Benjamin Dubansky

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.

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.

Cross-resistance in Gulf killifish (Fundulus grandis) populations resistant to dioxin-like compounds

The Houston Ship Channel (HSC) in Houston, Texas is an aquatic environment with a long history of contamination, including polychlorinated dibenzodioxins (PCDD), polychlorinated dibenzofurans (PCDF), polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), and heavy metals. Populations of Gulf killifish (Fundulus grandis) from the HSC have adapted to resist developmental cardiac deformities caused by dioxin-like compounds (DLCs). Contaminants in the HSC have acted as a strong selective pressure on resident Gulf killifish populations. Rapid adaptation can lead to fitness costs, some as a direct result of the mechanisms involved in the adaptive process, whereas other adaptations may be more general. To explore potential fitness costs, we evaluated two Gulf killifish populations with documented resistance to DLC-induced cardiac teratogenesis (Patrick Bayou and Vince Bayou), and one previously characterized reference population (Gangs Bayou). We also characterized a previously unstudied population from Galveston Bay as an additional reference population (Smith Point). We tested the sensitivity of F1 larvae from these four populations to two classes of pesticides (pyrethroid (permethrin) and carbamate (carbaryl)) and two model pro-oxidants (tert-butyl hydroquinone (tBHQ) and tert-butyl hydroperoxide (tBOOH)). In addition, we explored their responses to hypoxia and measured resting metabolic rates (M.O2). Both adapted populations were cross-resistant to the toxicity of carbaryl and both pro-oxidants tested. There were no population differences in sensitivity to permethrin. On the other hand, one reference population (Gangs Bayou) was less sensitive to hypoxia, and maintained a lower M.O2 . However, there were no differences in hypoxia tolerance or resting metabolic rate between the second reference and the two adapted populations. This investigation emphasizes the importance of including multiple reference populations to clearly link fitness costs or cross-resistance to pollution adaptation, rather than to unrelated environmental or ecological differences. When compared to previous literature on adapted populations of Fundulus heteroclitus, we see a mixture of similarities and differences, suggesting that F. grandis adapted phenotypes likely involve multiple mechanisms, which may not be completely consistent among adapted populations.

Response to Comment on “Multi-Tissue Molecular, Genomic, and Developmental Effects of the Deepwater Horizon Oil Spill on Resident Gulf Killifish (Fundulus grandis)”

