Effects of polycyclic aromatic hydrocarbons and abiotic stressors on Fundulus grandis cardiac transcriptomics.

Following the 2010 Deepwater Horizon oil spill, extensive research has been conducted on the toxicity of oil and polycyclic aromatic hydrocarbons (PAHs) in the aquatic environment. Many studies have identified the toxicological effects of PAHs in estuarine and marine fishes, however, only recently has work begun to identify the combinatorial effect of PAHs and abiotic environmental factors such as hypoxia, salinity, and temperature. This study aims to characterize the combined effects of abiotic stressors and PAH exposure on the cardiac transcriptomes of developing Fundulus grandis larvae. In this study, F. grandis larvae were exposed to varying environmental conditions (dissolved oxygen (DO) 2, 6 ppm; temperature 20, 30 °C; and salinity 3, 30 ppt) as well as to a single concentration of high energy water accommodated fraction (HEWAF) (∑PAHs 15 ppb). Whole larvae were sampled for RNA and transcriptional changes were quantified using RNA-Seq followed by qPCR for a set of target genes. Analysis revealed that exposure to oil and abiotic stressors impacts signaling pathways associated with cardiovascular function. Specifically, combined exposures appear to reduce development of the systemic vasculature as well as strongly impact the cardiac musculature through cardiomyocyte proliferation resulting in inhibited cardiac function and modulated blood pressure maintenance. Results of this study provide a holistic view of impacts of PAHs and common environmental stressors on the cardiac system in early life stage estuarine species. To our knowledge, this study is one of the first to simultaneously manipulate oil exposure with abiotic factors (DO, salinity, temperature) and the first to analyze cardiac transcriptional responses under these co-exposures.

Age-related central regulation of orexin and NPY in the short-lived African killifish Nothobranchius furzeri.

Orexin A (OXA) and neuropeptide Y (NPY) are two hypothalamic neuropeptides involved in the regulation of feeding behavior and food intake in all vertebrates. Accumulating evidences document that they undergo age-related modifications, with consequences on metabolism, sleep/wake disorders and progression of neurodegenerations. The present study addressed the age related changes in expression and distribution of orexin A (its precursor is also known as hypocretin-HCRT) and NPY, and their regulation by food intake in the short-lived vertebrate model Nothobranchius furzeri. Our experiments, conducted on male specimens, show that: (a) HCRT and OXA and NPY mRNA and protein are localized in neurons of diencephalon and optic tectum, as well as in numerous fibers projecting through the entire neuroaxis, and are colocalized in specific nuclei; (b) in course of aging, HCRT and NPY expressing neurons are localized also in telencephalon and rhombencephalon; (c) HCRT expressing neurons increased slightly in the diencephalic area of old animals and in fasted animals, whereas NPY increased sharply; (d) central HCRT levels are not regulated neither in course of aging nor by food intake; and (e) central NPY levels are augmented in course of aging, and regulated by food intake only in young. These findings represent a great novelty in the study of central orexinergic and NPY-ergic systems in vertebrates', demonstrating an uncommon and unprecedented described regulation of these two orexigenic neuropeptides.

Combined effects of Deepwater Horizon crude oil and environmental stressors on Fundulus grandis embryos.

