Characterization of the differential expressed genes and transcriptomic pathway analysis in the liver of sub-adult red drum (Sciaenops ocellatus) exposed to Deepwater Horizon chemically dispersed oil.

The Deepwater Horizon blowout resulted in the second-largest quantity of chemical dispersants used as a countermeasure for an open water oil spill in the Gulf of Mexico. Of which, the efficacy of dispersant as a mitigation strategy and its toxic effects on aquatic fauna remains controversial. To enhance our understanding of potential sub-lethal effects of exposure to chemically dispersed-oil, sub-adult red drum (Sciaenops ocellatus) were continuously exposed to a Corexit 9500: DWH crude oil chemically enhanced water accommodated fraction (CEWAF) for 3-days and transcriptomic responses were assessed in the liver. Differential expressed gene (DEG) analysis demonstrated that 63 genes were significantly impacted in the CEWAF exposed fish. Of these, 37 were upregulated and 26 downregulated. The upregulated genes were primarily involved in metabolism and oxidative stress, whereas several immune genes were downregulated. Quantitative real-time RT-PCR further confirmed upregulation of cytochrome P450 and glutathione S-transferase, along with downregulation of fucolectin 2 and chemokine C-C motif ligand 20. Ingenuity Pathway Analysis (IPA) predicted 120 pathways significantly altered in the CEWAF exposed red drum. The aryl hydrocarbon receptor pathway was significantly activated, while pathways associated with immune and cellular homeostasis were primarily suppressed. The results of this study indicate that CEWAF exposure significantly affects gene expression and alters signaling of biological pathways important in detoxification, immunity, and normal cellular physiology, which can have potential consequences on organismal fitness.

Shifts in the Skin-Associated Microbiota of Hatchery-Reared Common Snook Centropomus undecimalis During Acclimation to the Wild.

The skin-associated microbiota of fish competes against pathogens for space and nutrients, preventing colonization by harmful bacteria encountered during environmental transitions such as those faced during stock enhancement. Thus, alterations in bacterial community structure during release of cultured fish have important implications for health of these individuals. This study investigated microbiota structure during acclimation of juvenile hatchery-reared common snook Centropomus undecimalis to the wild by comparing skin-associated microflora among snook in captivity, after 48 h of acclimation at release sites, and from the wild. After two days of acclimation, the microbiota of hatchery-reared snook mirrored that observed on wild snook. Relative abundances of potential pathogens were higher in captive fish, whereas acclimated and wild fish harbored bacterial taxa influenced by geographical factors and water quality at release sites. Predicted microbiota function of acclimated and wild fish showed higher production of protective amino acids and antimicrobials, identifying a mechanism for microbial supplementation of the immune defense of these fish. The two-day transition to wild-type microbiota suggests a temporal scale of hours associated with bacterial succession indicating that the microbiota, whose structure is vital to fish health, aids in acclimation of fish to new environments during stock enhancement efforts.

Nonlethal Biomarkers of Oxidative Stress in Oiled Sediment Exposed Southern Flounder (Paralichthys lethostigma): Utility for Field-Base Monitoring Exposure and Potential Recovery.

The Deepwater Horizon (DWH) blowout resulted in the deposition of toxic polycyclic aromatic hydrocarbons (PAHs), in the coastal sediments of the Gulf of Mexico. The immediate effects on an ecosystem from an oil spill are clearly recognizable, however the long-term chronic effects and recovery after a spill are still not well understood. Current methodologies for biomonitoring wild populations are invasive and mostly lethal. Here, two potential nonlethal biomonitoring tools for the assessment of PAH toxicity and induced biological alterations in the field, were identified using laboratory-validated methods. In this study, subadult southern flounder (Paralichthys lethostigma) were chronically exposed to DWH surrogate oiled sediments for 35 days; a subset of these exposed flounder were then provided a clean nonexposure period to ascertain the utility of selected biomarkers to monitor recovery post exposure. After chronic exposure, there was an increase in gene expression of cytochrome P450 1A but not glutathione S-transferase. There was also a notable imbalance of oxidants to antioxidants, measured as reduced glutathione, oxidized glutathione, and their ratio in the blood. Evidence of subsequent oxidative damage due to chronic exposure was found through lipid peroxidation and DNA damage assessments of liver, gill, and blood.

