a suicide worthy of the Darwin Award.

I'm sure the reactions will be as stupid as the reaction I experienced here some time ago when a correspondent dug up one of my old posts to announce that the world was ending because a tunnel collapsed on the radioactively contaminated Hanford Nuclear Weapons Complex in Washington State, a collapse also widely reported in the media, albeit not as actively as the famous Fukushima tuna fish.

God bless the ignore list.

Anyway, the paper here is about another kind of fish, a fish contaminated by petroleum leaks, in this case, oil from the Deepwater Horizon Oil.

From the introduction to the paper:

Large-scale oil spills such as the 2010 Deepwater Horizon (DWH) spill in the Gulf of Mexico can have significant ecological impacts on marine populations, for example, via exposure to polycyclic aromatic hydrocarbons (PAHs). The DWH oil spill resulted in extensive oiling of spawning regions for commercially important pelagic species in the Gulf of Mexico, including mahi-mahi (Coryphaena hippurus). Sublethal exposure to environmentally relevant concentrations of crude oil-derived PAHs has been shown to impair cardiac development, swimming performance, craniofacial development, and behavior, leading to reduced fitness and increased mortality risk.1−4 Therefore, there is a need for the development of monitoring tools that can estimate environmental exposure and monitor the health status of individuals following a spill in a noninvasive manner to minimize further population disruptions.


As biological barriers go, the one with which I am most familiar is human skin, with particular focus on its molecular biology, human skin a fascinating organ, but the authors here are concerned with another complex biological barrier, the mucus layer coating fish, also a fascinating organ with fascinating molecular biology which, as the authors note...

Epidermal mucus provides the first line of defense against pathogens and toxicants and is secreted by goblet cells, sacciform cells, and club cells located throughout the epidermis.6 Mucus contains a wide range of molecules such as proteins, lipids, carbohydrates, mRNA, and DNA, with the most well-characterized being antimicrobial and immunerelated (sic) molecules.7,8 Furthermore, mucosal composition has been shown to be altered by a variety of stress conditions. For example, oil exposure in dusky splitfin (Goodea gracilis) elicited antioxidant responses in the epidermal mucus that were greater than those found in the liver, brain, and muscle.9 Other studies focused on aquaculture applications have demonstrated that immune-related proteins such as lectins, heat shock proteins, and complement factors are highly abundant and dynamically regulated following bacterial infection, food deprivation, and overcrowding stress.10−13 Thus, changes in mucosal composition could be used to identify biomarkers of exposure for environmental contaminants or stressors.


The authors focus on an usually studied subclass of biological barrier molecules, specifically RNA.

Recently there has been increased interest in what is called the "exposome" which is the molecular signatures of exposure to external molecules and stresses, many of which ultimately show up in toxicological syndromes. This is a relatively new undertaking and is proving to be quite fascinating. Here, for example is a link to a recent paper upon which I stumbled, the expsome associated with Alzheimer's disease: The Alzheimer's Disease Exposome. Publication of this paper, by the way, does not "prove" anything at all about Alzheimer's disease, but it offers an important area of inquiry worthy of study.

As they are sometimes and somewhat naively interpreted, the nucleic acids represent something like a computer program of life, and these "programs" are of two types, the germline type - that which is heritable - and the somatic type, that which is involved in the control and feedback loops of the molecular biology of living things. In somatic nucleic acids, a number of changes accumulate normally during life, these are called "epigenetic changes" and as such represent the exposome. These somatic changes are known to control normal and abnormal processes like, in the former case, aging and development, in the latter case, diseases like cancer and autoimmune diseases like lupus and rheumatoid arthritis.

The oil utilized in this study to examine the effects of Deepwater Horizon spill was real Deepwater Horizon Oil, collected from the sea and stored as such:

Oil Preparation and Exposures. High-energy water accommodated fractions (HEWAFs) were prepared from crude oil obtained during surface skimming (OFS) following the DWH oil spill and transferred to the University of Miami under the chain of custody (sample ID OFS-20100719- Juniper-001 A00884). The HEWAF solutions were prepared according to established methods and were diluted to nominal concentrations of 5% or 10% using ultraviolet-sterilized seawater for testing (Supplemental Methods).2 Juvenile mahi-mahi raised from captive wild mahi-mahi broodstock fish16 [F1 generation, ∼28 days of age, mass of 4.89 ± 0.14 g (standard error of the mean)] were placed into 10 L glass aquaria containing 8 L of either fresh seawater (control), 5% HEWAF (low), or 10% HEWAF (high) for 48 h. Four individuals were placed in each tank, with four replicate tanks in each treatment group. An 80% water change with fresh seawater or HEWAF dilution was performed on each aquarium ∼24 h after the beginning of exposure. Exposures were performed in a temperature-controlled environment at 27 °C with a 16 h:8 h light:dark photoperiod. None of the exposure concentrations elicited acute mortality.


