Carolyn S. Bentivegna

Effects of tributyltin on medaka (Oryzias latipes) embryos at different stages of development

Potential mechanisms of action for the toxicity of tributyltin (TBT) were studied in the freshwater fish embryos of medaka (Oryzias latipes). Toxic concentrations of TBT have been found in estuaries and freshwater systems, presumably due to their use as biocides in boat, antifoulant paints and in industry for plastics production. Medaka embryos were exposed to a single concentration of TBT at developmental stages that corresponded to the formation of structures and/or organs which might be potential targets. Times of exposure included day 0, oviposition, day 3, completion of somite formation, and day 5, liver formation. Endpoints for evaluating toxicity were acute embryo lethality (96 h), rate of embryo development, hatching success, gross abnormalities, as well as hatchling eye diameter and number of somites. The clear chorion of medaka embryos allowed staging and in ova observations. Results showed that the acute toxicity of TBT was stage related. The 96 h LC50 (LC50: lowest concentration to cause 50% lethality in the test population) for embryos exposed on day 0 was 159 nM, which was lower than that for days 3 and 5, 360 and 340 nM, respectively. Subchronic endpoints showed that toxicity was concentration related and that embryos exposed on day 0 were more sensitive than those exposed on days 3 and 5. Lowest observable effect levels (LOELs) for hatching success were 36 nM for day 0 and 143 nM for days 3 and 5. LOELs for the combined effects of hatching success and gross abnormalities were 36 nM for day 0 and 71 nM for days 3 and 5. Developmental rate was slowed by TBT in a concentration-related manner; however, embryos treated with 36 and 71 nM were able to recover and hatch at the same time as controls. Types of gross abnormalities were similar regardless of day of exposure and consisted of tails bent at the tip, curled, and/or shortened. These abnormalities corresponded with statistically significant reductions in numbers of somites in all three age groups exposed to 71 nM (P<0.05). Although day 0 embryos were the most sensitive, the similar abnormalities for all 3 days of exposure indicated that TBTs toxicity was not due to effects on an age-dependent target but one present throughout embryo development.

Comparison of De Novo Transcriptome Assemblers and k-mer Strategies Using the Killifish, Fundulus heteroclitus.

Background De novo assembly of non-model organism’s transcriptomes has recently been on the rise in concert with the number of de novo transcriptome assembly software programs. There is a knowledge gap as to what assembler software or k-mer strategy is best for construction of an optimal de novo assembly. Additionally, there is a lack of consensus on which evaluation metrics should be used to assess the quality of de novo transcriptome assemblies. Result Six different assembly strategies were evaluated from four different assemblers. The Trinity assembly was used in its default 25 single k-mer value while Bridger, Oases, and SOAPdenovo-Trans were performed with multiple k-mer strategies. Bridger, Oases, and SOAPdenovo-Trans used a small multiple k-mer (SMK) strategy consisting of the k-mer lengths of 21, 25, 27, 29, 31, and 33. Additionally, Oases and SOAPdenovo-Trans were performed using a large multiple k-mer (LMK) strategy consisting of k-mer lengths of 25, 35, 45, 55, 65, 75, and 85. Eleven metrics were used to evaluate each assembly strategy including three genome related evaluation metrics (contig number, N50 length, Contigs >1 kb, reads) and eight transcriptome evaluation metrics (mapped back to transcripts (RMBT), number of full length transcripts, number of open reading frames, Detonate RSEM-EVAL score, and percent alignment to the southern platyfish, Amazon molly, BUSCO and CEGMA databases). The assembly strategy that performed the best, that is it was within the top three of each evaluation metric, was the Bridger assembly (10 of 11) followed by the Oases SMK assembly (8 of 11), the Oases LMK assembly (6 of 11), the Trinity assembly (4 of 11), the SOAP LMK assembly (4 of 11), and the SOAP SMK assembly (3 of 11). Conclusion This study provides an in-depth multi k-mer strategy investigation concluding that the assembler itself had a greater impact than k-mer size regardless of the strategy employed. Additionally, the comprehensive performance transcriptome evaluation metrics utilized in this study identified the need for choosing metrics centered on user defined research goals. Based on the evaluation metrics performed, the Bridger assembly was able to construct the best assembly of the testis transcriptome in Fundulus heteroclitus.

