Bioenergetic Effects of Polycyclic Aromatic Hydrocarbon Resistance Manifest Later in Life in Offspring of Fundulus heteroclitus from the Elizabeth River.

Shifts in key physiological processes can confer resistance to chemical pollutants. However, these adaptations may come with certain trade-offs, such as altered energy metabolic processes, as evident in Atlantic killifish (Fundulus heteroclitus) in Virginia's Elizabeth River (ER) that have evolved resistance to polycyclic aromatic hydrocarbons (PAHs). We seek to understand the bioenergetic costs of PAH resistance among subpopulations of Atlantic killifish with differing contamination levels in order to examine how these changes manifest across multiple life stages and how these costs might be exacerbated by additional stressors. Bioenergetics data revealed differences in metabolic rates between offspring of PAH-resistant fish and reference fish were absent or minimal in both the embryo and larval stages but pronounced at the juvenile life stage, suggesting that bioenergetic changes in pollution-adapted killifish manifest later in life. We also provide evidence that killifish from remediated sites are more sensitive to PAH exposure than killifish from nonremediated sites, suggesting loss of PAH tolerance following relaxed selection. Collectively, our data suggest that the fitness consequences associated with evolved resistance to anthropogenic stressors may manifest differently over time and depend on the magnitude of the selection pressure. This information can be valuable in effective risk and remediation assessments as well as in broadening our understanding of species responses to environmental change.

Long-Term Immunological Alterations in Bottlenose Dolphin a Decade after the Deepwater Horizon Oil Spill in the Northern Gulf of Mexico: Potential for Multigenerational Effects.

Health assessments were conducted on bottlenose dolphins in Barataria Bay, Louisiana, USA, during 2011 to 2018, to assess potential health effects following the Deepwater Horizon oil spill, compared to the unoiled Sarasota Bay, Florida, USA, reference dolphin population. We previously reported significant increases in T-lymphocyte proliferation, as well as lower T helper 1 (Th1) cytokines, higher Th2 cytokine IL-4, and lower T regulatory (Treg) cytokine IL-10 in Barataria Bay in 2011 compared to Sarasota Bay, consistent with Deepwater Horizon oil exposure. Although values between 2013 and 2016 were more similar to those observed in Sarasota Bay, T-cell proliferation was again elevated and cytokine balance tilted toward Th2 in Barataria Bay during 2017-2018. In 2018, Barataria Bay dolphins had significantly more circulating Treg cells than Sarasota Bay dolphins. Mice experimentally exposed to oil also had significantly increased T-lymphocyte proliferation and circulating Treg cell number, including effects in their unexposed progeny. In vitro stimulation resulted in greater Th2 responsiveness in Barataria Bay compared to Sarasota Bay dolphins, and in vitro oil exposure of Sarasota Bay dolphin cells also resulted in enhanced Th2 responsiveness. Evidence points to Treg cells as a potential target for the immunomodulatory effects of oil exposure. The immunological trends observed in Barataria Bay appeared exaggerated in dolphins born after the spill, suggesting the possibility of continued oil exposure or multigenerational health consequences of exposure to oil, as observed in mice. Environ Toxicol Chem 2021;40:1308-1321. © 2021 SETAC.

Methyl mercury (MeHg) in vitro exposure alters mitogen-induced lymphocyte proliferation and cytokine expression in Steller sea lion (Eumetopias jubatus) pups.