In the present study, we examined how sensitivity to oil changes in combination with environmental stressors in Fundulus grandis embryos. We exposed embryos (<24 h post fertilization) to a range of high-energy water accommodated fraction (HEWAF) concentrations (0-50 parts per billion [ppb] total polycyclic aromatic hydrocarbons [PAHs]) made from Macondo crude oil in conjunction with various environmental conditions (temperature: 20 and 30 °C; salinity: 3, 7, and 30 practical salinity units [PSU]; and dissolved oxygen: 2 and 6 mg/L). Endpoints included mortality, hatching rates, and expression of cytochrome p450 1a and 1c (cyp1a, cyp1c) in hatched larvae. There was 100% mortality for all fish under the 2 parts per million (ppm) dissolved oxygen regimes. For the 6 mg/L dissolved oxygen treatments, mortality and median lethal time (LT50) were generally higher in the 30 °C treatments versus the 20 °C treatments. Oil increased mortality in fish exposed to the highest concentration in the 20-3-6 (°C-PSU-mg/L), 25-7-6, and 30-30-6 conditions. Hatching was driven by environmental conditions, with oil exposure having a significant impact on hatching in only the 25-7-6 and 30-30-6 groups at the greatest HEWAF exposure. Expression of cyp1a was up-regulated in most treatment groups versus the controls, with cyp1c expression exhibiting a similar pattern. These data suggest interactive effects among temperature, salinity, and PAHs, highlighting a need to further assess the effects of oil exposure under various environmental conditions. Environ Toxicol Chem 2018;37:1916-1925. © 2018 SETAC.

Antifungal and Ichthyotoxic Sesquiterpenoids from Santalum album Heartwood.

In our continuing study on a survey of biologically active natural products from heartwood of Santalum album (Southwest Indian origin), we newly found potent fish toxic activity of an n-hexane soluble extract upon primary screening using killifish (medaka) and characterized α-santalol and ß-santalol as the active components. The toxicity (median tolerance limit (TLm) after 24 h at 1.9 ppm) of α-santalol was comparable with that of a positive control, inulavosin (TLm after 24 h at 1.3 ppm). These fish toxic compounds including inulavosin were also found to show a significant antifungal effect against a dermatophytic fungus, Trichophyton rubrum. Based on a similarity of the morphological change of the immobilized Trichophyton hyphae in scanning electron micrographs between treatments with α-santalol and griseofulvin (used as the positive control), inhibitory effect of α-santalol on mitosis (the antifungal mechanism proposed for griseofulvin) was assessed using sea urchin embryos. As a result, α-santalol was revealed to be a potent antimitotic agent induced by interference with microtubule assembly. These data suggested that α-santalol or sandalwood oil would be promising to further practically investigate as therapeutic agent for cancers as well as fungal skin infections.

Genomic and genotoxic responses to controlled weathered-oil exposures confirm and extend field studies on impacts of the Deepwater Horizon oil spill on native killifish.

To understand the ecotoxicological impacts of the Deepwater Horizon oil spill, field studies provide a context for ecological realism but laboratory-based studies offer power for connecting biological effects with specific causes. As a complement to field studies, we characterized genome-wide gene expression responses of Gulf killifish (Fundulus grandis) to oil-contaminated waters in controlled laboratory exposures. Transcriptional responses to the highest concentrations of oiled water in the laboratory were predictive of field-observed responses that coincided with the timing and location of major oiling. The transcriptional response to the low concentration (∼ 10-fold lower than the high concentration) was distinct from the high concentration and was not predictive of major oiling in the field. The high concentration response was characterized by activation of the molecular signaling pathway that facilitates oil metabolism and oil toxicity. The high concentration also induced DNA damage. The low concentration invoked expression of genes that may support a compensatory response, including genes associated with regulation of transcription, cell cycle progression, RNA processing, DNA damage, and apoptosis. We conclude that the gene expression response detected in the field was a robust indicator of exposure to the toxic components of contaminating oil, that animals in the field were exposed to relatively high concentrations that are especially damaging to early life stages, and that such exposures can damage DNA.

Oxidative stress responses of gulf killifish exposed to hydrocarbons from the Deepwater Horizon oil spill: Potential implications for aquatic food resources.

