The impact of salinity and dissolved oxygen regimes on transcriptomic immune responses to oil in early life stage Fundulus grandis.

Understanding the effects of oil exposure on early life stage fish species is critical to fully assessing the environmental impacts of oil spills. Oil released from the 2010 Deepwater Horizon spill reached habitats where estuarine fish routinely spawn. In addition, estuaries are highly dynamic environments, therefore, fish in these areas are routinely exposed to varying salinity and dissolved oxygen (DO) levels, each of which are known to modulate transcriptional responses. Fish exposed to oil often display altered immune competence, and several studies have shown that Deepwater Horizon oil in particular causes modulation of various immune functions. However, few studies have directly examined how environmental parameters may affect oil-induced immunomodulation, particularly in early life stage fishes when the immune system is still developing. To this end, we examined transcriptional patterns of immune genes and pathways in Fundulus grandis larvae to various oil (0, 15 µg/L), salinity (3, 30 ppt), and DO (2.5, 6 mg/L) regimes in a fully factorial design. Our results suggest that immune pathways are generally activated in all treatment groups with the exception of the Low Salinity/No Oil/Hypoxia treatment where immune pathways are largely suppressed, and the High Salinity/No Oil/Hypoxia treatment where pathways are unchanged. The High Salinity/Oil/Hypoxia treatment had the largest number of enriched immune pathways (44 as defined by IPA and 43 as defined by ConsensusPathDB), indicating that oil under certain environmental conditions has the potential to further modulate immune-related genes, pathways, and responses in fish.

Combined effects of salinity, temperature, hypoxia, and Deepwater Horizon oil on Fundulus grandis larvae.

Oil spills have polluted the marine environment for decades and continue to be a major source of polycyclic aromatic hydrocarbons (PAHs) to marine ecosystems around the globe, for example during the 2010 Deepwater Horizon spill. Although the toxicity of PAHs to fish has been well studied, their effects combined with abiotic stressors are poorly understood. The goal of this study was to describe the combined impacts of crude oil and environmental stressors on fish larvae, a sensitive life stage. Gulf killifish (Fundulus grandis) larvae (<24 h post-hatch) were exposed for 48 h to high energy water accommodated fractions (HEWAF; total PAHs 0-125 ppb) of Macondo oil from the Deepwater Horizon spill under different combinations of environmental conditions (dissolved oxygen 2, 6 ppm; temperature 20, 25, 30 °C; salinity 3, 10, 30 ppt). Even under optimal environmental conditions (25 °C, 10 ppt, 6 ppm) larval survival and development were negatively affected by PAHs, starting with the lowest concentration tested (∼15 ppb). Hypoxia and high temperature each increased the adverse effects of HEWAF on development and mortality. In contrast, salinity had little effect on any of the endpoints measured. Importantly, expression of the detoxifying gene cyp1a was highly induced in PAH-exposed larvae under normoxic conditions, but not under hypoxic conditions, potentially explaining the enhanced toxicity observed under hypoxia. This work highlights the importance of considering how suboptimal environmental conditions can exacerbate the effects of pollution on fish early life stages.

Combined effects of cadmium exposure and temperature on the annual killifish (Nothobranchius furzeri).

Freshwater organisms are increasingly exposed to combinations of stressors. However, because it is time-consuming and costly, research on the interaction of stressors, such as compound toxicity and global warming on vertebrates, is scarce. Studies on multigenerational effects of these combined stressors are almost nonexistent. In the present study, we tested the combined effects of 4 °C warming and cadmium (Cd) exposure on life-history traits, biomarkers, bioaccumulation, and multigenerational tolerance in the turquoise killifish, Nothobranchius furzeri. The extremely short life cycle of this vertebrate model allows for assessment of sublethal and multigenerational effects within 4 mo. The applied Cd concentrations had only limited effects on the measured endpoints, which suggests that N. furzeri is more resistant to Cd than fathead minnow and rainbow trout. In contrast, the temperature increase of 4 °C was stressful: it delayed female maturation and lowered adult mass and fecundity. Finally, indications of synergistic effects were found on peak fecundity and embryonic survival. Overall, these results indicate the importance of studying chronic and multigenerational effects of combined stressors. Environ Toxicol Chem 2018;37:2361-2371. © 2018 SETAC.