Developmental Effects of the Deepwater Horizon Oil Spill on Resident Gulf Killifish (Fundulus grandis)” I our studies, functional genomic responses clearly indicate exposures of resident and experimental animals to the toxic components of oil spilled from the Deepwater Horizon oil spill (DHOS) disaster, in the field and laboratory. Such molecular responses are tightly linked with impacts on fitness parameters including embryonic survival and developmental deformities. Sublethal effects of crude oil exposure, such as the developmental abnormalities in embryonic and larval fish, can be causally linked to a reduction in fitness and a decrease in survival of the adult fish. As such, Gulf killifish (Fundulus grandis) populations, and other animals that rely on the same coastal marsh for reproduction, likely faced a considerable challenge to reproductive success in field sites heavily oiled during the DHOS. Exposure to sediments collected from heavily oiled coastal marsh in Southern Louisiana caused cardiovascular defects in Gulf killifish embryos during controlled laboratory studies, and developmental impacts such as delayed hatch and reduced percent hatch rate, collectively reducing the percent of the total embryos reaching the free-swimming larval stage. In fact, of the small percentage of surviving exposed embryonic fish that hatched during exposure to the Grande Terre, Louisiana sediments, all had severe cardiac edema and were significantly smaller and listless after hatch. We concur that no such fish could survive to adulthood in the field. Though we did not directly test for population-level effects, it is perfectly reasonable, contrary to the assertions of Pearson, that fitness impacts are likely to affect population vital rates, as we imply in our paper. We agree with much of Pearson’s comment that there is much to be learned about the causal links between organismallevel effects seen in the field and laboratory and ecosystem level impacts, or to the impacts on fisheries. We do not dispute that it remains a challenge to link biological effects at the organism level to the biology and economics of fisheries, considering the complexity of natural systems and the pragmatism that dictates our experimental reality. As such, we contend that efforts should continue well after this publication to monitor oiled sites in the northern Gulf of Mexico for evidence of population level effects; impacts that could take years to emerge. To this extent, integrated field-based and laboratory-based experiments that link molecular responses through to fitness end points represent the state-of-the-art for environmental impact assessment; such approaches identify regions and particular sediments oiled by the DHOS that are of potential concern to the health of resident individuals and populations. In our studies, sediments collected from Gulf killifish breeding habitat in regions directly oiled by the DHOS, were used in laboratory exposures. By using the same sediments that Gulf killifish were exposed to during the peak summer months of development in the field, our laboratory observations can be linked to the field, and the impacts that we observe in killifish are likely to be shared by other species that utilize that same habitat. Furthermore, by using oiled sediments collected during August 2010 and 2011, our observations relate findings in the lab to two successive breeding seasons in this spill-impacted bay. Pearson’s highlight of other studies that fail to indicate oil spill impacts on juvenile fish from sea grass beds bears little relevance on the implications of our research findings. The areas in those studies highlighted by Pearson were either not heavily oiled by the DHOS or were not suitable habitats for Fundulus grandis. In contrast, the locations sampled for our studies included the heavily oiled Grande Terre Island and Barataria Bay marshland of Southern Louisiana that were ideal habitat for Fundulus species, and which experienced some of the most lingering effects of the DHOS measured to date. Furthermore, chemical analysis of sediment samples used in the exposures was presented, and those data support a relationship between total polyaromatic hydrocarbons (and alkanes) in sediments and biological effects. Pearson also chose this forum to debate the relevance of decades of research findings on the Exxon Valdez oil spill (EVOS). We decline to debate the effects of the EVOS on fish populations in this forum, other than to highlight much evidence supports population-level impacts to fish, birds, and mammals in the years that followed the EVOS (see for review). Though the DHOS is unique in many ways compared to other spills, it is the largest marine oil spill in history, easily dwarfing the EVOS. Data from our studies are consistent with sediment contamination from the DHOS adversely affecting fitness parameters in exposed developing Gulf killifish, and such fitness impacts are likely to affect population vital rates. Whether these impacts ultimately emerge as population-level effects, as was evident in many species following the EVOS, remains to be seen. We have observed that industry-hired scientists consistently demand a completely connected chain of causality, from chemical exposure through population measurements, from individual studies, to assent to possible significant effects on natural resources. As a consequence, industry is unlikely to accept the findings of probable damage to fish populations from toxic chemicals, even when existing evidence is clearly consistent with such effects. The Gulf of Mexico holds much aesthetic, ecological, and economic importance for the region and the nation, as do other regions exploited for oil extraction. Because the lives and livelihoods of many people are intimately tied to these environments, the public should expect high standards of environmental stewardship and accountability from the corporations that are granted access to these globally important resources. Benjamin Dubansky*,†,∥ Andrew Whitehead‡

Natural development of dermal ectopic bone in the american alligator (Alligator mississippiensis) resembles heterotopic ossification disorders in humans: OSTEODERM FORMATION RESEMBLES HETEROTOPIC OSSIFICATION

Heterotopic ossification (HO) occurs when soft tissues are inappropriately converted to bony tissue. Several human diseases result in HO with few reliable treatment options. Animal models that naturally produce dermal ectopic bone (i.e., osteoderms), such as crocodilians, have never been utilized as models for studying these disorders in humans. Here, a histological evaluation and staging criteria for osteoderm development is described for the first time in the American alligator (Alligator mississipiensis). Differential staining and immunohistochemistry of alligator scales depict a progressive change during development, where woven bone forms from the differentiated dermis. Bone formation proceeds via intramembranous ossification, which is initiated in part by endothelial cell precursors that undergo endothelial‐to‐mesenchymal transition and eventually acquire an osteoblast phenotype. As such, the development of osteoderms in the American alligator bears morphological and mechanistic similarities to HO in humans, presenting a potential model for future study of soft tissue mineralization pathologies and providing insight into the morphological and molecular development of osteoderms in other vertebrate lineages. Anat Rec, 2017.

Is Exposure to Macondo Oil Reflected in the Otolith Chemistry of Marsh-Resident Fish?

Genomic and physiological responses in Gulf killifish (Fundulus grandis) in the northern Gulf of Mexico have confirmed oil exposure of resident marsh fish following the Macondo blowout in 2010. Using these same fish, we evaluated otolith microchemistry as a method for assessing oil exposure history. Laser-ablation inductively-coupled-plasma mass spectrometry was used to analyze the chemical composition of sagittal otoliths to assess whether a trace metal signature could be detected in the otoliths of F. grandis collected from a Macondo-oil impacted site in 2010, post-spill relative to pre-spill, as well as versus fish from areas not impacted by the spill. We found no evidence of increased concentrations of two elements associated with oil contamination (nickel and vanadium) in F. grandis otoliths regardless of Macondo oil exposure history. One potential explanation for this is that Macondo oil is relatively depleted of those metals compared to other crude oils globally. During and after the spill, however, elevated levels of barium, lead, and to a lesser degree, copper were detected in killifish otoliths at the oil-impacted collection site in coastal Louisiana. This may reflect oil contact or other environmental perturbations that occurred concomitant with oiling. For example, increases in barium in otoliths from oil-exposed fish followed (temporally) freshwater diversions in Louisiana in 2010. This implicates (but does not conclusively demonstrate) freshwater diversions from the Mississippi River (with previously recorded higher concentrations of lead and copper), designed to halt the ingress of oil, as a mechanism for elevated elemental uptake in otoliths of Louisiana marsh fishes. These results highlight the potentially complex and indirect effects of the Macondo oil spill and human responses to it on Gulf of Mexico ecosystems, and emphasize the need to consider the multiple stressors acting simultaneously on inshore fish communities.

