Cardiac remodeling in response to embryonic crude oil exposure involves unconventional NKX family members and innate immunity genes.

Cardiac remodeling results from both physiological and pathological stimuli. Compared with mammalian hearts, fish hearts show a broader array of remodeling changes in response to environmental influences, providing exceptional models for dissecting the molecular and cellular bases of cardiac remodeling. We recently characterized a form of pathological remodeling in juvenile pink salmon (Oncorhynchus gorbuscha) in response to crude oil exposure during embryonic cardiogenesis. In the absence of overt pathology (cardiomyocyte death or inflammatory infiltrate), cardiac ventricles in exposed fish showed altered shape, reduced thickness of compact myocardium and hypertrophic changes in spongy, trabeculated myocardium. Here, we used RNA sequencing to characterize molecular pathways underlying these defects. In juvenile ventricular cardiomyocytes, antecedent embryonic oil exposure led to dose-dependent upregulation of genes involved in innate immunity and two NKX homeobox transcription factors not previously associated with cardiomyocytes, nkx2.3 and nkx3.3 Absent from mammalian genomes, the latter is largely uncharacterized. In zebrafish embryos, nkx3.3 demonstrated a potent effect on cardiac morphogenesis, equivalent to that of nkx2.5, the primary transcription factor associated with ventricular cardiomyocyte identity. The role of nkx3.3 in heart growth is potentially linked to the unique regenerative capacity of fish and amphibians. Moreover, these findings support a cardiomyocyte-intrinsic role for innate immune response genes in pathological hypertrophy. This study demonstrates how an expanding mechanistic understanding of environmental pollution impacts - i.e. the chemical perturbation of biological systems - can ultimately yield new insights into fundamental biological processes.

Temperature dependent pre- and postprandial activity in Pacific bluefin tuna (Thunnus orientalis).

Bluefin tunas are highly specialized fish with unique hydrodynamic designs and physiological traits. In this study, we present results in a captive population that demonstrate strong effects of ambient temperature on the tail beat frequency and swimming speed of a pelagic fish in both pre- and post-prandial states. We measured the responses of a ram ventilator, the Pacific bluefin tuna (Thunnus orientalis), after digestion of a meal to explore the impacts of the metabolic costs of digestion on behavior and respiration. A combination of respirometry, physiological biologging of visceral temperatures, and activity monitoring with accelerometry were used to explore the metabolic costs of digestion and the impacts on ventilation and swimming speed. Experiments were conducted at temperatures that are within the metabolic optimum for Pacific bluefin tuna (17 °C), and at a second temperature corresponding to the upper distributional limit of the species in the California Current (24 °C). Warmer temperatures resulted in higher tail-beat frequency and greater elevation of body temperature in pre-prandial Pacific bluefin tuna. Specific dynamic action (SDA) events resulted in a significant postprandial increase in tail-beat frequency of ~0.2 Hz, compared to pre-prandial levels of 1.5 Hz (17 °C) and 1.75 Hz (24 °C), possibly resulting from ventilatory requirements. Data of fish exercised in a swim-tunnel respirometer suggest that the observed increase in tail-beat frequency comprise 5.5 and 6.8% of the oxygen demand during peak SDA at 24 °C and 17 °C respectively. The facultative increase in swimming speed might increase oxygen uptake at the gills to meet the increasing demand by visceral organs involved in the digestive process, potentially decreasing the available energy of each meal for other metabolic processes, such as growth, maturation, and reproduction. We hypothesize that these post-prandial behaviors allow tuna to evacuate their guts more quickly, ultimately permitting fish to feed more frequently when prey is available.

Phylotranscriptomic Insights into the Diversification of Endothermic Thunnus Tunas.