Health Characteristics and Blood Bacterial Assemblages of Healthy Captive Red Drum: Implications for Aquaculture and Fish Health Management.

The newly emerging tissue microbiota hypothesis suggests that bacteria found in blood and tissues play a role in host health, as these bacterial communities have been associated with various noncommunicable diseases such as obesity, liver disease, and cardiovascular disease. Numerous reports have identified bacteria in the blood of healthy finfish, indicating bacteremia may not always indicate disease. Current research priorities in aquaculture include the development of technologies and practices that will allow for an effective reduction in antibiotic use for the prevention and treatment of disease. Overall, a better understanding of fish health is needed, particularly among species selected for commercial-scale production. This study investigated blood characteristics of cultured Red Drum Sciaenops ocellatus with the tissue microbiota hypothesis in mind. Bacterial assemblages within the blood were characterized using next-generation sequencing and compared with other various blood characteristics, including innate immune function enzymes, between two fish cohorts reared in aquaculture. A total of 137 prokaryotic operational taxonomic units (OTUs) were identified from the blood of Red Drum. Microbiota diversity and structure varied greatly among individuals, for which the number of OTUs ranged from 4 to 58; however, predicted metagenomic function was highly similar between individuals and was dominated by the metabolism of carbohydrates and amino acids and membrane transport. Communities were dominated by Proteobacteria, followed by Bacteroidetes, Firmicutes, and Actinobacteria. The most commonly identified genera included Acinetobacter, Bacillus, Corynebacterium, and Pseudomonas. Three genera previously identified as containing marine fish pathogens were detected: Corynebacterium, Pantoea, and Chryseobacterium. A subset of bacterial OTUs were positively correlated with superoxide dismutase activity and negatively correlated with lysozyme activity, indicating a relationship between blood microbiota and the innate immune system. The results of this study provide further evidence for the tissue microbiota hypothesis and demonstrate the potential for these bacterial communities to be linked to immunological characteristics often used as biomarkers for fish health.

Microbiota of wild-caught Red Snapper Lutjanus campechanus.

BACKGROUND: The microbiota plays an essential role in host health, particularly through competition with opportunistic pathogens. Changes in total bacterial load and microbiota structure can indicate early stages of disease, and information on the composition of bacterial communities is essential to understanding fish health. Although Red Snapper (Lutjanus campechanus) is an economically important species in recreational fisheries and a primary aquaculture candidate, no information is available on the microbial communities of this species. The aim of this study was to survey the microbiota of apparently healthy, wild-caught Red Snapper from the Gulf of Mexico. Sampled Red Snapper showed no physical signs of disease. Tissues that are either primary entry routes for pathogens (feces, gill) or essential to disease diagnosis (blood) were sampled. Bacteria were enumerated using culture-based techniques and characterized by pyrosequencing. RESULTS: Aerobic counts of feces and gill samples were 107 and 104 CFU g-1, respectively. All individuals had positive blood cultures with counts up to 23 CFU g-1. Gammaproteobacteria dominated the microbiota of all sample types, including the genera Pseudoalteromonas and Photobacterium in feces and Pseudomonas in blood and gill. Gill samples were also dominated by Vibrio while blood samples had high abundances of Nevskia. High variability in microbiota composition was observed between individuals, with percent differences in community composition ranging from 6 to 76 % in feces, 10 to 58 % in gill, and 52 to 64 % in blood. CONCLUSIONS: This study provides the first characterization of the microbiota of the economically significant Red Snapper via pyrosequencing. Its role in fish health highlights the importance of understanding microbiota composition for future work on disease prevention using microbial manipulation.

Dispersed Crude Oil Induces Dysbiosis in the Red Snapper Lutjanus campechanus External Microbiota.