Incubation relative humidity induces renal morphological and physiological remodeling in the embryo of the chicken (Gallus gallus domesticus)

The metanephric kidneys of the chicken embryo, along with the chorioallantoic membrane, process water and ions to maintain osmoregulatory homeostasis. We hypothesized that changes in relative humidity (RH) and thus osmotic conditions during embryogenesis would alter the developmental trajectory of embryonic kidney function. White leghorn chicken eggs were incubated at one of 25-30% relative humidity, 55-60% relative humidity, and 85-90% relative humidity. Embryos were sampled at days 10, 12, 14, 16, and 18 to examine embryo and kidney mass, glomerular characteristics, body fluid osmolalities, hematological properties, and whole embryo oxygen consumption. Low and especially high RH elevated mortality, which was reflected in a 10-20% lower embryo mass on D18. Low RH altered several glomerular characteristics by day 18, including increased numbers of glomeruli per kidney, increased glomerular perfusion, and increased total glomerular volume, all indicating potentially increased functional kidney capacity. Hematological variables and plasma and amniotic fluid osmolalities remained within normal physiological values. However, the allantoic, amniotic and cloacal fluids had a significant increase in osmolality at most developmental points sampled. Embryonic oxygen consumption increased relative to control at both low and high relative humidities on Day 18, reflecting the increased metabolic costs of osmotic stress. Major differences in both renal structure and performance associated with changes in incubation humidity occurred after establishment of the metanephric kidney and persisted into late development, and likely into the postnatal period. These data indicate that the avian embryo deserves to be further investigated as a promising model for fetal programming of osmoregulatory function, and renal remodeling during osmotic stress.

Reply to Jenkins et al.: Evidence for contaminating oil exposure is closely linked in space and time to biological effects

Our original article (1) linked exposure of resident killifish to contaminating oil from the Deepwater Horizon (DWH) oil spill with significant biological responses, including genome expression, protein expression, and tissue morphology. Given decades of laboratory studies on the effects of crude oil in many species, including fish, and after extensive field studies following the Exxon Valdez spill, some of the responses we captured are recognized as diagnostic of exposure to, and effects from, the toxic components of weathered crude oil (e.g., ref. 2). Jenkins et … [↵][1]1To whom correspondence should be addressed. E-mail: awhitehead{at}ucdavis.edu. [1]: #xref-corresp-1-1

The Nexus of Development and Environment

The relationship between environment and animal development has been recognized since the time of Aristotle, but the urgency of creating a thorough understanding of this relationship is emerging as environments in which animals develop are changing as a result of pollution, climate change and other anthropogenic activities. This book, an overview of which is provided in this first chapter, is organized along three key themes. Each theme predominates in its own section – Part I: Plasticity in developmental and evolutionary time and space, Part II: Contemporary experimental approaches, and Part III. Environmental effects and experimental outcomes. Each of its chapters provides a comprehensive, up-to-date assessment of how development and environment are inextricably woven together, and points to future directions for research at their nexus.

Methodology for exposing avian embryos to quantified levels of airborne aromatic compounds associated with crude oil spills

Oil spills on birds and other organisms have focused primarily on direct effects of oil exposure through ingestion or direct body fouling. Little is known of indirect effects of airborne volatiles from spilled oil, especially on vulnerable developing embryos within the bird egg. Here a technique is described for exposing bird embryos in the egg to quantifiable amounts of airborne volatile toxicants from Deepwater Horizon crude oil. A novel membrane inlet mass spectrometry system was used to measure major classes of airborne oil-derived toxicants and correlate these exposures with biological endpoints. Exposure induced a reduction in platelet number and increase in osmolality of the blood of embryos of the chicken (Gallus gallus). Additionally, expression of cytochrome P4501A, a protein biomarker of oil exposure, occurred in renal, pulmonary, hepatic and vascular tissues. These data confirm that this system for generating and measuring airborne volatiles can be used for future in-depth analysis of the toxicity of volatile organic compounds in birds and potentially other terrestrial organisms.