Sharon E. Hook

The role of biomarkers in the assessment of aquatic ecosystem health.

Ensuring the health of aquatic ecosystems and identifying species at risk from the detrimental effects of environmental contaminants can be facilitated by integrating analytical chemical analysis with carefully selected biological endpoints measured in tissues of species of concern. These biological endpoints include molecular, biochemical, and physiological markers (i.e., biomarkers) that when integrated, can clarify issues of contaminant bioavailability, bioaccumulation, and ecological effects while enabling a better understanding of the effects of nonchemical stressors. In the case of contaminant stressors, an understanding of chemical modes of toxicity can be incorporated with diagnostic markers of aquatic animal physiology to help understand the health status of aquatic organisms in the field. Furthermore, new approaches in functional genomics and bioinformatics can help discriminate individual chemicals, or groups of chemicals among complex mixtures that may contribute to adverse biological effects. Although the use of biomarkers is not a new paradigm, such approaches have been underused in the context of ecological risk assessment and natural resource damage assessment. From a regulatory standpoint, these approaches can help better assess the complex effects from coastal development activities to assessing ecosystem integrity pre- and post development or site remediation.

Comparison of toxicity and transcriptomic profiles in a diatom exposed to oil, dispersants, dispersed oil

Dispersants are commonly used to mitigate the impact of oil spills, however, the ecological cost associated with their use is uncertain. The toxicity of weathered oil, dispersed weathered oil, and the hydrocarbon-based dispersant Slickgone NS(®), to the diatom Phaeodactylum tricornutum has been examined using standardized toxicity tests. The assumption that most toxicity occurs via narcosis was tested by measuring membrane damage in diatoms after exposure to one of the petroleum products. The mode of toxic action was determined using microarray-based gene expression profiling in diatoms after exposure to one of the petroleum products. The diatoms were found to be much more sensitive to dispersants than to the water accommodated fraction (WAF), and more sensitive to the chemically enhanced WAF (CEWAF) than to either the WAF itself or the dispersants. Exposure to dispersants and CEWAF caused membrane damage, while exposure to WAF did not. The gene expression profiles resulting from exposure to all three petroleum mixtures were highly similar, suggesting a similar mode of action for these compounds. The observed toxicity bore no relationship to PAH concentrations in the water column or to total petroleum hydrocarbon (TPH), suggesting that an undescribed component of the oil was causing toxicity. Taken together, these results suggest that the use of dispersants to clean up oil spills will dramatically increase the oil toxicity to diatoms, and may have implications for ecological processes such as the timing of blooms necessary for recruitment.

Estrogen receptor mRNA expression patterns in the liver and ovary of female rainbow trout over a complete reproductive cycle.

Estrogens are critical hormones involved in reproduction and need to bind to estrogen receptors in target organs for biological activity. Fishes have two distinct estrogen receptor subtypes, alpha (α) and beta (β), with variable combinations of additional isoforms of each subtype dependent on the history of genome duplication within a taxon. The comparative expression patterns of estrogen receptor isoforms during the female reproductive cycle will provide important insights into the unique function and importance of each. The purpose of this study was to measure the mRNAs for the four estrogen receptor isoforms (erα1, erα2, erβ1, erβ2) in the liver and ovary of adult, female rainbow trout over the course of an annual reproductive cycle. The expression of estrogen receptor mRNA isoforms was measured by quantitative real-time RT-PCR. Several reproductive indices (gonadosomatic index, maximum oocyte diameter, plasma estradiol-17β, plasma vitellogenin, and ovulation) were also quantified for comparison and used in a correlation analysis to examine any inter-relationships. Of the four isoforms, the expression of erα1 was highest in the liver, and had a significant positive correlation with liver erβ1 expression. Liver expression of erα2 mRNA was the lowest, but showed a significant positive correlation with maximum oocyte diameter in the ovary. The pattern of the erβ isoforms in liver was one of initially elevated mRNA expression followed by a gradual decrease as reproductive development proceeded. In the ovary the erβ1 isoform had the highest mRNA expression of all estrogen receptor isoforms, at the beginning of the reproductive cycle, but then decreased afterward. Both ovarian erβ isoforms had a significant positive correlation with one another. In contrast, erα2 mRNA expression showed a high maximum level in the ovary near the end of the cycle along with a significant positive correlation with plasma estradiol-17β levels; the highest gonadosomatic indices, maximum oocyte diameter, and vitellogenin levels occurred then too.