Birds, mammals, and certain fishes, including tunas, opahs and lamnid sharks, are endothermic, conserving internally generated, metabolic heat to maintain body or tissue temperatures above that of the environment. Bluefin tunas are commercially important fishes worldwide, and some populations are threatened. They are renowned for their endothermy, maintaining elevated temperatures of the oxidative locomotor muscle, viscera, brain and eyes, and occupying cold, productive high-latitude waters. Less cold-tolerant tunas, such as yellowfin tuna, by contrast, remain in warm-temperate to tropical waters year-round, reproducing more rapidly than most temperate bluefin tuna populations, providing resiliency in the face of large-scale industrial fisheries. Despite the importance of these traits to not only fisheries but also habitat utilization and responses to climate change, little is known of the genetic processes underlying the diversification of tunas. In collecting and analyzing sequence data across 29,556 genes, we found that parallel selection on standing genetic variation is associated with the evolution of endothermy in bluefin tunas. This includes two shared substitutions in genes encoding glycerol-3 phosphate dehydrogenase, an enzyme that contributes to thermogenesis in bumblebees and mammals, as well as four genes involved in the Krebs cycle, oxidative phosphorylation, ß-oxidation, and superoxide removal. Using phylogenetic techniques, we further illustrate that the eight Thunnus species are genetically distinct, but found evidence of mitochondrial genome introgression across two species. Phylogeny-based metrics highlight conservation needs for some of these species.

Quantification of Environmental DNA (eDNA) Shedding and Decay Rates for Three Marine Fish.

Analysis of environmental DNA (eDNA) to identify macroorganisms and biodiversity has the potential to significantly augment spatial and temporal biological monitoring in aquatic ecosystems. Current monitoring methods relying on the physical identification of organisms can be time consuming, expensive, and invasive. Measuring eDNA shed from organisms provides detailed information on the presence and abundance of communities of organisms. However, little is known about eDNA shedding and decay in aquatic environments. In the present study, we designed novel Taqman qPCR assays for three ecologically and economically important marine fish-Engraulis mordax (Northern Anchovy), Sardinops sagax (Pacific Sardine), and Scomber japonicas (Pacific Chub Mackerel). We subsequently measured fish eDNA shedding and decay rates in seawater mesocosms. eDNA shedding rates ranged from 165 to 3368 pg of DNA per hour per gram of biomass. First-order decay rate constants ranged from 0.055 to 0.101 per hour. We also examined the size fractionation of eDNA and concluded eDNA is both intra- and extracellular. Finally, we derived a simple mass-balance model to estimate fish abundance from eDNA concentration. The mesocosm-derived shedding and decay rates inform the interpretation of eDNA concentrations measured in environmental samples and future use of eDNA as a monitoring tool.

Assessing niche width of endothermic fish from genes to ecosystem.

Endothermy in vertebrates has been postulated to confer physiological and ecological advantages. In endothermic fish, niche expansion into cooler waters is correlated with specific physiological traits and is hypothesized to lead to greater foraging success and increased fitness. Using the seasonal co-occurrence of three tuna species in the eastern Pacific Ocean as a model system, we used cardiac gene expression data (as a proxy for thermal tolerance to low temperatures), archival tag data, and diet analyses to examine the vertical niche expansion hypothesis for endothermy in situ. Yellowfin, albacore, and Pacific bluefin tuna (PBFT) in the California Current system used more surface, mesopelagic, and deep waters, respectively. Expression of cardiac genes for calcium cycling increased in PBFT and coincided with broader vertical and thermal niche utilization. However, the PBFT diet was less diverse and focused on energy-rich forage fishes but did not show the greatest energy gains. Ecosystem-based management strategies for tunas should thus consider species-specific differences in physiology and foraging specialization.

Deepwater Horizon crude oil impacts the developing hearts of large predatory pelagic fish.

The Deepwater Horizon disaster released more than 636 million L of crude oil into the northern Gulf of Mexico. The spill oiled upper surface water spawning habitats for many commercially and ecologically important pelagic fish species. Consequently, the developing spawn (embryos and larvae) of tunas, swordfish, and other large predators were potentially exposed to crude oil-derived polycyclic aromatic hydrocarbons (PAHs). Fish embryos are generally very sensitive to PAH-induced cardiotoxicity, and adverse changes in heart physiology and morphology can cause both acute and delayed mortality. Cardiac function is particularly important for fast-swimming pelagic predators with high aerobic demand. Offspring for these species develop rapidly at relatively high temperatures, and their vulnerability to crude oil toxicity is unknown. We assessed the impacts of field-collected Deepwater Horizon (MC252) oil samples on embryos of three pelagic fish: bluefin tuna, yellowfin tuna, and an amberjack. We show that environmentally realistic exposures (1-15 µg/L total PAH) cause specific dose-dependent defects in cardiac function in all three species, with circulatory disruption culminating in pericardial edema and other secondary malformations. Each species displayed an irregular atrial arrhythmia following oil exposure, indicating a highly conserved response to oil toxicity. A considerable portion of Gulf water samples collected during the spill had PAH concentrations exceeding toxicity thresholds observed here, indicating the potential for losses of pelagic fish larvae. Vulnerability assessments in other ocean habitats, including the Arctic, should focus on the developing heart of resident fish species as an exceptionally sensitive and consistent indicator of crude oil impacts.