The fish external microbiota competitively excludes primary pathogens and prevents the proliferation of opportunists. A shift from healthy microbiota composition, known as dysbiosis, may be triggered by environmental stressors and increases host susceptibility to disease. The Deepwater Horizon (DWH) oil spill was a significant stressor event in the Gulf of Mexico. Despite anecdotal reports of skin lesions on fishes following the oil spill, little information is available on the impact of dispersed oil on the fish external microbiota. In this study, juvenile red snapper (Lutjanus campechanus) were exposed to a chemically enhanced water-accommodated fraction (CEWAF) of Corexit 9500/DWH oil (CEWAF) and/or the bacterial pathogen Vibrio anguillarum in treatments designed to detect changes in and recovery of the external microbiota. In fish chronically exposed to CEWAF, immunoglobulin M (IgM) expression significantly decreased between 2 and 4 weeks of exposure, coinciding with elevated liver total polycyclic aromatic hydrocarbons (PAHs). Dysbiosis was detected on fish chronically exposed to CEWAF compared to seawater controls, and addition of a pathogen challenge altered the final microbiota composition. Dysbiosis was prevented by returning fish to clean seawater for 21 days after 1 week of CEWAF exposure. Four fish exhibited lesions during the trial, all of which were exposed to CEWAF but not all of which were exposed to V. anguillarum. This study indicates that month-long exposure to dispersed oil leads to dysbiosis in the external microbiota. As the microbiota is vital to host health, these effects should be considered when determining the total impacts of pollutants in aquatic ecosystems. IMPORTANCE Fish skin is an immunologically active tissue. It harbors a complex community of microorganisms vital to host homeostasis as, in healthy fish, they competitively exclude pathogens found in the surrounding aquatic environment. Crude oil exposure results in immunosuppression in marine animals, altering the relationship between the host and its microbial community. An alteration of the healthy microbiota, a condition known as dysbiosis, increases host susceptibility to pathogens. Despite reports of external lesions on fishes following the DWH oil spill and the importance of the external microbiota to fish health, there is little information on the effect of dispersed oil on the external microbiota of fishes. This research provides insight into the impact of a stressor event such as an oil spill on dysbiosis and enhances understanding of long-term sublethal effects of exposure to aid in regulatory decisions for protecting fish populations during recovery.

Effect of copper sulfate on the external microbiota of adult common snook (Centropomus undecimalis).

BACKGROUND: The environment exerts a strong influence on the fish external microbiota, with lower diversity and increased abundances of opportunistic bacterial groups characterizing cultured fish compared to their wild counterparts. Deviation from a healthy external microbiota structure has been associated with increased susceptibility to bacterial pathogens. Treatment of wild-caught broodstock with copper sulfate for the removal of external parasites is a common aquaculture practice. Despite the microbiota's importance to fish health, the effects of copper sulfate on mucosal bacterial communities and their ability to recover following this chemical treatment have not been examined. The skin microbiota of adult common snook was characterized from wild individuals (Wild), and wild-caught fish maintained in recirculating aquaculture systems (RAS) immediately following a month-long copper sulfate treatment (Captive-1), and then two-weeks (Captive-2) and 2 years (Captive-3) after cessation of copper treatment. RESULTS: The skin microbiota of wild fish were characterized by high diversity and taxa including Synechocococcus, SAR11, and a member of the Roseobacter clade. Bacterial diversity decreased in Captive individuals during the 2-year sampling period. Captive fish harbored greater abundances of Firmicutes, which may reflect glycan differences between aquaculture and natural feeds. Bacterial taxa with copper resistance mechanisms and indicative of metal contamination were enriched in Captive-1 and Captive-2 fish. Vibrionaceae were dominant in Captive fish, particularly immediately and 2 weeks following copper treatment. Based on our observations and previous literature, our results suggest putatively beneficial taxa amass over time in captivity. Within 2 years, Captive individuals harbored Bacillus which contains numerous probiotic candidates and the complex carbon degraders of the family Saprospiraceae. Predicted butanoate metabolism exceeded that of Wild fish, and its reported roles in immunity and energy provision suggest a prebiotic effect for fishes. CONCLUSIONS: The mucosal microbiota contains bacterial taxa that may act as bioindicators of environmental pollution. Increases in mutualistic groups indicate a return to a beneficial skin microbiota following copper sulfate treatment. Our data also suggests that vastly different taxa, influenced by environmental conditions, can be associated with adult fish without noticeable health impairment, perhaps due to establishment of various mutualists to maintain fish mucosal health.