Steller sea lions (Eumetopias jubatus, SSLs) are managed as two distinct population segments within U.S. waters: the endangered western distinct population segment and the recently delisted eastern distinct population segment. Recent studies reported concentrations of mercury in several tissues collected from young SSLs in the Aleutian Islands that were at or above concentrations found to negatively impact health in other fish-eating mammals. However, there are limited studies which have investigated the range of mercury concentrations that may negatively influence the SSL immune system. This study assessed relationships between methyl mercury (MeHg+) concentrations and two immune functions, lymphocyte proliferation and cytokine expression. Peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved from pups on three rookeries within the western distinct population segment: Chiswell Island, Ulak, and Agattu Islands. Lymphocyte proliferation and cytokine expression were assessed in vitro using thawed PBMCs with exposure to MeHg+ (unexposed control, 0.001, 0.01, and 0.1 µg/ml). Lymphocyte proliferation was measured without and with stimulation with a T cell mitogen (ConA) and B cell mitogen (LPS) and the concentration of cytokines was measured in the cell culture supernatant (with and without ConA or LPS). Spontaneous lymphocyte proliferation was significantly increased at 0.01 and 0.1 µg/ml. T lymphocyte proliferation was significantly increased at 0.001 µg/ml and 0.1 µg/ml, while B lymphocyte proliferation was decreased at 0.1 µg/ml. Cytokine concentrations for INFγ, IL-10, IL-6, and TNFα were reduced at 0.1 µg/ml upon either T or B cell mitogen stimulation, with the exception for IL-10, where 0.1 µg/ml reduced IL-10 concentration compared to unstimulated cells. These data suggest immune functions were affected by MeHg+ exposure requiring in vivo follow up investigations. The observed modulation of immune functions is of concern as any toxicant-induced modulation may adversely affect the health of individuals, particularly younger animals undergoing periods of critical development.

T Helper Cell Subsets and Their Functions in Common Bottlenose Dolphins (Tursiops truncatus).

Considerable efforts have been made to better understand the immune system of bottlenose dolphins in view of the common environmental challenges they encounter, such as exposure to polychlorinated biphenyls, oil spills, or harmful algal bloom biotoxins. However, little is known about the identity and functionality of the Th1, Th2, and Treg T helper cell subsets in bottlenose dolphins. The present study aimed at validating assays and reagents to identify T helper cell subsets and their functions in a subset of dolphins from Sarasota Bay, Florida, USA, which have been long studied and often used as a reference population. A population of CD4+ FOXP3+ lymphocytes was identified representing an average <1% of blood lymphocyte population, which is in the range observed in for Treg cells in other species. The use of porcine reagents to measure TGFß, one of the key Treg cytokines, was further validated using the relatively high-throughput and highly standardized Luminex technology. The proportion of circulating Treg cells was not correlated with the serum concentrations of the Treg effector cytokines TGFß and IL-10, nor could it significantly contribute to predicting the variability of T lymphocyte proliferation, suggesting that not all dolphin circulating Treg cells are functional and active. However, stimulation of dolphin lymphocytes with TGFß and IL-2 increased the expression of the gene for TGFß and IL-10, and stimulation with IL-12 and IFNγ induced a robust increase in the expression of the gene for IFNγ, suggesting the potential for polarization and differentiation of dolphin T helper cells toward a Treg and Th1 response, respectively. The lack of an increase in the expression of the genes for the Th2 cytokines IL-4 and IL-13 upon stimulation with IL-4 may be due to the requirement for IL-2 for a Th2 polarization as described in mice. However, regression analysis and PCA suggested the potential ability of both the Th1 and Th2 response to be triggered upon acute inflammatory signals. These results may be useful in better understanding the mechanisms by which the dolphin immune system is affected upon exposure to environmental challenges and how it responds to pathogen challenges.

Parental exposure to Deepwater Horizon oil in different environmental scenarios alters development of sheepshead minnow (Cyprinodon variegatus) offspring.

