Larval Red Drum (Sciaenops ocellatus) Sublethal Exposure to

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Larval red drum (Sciaenops ocellatus) sublethal exposure to weathered Deepwater Horizon crude oil: Developmental and transcriptomic consequences Elvis Genbo Xu, Alexis Khursigara, Jason Magnuson, Edward Starr Hazard, Gary T Hardiman, Andrew Esbaugh, Aaron P. Roberts, and Daniel Schlenk Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b02037 • Publication Date (Web): 02 Aug 2017 Downloaded from http://pubs.acs.org on August 3, 2017

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Environmental Science & Technology

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Larval red drum (Sciaenops ocellatus) sublethal exposure to weathered Deepwater Horizon

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crude oil: Developmental and transcriptomic consequences

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Elvis Genbo Xu1*, Alex J. Khursigara2, Jason Magnuson3, E. Starr Hazard4,5, Gary Hardiman5,6,

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Andrew J. Esbaugh2, Aaron. P. Roberts3, Daniel Schlenk1

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Department of Environmental Sciences, University of California, Riverside, CA 92521

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Marine Science Institute, University of Texas at Austin, TX 78373

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Department of Biological Sciences & Advanced Environmental Research Institute, University of North Texas, TX 76203 4

Center for Genomics Medicine, Medical University of South Carolina, Charleston, SC 29403

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Computational Biology Resource Center, Medical University of South Carolina, Charleston, SC 29403 6

Departments of Medicine & Public Health Sciences, Medical University of South Carolina, Charleston, SC 29403

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*Corresponding author

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Corresponding author: Elvis Genbo Xu

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Corresponding email: [email protected]

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Corresponding address: Department of Environment Sciences, University of California,

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Riverside, CA 92521, USA

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Corresponding Tel.: 1-951-313-7643

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Fax No. 1-951-827-3993 1 ACS Paragon Plus Environment


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Abstract

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The Deepwater Horizon (DWH) incident resulted in extensive oiling of the pelagic zone and

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shoreline habitats of many commercially important fish species. Exposure to water

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accommodated fraction (WAF) of oil from the spill causes developmental toxicity through

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cardiac defects in pelagic fish species. However, few studies have evaluated the effects of the oil

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on near-shore estuarine fish species such as red drum (Sciaenops ocellatus). Following exposure

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to a certified weathered slick oil (4.74 µg/L ∑PAH50) from the DWH event, significant sub-

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lethal impacts were observed ranging from impaired nervous system development (average 17%

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and 22% reduction of brain and eye area at 48 hpf, respectively) to abnormal cardiac morphology

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(100% incidence at 24, 48 and 72 hpf) in red drum larvae. Consistent with the phenotypic

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responses, significantly differentially expressed transcripts, enriched gene ontology, and altered

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functions and canonical pathways predicted adverse outcomes in nervous and cardiovascular

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systems, with more pronounced changes at later larval stages. Our study demonstrated that the

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WAF of weathered slick oil of DWH caused morphological abnormalities predicted by a suite of

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advanced bioinformatic tools in early developing red drum, and provided also the basis for a

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better understanding of molecular mechanisms of crude oil toxicity in fish.

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2 ACS Paragon Plus Environment

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Table of Contents (TOC) Art

Introduction The Deepwater Horizon (DWH) incident in 2010 was the largest marine oil spill in U.S. history,

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resulting in the release of approximately 700 million liters of crude oil, and extensively oiling the

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pelagic zone and shoreline habitats.1 Previous studies have investigated the developmental

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toxicity of crude oil to different fish and identified a variety of abnormalities in cardiac function,

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formation of the craniofacial skeleton, nervous system as well as reduced swimming

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performance.2-9 Most of the previous studies were performed on pelagic species, but little is

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known about the effects on local fast-developing estuarine fish species such as red drum

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(Sciaenops ocellatus). The red drum inhabits the Southern Atlantic and Gulf of Mexico coasts, 3 ACS Paragon Plus Environment


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and generally spend their initial first 3-4 years within estuarine and nearshore waters.

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Importantly, this species is a highly valued recreational species in Gulf of the Mexico, which

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represents significant economic importance for Gulf coast communities.10 Red drum develops

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faster than many of the studied pelagic species and other estuarine species,6,8 which may enhance

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susceptibility to the developmental impacts of oil. Unlike mahi-mahi (Coryphaena hippurus),

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red drum embryos were less sensitive to naturally weathered oil than oil directly obtained from

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source well.11 Consequently, further investigation on molecular events and pathways responsible

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for developmental toxicity is essential to understand different mechanisms of crude oil toxicity to

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the different fish species.

