Connecting gut microbiome changes with fish health conditions in juvenile Atlantic cod (Gadus morhua) exposed to dispersed crude oil.

Polycyclic aromatic hydrocarbons found in crude oil can impair fish health following sublethal exposure. However, the dysbiosis of microbial communities within the fish host and influence it has on the toxic response of fish following exposure has been less characterized, particularly in marine species. To better understand the effect of dispersed crude oil (DCO) on juvenile Atlantic cod (Gadus morhua) microbiota composition and potential targets of exposure within the gut, fish were exposed to 0.05 ppm DCO for 1, 3, 7, or 28 days and 16 S metagenomic and metatranscriptomic sequencing on the gut and RNA sequencing on intestinal content were conducted. In addition to assessing species composition, richness, and diversity from microbial gut community analysis and transcriptomic profiling, the functional capacity of the microbiome was determined. Mycoplasma and Aliivibrio were the two most abundant genera after DCO exposure and Photobacterium the most abundant genus in controls, after 28 days. Metagenomic profiles were only significantly different between treatments after a 28-day exposure. The top identified pathways were involved in energy and the biosynthesis of carbohydrates, fatty acids, amino acids, and cellular structure. Biological processes following fish transcriptomic profiling shared common pathways with microbial functional annotations such as energy, translation, amide biosynthetic process, and proteolysis. There were 58 differently expressed genes determined from metatranscriptomic profiling after 7 days of exposure. Predicted pathways that were altered included those involved in translation, signal transduction, and Wnt signaling. EIF2 signaling was consistently dysregulated following exposure to DCO, regardless of exposure duration, with impairments in IL-22 signaling and spermine and spermidine biosynthesis in fish after 28 days. Data were consistent with predictions of a potentially reduced immune response related to gastrointestinal disease. Herein, transcriptomic-level responses helped explain the relevance of differences in gut microbial communities in fish following DCO exposure.

Transcriptomic profiling of miR-203a inhibitor and miR-34b-injected zebrafish (Danio rerio) validates oil-induced neurological, cardiovascular and eye toxicity response pathways.

The global sequencing of microRNA (miRNA; miR) and integration to downstream mRNA expression profiles in early life stages (ELS) of fish following exposure to crude oil determined consistently dysregulated miRNAs regardless of the oil source or fish species. The overlay of differentially expressed miRNAs and mRNAs into in silico software determined that the key roles of these miRNAs were predicted to be involved in cardiovascular, neurological and visually-mediated pathways. Of these, altered expression of miRNAs, miR-203a and miR-34b were predicted to be primary targets of crude oil. To better characterize the effect of these miRNAs to downstream transcript changes, zebrafish embryos were microinjected at 1 h post fertilization (hpf) with either a miR-203a inhibitor or miR-34b. Since both miRs have been shown to be associated with aryl hydrocarbon receptor (AhR) function, benzo(a)pyrene (BaP), a potent AhR agonist, was used as a potential positive control. Transcriptomic profiling was conducted on injected and exposed larvae at 7 and 72 hpf, and eye morphology assessed following exposure at 72 hpf. The top predicted physiological system disease and functions between differentially expressed genes (DEGs) shared with miR-203a inhibitor-injected and miR-34b-injected embryos were involved in brain formation, and the development of the central nervous system and neurons. When DEGs of miR-203a inhibitor-injected embryos were compared with BaP-exposed DEGs, alterations in nervous system development and function, and abnormal morphology of the neurosensory retina, eye and nervous tissue were predicted, consistent with both AhR and non-AhR pathways. When assessed morphologically, the eye area of miR-203a inhibitor and miR-34b-injected and BaP-exposed embryos were significantly reduced. These results suggest that miR-203a inhibition and miR-34b overexpression contribute to neurological, cardiovascular and eye toxicity responses that are caused by oil and PAH exposure in ELS fish, and are likely mediated through both AhR and non-AhR pathways.

Transcriptomic responses and apoptosis in larval red drum (Sciaenops ocellatus) co-exposed to crude oil and ultraviolet (UV) radiation.

Ultraviolet (UV) radiation can significantly increase the toxicity of polycyclic aromatic hydrocarbons (PAHs) in crude oil to early life stage (ELS) fishes through photo-induced /photo-enhanced toxicity. However, little is known about the sub-lethal effects and mechanisms of photo-induced PAH toxicity in ELS fishes. The present study investigated apoptosis and global transcriptomic effects in larval red drum (Sciaenops ocellatus) (24-72 h post-fertilization) following co-exposure to oil (0.29-0.30 µg/L ∑PAH50) and UV. Apoptosis was quantified using the TUNEL assay, and transcriptomic effects were assessed using RNA sequencing analysis. Apoptotic fluorescence was greatest in the eyes and skin following 24 and 48 h co-exposure to oil and UV, indicating photo-induced toxicity. Consistent with these phenotypic responses, pathways associated with phototransduction, eye development, and dermatological disease were among the top predicted pathways impacted. The present study is the first to provide global transcriptomic analysis of UV and oil co-exposure in an ELS fish.

Antibiotic Chlortetracycline Causes Transgenerational Immunosuppression via NF-κB.