Assessment of gonadal and thyroid histology in Gulf killifish (Fundulus grandis) from Barataria Bay Louisiana one year after the Deepwater Horizon oil spill.

We examined gonads and thyroid glands of Gulf killifish (Fundulus grandis) 1yr after the Deepwater Horizon oil spill. F. grandis were trapped from two impacted sites in Barataria Bay (Bayou St. Denis, Bay Jimmy) and an un-impacted site in East Texas (Sabine Pass). The greatest number of F. grandis were collected at Sabine Pass. F. grandis collected at Bayou St. Denis were smaller and had smaller Fulton condition factor scores than fish collected at Sabine Pass. Sex ratios were biased roughly 2:1 in favor of females at Sabine Pass and Bayou St. Denis. Gonad-somatic index (GSI) in males from Sabine Pass was double that of fish from Bay Jimmy while germinal epithelium thickness of the testes was 2.7 fold smaller in males from the impacted site. GSI and oocyte diameters in females from Bayou St. Denis were significantly smaller than females from Bay Jimmy or the reference site. There were no differences in thyroid follicle cell height. While total polyaromatic hydrocarbons at the impacted sites were no different from the reference site, the impacted sites did have greater concentrations of benzo[a]pyrene in sediment pore water. The finding of smaller GSI and testicular germinal epithelium in males from an impacted site suggest that exposure to a combination of oil and dispersants may adversely impact testicular function.

Transcriptomic alterations during ageing reflect the shift from cancer to degenerative diseases in the elderly.

Disease epidemiology during ageing shows a transition from cancer to degenerative chronic disorders as dominant contributors to mortality in the old. Nevertheless, it has remained unclear to what extent molecular signatures of ageing reflect this phenomenon. Here we report on the identification of a conserved transcriptomic signature of ageing based on gene expression data from four vertebrate species across four tissues. We find that ageing-associated transcriptomic changes follow trajectories similar to the transcriptional alterations observed in degenerative ageing diseases but are in opposite direction to the transcriptomic alterations observed in cancer. We confirm the existence of a similar antagonism on the genomic level, where a majority of shared risk alleles which increase the risk of cancer decrease the risk of chronic degenerative disorders and vice versa. These results reveal a fundamental trade-off between cancer and degenerative ageing diseases that sheds light on the pronounced shift in their epidemiology during ageing.

The P-type ATPase inhibiting potential of polyoxotungstates.

Polyoxometalates (POMs) are transition metal complexes that exhibit a broad diversity of structures and properties rendering them promising for biological purposes. POMs are able to inhibit a series of biologically important enzymes, including phosphatases, and thus are able to affect many biochemical processes. In the present study, we analyzed and compared the inhibitory effects of nine different polyoxotungstates (POTs) on two P-type ATPases, Ca2+-ATPase from skeletal muscle and Na+/K+-ATPase from basal membrane of skin epithelia. For Ca2+-ATPase inhibition, an in vitro study was performed and the strongest inhibitors were determined to be the large heteropolytungstate K9(C2H8N)5[H10Se2W29O103] (Se2W29) and the Dawson-type POT K6[α-P2W18O62] (P2W18) exhibiting IC50 values of 0.3 and 0.6 µM, respectively. Promising results were also shown for the Keggin-based POTs K6H2[CoW11TiO40] (CoW11Ti, IC50 = 4 µM) and Na10[α-SiW9O34] (SiW9, IC50 = 16 µM), K14[As2W19O67(H2O)] (As2W19, IC50 = 28 µM) and the lacunary Dawson K12[α-H2P2W12O48] (P2W12, IC50 = 11 µM), whereas low inhibitory potencies were observed for the isopolytungstate Na12[H4W22O74] (W22, IC50 = 68 µM) and the Anderson-type Na6[TeW6O24] (TeW6, IC50 = 200 µM). Regarding the inhibition of Na+/K+-ATPase activity, for the first time an ex vivo study was conducted using the opercular epithelium of killifish in order to investigate the effects of POTs on the epithelial chloride secretion. Interestingly, 1 µM of the most potent Ca2+-ATPase inhibitor, Se2W29, showed only a minor inhibitory effect (14% inhibition) on Na+/K+-ATPase activity, whereas almost total inhibition (99% inhibition) was achieved using P2W18. The remaining POTs exhibited similar inhibition rates on both ATPases. These results reveal the high potential of some POTs to act as P-type ATPase inhibitors, with Se2W29 showing high selectivity towards Ca2+-ATPase.