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The morphological and functional impacts of crude oil on fish embryonic development have

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been intensively investigated. However, molecular initiating events preceding these defects are

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not well understood.2,5-7,9 Studies in scombrid fish species have indicated inhibition of plasma

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membrane potassium ion channels and intracellular calcium transporters in cardiac myocytes.12

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Reductions in expression of these genes and others controlling intracellular calcium have also

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been noted in different fish species.3,13 However, it is unclear how oil or PAHs within oil impact

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the regulation and function of these channels and transporters. In addition to cardiotoxicity, our

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previous study by RNA sequencing and physiological assessment in mahi-mahi demonstrated

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that weathered oil also resulted in significant perturbations in metabolism, steroid biosynthesis,

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vision, and AhR pathway suggesting other targets in addition to the heart may be involved in the

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developmental toxicity of DWH oil.3 A more recent study in Atlantic haddock (Melanogrammus

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aeglefinus) explored the transcriptional basis for cardiac formation, craniofacial development,

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ionoregulation and cholesterol homeostasis, confirming a key role of intercellular calcium

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cycling and excitation-contraction coupling after crude oil exposure.14 While cardiotoxicity of


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crude oil and PAHs have long been studied, central nervous system development is also affected

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by PAH mixtures, resulting in behavioral dysfunction, alterations in locomotion, impaired visual

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acuity, and reduced foraging efficiency.15,16 However, the underlying molecular mechanisms for

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these effects have yet to be identified for early life stages of fish. Given that crude oil is a

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complex chemical mixture, there likely are multiple targets involving multiple interacting

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molecular mechanisms. High throughput sequencing (HTS) allows relatively unbiased

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quantification of expression levels of transcripts with a high sensitivity and broad genome

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coverage at one time of assessment, compared to other methods such as microarray and targeted

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PCR, and downstream bioinformatic assessment has the potential to predict phenotypic outcomes

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during developmental processes after toxicant exposure.

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Here, we exposed red drum larvae to weathered DWH slick oil and evaluated morphological

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anchoring transcriptional effects at three different critical windows (24, 48 and 72 hour post

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fertilization (hpf)) of larval development using a HTS approach coupled with advanced

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bioinformatic tools.3,4 Based on genome-wide differentially expressed transcripts, the most

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enriched gene ontology, impacted biological processes, and canonical pathways in nervous,

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cardiovascular and ocular systems, as well as upstream regulators were identified. These

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molecular responses were compared at different developmental stages with phenotypical

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measurements, providing novel insights into the mechanisms of DWH oil-induced

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developmental toxicities in this rapidly growing estuarine species. This study also provides

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potential phenotype-specific biomarkers through linking DEGs to toxicity endpoints, which

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could be useful in ecological risk assessment of oil pollution.

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Materials and Methods



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Animals and DWH oil exposure

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Embryonic red drum were collected from brood stock tanks at the Texas Parks and Wildlife –

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CCA Marine Development Center in Corpus Christi, Texas and transported under constant

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aeration to the University of Texas Marine Science Institute. Embryos were subsequently treated

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with formalin during aeration. Embryos were then rinsed with sterilized seawater and checked

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for buoyancy and coloration using a Nikon SMZ2800N microscope. Spawns with low

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fertilization rates or poor egg quality were not used. The oil was a weathered oil collected from

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a slick in the Gulf of Mexico on June 29th, 2010 from the hold of barge number CT02404. Oil

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exposures were generated according to standard protocols for high energy water accommodated

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fractions (HEWAF), as previously described.7 Oil loading rate was 1 g per 1 liter of seawater (35

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ppt). Red drum spawned at night and eggs were collected in the next morning. Treatments

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started at 12 hpf, and red drum eggs typically hatch at 24 hpf. Time course exposure to WAF of

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weathered slick oil (2.5%; 4.74 µg/L PAHs) was at 24, 48 and 72 hpf with four replicates. The

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test was performed in an environmental control chamber set at 25 °C and 30 ppt salinity with a

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14:10 h light: dark photoperiod. Survival was assessed daily. A minimum of a 70% hatching

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success was required followed by a 80% survival for all tests. All experiments were approved by

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the institutional animal care and use committee (IACUC) at the University of Texas at Austin

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(AUP-2014-00375).

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Water chemistry analysis

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A sub-sample of the initial diluted HEWAF was collected in a 250 mL amber bottle and stored at

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4°C

until

analyzed

by

ALS

Environmental

(Kelso,


WA)

for

PAHs

using

gas

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chromatography/mass spectrometry-selective ion monitoring (GC/MS-SIM; based on EPA

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method 8270D). Individual measurements (µgL-1) for 76 PAHs were quantified from initial

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samples collected from HEWAF dilutions. Reported ΣPAH values represent the sum of 50 select

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PAH analytes. Temperature, pH, dissolved oxygen, and salinity were measured at test start (0

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hour), and subsequent days (24, 48, and 72 hpf). A summary of all measured water quality

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parameters and ΣPAH concentrations are provided in Supporting Information Tables S1, S2.

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Morphological Characteristics

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Initial range finding morphological measurements were conducted on 24, 48 and 72 hpf larvae

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treated with the WAF of slick oil (15.8 µg/L PAHs) as well as controls with one replicate (n = 10)

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to characterize developmental features. Given more pronounced morphological changes were

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observed at 48 hpf, a second treatment was carried out on larvae treated with the slick oil (4.74

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µg/L PAHs) and controls collected at 48 hpf from four replicate beakers.