454 pyrosequencing-based analysis of gene expression profiles in the amphipod Melita plumulosa: transcriptome assembly and toxicant induced changes.

Next generation sequencing using Roches 454 pyrosequencing platform can be used to generate genomic information for non-model organisms, although there are bioinformatic challenges associated with these studies. These challenges are compounded by a lack of a standardized protocol to either assemble data or to evaluate the quality of a de novo transcriptome. This study presents an assembly of the control and toxicant responsive transcriptome of Melita plumulosa, an Australian amphipod commonly used in ecotoxicological studies. RNA was harvested from control amphipods, juvenile amphipods, and from amphipods exposed to either metal or diesel contaminated sediments. This RNA was used as the basis for a 454 based transcriptome sequencing effort. Sequencing generated 1.3 million reads from control, juvenile, metal-exposed and diesel-exposed amphipods. Different read filtering and assembly protocols were evaluated to generate an assembly that (i) had an optimal number of contigs; (ii) had long contigs; (iii) contained a suitable representation of conserved genes; and (iv) had long ortholog alignment lengths relative to the length of each contig. A final assembly, generated using fixed-length trimming based on the sequence quality scores, followed by assembly using the MIRA algorithm, produced the best results. The 26,625 contigs generated via this approach were annotated using Blast2GO, and the differential expression between treatments and control was determined by mapping with BWA followed by DESeq. Although the mapping generated low coverage, many differentially expressed contigs, including some with known developmental or toxicological function, were identified. This study demonstrated that 454 pyrosequencing is an effective means of generating reference transcriptome information for organisms, such as the amphipod M. plumulosa, that have no genomic information available in databases or in closely related sequenced species. It also demonstrated how optimization of read filtering protocols and assembly approaches changes the utility of results obtained from next generation sequencing studies, and establishes criteria to determine the quality of a de novo assembly in species lacking a reference genome. This new transcriptomic knowledge provides the genomic foundation for the creation of microarray and qPCR assays, serving as a reference transcriptome in future RNAseq studies, and allowing both the biology and ecotoxicology of this organism to be better understood. This approach will allow genomics-based methodology to be applied to a wider range of environmentally relevant species.

Temporal patterns in the transcriptomic response of rainbow trout, Oncorhynchus mykiss, to crude oil

Time is often not characterized as a variable in ecotoxicogenomic studies. In this study, temporal changes in gene expression were determined during exposure to crude oil and a subsequent recovery period. Juvenile rainbow trout, Oncorhynchus mykiss, were exposed for 96 h to the water accommodated fractions of 0.4, 2 or 10 mgl(-1) crude oil loadings. Following 96 h of exposure, fish were transferred to recovery tanks. Gill and liver samples were collected after 24 and 96 h of exposure, and after 96 h of recovery for RNA extraction and microarray analysis. Fluorescently labeled cDNA was hybridized against matched controls, using salmonid cDNA arrays. Each exposure scenario generated unique patterns of altered gene expression. More genes responded to crude oil in the gill than in the liver. In the gill, 1137 genes had altered expression at 24 h, 2003 genes had altered expression levels at 96 h of exposure, yet by 96 h of recovery, no genes were significantly altered in expression. In the liver at 10 mgl(-1), only five genes were changed at 24 h, yet 192 genes had altered expression after 96 h recovery. At 2 mgl(-1) in the liver, many genes had altered regulation at all three time points. The 0.4 mgl(-1) loading also showed 289 genes upregulated at 24 h after exposure. The Gene Ontology terms associated with altered expression in the liver suggested that the processes of protein synthesis, xenobiotic metabolism, and oxidoreductase activity were altered. The concentration-responsive expression profile of cytochrome P450 1A, a biomarker for oil exposure, did not predict the majority of gene expression profiles in any tissue or dose, since direct relationships with dose were not observed for most genes. While the genes and their associated functions agree with known modes of toxic action for crude oil, the gene lists obtained do not match our previously published work, presumably due to array analysis procedures. These results demonstrate that changes in gene expression with time and dose may be complicated, and should be characterized in controlled laboratory settings before attempts are made to interpret responses in field-collected organisms. Further, processes for analyzing microarray data need to be developed such that standardized gene lists are developed, or that analysis does not rely on lists of significantly altered genes before arrays can be further evaluated as a monitoring tool.