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.

Benzo[a]pyrene effects on reproductive endpoints in Fundulus heteroclitus.

Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon (PAH) that has been implicated in modulating aromatase enzyme function with the potential to interrupt normal reproductive function. The aim of this study was to use a fish model, Fundulus heteroclitus, to assess whether BaP exposure adversely impacts reproduction. Adult fish were exposed to waterborne BaP nominal concentrations of (0, 1, or 10 µg/l) for 28 days. Males and females were combined for the second half of the exposure (days 14-28) in order to quantitate egg production and fertilization success. Egg fertilization and subsequent hatching success of F1 embryos was significantly decreased by the high dose of BaP. In males, both gonad weight and plasma testosterone concentrations were significantly reduced compared to controls by 10 µg/l BaP. Histopathological examination of testes including spermatogonia, spermatocyte and spermatid cyst areas, percentage of cysts per phase, and area of spermatozoa per seminiferous tubule were not significantly affected. Other biomarkers, including male liver weight, liver vitellogenin (vtg) mRNA expression and sperm concentrations, were also not affected. In females, estradiol concentrations were significantly reduced after BaP exposure, but egg production, gonad weight, liver weight, vtg expression and oocyte maturation were not altered. Steroid concentrations in Fundulus larvae from exposed parents at 1 and 3 weeks posthatch were not significantly changed. BaP exposure at these environmentally relevant concentrations caused negative alterations particularly in male fish to both biochemical and phenotypic biomarkers associated with reproduction and multigenerational embryo survival.

Alternative intrapopulation life-history strategies and their trade-offs in an African annual fish.

In ephemeral habitats, the same genotypes cope with unpredictable environmental conditions, favouring the evolution of developmental plasticity and alternative life-history strategies (ALHS). We tested the existence of intrapopulation ALHS in an annual killifish, Nothobranchius furzeri, inhabiting temporary pools. The pools are either primary (persisting throughout the whole rainy season) or secondary (refilled after desiccation of the initial pool), representing alternative niches. The unpredictable conditions led to the evolution of reproductive bet-hedging with asynchronous embryonic development. We used a common garden experiment to test whether the duration of embryonic period is associated with post-embryonic life-history traits. Fish with rapid embryonic development (secondary pool strategy, high risk of desiccation) produced phenotypes with more rapid life-history traits than fish with slow embryonic development (primary pool strategy). The fast fish were smaller at hatching but had larger yolk sac reserves. Their post-hatching growth was more rapid, and they matured earlier. Further, fast fish grew to a smaller body size and died earlier than slow fish. No differences in fecundity, propensity to mate or physiological ageing were found, demonstrating a combination of plastic responses and constraints. Such developmentally related within-population plasticity in life history is exceptional among vertebrates.

Production and characterization of microbial biosurfactants for potential use in oil-spill remediation.