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anesthetized using 250 mgL-1 of MS222 (buffered with 500 mgL-1 NaHCO3). Individuals were

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then mounted in left lateral view onto 3 % methylcellulose in a Petri dish for image collection.

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All images were collected using a Nikon SMZ800N microscope and Nikon Digital Sight DS U-3

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and associated software. Still frames were imported into Image J to quantify individual

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morphological endpoints, including brain area, eye area, iris area, pericardial area spine length

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and total body length.

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RNA sequencing and de novo assembly


Larvae were


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A detailed description of RNA isolation, cDNA library construction and sequencing is presented

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in Xu et al.3 Larvae collected in triplicate at 24, 48 and 72hpf were used for sequencing. Briefly,

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the 25 red drum larvae from each replicate were pooled and total RNA was isolated and purified

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with RNeasy Mini Kit (Qiagen, Valencia, California). The 200 ng of total RNA were used to

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prepare RNA-Seq libraries using the TruSeq RNA Sample Prep kit following the Illumina

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protocol (Illumina, San Diego, CA). Single read sequencing (1X50; 50 million reads per sample)

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was performed on an Illumina HiSeq 2500. Data were subjected to Illumina quality control (QC)

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procedures (>80% of the data yielded a Phred score of 30). The read data were deposited in the

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NCBI database (Accession Number: GSE90113). Adapter sequences were trimmed off from the

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raw sequences and filtered using Trimmomatic (version 0.33).17 Trinity (version 2.2.0) was used

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for de novo assembly.18 Trans-decoder (version 3.0.0) for coding sequence prediction, and

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Trinotate (2.0.2) for functional annotation. A detailed assembly and annotation protocol can be

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found at Xu et al.4

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Time-course transcriptomic analysis

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A time-course (24, 48 and 72 hpf) transcriptomic analysis was carried out on an OnRamp

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Bioinformatics Genomics Research Platform (OnRamp Bioinformatics, San Diego, CA).

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Basically, OnRamp mapped reads to the Fugu (Takifugu rubripes) transcriptome (FUGU4) using

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BLASTX, generated gene-level count data, and performed differential expression analysis with

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DEseq2. The protein FASTA sequences from Ensembl for Fugu were compared using Ensembl's

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homology to create an Entrez gene list that mapped via Fugu to red drum. A detailed description

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of these methods is presented in Xu et al.3 Statistical bioinformatics analysis was carried out


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using the sorted gene list by DAVID Bioinformatics Resources, Advaita Pathway Guide and

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Ingenuity Pathway Analysis (IPA, Qiagen, Valencia, CA). The gene lists were first examined on

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GO terms against Fugu reference using DAVID Bioinformatics Resources. The analysis was

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performed

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(GOTERM_CC_DIRECT)

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KEGG_PATHWAY using the functional annotation tool with a corrected Fisher exact p-value

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(EASE score) < 0.1. The rationale behind using Advaita and IPA is that this approach has been

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demonstrated to improve functional analysis of fish genes with a more sensitive systems level

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interrogation, by providing access to the best-annotated databases for human/mouse/rat

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models,19,20 while limitations of the mapping due to the extra genome duplication events in

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teleost fish and species differences in gene functions still exist. Biological process, microRNA

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and disease were enriched and predicted by Advaita. IPA was used to examine the toxicity

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pathways and reveal biological pathways/mechanisms underlying toxicity-specific phenotypes,

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as well as to provide insights into phenotype-specific biomarkers through linking DEGs to their

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known role in specific toxicity endpoints.4 Canonical pathways were further overlapped and

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clustered using IPA to understand how pathways may impact function, and whether there are any

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linkages among significantly disturbed pathways.

on

biological

process

(GOTERM_BP_DIRECT),

molecular

function

cellular

component

(GOTERM_MF_DIRECT)

and

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Results

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Chemical composition of weathered slick oil

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The weathered slick oil predominantly consisted of 3-ring (72%) and 4-ring (22%) PAHs (Fig.

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S1). The most abundant compounds were Phenanthrenes and Anthracenes, following by 9 ACS Paragon Plus Environment


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Dibenzothiophene. The profile is similar to those obtained from HEWAF preparations using

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other sources of slick oil from the DWH spill.3,5

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Morphological abnormalities from slick oil exposure

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Developmental stages of red drum are similar to those of many warm water marine fishes with

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early cell divisions essentially complete by 1.5 hours and an early embryo formed by 12 h. At a

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rearing temperature of 25°C, red drum embryos began hatching at 21 hpf, and most hatched at 24

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hpf. At 48 hpf, the liver, stomach and intestine were well-differentiated, and the eyes were

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pigmented. The mouth was functional and the yolk-sac was lost between 72 and 96 hpf. The

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growth in total length was minimal from 24 to 72 hpf, but significant growth in area of eye and

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brain during 24-72 hpf indicated the larvae were undergoing accelerated head development (see

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controls in Table S3). At 48 hpf, the slick oil treatment significantly increased pericardial area

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(45%), and decreased brain area (17%) and eye area (22%) in larvae compared to controls. The

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total body length was also significantly reduced by 12% (Table 1). The slick oil treatment

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increased pericardial area and decreased tectum area in larvae at 24, 48 and 72 hpf compared to

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controls, respectively (one replicate; Table S3). The slick oil treatment decreased the length of

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body and notochord at 24, 48 and 72 hpf, respectively, compared to time-matched controls.