The extensive and increasing global use of antibiotics results in the ubiquitous presence of antibiotics in the environment, which has made them "pseudo persistent organic contaminants." Despite numerous studies showing wide adverse effects of antibiotics on organisms, the chronic environmental risk of their exposure is unknown, and the molecular and cellular mechanisms of antibiotic toxicity remain unclear. Here, we systematically quantified transgenerational immune disturbances after chronic parental exposure to environmental levels of a common antibiotic, chlortetracycline (CTC), using zebrafish as a model. CTC strongly reduced the antibacterial activities of fish offspring by transgenerational immunosuppression. Both innate and adaptive immunities of the offspring were suppressed, showing significant perturbation of macrophages and neutrophils, expression of immune-related genes, and other immune functions. Moreover, these CTC-induced immune effects were either prevented or alleviated by the supplementation with PDTC, an antagonist of nuclear factor-κB (NF-κB), uncovering a seminal role of NF-κB in CTC immunotoxicity. Our results provide the evidence in fish that CTC at environmentally relevant concentrations can be transmitted over multiple generations and weaken the immune defense of offspring, raising concerns on the population hazards and ecological risk of antibiotics in the natural environment.

Exposure to Deepwater Horizon crude oil increases free cholesterol in larval red drum (Sciaenops ocellatus).

The 2010 Deepwater Horizon oil spill impacted over 2100 km of shoreline along the northern Gulf of Mexico, which coincided with the spawning season of many coastal species, including red drum (Sciaenops ocellatus). Red drum develop rapidly and are sensitive to crude oil exposure during the embryonic and larval periods. This study investigates the predictions from recent transcriptomic studies that cholesterol biosynthetic processes are impacted by oil exposure in fish early life stages. We found that red drum larvae exposed for 72 h to ΣPAH50 3.55-15.45 µg L-1 exhibited significantly increased pericardial area, a cardiotoxicity metric, but the expression of several genes targeted in the cholesterol synthesis pathway was not affected. However, whole-mount staining revealed significant increases in free cholesterol throughout the larval body (ΣPAH50 4.71-16.15 µg L-1), and total cholesterol followed an increasing trend (ΣPAH50 3.55-15.45 µg L-1). Cholesterol plays a critical role in fish embryo development and ion channel function. Therefore, the disruption of cholesterol homeostasis, as observed here, could play a role in the oil toxicity phenotype observed across many fish species.

Exposure to Crude Oil Induces Retinal Apoptosis and Impairs Visual Function in Fish.

Polycyclic aromatic hydrocarbons (PAHs) present in crude oil are known to impair visual development in fish. However, the underlying mechanism of PAH-induced toxicity to the visual system of fish is not understood. Embryonic zebrafish (Danio rerio) at 4 h post fertilization were exposed to weathered crude oil and assessed for visual function using an optokinetic response, with subsequent samples taken for immunohistochemistry and gene expression analysis. Cardiotoxicity was also assessed by measuring the heart rate, stroke volume, and cardiac output, as cardiac performance has been proposed to be a contributing factor to eye-associated malformations following oil exposure. Larvae exposed to the highest concentrations of crude oil (89.8 µg/L) exhibited an increased occurrence of bradycardia, though no changes in stroke volume or cardiac output were observed. However, genes important in eye development and phototransduction were downregulated in oil-exposed larvae, with an increased occurrence of cellular apoptosis, reduced neuronal connection, and reduced optokinetic behavioral response in zebrafish larvae.

Changes in microRNA-mRNA Signatures Agree with Morphological, Physiological, and Behavioral Changes in Larval Mahi-Mahi Treated with Deepwater Horizon Oil.

In this study, we performed a systematic evaluation of global microRNA-mRNA interactions associated with the developmental toxicity of Deepwater Horizon oil using a combination of integrated mRNA and microRNA deep sequencing, expression profiling, gene ontology enrichment, and functional predictions by a series of advanced bioinformatic tools. After exposure to water accommodated fraction (WAF) of both weathered slick oil (0.5%, 1%, and 2%) and source oil (0.125%, 0.25%, and 0.5%) from the Deep Water Horizon oil spill, four dose-dependent miRNAs were identified, including three up-regulated (miR-23b, miR-34b, and miR-181b) and one down-regulated miRNAs (miR-203a) in mahi-mahi hatchings exposed from 6 h postfertilization (hpf) to 48 hpf. Consistent with morphological, physiological, and behavioral changes, the target genes of these miRNAs were largely involved in the development of the cardiovascular, visual, nervous system and associated toxicity pathways, suggesting that miRNAs play an essential role in regulating the responses to oil exposure. The results obtained from this study improve our understanding of the role of miRNAs and their target genes in relation to dose-dependent oil toxicity and provide the potential of using miRNAs as novel biomarkers in future oil studies.

Effects of Deepwater Horizon crude oil on ocular development in two estuarine fish species, red drum (Sciaenops ocellatus) and sheepshead minnow (Cyprinodon variegatus).

Polycyclic aromatic hydrocarbons (PAHs) present in crude oil have been shown to cause the dysregulation of genes important in eye development and function, as well as morphological abnormalities of the eye. However, it is not currently understood how these changes in gene expression are manifested as deficits in visual function. Embryonic red drum (Sciaenops ocellatus) and sheepshead minnow (Cyprinodon variegatus) were exposed to water accommodated fractions (WAFs) of weathered crude oil and assessed for visual function using an optomotor response assay in early life-stage larvae, with subsequent samples taken for histological analysis of the eyes. Larvae of both species exposed to increasing concentrations of oil exhibited a reduced optomotor response. The mean diameters of retinal layers, which play an important role in visual function and image processing, were significantly reduced in oil-exposed sheepshead larvae, though not in red drum larvae. The present study provides evidence that weathered crude oil has a significant effect on visual function in early life-stage fishes.