Two biosurfactants, surfactin and fatty acyl-glutamate, were produced from genetically-modified strains of Bacillus subtilis on 2% glucose and mineral salts media in shake-flasks and bioreactors. Biosurfactant synthesis ceased when the main carbohydrate source was completely depleted. Surfactin titers were ∼30-fold higher than fatty acyl-glutamate in the same medium. When bacteria were grown in large aerated bioreactors, biosurfactants mostly partitioned to the foam fraction, which was recovered. Dispersion effectiveness of surfactin and fatty acyl-glutamate was evaluated by measuring the critical micelle concentration (CMC) and dispersant-to-oil ratio (DOR). The CMC values for surfactin and fatty acyl-glutamate in double deionized distilled water were 0.015 and 0.10 g/L, respectively. However, CMC values were higher, 0.02 and 0.4 g/L for surfactin and fatty acyl-glutamate, respectively, in 12 parts per thousand Instant Ocean®[corrected].sea salt, which has been partly attributed to saline-induced conformational changes in the solvated ionic species of the biosurfactants. The DORs for surfactin and fatty acyl-glutamate were 1:96 and 1:12, respectively, in water. In Instant Ocean® solutions containing 12 ppt sea salt, these decreased to 1:30 and 1:4, respectively, suggesting reduction in oil dispersing efficiency of both surfactants in saline. Surfactant toxicities were assessed using the Gulf killifish, Fundulus grandis, which is common in estuarine habitats of the Gulf of Mexico. Surfactin was 10-fold more toxic than fatty acyl-glutamate. A commercial surfactant, sodium laurel sulfate, had intermediate toxicity. Raising the salinity from 5 to 25 ppt increased the toxicity of all three surfactants; however, the increase was the lowest for fatty acyl-glutamate.

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.

Long-Term survival of anoxia despite rapid ATP decline in embryos of the annual killifish Austrofundulus limnaeus.

Embryos of the annual killifish Austrofundulus limnaeus can survive for months in the complete absence of oxygen. Survival of anoxia is associated with entry into a state of metabolic dormancy known as diapause. However, extreme tolerance of anoxia is retained for several days of post-diapause development. Rates of heat dissipation in diapause II and 4 days post-diapause II embryos were measured under aerobic conditions and during the transition into anoxia. Phosphorylated adenylate compounds were quantified in embryos during entry into anoxia and after 12 hr of aerobic recovery. Rates of heat dissipation were not affected by exposure to anoxia in diapause II embryos, while post-diapause II embryos experienced a profound decrease in heat dissipation. ATP decreased substantially in both developmental stages upon exposure to anoxia, and all indicators of cellular energetic status indicated energetic stress, at least based on the mammalian paradigm. The rate of decline in ATP is the most acute reported for any vertebrate. The mechanisms responsible for cellular survival despite a clear dysregulation between energy production and energy consumption remain to be identified. Necrotic and apoptotic cell death in response to hypoxia contribute to poor survival during many diseases and pathological conditions in mammals. Understanding the mechanisms that are in place to prevent maladaptive cell death in embryos of A. limnaeus may greatly improve treatment strategies in diseases that involve hypoxia and reperfusion injuries.

Effects of low salinity media on growth, condition, and gill ion transporter expression in juvenile Gulf killifish, Fundulus grandis.

The Gulf killifish, Fundulus grandis, is a euryhaline teleost which has important ecological roles in the brackish-water marshes of its native range as well as commercial value as live bait for saltwater anglers. Effects of osmoregulation on growth, survival, and body condition at 0.5, 5.0, 8.0 and 12.0‰ salinity were studied in F. grandis juveniles during a 12-week trial. Relative expression of genes encoding the ion transport proteins Na(+)/K(+)-ATPase (NKA), Na(+)/K(+)/2Cl(-) cotransporter(NKCC1), and cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel was analyzed. At 0.5‰, F. grandis showed depressed growth, body condition, and survival relative to higher salinities. NKA relative expression was elevated at 7 days post-transfer but decreased at later time points in fish held at 0.5‰ while other salinities produced no such increase. NKCC1, the isoform associated with expulsion of ions in saltwater, was downregulated from week 1 to week 3 at 0.5‰ while CFTR relative expression produced no significant results across time or salinity. Our results suggest that Gulf killifish have physiological difficulties with osmoregulation at a salinity of 0.5‰ and that this leads to reduced growth performance and survival while salinities in the 5.0-12.0‰ are adequate for normal function.