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Notably, eyes were smaller in oil-treated larvae indicated by reduced lens diameter at 24, 48 and

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72 hpf (Table S3). The pupil diameter was also reduced at 48 and 72 hpf, and the effect was

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more pronounced at 48 hpf (Table S3).

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De novo assembly of red drum transcriptome and annotation


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A total of 2,168,925,036 Illumina HiSeq reads from red drum larvae were generated. After

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trimming the adapters, 181,576,332 bases were assembled with Trinity resulting in 257,434

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transcript contigs with an average length of 705 bp and an N50 of 1,071 bases. 30,950

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HMMER/PFAM protein domains (Pfam), 9,219 predicted transmembrane regions (TmHMM),

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34,080 non-supervised orthologous groups of genes (eggNOG), and 38,647 GO_blast were

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determined using Trinotate pipeline (Table S4). The final transcriptome assembly provided a

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valuable resource for wider and deeper genetic research on red drum.

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Transcriptional responses at 24 hpf

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In 24 hpf animals exposed to slick oil, 104 genes were significantly differentially expressed at an

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false discovery rate (FDR) < 0.4, with 78% up-regulated (Fig. S2a). The significant Gene

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Ontology (GO) terms (biological process, cellular component, molecular function) and Kyoto

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Encyclopedia of Genes and Genomes (KEGG) pathways were enriched by DAVID against Fugu

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background (Fig. S3). Biological processes and pathways changed by oil exposure included

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transcripts involved in fatty acid metabolism, lipid homeostasis, cell redox homeostasis, ECM-

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receptor interaction, focal adhesion, metabolic pathways (tyrosine, arachidonic acid, glutathione,

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carbon), and ubiquinone biosynthesis. For the ontology of molecular function, Flavin adenine

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dinucleotide binding and oxidoreductase activity were the predominant transcripts altered by oil

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exposure. Consistent with DAVID assessments, Advaita analysis revealed metabolic pathways,

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protein digestion, metabolism of xenobiotics by cytochrome P450, extracellular matrix (ECM)-

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receptor interaction, steroid biosynthesis, and focal adhesion as the most highly ranked bio-

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pathways (Table S5). IPA predicted significant activation of aryl hydrocarbon receptor (AhR)



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signaling, NRF2-mediated oxidative stress responses, fatty acid metabolism, hepatic fibrosis,

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xenobiotic metabolism, cholesterol biosynthesis, and transmembrane potential of mitochondria

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by slick oil at 24 hpf (Fig. 1).

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Compared to 24 hpf, the expression of more genes (769 genes) was significantly altered at 48 hpf

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after slick oil exposure. The enriched GO terms were generally different between 24 hpf and 48

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hpf after slick oil exposure, although some common terms were enriched at both 24 and 48 hpf,

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such as lipid homeostasis, proteinaceous extracellular matrix, collagen trimmer, mitochondrion,

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oxidoreductase activity, ECM-receptor interaction, and carbon metabolism. The most

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representative GO terms by slick oil exposure at 48 hpf included proteolysis and proteasome-

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related terms. Notably, some neuron-associated terms started to be highly enriched at this stage,

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such as axonogenesis, neural crest cell migration, neuron projection, and synapse, suggesting the

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disruption of nervous system development. Similar to DAVID, Advaita revealed proteasome,

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protein digestion, and metabolic pathways as the highest ranked bio-pathways. Several genes

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(e.g. rho, grm6, gnat1,gnat2, cnga3, cngb3, cacna1f) that were significantly differentially

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downregulated after slick oil exposure were linked to eye diseases, e.g. retinitis pigmentosa,

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congenital stationary night blindness, cataracts, and cone-rod dystrophy

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nervous system development and function (174 genes) was the highest ranked physiological

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phenotype at 48 hpf predicted by IPA. The highest ranked toxicity predictions included NRF-

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mediated oxidative stress response (ranked No.1; 26 genes; p-value = 1.65E-08), cardiac


(Table S5).

The

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hypertrophy, mitochondrial dysfunction, hepatic fibrosis, xenobiotic metabolism, AhR signaling,

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increased heart failure, liver proliferation, and PPARα/RXRα activation (Fig. 1).