Characterizing transcriptomic responses of southern flounder (Paralichthys lethostigma) chronically exposed to Deepwater Horizon oiled sediments.

To obtain a deeper understanding of the transcriptomic responses to oil in southern flounder (Paralichthys lethostigma), we performed quantitative PCR and RNA sequencing on liver and gill tissue after a chronic exposure (35 days) to Deepwater Horizon crude oiled sediment and after a 30-day recovery period. We wanted to understand which specific genes are differentially expressed in liver and gill tissues directly after oiled sediment exposure and with the addition of a recovery period. Furthermore, we wanted to examine specific enriched pathways in these two tissues to determine the impact of exposure with and without a recovery period on biological processes (e.g. immune function). Liver and gill tissues were chosen because they represent two distinct organs that are highly important to consider when examining the impacts of oiled sediment exposure. The liver is the classic detoxification organ, while the gill is in direct contact with sediment in benthic fishes. Examination of these two tissues, therefore, generates a broad understanding of the transcriptomic consequences of oil exposure across an organism. Gene expression for interleukin 8 (il8) and interleukin 1B (il1ß) was significantly increased versus control measurements for fish exposed to oiled sediments for 35 days in gill tissue. Hierarchical clustering of gene expression showed that tissue type was the main driver of gene expression (rather than treatment). The inclusion of a 30-day post-exposure recovery period showed a return of il8 and il1ß gene expression in the gill to baseline expression levels. However, the recovery period increased the number of differentially expressed genes and significantly affected canonical pathways in both tissue types. Pathways related to cholesterol biosynthesis were significantly suppressed in oil-exposed flounder with a recovery period, but not in the exposed flounder without a recovery period. At the end of the exposure, 17 pathways were significantly affected in the gill, including thyroid hormone metabolism-related pathways, which were the most influenced. Liver tissue from the recovered fish had the greatest number of enriched pathways for any tissue or time point (187). Cellular and humoral immune response pathways were considerably impacted in the liver after the recovery period, suggesting that the immune system was attempting to respond to potential damage caused from the chronic oil exposure. Our results demonstrate that liver and gill tissues from southern flounder were differentially altered by Deepwater Horizon oiled sediment exposure and that a 30-day recovery period after exposure substantially shifted gene expression and canonical pathway profiles.

Benefits of a Bacillus probiotic to larval fish survival and transport stress resistance.

The need for sustainable bacterial management approaches in aquaculture is crucial for advancement of the industry. Probiotics are a promising strategy as evidenced by benefits demonstrated in intensive larviculture of various marine fish species. In this study we investigate the effects of a mixed Bacillus species (B. licheniformis and B. amyloliquefaciens) probiotic on rearing of larval common snook (Centropomus undecimalis). Experimental treatments included (1) probiotics supplemented to the water and live feed, (2) probiotics supplemented to the water only, and (3) no probiotic controls. Data from two separate trials indicated up to 2.5 times higher survival with probiotic addition, as well as 20% higher survival 7 days following a transport event. These benefits were not explained by faster growth, measured water quality parameters, or innate immune enzyme activities. Microbiota analysis indicated the importance of system stabilization prior to larval stocking to improve rearing success and probiotic performance. ied Potential probiotic benefits include accelerated gastrointestinal tract development, enhanced immunity, inhibition of opportunistic bacteria, and improvements to water quality parameters. Results suggest this probiotic should be tested in other marine fish species in order to reduce larval rearing bottlenecks.