Detection of polycyclic aromatic hydrocarbons (PAHs) in raw menhaden fish oil using fluorescence spectroscopy: Method development

Raw menhaden fish oil was developed for biomonitoring polycyclic aromatic hydrocarbons (PAHs) using fluorescence spectroscopy. Menhaden (Genus Brevoortia) were collected in 2010 and/or 2011 from Delaware Bay, New Jersey, USA; James River, Virginia, USA; Vermillion Bay, Louisiana, USA (VBLA); and Barataria Bay, Louisiana, USA (BBLA). Barataria Bay, Louisiana received heavy oiling from the Deepwater Horizon oil spill. Method development included determining optimal wavelengths for PAH detection, fish oil matrix interferences, and influence of solvent concentration on extraction. Results showed that some fish oils contained high molecular weight PAH-like compounds in addition to other fluorescent compounds such as albumin and vitamin A and vitamin E. None of these naturally occurring compounds interfered with detection of high molecular weight PAHs. However, data suggested that the lipid component of fish oil was altering fluorescence spectra by supporting the formation of PAH excimers. For example, the most intense excitation wavelength for hydroxypyrene shifted from Ex285/Em430 to Ex340/Em430. Comparison of Deepwater Horizon crude oil and fish oil spectra indicated that some fish oils contained crude oil-like PAHs. Using wavelengths of Ex360/Em430, fish oil concentrations were calculated as 3.92 μg/g, 0.61 μg/g, and 0.14 μg/g for a Delaware Bay sample, BBLA 2011, and VBLA 2011, respectively. Overall, these results supported using menhaden fish oil to track PAH exposures spatially and temporally.

Chemical and histological comparisons between Brevoortia sp. (menhaden) collected in fall 2010 from Barataria Bay, LA and Delaware Bay, NJ following the DeepWater Horizon (DWH) oil spill.

Body burdens of PAHs were compared to histological effects in menhaden (Family: Clupeidae, Genus: Brevoortia) collected in fall 2010 from Barataria Bay, LA (BBLA) and Delaware Bay, NJ (DBNJ). Barataria Bay was heavily oiled during the DeepWater Horizon (DWH) oil spill, while Delaware Bay although urbanized had no reported recent oil spills. GCMS analyses of pre-spill 2009, BBLA and DBNJ fish found predominantly C2/C3 phenanthrene (1.28-6.52 ng/mg). However, BBLA also contained five higher molecular weight PAHs (0.06-0.34 ng/mg DW). Fluorescent aromatic compound spectroscopy (FACS) of gastrointestinal (GI) tract tissue showed statistically higher levels of hydroxypyrene-like PAHs in DBNJ than BBLA fish. Histopathologic lesions were more prevalent in BBLA than DBNJ fish. The lesion prevalence (gill, trunk kidney, epidermis, stomach) in the BBLA menhaden were significantly higher and more severe than observed in the DBNJ menhaden. Reversible lesions included gill lamellar hyperplasia, adhesions, edema, and epidermal hyperplasia. The increased pigmented macrophage centers were indicative of activated macrophages responding to connective tissue damage or other antigens. The liver hepatic necrosis and renal tissue mineralization may well have undergone repair, but damage to the kidney nephrons and hepatic/biliary regions of the liver would be slower to resolve and apparently remained after elimination of PAHs. Therefore, a direct cause and effect between DWH oil spill and increased lesion prevalence in BBLA menhaden could not be established.

Advancing monosaccharides as biomarkers: Part II. Effects of starvation and cadmium in Chironomus riparius as detected by fluorophore-assisted carbohydrate-electrophoresis.

Saccharides were evaluated as biomarkers for cadmium (Cd) and starvation using fluorophore-assisted carbohydrate-electrophoresis (FACE) in 4th instar Chironomus riparius. FACE allowed different types of saccharides in whole larval homogenate to be analyzed simultaneously and in parallel with other larval samples. Larval homogenates showed seven principle bands labeled A, B, C, D, E, F and G. Previous work found that the migration patterns of bands A, C, D and F matched those of ribose, glucose, galactose and fructose, respectively. Four of the bands, B, C, E and G were generated from glucose-based mono, oligo, and polysaccharides. Band B was primarily derived from glucose and band E from glycogen. Experiments (0-72 h) with starved larvae showed a time dependent reduction in bands B and E that was statistically significant at 72 h. Experiments with Cd (0.01-1000 microM) showed a concentration and time dependent reduction in band E with a LOEL of 1 microM and NOEL of 0.01 microM at 48 h. The LOEL was 0.014% of the 48 h LC50. Significant reduction of band E only occurred in fed larvae indicating that food was an important route of exposure. Reductions in saccharides were independent of larval weight loss at 48 h. This suggested that major changes in saccharides were not due to weight loss but metabolic stress in the presence of Cd.