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The number of significantly differentially expressed genes (FDR < 0.4) greatly increased from

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769 at 48 hpf to 2,564 genes at 72 hpf after slick oil exposure. Compared to 24 and 48 hpf, the

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significantly enriched GO terms were vastly different indicated by DAVID in 72 hpf after slick

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oil exposure. Protein translation, cytosolic ribosomal subunits, structural constituents of the

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ribosome, and ribosome were the most significantly enriched biological processes, cellular

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components, molecular functions and KEGG pathways in 72 hpf after slick oil exposure,

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respectively (Fig. S3). Ribosome and metabolic pathways were the highest ranked bio-pathways

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identified by Advaita in 72 hpf animals after slick oil exposure (Table S5). Comparing DAVID

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and Advaita Pathway Guide analyses, both approaches were consistent with regard to the

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enriched GO pathways identified, with ribosomal and metabolic pathways being the highest

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ranked pathways (Fig. 1). Advaita also predicted that other biological processes and molecular

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functions were altered, including phototransduction, steroid biosynthesis, retinol metabolism as

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well as the disease pathways, diamond-blackfan anemia (faulty ribosome biogenesis), retinitis

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pigmentosa, and cytochrome c oxidase deficiency. Notably, at 72 hpf after slick oil exposure,

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the genes involved in ribosomal pathway were all significantly downregulated. IPA analysis

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further predicted the activation of cardiac hypertrophy (ranked No.1; 95 genes; p-value = 1.49E-

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12), mitochondrial dysfunction, and xenobiotic metabolism, renal necrosis, TR/RXR activation,

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cardiac necrosis, FXR/RXR activation, AhR signaling, hypoxia-inducible factor signaling, liver



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proliferation, increases heart failure, cholesterol biosynthesis, and decreases in transmembrane

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potential of mitochondria by slick oil exposure (Fig. 1).

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Upstream analysis

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The upstream analysis by IPA was used to identify the activation state of upstream regulators

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that could explain the observed gene expression profile alterations in slick oil-treated red drum

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larvae at 24, 48 and 72 hpf. The predicted top activated or down-regulated regulators in red drum

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larvae treated with slick oil are shown in Fig. 2. More similar regulators were observed between

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48 and 72 hpf groups with fewer at 24 hpf. Nuclear factor nfe2l2 was predicted as the most

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relevant activated upstream regulator across all developmental stages. nfe2l2 regulates genes

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which contain antioxidant response elements (ARE) in their promoters, and many of these genes

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are known to encode proteins involved in cellular responses to oxidative stress, immune system

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processes, metabolism of xenobiotics, oxidoreductase activity and apoptosis. Examples include

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sqstm1, gsto1, prdx1, hspa9, creg1 and osgin1, which were all up-regulated in the present study.

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Another upstream regulator that was highly ranked was AhR. Its target genes, cyp1a1, ahrr,

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cyp3a4 were among the most upregulated genes in our dataset; a number of collagens that play

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an important role in maintaining the integrity of various tissues were significantly downregulated

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also via AhR activation. One mature microRNA was also predicted as a top regulator, miR-124,

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that targets ahrr mRNA in humans.21 A list of microRNAs was predicted based on differential

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expression of their target mRNA by Advaita (Table S5), and deserve further investigation.

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Discussion 14 ACS Paragon Plus Environment

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The global changes in gene expression in embryonic exposures using transcriptomic and

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bioinformatic tools can help target conventional morphological assessment in detecting

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subsequent phenotypic effects in organisms exposed to various stressors. Assessments with

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these tools may improve predictability, sensitivity and efficiency of toxicity assessments in

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embryonic fish and other species.

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opportunity to systematically identify most of the genes that are responsive in stressed embryos

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or larvae. Embryonic gene expression is a highly dynamic process with unique changes at each

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developmental stage. The patterns of gene expression across major developmental stages have

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been known to have lasting impacts on the development of principal physiological systems in

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vertebrate species.22-24 In particular, the embryo-to-larval stage is a crucial period in the life of

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fish, and is more sensitive to environmental stress than adult stages.25 Our previous study in

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mahi-mahi embryos and larvae using toxicogenomic tools demonstrated the toxicity of crude oil

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is time- and oil type-dependent.3 To our knowledge, the present study is the first to de novo

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assemble the transcriptome of red drum exposed to DWH crude oil. A list of phenotype-

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associated DEGs for red drum was identified at different developmental stages (Table S6). This

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DEG analysis illustrated similarities and differences in responses to DWH crude oil at different

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developmental times at individual gene levels.

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Consistent with studies for other fish species,2,3,13 our results also support evidence for the most

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dominant malformations observed being abnormal development of nervous and cardiovascular

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systems, which anchor changes in gene expression to predicted phenotypes by a suite of

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bioinformatic tools (i.e., DAVID, Advaita and IPA). This agrees well with the studies in normal

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developing fish species indicating that the primary differentially expressed functional groups

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during the embryo-to-larval transition are nervous, muscular, and cardiovascular system

The advent of HTS technology provides an unbiased



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development.25 Similar to mahi-mahi,3 the transcriptional responses in red drum induced by oil

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exposure is also time- dependent, and this could due to transcriptional changes associated with

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normal embryonic developmental processes.26

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PAHs have long been studied for their carcinogenic properties and cardiotoxicity, and more

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recent studies have highlighted the neurotoxicity of individual PAHs and mixtures. Prenatal