Genomic and physiological footprint of the Deepwater Horizon oil spill on resident marsh fishes.

The biological consequences of the Deepwater Horizon oil spill are unknown, especially for resident organisms. Here, we report results from a field study tracking the effects of contaminating oil across space and time in resident killifish during the first 4 mo of the spill event. Remote sensing and analytical chemistry identified exposures, which were linked to effects in fish characterized by genome expression and associated gill immunohistochemistry, despite very low concentrations of hydrocarbons remaining in water and tissues. Divergence in genome expression coincides with contaminating oil and is consistent with genome responses that are predictive of exposure to hydrocarbon-like chemicals and indicative of physiological and reproductive impairment. Oil-contaminated waters are also associated with aberrant protein expression in gill tissues of larval and adult fish. These data suggest that heavily weathered crude oil from the spill imparts significant biological impacts in sensitive Louisiana marshes, some of which remain for over 2 mo following initial exposures.

Toxicity of aqueous fullerene in adult and larval Fundulus heteroclitus.

Aqueous suspensions of fullerene aggregates (aqua-nC60) were used to investigate the movement of carbon-based nanomaterials in a marine water column and to determine their effects on different life stages of a marine teleost. Fullerene aggregates formed precipitates as a result of mixing in natural seawater, and levels of aqua-nC60 were significantly increased in bottom waters after 24 h. Exposure of Fundulus heteroclitus embryos, larvae, and adults to increasing concentrations of aqua-nC60 resulted in very little mortality, and no median lethal concentrations could be calculated at < or = 10 mg/L. Aggregates of aqua-nC60 did adhere to the chorion but did not affect development of the embryos or their hatching success. Movements of aqua-nC60 through the chorion and into the embryo tended to increase with higher exposure levels; however, the concentrations were extremely low and did not differ significantly. Larvae exposed to increasing concentrations of aqua-nC60 exhibited a significant dose-dependent increase in total glutathione (GSH). This was accompanied by a decreasing trend in lipid peroxidation (LPO), but LPO was not statistically different between treatments. Adult F. heteroclitus exposed to increasing concentrations of aqua-nC60 exhibited an increase in total GSH in liver tissue but not in the gill. No significant effects on LPO were observed in either tissue. Thus, we conclude that aqua-nC60 affects the oxidative stress response of F. heteroclitus and that increased antioxidant defenses provide some physiological tolerance for these materials. Environmental factors influencing uptake, metabolism, and physiological response following exposure, however, need further investigation.

Long term hypoxia suppresses reproductive capacity in the estuarine fish, Fundulus grandis.

Human nutrient input has significantly altered dissolved oxygen (DO) cycles in coastal waters such that summertime hypoxia (DO <2 mg/L) and anoxia of bottom water are common worldwide. Prolonged hypoxia usually reduces metabolic rate in fish and potentially reduces reproduction, particularly in a spring and summer spawning species such as the Gulf killifish, Fundulus grandis. To evaluate the effects of long term hypoxia on reproduction, Gulf killifish were subjected to either normoxia (6.68+/-2.1 mg/L DO) or hypoxia (1.34+/-0.45 mg/L DO) for one month. Fecundity, growth, gonadosomatic index (GSI), circulating sex steroids (testosterone, T; 11-ketotestosterone, 11KT; and estradiol-17beta, E2), and egg yolk protein (vitellogenin, VTG) were measured. Hypoxia significantly reduced growth and reproduction. E2 was 50% lower in females and 11KT was 50% lower in males, although the precursor hormone T was unchanged in either sex after hypoxic exposure. Hypoxia-exposed females produced significantly fewer eggs and initiated spawning later than control fish. Plasma VTG concentration was unchanged, suggesting that hypoxia may delay VTG uptake by oocytes. Long term laboratory exposure clearly suppressed reproductive capacity in Gulf killifish. Wild populations experience cyclic hypoxia which could have equivalent effects if daily hypoxic periods are long and frequent - a potential consequence of anthropogenic nutrient enrichment in marsh systems.