Hypotheses concerning the decline and poor recovery of Pacific herring in Prince William Sound, Alaska

This paper updates previous reviews of the 1993 stock decline of Pacific herring (Clupea pallasi) in Prince William Sound, Alaska, and focuses on hypotheses about subsequent poor recovery. Recent age structured assessment modeling with covariate analysis indicates that the population dynamics of the sound’s herring are influenced by oceanic factors, nutrition, and, most substantially, hatchery releases of juvenile pink salmon. For the 1993 decline, poor nutrition remains the most probable cause with disease a secondary response. Concerning poor recovery, we examined 16 potential factors and found three to be causal: oceanic factors, poor nutrition, and hatchery releases of juvenile pink salmon. Absences of strong year classes at both Sitka and Prince William Sound after 1993 indicate the action of large-scale ocean processes. Beyond regional-scale environmental factors, two factors specific to the sound influence the population dynamics of herring and are likely impeding recovery. First, pink salmon fry releases have increased to about 600 million annually and may disrupt feeding in young herring, which require adequate nutrition for growth and overwintering survival. Juvenile pink salmon and age-1 herring co-occur in nearshore areas of bays in late spring and summer, and available data on dietary overlap indicates potential competition between the age-1 juvenile herring and juvenile pink salmon. Field studies demonstrate that juvenile herring reduce food intake substantially in the presence of juvenile pink salmon. Second, overwintering humpback whales may consume potentially large amounts of adult herring, but further studies must confirm to what extent whale predation reduces herring biomass.

Effects of atrazine on endocrinology and physiology in juvenile barramundi, Lates calcarifer (Bloch)

Exposure to certain environmental contaminants such as agricultural pesticides can alter normal endocrine and reproductive parameters in wild fish populations. Recent studies have found widespread pesticide contamination across the rivers that discharge into the Great Barrier Reef lagoon. Potential impacts on native fish species exposed to known endocrine disrupting chemicals such as atrazine, simazine, and diuron have not been assessed. In the present study, the authors examined the endocrine and physiological effects of short-term, acute exposure of environmentally relevant concentrations of analytical grade atrazine in juvenile barramundi (Lates calcarifer) in a controlled laboratory experiment. Expression of hepatic vitellogenin was not affected, supporting results of previous studies that showed that atrazine does not have a direct estrogenic effect via mediation of estrogen receptors. The lack of effect on brain cytochrome P19B (CYP19B) expression levels, combined with increases in testosterone (T) and 17β estradiol and a stable T:17β estradiol ratio, does not support the hypothesis that atrazine has an indirect estrogenic effect via modulation of aromatase expression. Gill ventilation rate, a measure of oxidative stress, did not change in contrast to other studies finding enhanced osmoregulatory disturbance and gill histopathology after atrazine exposure. To more closely reflect field conditions, the authors recommend that laboratory studies should focus more on examining the effects of commercial pesticide formulations that contain additional ingredients that have been found to be disruptive to endocrine function.