Exxon Valdez to Deepwater Horizon: comparable toxicity of both crude oils to fish early life stages.

The 2010 Deepwater Horizon disaster in the Gulf of Mexico was the largest oil spill in United States history. Crude oils are highly toxic to developing fish embryos, and many pelagic fish species were spawning in the northern Gulf in the months before containment of the damaged Mississippi Canyon 252 (MC252) wellhead (April-July). The largest prior U.S. spill was the 1989 grounding of the Exxon Valdez that released 11 million gallons of Alaska North Slope crude oil (ANSCO) into Prince William Sound. Numerous studies in the aftermath of the Exxon Valdez spill defined a conventional crude oil injury phenotype in fish early life stages, mediated primarily by toxicity to the developing heart. To determine whether this type of injury extends to fishes exposed to crude oil from the Deepwater Horizon - MC252 incident, we used zebrafish to compare the embryotoxicity of ANSCO alongside unweathered and weathered MC252 oil. We also developed a standardized protocol for generating dispersed oil water-accommodated fractions containing microdroplets of crude oil in the size range of those detected in subsurface plumes in the Gulf. We show here that MC252 oil and ANSCO cause similar cardiotoxicity and photo-induced toxicity in zebrafish embryos. Morphological defects and patterns of cytochrome P450 induction were largely indistinguishable and generally correlated with polycyclic aromatic compound (PAC) composition of each oil type. Analyses of embryos exposed during different developmental windows provided additional insight into mechanisms of crude oil cardiotoxicity. These findings indicate that the impacts of MC252 crude oil on fish embryos and larvae are consistent with the canonical ANSCO cardiac injury phenotype. For those marine fish species that spawned in the northern Gulf of Mexico during and after the Deepwater Horizon incident, the established literature can therefore inform the assessment of natural resource injury in the form of potential year-class losses.

Effects of temperature acclimation on Pacific bluefin tuna (Thunnus orientalis) cardiac transcriptome.

Little is known about the mechanisms underpinning thermal plasticity of vertebrate hearts. Bluefin tuna hearts offer a unique model to investigate processes underlying thermal acclimation. Their hearts, while supporting an endothermic physiology, operate at ambient temperature, and are presented with a thermal challenge when migrating to different thermal regimes. Here, we examined the molecular responses in atrial and ventricular tissues of Pacific bluefin tuna acclimated to 14°C, 20°C, and 25°C. Quantitative PCR studies showed an increase in sarcoplasmic reticulum Ca(2+) ATPase gene expression with cold acclimation and an induction of Na(+)/Ca(2+)-exchanger gene at both cold and warm temperatures. These data provide evidence for thermal plasticity of excitation-contraction coupling gene expression in bluefin tunas and indicate an increased capacity for internal Ca(2+) storage in cardiac myocytes at 14°C. Transcriptomic analysis showed profound changes in cardiac tissues with acclimation. A principal component analysis revealed that temperature effect was greatest on gene expression in warm-acclimated atrium. Overall data showed an increase in cardiac energy metabolism at 14°C, potentially compensating for cold temperature to optimize bluefin tuna performance in colder oceans. In contrast, metabolic enzyme activity and gene expression data suggest a decrease in ATP production at 25°C. Expression of genes involved in protein turnover and molecular chaperones was also decreased at 25°C. Expression of genes involved in oxidative stress response and programmed cell death suggest an increase in oxidative damage and apoptosis at 25°C, particularly in the atrium. These findings provide insights into molecular processes that may characterize cardiac phenotypes at upper thermal limits of teleosts.