Cool, I think. I'm glad scientists collected "DWH" oil samples and stored them for study years after the fact. Excellent scientific forethought!

Some graphics from the paper:



Figure 1. Results from RNA sequencing of mahi-mahi epidermal mucus, including (A) a heat map of Euclidean distances between samples in the control, low-HEWAF (∑PAH = 16.55 μg/L), and high-HEWAF (∑PAH = 23.03 μg/L) exposure groups calculated from DEseq2 variance stabilizing transformation of the RSEM count data, (B) a volcano plot of differentially expressed transcripts within the low- and high-HEWAF exposure groups, and (C) the top canonical pathways, physiological system development and function (D & F) pathways, and toxicity functions predicted to be altered in the low-oil exposure group by Ingenuity Pathway Analysis. Full lists of all pathways for both low- and high-HEWAF exposures can be found in Tables S8–S12.



Figure 2. (A) Immune system and (B) cardiovascular system and Ca2+ homeostasis functions, as well as associated genes, that were predicted to be altered in mahi-mahi epidermal mucus following low-HEWAF (∑PAH = 16.55 μg/L) exposure based on analysis with IPA.


The authors studied, using modern technology, 194,282 fish genes in the mucus barrier.

They found:

Analysis of differentially expressed transcripts showed that the mucosal transcriptome was significantly altered following PAH exposure. There were 501 differentially expressed transcripts in the low-HEWAF exposure (227 upregulated, 274 downregulated) and 196 differentially expressed transcripts in the high-HEWAF exposure (121 downregulated, 75 upregulated) (Figure 1B). The low and high treatments shared 136 of the same differentially expressed transcripts (Figure S1), suggesting common transcriptional responses occur within the range of PAH concentrations tested. Cytochrome P450 enzymes (cyp1a1 and cyp1b1) are well-established biomarkers of PAH exposure27 and were among the most highly upregulated transcripts in both the low- and high-HEWAF exposures (Table S7). The efficacy of cyp1a1 induction as a biomarker for PAH exposure has also been examined with other nonlethal sampling methods, with greater cyp1a1 upregulation observed in the caudal fin than in the liver of marine diesel-exposed juvenile coho salmon (Oncorhynchus kisutch).28 Thus, upregulation of cyp1a1 appears to show a robust molecular response using nonlethal sampling methods and may be a useful biomarker candidate for PAH exposure...

... Together, our data suggest that mucosal mRNA abundance may be indicative of whole-animal changes in immune-related function in PAH-exposed fish. IPA also predicted alterations in toxicity functions induced by oil exposure. The top-ranked toxicity functions in the low- HEWAF exposure were liver fibrosis, liver hyperplasia, congenital heart anomaly, and cardiac enlargement (Figure 1C and Table S12). Similarly, liver enlargement, pulmonary hypertension, liver hyperbilirubinemia, and liver hyperplasia were the top-ranked toxicity functions in the high-HWAF exposure. Of all toxicity functions, 17 cardiac functions were predicted to be altered in the low-HEWAF exposure and 11 in the high-HEWAF exposure, with many overlapping categories such as cardiac enlargement, cardiac arrhythmia, cardiac fibrosis, and cardiac necrosis/cell death (Table S12). In addition, there was a predicted inhibition of cardiac muscle function, cardiac muscle contractility, and abnormality of the heart ventricle (Figure 2B), which are cardiac phenotypes known to be altered by crude oil-derived PAH exposure in fish.37,38 Alterations in Ca2+ homeostasis were also predicted from the mucus transcriptional profile, such as decreased mobilization of Ca2+ and an increased quantity of Ca2+ (Figure 2B). Transcripts of ryanodine receptor 2 (ryr2), the primary mediator of calcium-induced Ca2+ release required for cardiomyocyte contraction, were upregulated in the mucus and have also been shown to be dysregulated in oil-exposed mahi-mahi and Atlantic haddock (Melanogrammus aeglef inus) embryos (Table S11).39,40


IPA here refers to "Ingenuity Pathway Analysis" a bioinformatics software tool. IPA, Qiagen

The authors state that to their knowledge, this is the first paper to look at this particular pathway in determining the exposome of oil spills on marine life.

This paper will get no attention from our distracted media with its selective attention, the same media that gave the intellectually and morally challenged awful criminal Donald J. Trump the White House, "...because...her emails..."

In reality the "election" of Donald J. Trump is a trivial, if wholly unfortunate, blip in world history. The destruction of the planetary atmosphere and oceans by dangerous fossil fuels like, but not limited to, petroleum, "...because...Fukushima..." is not trivial.

I wish you a pleasant and safe Friday the 13th.

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