The explosion of the Deepwater Horizon (DWH) oil exploration platform on April 20, 2010 began a catastrophic leak of approximately 640 million liters crude oil into the northern Gulf of Mexico (GOM), affecting more than 2100 km of coastline, including wetlands and estuaries that provide habitat and nursery for many aquatic species. Estuaries of the GOM are dynamic environments, with constant fluctuations in salinity and dissolved oxygen, including large hypoxic zones during summer months. Spawning fish in northern GOM estuaries following the DWH incident were at significant risk of oil exposure, and adverse environmental conditions at the time of exposure, such as hypoxia and low salinity, could have exacerbated developmental effects in the offspring. The present study investigated the effects of F0 parental oil exposure in different environmental scenarios on development of F1 sheepshead minnow (SHM) offspring. Adult SHM were exposed to the high-energy water accommodated fraction (HEWAF) of crude oil in three environmental scenarios: normoxic (NORM), hypoxic (HYP), and hypoxic with low salinity (HYP-LS). Parental HEWAF exposure in the NORM scenario resulted in developmental effects in F1 offspring, including altered heart rate, decreased length at hatch, and impaired prey capture. Co-exposure of F0 SHM to HEWAF and adverse environmental conditions altered HEWAF effects on F1 heart rate, hatch rate, prey capture, and survival. Time to hatch was not significantly impacted by parental HEWAF in any environmental scenario. The present study demonstrates that parental exposure to HEWAF results in developmental changes in F1 embryos, and co-exposure to adverse environmental conditions altered the effects for several developmental endpoints. These data suggest that SHM exposed to oil in estuaries experiencing hypoxia or low salinity may produce offspring with worsened outcomes. These developmental effects, in addition to previously reported reproductive effects in adult fish, could lead to long-term population level impacts for SHM.

Hypoxia and reduced salinity exacerbate the effects of oil exposure on sheepshead minnow (Cyprinodon variegatus) reproduction.

Estuaries of the northern Gulf of Mexico are dynamic environments, with fluctuations in salinity and dissolved oxygen, including areas of seasonal hypoxia. Fish that reside and reproduce in these estuaries, including sheepshead minnow (Cyprinodon variegatus; SHM), were at significant risk of oil exposure following the Deepwater Horizon oil spill. It is poorly understood how differences in environmental conditions during oil exposure impact its toxicity. The present study investigated the effects of crude oil high-energy water accommodated fraction (HEWAF) on SHM reproduction in three environmental scenarios (normoxic, hypoxic, and hypoxic with low salinity) to determine if differences in salinity (brackish vs low salinity) and dissolved oxygen (normoxia vs hypoxia) could exacerbate the effects of HEWAF-derived polycyclic aromatic hydrocarbons (PAHs). We observed that HEWAF exposure significantly increased liver somatic index of SHM compared to control, but this effect was not exacerbated by hypoxia or low salinity. HEWAF exposure also significantly decreased egg production and egg fertilization rate, but only in the hypoxic and hypoxic with low salinity scenarios. A significant correlation existed between body burdens of PAHs and reproductive endpoints, providing substantial evidence that oil exposure reduced reproductive capacity in SHM, across a range of environmental conditions. These data suggest that oil spill risk assessments that fail to consider other environmental stressors (i.e. hypoxia and salinity) may be underestimating risk.

Transgenerational effects of polycyclic aromatic hydrocarbon exposure on sheepshead minnows (Cyprinodon variegatus).

The Deepwater Horizon oil spill resulted in the release of over 640 million L of crude oil into the Gulf of Mexico, affecting over 2000 km of shoreline, including estuaries that serve as important habitats and nurseries for aquatic species. Cyprinodon variegatus (sheepshead minnow) are small-bodied fish that inhabit northern Gulf of Mexico estuaries, are easily adaptable to laboratory conditions, and are commonly used in toxicological assessment studies. The purpose of the present study was to determine the somatic, reproductive, and developmental effects of an environmentally relevant polycyclic aromatic hydrocarbon (PAH) mixture, the oil high-energy water accommodated fraction (HEWAF), on experimentally exposed sheepshead minnow (F0 ) as well as 2 generations of offspring (F1 and F2 ) without additional exposure. The F0 generation exposed to HEWAF had increased liver somatic indices, altered egg production, and decreased fertilization. Several developmental endpoints in the F1 were altered by F0 HEWAF exposure. As adults, low HEWAF-exposed F1 females demonstrated decreased weight and length. Both the F1 and F2 generations derived from high HEWAF-exposed F0 had deficits in prey capture compared to control F1 and F2 , respectively. Correlations between endpoints and tissue PAHs provide evidence that the physiological effects observed were associated with hydrocarbon exposure. These data demonstrate that PAHs were capable of causing physiological changes in exposed adult sheepshead minnow and transgenerational effects in unexposed offspring, both of which could have population-level consequences. Environ Toxicol Chem 2019;38:638-649. © 2018 SETAC.