Microarray gene expression profiles from mature gonad tissues of Atlantic bluefin tuna, Thunnus thynnus in the Gulf of Mexico.

BACKGROUND: Bluefin tunas are highly prized pelagic fish species representing a significant economic resource to fisheries throughout the world. Atlantic bluefin tuna (Thunnus thynnus) populations have significantly declined due to overexploitation. As a consequence of their value and population decline, T. thynnus has been the focus of considerable research effort concerning many aspects of their life history. However, in-depth understanding of T. thynnus reproductive biology is still lacking. Knowledge of reproductive physiology is a very important tool for determining effective fisheries and aquaculture management. Transcriptome techniques are proving powerful and provide novel insights into physiological processes. Construction of a microarray from T. thynnus ESTs sourced from reproductive tissues has provided an ideal platform to study the reproductive physiology of bluefin tunas. The aim of this investigation was to compare transcription profiles from the ovaries and testes of mature T. thynnus to establish sex specific variations underlying their reproductive physiology. RESULTS: Male and females T. thynnus gonad tissues were collected from the wild and histologically staged. Sub-samples of sexually mature tissues were also measured for their mRNA differential expression among the sexes using the custom microarray design BFT 4X44K. A total of 7068 ESTs were assessed for differential expression of which 1273 ESTs were significantly different (p<0.05) with >2 fold change in expression according to sex. Differential expression for 13 of these ESTs was validated with quantitative PCR. These include genes involved in egg envelope formation, hydration, and lipid transport/accumulation more highly expressed in ovaries compared with testis, while genes involved in meiosis, sperm motility and lipid metabolism were more highly expressed in testis compared with ovaries. CONCLUSIONS: This investigation has furthered our knowledge of bluefin tunas reproductive biology by using a contemporary transcriptome approach. Gene expression profiles in T. thynnus sexually mature testes and ovaries were characterized with reference to gametogenesis and potential alternative functions. This report is the first application of microarray technology for bluefin tunas and demonstrates the efficacy by which this technique may be used for further characterization of specific biological aspects for this valuable teleost fish.

Spatial analysis of biomineralization associated gene expression from the mantle organ of the pearl oyster Pinctada maxima.

BACKGROUND: Biomineralization is a process encompassing all mineral containing tissues produced within an organism. One of the most dynamic examples of this process is the formation of the mollusk shell, comprising a variety of crystal phases and microstructures. The organic component incorporated within the shell is said to dictate this architecture. However general understanding of how this process is achieved remains ambiguous. The mantle is a conserved organ involved in shell formation throughout molluscs. Specifically the mantle is thought to be responsible for secreting the protein component of the shell. This study employs molecular approaches to determine the spatial expression of genes within the mantle tissue to further the elucidation of the shell biomineralization. RESULTS: A microarray platform was custom generated (PmaxArray 1.0) from the pearl oyster Pinctada maxima. PmaxArray 1.0 consists of 4992 expressed sequence tags (ESTs) originating from mantle tissue. This microarray was used to analyze the spatial expression of ESTs throughout the mantle organ. The mantle was dissected into five discrete regions and analyzed for differential gene expression with PmaxArray 1.0. Over 2000 ESTs were determined to be differentially expressed among the tissue sections, identifying five major expression regions. In situ hybridization validated and further localized the expression for a subset of these ESTs. Comparative sequence similarity analysis of these ESTs revealed a number of the transcripts were novel while others showed significant sequence similarities to previously characterized shell related genes. CONCLUSIONS: This investigation has mapped the spatial distribution for over 2000 ESTs present on PmaxArray 1.0 with reference to specific locations of the mantle. Expression profile clusters have indicated at least five unique functioning zones in the mantle. Three of these zones are likely involved in shell related activities including formation of nacre, periostracum and calcitic prismatic microstructure. A number of novel and known transcripts have been identified from these clusters. The development of PmaxArray 1.0, and the spatial map of its ESTs expression in the mantle has begun characterizing the molecular mechanisms linking the organics and inorganics of the molluscan shell.