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exposure to benzo(a)pyrene (BaP) has been shown to induce neurological abnormalities like

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cognitive impairment, learning difficulties, and loss of short-term memory in human.27 BaP

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decreased

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neurodegeneration in zebrafish.28 Exposure to PAH mixtures impaired neurodifferentiation and

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caused corresponding neurobehavioral impairment in fish.16 A more recent study shows a

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complex PAH mixture (mostly naphthalene, phenanthrene, fluoranthene and acenapthene,

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similar to the composition of the slick oil in the present study) significantly impaired early-stage

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neurodevelopment, resulting in a profound drop (up to 30%) in the glia-to-neuron ratio in

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embryonic neural stem cells, which can affect brain homeostasis and function.29 In the present

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study, nervous system development and function was predicted as the top-ranked physiological

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phenotype in 48 hpf red drum exposed to slick oil, and included significant inhibition of synapse

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development (p=1.43E-6, z score = -2.907), quantity of nervous tissue (p = 8.05E-05, z score = -

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2.759), quantity of neurons (p = 1.56E-04; z score = -2.624), long term depression of synapse (p

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= 5.72E-05; z score = -2.2), and impairment of synaptic transmission of nervous tissue (p =

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1.44E-05, z sore = -2.138).

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signaling pathways in the nervous system as top-ranked pathways in red drum larvae after slick

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oil exposure at both 48 and 72 hpf (Fig. 3). These results are consistent with other studies in

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embryonic fish species that demonstrated nervous system development and function were the

brain

mass,

locomotor

activity,

dopaminergic

neurons

and

resulted

in

Predictions by IPA also included impairment of a number of


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most significantly enriched biological functions during normal embryo-to-larva transition.24,30 In

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our study, the AhR pathway remained the top-ranked toxicity pathway in all three stages of red

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drum larvae treated by slick oil, with the least significance (largest adjusted p-value) at 48 hpf

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(Fig. 1), but a direct linkage from AhR pathways to neural signaling pathway and neurotoxicity

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of slick was not identified in the present study. Activation of the AhR contributes to adverse

361

effects of PAHs on proliferation and migration of mouse but not human neural stem cells.31 The

362

molecular connections between AhR activation and neurotoxicity of oil is still unclear,

363

particularly considering PAHs with strong affinity to AhR were not effective in altering mouse

364

neural stem cells.32 Thus, it is likely that other mechanisms may contribute to the adverse effects

365

rather than just those mediated by the AhR. For example, the cholinergic system influences

366

cognition, anxiety, locomotion, and behavior by acting upon nicotinic acetylcholine receptors

367

(nAChRs), and diminishment of nAChRs can impair passive avoidance in mice.33 It has also

368

been established that the loss of nAChRs in the cerebral cortex and certain subcortical regions is

369

associated with brain dysfunction and degenerative diseases such as Alzheimer’s, Parkinson’s

370

and Lewy body diseases.34 We found three genes encoding nAChR subunits (chrna6, chrnb2 and

371

chrnd) that were all significantly downregulated in 48 hpf red drum larvae after slick oil

372

exposure (Fig. 4).

373

Cannabinoid receptor 1 (cnr1) is a G protein-coupled receptor located primarily in the central

374

and peripheral nervous systems. In medium-size spiny neurons from rat striatum, activation of

375

cnr1 is necessary for long term depression of excitatory synapses.35 In astroglial cells from

376

mouse brain, mutant mouse cnr1 knockout as well as Ca2+/ calmodulin dependent kinase camk2a

377

gene knockout decreases long term depression of synapses,36,37 and causes hyperactivity

378

behavior in mouse.38,39

In embryos of rockfish (Sebastiscus marmoratus), the decrease of



Page 18 of 37

379

camk2a mRNA expression might contribute to altered synaptic plasticity and neuronal survival

380

following exposure to the five-ring PAH, Benzo(a)pyrene.40 Neuronal signaling is also

381

diminished by glutamate ionotropic receptor gria1,41,42 and mutant gria1 gene knockouts

382

increased hyperactivity in mouse.43 The cnr1, camk2a, gria1and cholinergic receptor genes were

383

all significantly downregulated in 48 hpf red drum larvae after slick oil exposure (Fig. 4). This is

384

consistent with our previous study in mahi-mahi after slick oil exposure,3 suggesting

385

neurotoxicity of slick oil through depression of neurotrophin as well as changes in glutamate,

386

cholinergic, cannabinoid receptors and also Ca2+ homeostasis (Fig. 4). Since Ca2+ appears to also

387

play a significant role in cardiac toxicity, impairment of multiple targets may be responsible for a

388

number of phenotypic responses besides that of cardiotoxicity that may adversely affect

389

development at later life stages in red drum.