Excitation-emission matrix scan analysis of raw fish oil from coastal New Jersey menhaden collected before and after Hurricane Sandy.

The impact of Hurricane Sandy (October 29, 2012) on PAH exposure was investigated in adult Atlantic menhaden (Brevoortia tyrannus) collected along the NJ coast. Collections were made in August, September and/or October of 2011, 2012 and 2013. PAHs were monitored in raw fish oil using excitation-emission matrix (EEM) spectroscopy. Results showed that raw fish oils had relatively high levels of high molecular weight, PAH-like compounds (173 to 24,421ng/mL) compared to values reported for bile in other species. EEM profiles resembled that of crude oil and excluded matrix interference by some common biological molecules that also fluoresce. Concentrations and EEM profiles varied by collection; however, collection ship, month, year and fish size did not account for the data. Replicates showed that fish from the same catch had similar PAH exposure. Overall, Hurricane Sandy did not alter body burdens of PAHs in raw fish oil of menhaden.

Advancing monosaccharides as biomarkers: part I. Development of fluorophore-assisted carbohydrate-electrophoresis in Chironomus riparius

Fluorophore-assisted carbohydrate-electrophoresis (FACE) was developed as a bioassay for environmental stressors in larval Chironomus riparius. This quantitative technique involved acid hydrolysis and 2-aminoacridone labeling of monosaccharides followed by carbohydrate gel electrophoresis. Methods for carbohydrate isolation from whole tissue homogenates as well as migration distances for 23 different monosaccharides and 4 disaccharides were established. Sensitivity of the technique (5 microg/ml saccharide) exceeded those of other detection methods. Results showed seven distinct bands in larvae. Four migrated distances similar to those of ribose, glucose, galactose, and fructose. Three proved to be alternative reaction products (ARP). Experiments determined that two of the ARPs were primarily from glucose and one was from glycogen. FACE allowed different saccharides from multiple larval samples to be analyzed in parallel. Effects of toxicants and diet on bioenergetics could be studied using this technique.

Quantitative Analysis of Polycyclic Aromatic Hydrocarbons at Part Per Billion Levels in Fish Oil by Headspace Solid-Phase Microextraction and Gas Chromatography-Mass Spectrometry (HS-SPME–GC–MS)

Headspace solid-phase microextraction (HS-SPME) was used in combination with gas chromatography-mass spectrometry (GC-MS) for the analysis of polycyclic aromatic hydrocarbons (PAH) at part per billion levels in fish oil samples collected from menhaden fish. The method was initially developed using fish oil from capsules spiked with a standard PAH mixture. The final HS-SPME-GC-MS method presented a linear range from 3 to 1,500 ng/g, with precision for most analytes <10% relative standard deviation. The limits of detection varied from 1 to 7 ng/g depending on the analyte. Real sample analysis was done on menhaden fish oil extracted from fish collected off the coasts of New Jersey and Louisiana. Naphthalene, fluorene, fluoranthene, pyrene, anthracene were detected at low levels of 70-180 ng/g in the real samples. The concentrations of PAHs detected in the real samples were well below established levels of concern for PAHs in finfish.

De Novo Assembly and Analysis of the Testes Transcriptome from the Menhaden, Bervoortia tyrannus

Background: The menhaden, Bervoortia tyrannus, is one of the most important fish within the oceanic ecosystem and a crucial species supporting major fisheries along the Atlantic and Gulf coasts. However, little is known about menhaden from a genetic aspect. The objective of this project is to apply high throughput sequencing to the testes of menhaden to provide the genetic tools required to further study their population dynamics Result: We applied Illumina Next Seq 500 technology to two different testes and used Velvet/Oases to perform the de novo assembly that resulted in the construction of 254,462 contigs. Applying BLASTX to annotate the contigs against the non-redundant protein database resulted in 46.89% matches. To validate the accuracy of the assembly, the reads were mapped back to transcripts (RMBT) with a percentage of 87.83%. To experimentally verify the assembly results, primers were designed based on the assembled transcriptome, and PCR products were verified by Sanger sequencing. To enhance the functional categorization of the annotated contigs, they were further classified using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Clusters of Orthologous Groups (COG) databases. Conclusion: This research is the first report of an annotated overview of the testes transcriptomes in B. tyrannus, resulting in the most comprehensive genetic resource available for menhaden to date. This work can provide a repository for future gene expression analysis, functional studies, and reproductive investigations in B. tyrannus. This will enhance the capabilities of population monitoring and can be used as a benchmark in comparative studies in other fish models. Overall, this research will open new opportunities and bring new insights for researchers studying B. tyrannus.