Ecosystem effects of polycyclic aromatic hydrocarbons (PAHs) remain under investigation following the Gulf of Mexico Deepwater Horizon oil spill. Fundulus grandis, an established indicator of aquatic ecosystem health, was investigated because this species shares genes and biochemical pathways with higher trophic-level fish and plays an important role in the gulf food chain. Oxidative stress responses including hepatic cytochrome P4501A (CYP1A) and serum antioxidant capacity were evaluated in fish exposed to PAHs. Fish were exposed to water-accommodated fractions (WAFs) of crude oil (7.0 ± 0.10 mg/L C6-C28) after which solutions were diluted below the level of detection over 8 h using 15 ppt aerated artificial seawater. Before euthanasia, fish remained in aquaria for 12 h, 24 h, or 48 h. Three replicate experiments were conducted at each time point using unexposed fish as experimental controls. Significant differences (p < 0.05) in CYP1A induction were observed in exposed versus control fish at 24 h. Expression of CYP1A increased by 25%, 66%, and 23% in exposed fish at 12 h, 24 h, and 48 h, respectively. Significant increases were observed in antioxidant capacity of nonenzymatic antioxidants in exposed versus control fish at each time point. Given the activity of CYP1A, radicals formed during PAH detoxification likely resulted in increased oxidant load requiring elevated antioxidant defenses. Research is needed to determine the duration of oxidative stress responses considering the potential for lipid oxidation in exposed fish or species feeding on exposed fish.

Multitissue molecular, genomic, and developmental effects of the Deepwater Horizon oil spill on resident Gulf killifish (Fundulus grandis).

The Deepwater Horizon oil rig disaster resulted in crude oil contamination along the Gulf coast in sensitive estuaries. Toxicity from exposure to crude oil can affect populations of fish that live or breed in oiled habitats as seen following the Exxon Valdez oil spill. In an ongoing study of the effects of Deepwater Horizon crude oil on fish, Gulf killifish ( Fundulus grandis ) were collected from an oiled site (Grande Terre, LA) and two reference locations (coastal MS and AL) and monitored for measures of exposure to crude oil. Killifish collected from Grande Terre had divergent gene expression in the liver and gill tissue coincident with the arrival of contaminating oil and up-regulation of cytochrome P4501A (CYP1A) protein in gill, liver, intestine, and head kidney for over one year following peak landfall of oil (August 2011) compared to fish collected from reference sites. Furthermore, laboratory exposures of Gulf killifish embryos to field-collected sediments from Grande Terre and Barataria Bay, LA, also resulted in increased CYP1A and developmental abnormalities when exposed to sediments collected from oiled sites compared to exposure to sediments collected from a reference site. These data are predictive of population-level impacts in fish exposed to sediments from oiled locations along the Gulf of Mexico coast.

Effects of potassium ion supplementation on survival and ion regulation in Gulf killifish Fundulus grandis larvae reared in ion deficient saline waters.

Teleost fish often live in an environment in which osmoregulatory mechanisms are critical for survival and largely unknown in larval fish. The effects of a single important marine ion (K(+)) on survival and ion regulation of larval Gulf killifish, an estuarine, euryhaline teleost, were determined. A four-week study was completed in four separate recirculating systems with newly hatched larvae. Salinity in all four systems was maintained between 9.5 and 10‰. Two systems were maintained using crystal salt (99.6% NaCl) with K(+) supplementation (1.31±0.04mmol/L and 2.06±0.04mmol/L K(+); mean±SEM), one was maintained with crystal salt and no K(+) supplementation (0.33±0.05mmol/L K(+)), the fourth system was maintained using a standard marine mix salt (2.96±0.04mmol/L K(+)), the salt mix also included standard ranges of other ions such as calcium and magnesium. Larvae were sampled throughout the experiment for dry mass, Na(+)/K(+)-ATPase (NKA) activity, whole body ion composition, relative gene expression (NKA, Na(+)/K(+)/2Cl(-) cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR)), and immunocytochemistry staining for NKA, NKCC, and CFTR. Larvae stocked into water with no K(+) supplementation resulted in 100% mortality within 24h. Mortality and dry mass were significantly influenced by K(+) concentration (P≤0.05). No differences were observed among treatment groups for NKA activity. At 1dph NKA mRNA expression was higher in the 0.3mmol [K(+)] group than in other treatment groups and at 7dph differences in intestinal NKA and CFTR staining were observed. These data indicate that the rearing of larval Gulf killifish may be possible in ion deficient water utilizing specific ion supplementation.