390

Although other targets may be possible, the present findings are consistent with previous studies

391

indicating that pericardial edema is a profound response in fish species after crude oil

392

exposure.3,7,11,13,44 The slick oil treatment significantly increased pericardial area in red drum

393

larvae at 24, 48 and 72 hpf compared to controls. Chemical blockade of IKr repolarizing

394

potassium currents and disruption of intra-cellular Ca2+ was suggested as the major initiating

395

events for cardiac defects in fish exposed to crude oil.12 A more recent study demonstrated a

396

transcriptional cascade that is tightly linked to defects in cardiomyocyte intracellular calcium

397

cycling and heart chamber growth through bmp10 in Atlantic haddock.14 However, mRNA of

398

bmp 10 was not changed in the current study in red drum, nor in previous studies with mahi-

399

mahi treated with slick oil that caused abnormal cardiac phenotypes. Some 4- and 6-ring PAHs

400

and oxygen-substituted PAHs can inappropriately activate the AhR in developing

401

cardiomyocytes, leading to primary defects in cardiac morphogenesis in fish species,45-47 and this


Page 19 of 37


402

form of cardiac toxicity is dependent on the AhR and is prevented by AhR gene knockdown.47,48

403

In contrast, the cardiotoxicity of weathered crude oil and single non-alkylated tricyclic PAHs

404

occurs without activation of the AhR in fish cardiomyocytes,49,50 and is not prevented by AhR

405

gene knockdown.51 Since the slick DHW oil is a complex mixture, multiple toxic mechanisms

406

were possibly involved in the cardiotoxicity, including both AhR-dependent and AhR-

407

independent pathways. Our data demonstrated several AhR-independent canonical pathways

408

associated with heart development and function were modified at the transcriptional level. For

409

example, IPA analysis predicted the significant activation of NFAT (nuclear factor of activated

410

T-cells) in cardiac hypertrophy (Rank #25 at 48 hpf and Rank #6 at 72 hpf; Fig. 3). Previous

411

studies have recognized the importance of Ca2+-sensitive signaling molecules, including

412

calcineurin, CalmK, and MAPK in hypertrophic pathways, in which NFAT plays a critical role,

413

being the best-characterized target for the development of cardiac hypertrophy.52 Prolonged

414

cardiac hypertrophy is associated with arrhythmia, sudden death, decompensation, and dilated

415

cardiomyopathy.53 The hypertrophic response is orchestrated by growth factors and cytokines

416

acting through several interdependent signaling cascades whose molecules include G-proteins

417

such GNAI1, GTPases such as SOS Ras, and kinases such as MAPK and CAMK, as well as

418

transcription factors HAND1, HDAC, which are essential for cardiac development and altered by

419

slick oil exposure (Fig. S4).

420

In addition to pericardial edema, impaired eye formation was the most noted indicator at 48 and

421

72 hpf. Forty-four significantly differentially expressed genes were identified in the pathway of

422

eye formation and retinal degeneration uniquely associated with the 48 hpf larvae after slick oil

423

exposure (Fig. S5).

424

oxidation producing numerous oxygenated PAHs.54 Eye malformation was induced in zebrafish

DWH slick oil has been weathered and undergone significant photo-



Page 20 of 37

425

embryos treated with oxygenated PAHs (such as 1,9-benz-10-anthrone ), and mRNAs associated

426

with visual perception were also altered.46,55 IPA predicted inhibition of phototransduction and

427

retinal degeneration by slick oil at 48 hpf (Fig. S5), and retinitis pigmentosa (RP) was the top

428

ranked disease by Advaita at both 48 and 72 hpf (Table S5). Photoreceptors respond to light by

429

the closure of a cyclic nucleotide-gated (CNG) channel (activated by binding of cGMP/cAMP),

430

causing hyperpolarization of the plasma membrane and decrease of the synaptic glutamate

431

release. CNG channels provide the only source for Ca2+ influx into rod and cone outer segments

432

for calcium, the lack of this channel would result in a decrease of cellular calcium

433

concentration.56,57 At low calcium concentrations, the membrane guanylate cyclase may be

434

stimulated permanently, leading to elevated levels of cGMP, which may trigger the degeneration

435

of photoreceptors.58 The absence of functional CNG channels is equivalent to the permanent

436

closure of channels which occur under continuous bright light conditions. Continuous exposure

437

of experimental animals to light has been shown to result in photoreceptor degeneration.59,60

438

Consistent with previous study in mahi-mahi, cyclic nucleotide-gated channel alpha 3 (cnga3),

439

retinoid isomerohydrolase (rpe65), guanylate cyclase 2D (gucy2d), transducin (gnat2), and their

440

upstream regulator orthodenticle homeobox 1 (otx1) were significantly downregulated, which

441

could lead to the perturbation of rhodopsin regeneration and phototransduction by slick oil (Fig.

442

S5). Similarly, both BaP and crude oil caused ocular toxicity and adverse affected visual-

443

associated behavior in fish species.61,62

444

exposure by disturbing 14 different pathways that interact with phototransduction pathways (Fig.

445

S6), including relaxin signaling, a-adrenergic signaling, CXCR4 signaling, CREB signaling in

446

neurons and androgen signaling through molecular connections such as G protein transducins

447

and guanine nucleotide binding protein (Fig. 4). The observed eye morphology would cause

Such ocular toxicity may be induced by slick oil


Page 21 of 37


448

severe disadvantages during fish life cycles by affecting feeding, competing, escaping, migration

449

responses and survival.