Lack of cross-reactivity of human and porcine reagents to quantify manatee (Trichechus manatus) cytokines.

Veterinary medical examinations, including both physical examination and diagnostic tests, are important to monitor the health of both managed-care and wild marine mammals. However, limited species-specific reagents and assays are available that may contribute to a broader medical examination. This project evaluated if commercially available human and porcine antibodies and reagents would cross-react with manatee (Trichechus manatus) cytokines as the first step to validate a new diagnostic tool for manatees. Overall, as a result of limited cross-reactivity, human and porcine commercial reagents did not allow for the quantification of manatee cytokines. At this point, caution must be exercised when using human or porcine immunoassay reagents to quantify manatee cytokines if the reagents have not been fully validated. Future efforts will continue to explore and test the cross-reactivity of reagents to measure manatee cytokines as new species-specific and commercial reagents become available.

Comparative toxicity of Corexit® 9500, oil, and a Corexit®/oil mixture on the eastern oyster, Crassostrea virginica (Gmelin).

Given their particle feeding behavior, sessile nature, and abundance in coastal zones, bivalves are at significant risk for exposure to oil and oil dispersant following environmental disasters like the Deepwater Horizon oil spill. However, the effects of oil combined with oil dispersants on the health of oysters are not well studied. Therefore, eastern oysters (Crassostrea virginica) were exposed in vivo to Corexit® 9500, crude oil (high-energy water accommodated fraction; HEWAF), and a Corexit®/oil mixture (chemically-enhanced water accommodated fraction; CEWAF) to evaluate potential toxic effects on immunological (phagocytosis and respiratory burst), physiological (feeding rate), and histological endpoints. Phagocytosis was significantly increased following CEWAF exposure only. Respiratory burst was significantly decreased following Corexit® exposure, but significantly increased following exposure to the highest concentration of CEWAF. Oyster feeding rates were significantly decreased following exposure to Corexit®, HEWAF, and CEWAF, and were most sensitive to CEWAF exposure. These modulations of important immunological and physiological functions could result in serious health outcomes for oysters, such as increased parasitism and decreased growth. Our experiments showed that subtle, sub-lethal effects occurred following acute in vivo exposure to Corexit®, HEWAF, and CEWAF, though oysters were not equally sensitive to the three components. Data from this study can be used for more accurate risk assessment concerning the impact of oil and Corexit® on the health of oysters.

Immune function in arctic mammals: Natural killer (NK) cell-like activity in polar bear, muskox and reindeer.

Natural killer (NK) cells are a vital part of the rapid and non-specific immune defense against invading pathogens and tumor cells. This study evaluated NK cell-like activity by flow cytometry for the first time in three ecologically and culturally important Arctic mammal species: polar bear (Ursus maritimus), muskox (Ovibos moschatus) and reindeer (Rangifer tarandus). NK cell-like activity for all three species was most effective against the mouse lymphoma cell line YAC-1, compared to the human leukemia cell line K562; NK cell response displayed the characteristic increase in cytotoxic activity when the effector:target cell ratio increased. Comparing NK activity between fresh and cryopreserved mouse lymphocytes revealed little to no difference in function, highlighting the applicability of cryopreserving cells in field studies. The evaluation of this important innate immune function in Arctic mammals can contribute to future population health assessments, especially as pollution-induced suppression of immune function may increase infectious disease susceptibility.

Effects of Polar Bear and Killer Whale Derived Contaminant Cocktails on Marine Mammal Immunity.