Dissolved and Particulate Copper Exposure Induces Differing Gene Expression Profiles and Mechanisms of Toxicity in the Deposit Feeding Amphipod Melita plumulosa

Uptake of metals via ingestion is an important route of exposure for many invertebrates, and it has been suggested that the toxic response to metals accumulated via food differs from that of metals accumulated via the dissolved phase. To test this hypothesis, the deposit-feeding epibenthic amphipod Melita plumulosa was exposed to nontoxic or reproductively toxic concentrations of copper via the overlying water, via ingestion of sediment, or via a combination of the two. Rates of copper uptake from the two exposure routes were predicted using a biokinetic model. Gene expression profiles were measured via microarray analysis and confirmed via quantitative polymerase chain reaction. Differences in expression profiles were related to the exposure route more than to individual or combined rates of copper uptake. Chitinase and digestive protease transcript expression levels correlated to the copper uptake rate from sediment, rather than from the dissolved phase or combined total uptake rate. Overall, this study supports the hypothesis that metals accumulated via ingestion have a different mode of toxic action than metals taken up from water. Consequently, guidelines that only consider dissolved metal exposure, including equilibrium-partitioning-based guidelines, may underestimate the potential effects from deposited or resuspended metal-contaminated sediments.

Assessing mechanisms of toxicant response in the amphipod Melita plumulosa through transcriptomic profiling

This study describes the function of transcripts with altered abundance in the epibenthic amphipod, Melita plumulosa, following whole-sediment exposure to a series of common environmental contaminants. M. plumulosa were exposed for 48 h to sediments spiked and equilibrated with the following contaminants at concentrations predicted to cause sublethal effects to reproduction: porewater ammonia 30 mg L(-1); bifenthrin at 100 μg kg(-1); fipronil at 50 μg kg(-1); 0.6% diesel; 0.3% crude oil; 250 mg Cu kg(-1); 400 mg Ni kg(-1); and 400 mg Zn kg(-1). RNA was extracted and hybridized against a custom Agilent microarray developed for this species. Although the microarray represented a partial transcriptome and not all features on the array could be annotated, unique transcriptomic profiles were generated for each of the contaminant exposures. Hierarchical clustering grouped the expression profiles together by contaminant class, with copper and zinc, the petroleum products and nickel, and the pesticides each forming a distinct cluster. Many of the transcriptional changes observed were consistent with patterns previously described in other crustaceans. The changes in the transcriptome demonstrated that contaminant exposure caused changes in digestive function, growth and moulting, and the cytoskeleton following metal exposure, whereas exposure to petroleum products caused changes in carbohydrate metabolism, xenobiotic metabolism and hormone cycling. Functional analysis of these gene expression profiles can provide a better understanding of modes of toxic action and permits the prediction of mixture effects within contaminated ecosystems.

Using transcriptomic profiles in the diatom Phaeodactylum tricornutum to identify and prioritize stressors

The transcriptomic profile of the marine diatom, Phaeodactylum tricornutum, exposed to several ecologically relevant stressors, was used to develop toxicity identification evaluation (TIE)-like gene expression assays. Algal growth inhibition was measured by flow cytometry to determine exposure concentrations that elicited a sublethal toxic response. P. tricornutum was exposed to concentrations of copper (2 μg L⁻¹), cadmium (5 μg L⁻¹), silver (20 μg L⁻¹), simazine (75 μg L⁻¹), the water accommodated fraction (WAF) of weathered crude oil (5 mg L⁻¹), 50 μg L⁻¹ ammonia, a decreased salinity treatment (15‰), and a mixture exposure of ammonia, decreased salinity and cadmium (10 μg L⁻¹). Analysis of the gene expression via microarray indicated that unique transcriptomic signals were generated for each of the individual treatments. Transcriptomic profiles of ammonia and the mixture treatment overlapped substantially. Photosynthesis related transcripts were altered in the simazine (herbicide) treatment. A transcript involved in degrading hydrocarbons, dioxygenase, had increased abundance after crude oil exposure. Overall, transcriptomic responses in the different treatments were associated with stress responses, membrane transport, transcription and translation and could be linked to contaminant mode of action. The transcriptomic profiles were used to design real-time (quantitative) polymerase chain reaction (qPCR) assays that would link changes in transcript abundance to a particular stressor in a TIE-based approach. At least one transcript for each contaminant tested (copper, cadmium, silver, salinity and ammonia) responded exclusively to that contaminant. With further development of additional transcriptomic markers for each contaminant, this new approach has potential to enhance traditional toxicology bioassays by providing additional lines of evidence to identify biologically relevant stressors within a contaminated ecosystem based on changes in the transcriptomic profile.