450

In conclusion, this is the first study to de novo assemble the transcriptome and investigate the

451

time-course transcriptomic responses in estuarine fish larvae exposed to DWH oil. Comparisons

452

of phenotypic responses with transcriptomic profiles were consistent with significantly

453

differentially expressed genes, enriched gene ontology, functions and canonical pathways mostly

454

involved in nervous and cardiovascular systems, both of which were validated at the phonotypic

455

level using morphometric analysis, and with more pronounced changes at later larval stages.

456

Overall, the present study has provided valuable molecular resources for expanding our

457

understanding of the developmental toxicity of DWH oil in fish. The developed de novo and

458

comparative toxicity pathway methods will also be useful for toxicogenomic study on other non-

459

model fish species. This study calls for follow-up studies to confirm the initial transcriptomic

460

results with additional methods (e.g. in situ expression in specific organ and tissue, dose-

461

response assays) at an expanded range of oil concentrations, and to assess the later life

462

consequences of larval exposure at the population level.

463 464

Supporting Information

465

Further information is available that provides PAH measurements (Fig. S1; Table S2), water

466

quality measurements (Table S1), plots showing relative expression of genes (Fig. S2),

467

significantly enriched gene ontology (GO) terms and KEGG pathways, diseases and miRNAs

468

(Fig. S3; Table S5), morphological measurements (Table S3), statistics of transcriptome

469

assembly and annotation (Table S4), plot showing the role of NFAT in cardiac hypertrophy (Fig.

470

S4), inhibition of eye formation and induced retinal degeneration ribosome biosynthesis (Fig. S5), 21 ACS Paragon Plus Environment


Page 22 of 37

471

overlapping canonical pathways (Fig. S6), the top predicted canonical pathways with

472

corresponding genes (Table S6).

473 474

Acknowledgments

475

This research was made possible by a grant from The Gulf of Mexico Research Initiative. Grant

476

No: SA-1520; Name: Relationship of Effects of Cardiac Outcomes in fish for Validation of

477

Ecological Risk (RECOVER). The authors are grateful to the Gulf of Mexico Research Initiative

478

Information and Data Cooperative (GRIIDC) for supporting data management system to store

479

the data generated ( doi:10.7266/N7S180KJ). G. Hardiman acknowledges Medical University of

480

South Carolina College of Medicine start-up funds. This research was supported in part by the

481

Genomics Shared Resource, Hollings Cancer Center.

482

483

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Figure legends

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Fig. 1 Toxicity predictions determined from differentially expressed genes between controls

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versus slick oil-treated red drum larvae for 24, 48 and 72 hpf. The left Y-axis indicates the –log

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(p-value; red dots in upper line) determined by Fisher's exact test, and the right Y-axis indicates

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the ratio (lower line), which is calculated as the number of genes found in each given pathway

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divided by the total number of genes in that pathway.

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Fig. 2 Activated and inhibited upstream regulators in slick oil treated red drum larvae predicted

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by Upstream Analysis in IPA. The prediction of activation is based on the global direction of

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changes of the modulated genes. The activation Z-score indicates whether the observed gene

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responses to upstream regulators agree with expected changes derived from the literature that

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accrued in IPA database, was used to predict the activation state. Z-scores ≥ 2 or ≤ − 2 indicates

690

that the upstream regulator was predicted to be activated or inhibited, respectively. A Fisher’s

691

Exact Test was used to determine the significance of the overlap between the regulator and the

692

responsive genes.

693

Fig. 3 Canonical Pathways for 48 hpf (a) and 72 hpf (b). The y-axis displays the -log of p-value

694

which is calculated by Fisher's exact test right-tailed. The numbers on the right indicate the

695

number of genes affiliated with that pathway. Red bar, number of up-regulated genes; green bar:

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down-regulated; gray bar, unchanged genes.

697

Fig. 4 Predicted mechanisms through Ingenuity Pathway Analysis showing how slick oil may

698

lead to the suppression of long term depression of synaptic transmission, decreased quantity of

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nervous tissue, and hyperactive behavior.

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Fig. 1 33 ACS Paragon Plus Environment


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Fig. 2

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Fig. 3

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709

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Fig. 4

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Table 1 Morphological measurements: brain area, eye area, pericardial area, Iris area, spine and

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total body length in control and oil treated red drum larvae at 48 hpf. Data were analyzed by t-

716

test and a two tailed p- value indicates the significant effect of slick oil exposure at p < 0.05 (N =

717

4). Morphological

Control

Slick oil

p-value

Brain Area (µm2)

15057 ± 429

12556 ± 828

0.029

Eye Area (µm2)

13138 ± 282

10296 ± 788

0.001

Iris Area (µm2)

1703 ± 63

1370 ± 100

0.090

Pericardial Area (µm2)

6444 ± 464

9343 ± 1200

Larval Red Drum (Sciaenops ocellatus) Sublethal Exposure to

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