Most controlled toxicity studies use single chemical exposures that do not represent the real world situation of complex mixtures of known and unknown natural and anthropogenic substances. In the present study, complex contaminant cocktails derived from the blubber of polar bears (PB; Ursus maritimus) and killer whales (KW; Orcinus orca) were used for in vitro concentration-response experiments with PB, cetacean and seal spp. immune cells to evaluate the effect of realistic contaminant mixtures on various immune functions. Cytotoxic effects of the PB cocktail occurred at lower concentrations than the KW cocktail (1 vs 16 µg/mL), likely due to differences in contaminant profiles in the mixtures derived from the adipose of each species. Similarly, significant reduction of lymphocyte proliferation occurred at much lower exposures in the PB cocktail (EC50: 0.94 vs 6.06 µg/mL; P < 0.01), whereas the KW cocktail caused a much faster decline in proliferation (slope: 2.9 vs 1.7; P = 0.04). Only the KW cocktail modulated natural killer (NK) cell activity and neutrophil and monocyte phagocytosis in a concentration- and species-dependent manner. No clear sensitivity differences emerged when comparing cetaceans, seals and PB. Our results showing lower effect levels for complex mixtures relative to single compounds suggest that previous risk assessments underestimate the effects of real world contaminant exposure on immunity. Our results using blubber-derived contaminant cocktails add realism to in vitro exposure experiments and confirm the immunotoxic risk marine mammals face from exposure to complex mixtures of environmental contaminants.

Saxitoxin increases phocine distemper virus replication upon in-vitro infection in harbor seal immune cells.

Several marine mammal epizootics have been closely linked to infectious diseases, as well as to the biotoxins produced by harmful algal blooms (HABs). In two of three saxitoxin (STX) associated mortality events, dolphin morbillivirus (DMV) or phocine distemper virus (PDV) was isolated in affected individuals. While STX is notorious for its neurotoxicity, immunotoxic effects have also been described. This study investigated the role of STX in altering immune function, specifically T lymphocyte proliferation, in harbor seals (Phoca vitulina concolor) upon in-vitro exposure. In addition, the study also examined whether exposure to STX could alter the susceptibility of harbor seal immune cells to PDV infection upon in-vitro exposure. STX caused an increase in harbor seal lymphocyte proliferation at 10ppb and exposure to STX significantly increased the amount of virus present in lymphocytes. These results suggest that low levels of STX within the range of those reported in northeast U.S. seals may affect the likelihood of systemic PDV infection upon in-vivo exposure in susceptible seals. Given the concurrent increase in morbillivirus epizootics and HAB events in the last 25 years, the relationship between low level toxin exposure and host susceptibility to morbillivirus needs to be further explored.

Immunomodulatory effects of exposure to polychlorinated biphenyls and perfluoroalkyl acids in East Greenland ringed seals (Pusa hispida).

To better elucidate the potential immune-related health effects of exposure to environmentally persistent organic pollutants (POP), such as polychlorinated biphenyls (PCBs) and perfluoroalkyl substances (PFASs), in ringed seals (Pusa hispida), a sentinel Arctic species, we assessed 1) associations between mitogen-induced lymphocyte proliferation and in vivo tissue contaminant burdens, and 2) the concentration-response effects of in vitro exposure to PFASs and PCB congeners on mitogen-induced lymphocyte proliferation. Upon in vitro contaminant exposure, the non-coplanar PCB congeners CB 138, 153, and 180, but not the coplanar CB 169, significantly reduced lymphocyte proliferation between 10 and 20µgg-1 ww. The respective in vitro EC50 values for these congeners were 13.3, 20.7, 20.8, and 54.6µgg-1 ww. No modulation of lymphocyte proliferation was observed upon in vitro exposure to two individual PFASs, perfluorooctane sulphonic acid (PFOS) and perfluorooctanoic acid (PFOA), at concentrations up to 1000ngg-1. In addition, no significant correlations were found between lymphocyte proliferation and any blood or blubber contaminant measured. Taken together, these data suggest this population of ringed seals is not currently at high risk of altered lymphocyte proliferation from exposure to the POPs or PFASs in this study.