Stephen P. Glaholt

Gene response profiles for Daphnia pulex exposed to the environmental stressor cadmium reveals novel crustacean metallothioneins

BackgroundGenomic research tools such as microarrays are proving to be important resources to study the complex regulation of genes that respond to environmental perturbations. A first generation cDNA microarray was developed for the environmental indicator species Daphnia pulex, to identify genes whose regulation is modulated following exposure to the metal stressor cadmium. Our experiments revealed interesting changes in gene transcription that suggest their biological roles and their potentially toxicological features in responding to this important environmental contaminant.ResultsOur microarray identified genes reported in the literature to be regulated in response to cadmium exposure, suggested functional attributes for genes that share no sequence similarity to proteins in the public databases, and pointed to genes that are likely members of expanded gene families in the Daphnia genome. Genes identified on the microarray also were associated with cadmium induced phenotypes and population-level outcomes that we experimentally determined. A subset of genes regulated in response to cadmium exposure was independently validated using quantitative-realtime (Q-RT)-PCR. These microarray studies led to the discovery of three genes coding for the metal detoxication protein metallothionein (MT). The gene structures and predicted translated sequences of D. pulex MTs clearly place them in this gene family. Yet, they share little homology with previously characterized MTs.ConclusionThe genomic information obtained from this study represents an important first step in characterizing microarray patterns that may be diagnostic to specific environmental contaminants and give insights into their toxicological mechanisms, while also providing a practical tool for evolutionary, ecological, and toxicological functional gene discovery studies. Advances in Daphnia genomics will enable the further development of this species as a model organism for the environmental sciences.

ZOOPLANKTON COMMUNITY STRUCTURE AFFECTS HOW PHYTOPLANKTON RESPOND TO NUTRIENT PULSES

Ecological theory suggests that community structure determines how eco- systems respond to perturbation. However, this idea is not well-tested empirically, in part because it is difficult to define the appropriate reference state for evaluating post-pertur- bation dynamics in field experiments. In this study, we (1) evaluated two different methods of determining the reference state, and (2) tested hypotheses derived from theory and previous experiments about the effects of herbivore community structure on autotroph responses to nutrient pulses in freshwater plankton communities. We hypothesized that (1) phytoplankton are less sensitive to a nutrient pulse with increasing zooplankton biomass, size, and Daphnia abundance, and (2) phytoplankton recover more quickly from the nutrient pulse with increased zooplankton biomass and increased abundance of taxa with rapid numerical responses, especially small-bodied cladocerans. We tested these hypotheses in field mesocosm experiments, which crossed four zooplankton community structures with two nutrient pulse treatments in a fully randomized factorial design. We found that phy- toplankton sensitivity decreased with increased zooplankton biomass, size, and Daphnia biomass regardless of the method used to define the reference state, supporting our first hypothesis and suggesting that results for sensitivity are robust to methodology. This result, together with previous work, clearly demonstrates that zooplankton have a strong effect on phytoplankton sensitivity. Further, as predicted, return times were shorter with increased zooplankton biomass and biomass of small cladocerans. However, contrary to expectations, return times also declined with Daphnia and copepod biomass, suggesting that the effects of zooplankton on return time are more complex than we hypothesized. Moreover, con- clusions for return time depended somewhat on methodology, indicating that we may need to reevaluate whether the concept of return time is relevant for field experiments.

Identification of Pathways, Gene Networks, and Paralogous Gene Families in Daphnia pulex Responding to Exposure to the Toxic Cyanobacterium Microcystis aeruginosa

Although cyanobacteria produce a wide range of natural toxins that impact aquatic organisms, food webs, and water quality, the mechanisms of toxicity are still insufficiently understood. Here, we implemented a whole-genome expression microarray to identify pathways, gene networks, and paralogous gene families responsive to Microcystis stress in Daphnia pulex . Therefore, neonates of a sensitive isolate were given a diet contaminated with Microcystis to contrast with those given a control diet for 16 days. The microarray revealed 2247 differentially expressed (DE) genes (7.6% of the array) in response to Microcystis , of which 17% are lineage-specific (i.e., these genes have no detectable homology to any other gene in currently available databases) and 49% are gene duplicates (paralogues). We identified four pathways/gene networks and eight paralogous gene families affected by Microcystis . Differential regulation of the ribosome, including three paralogous gene families encoding 40S, 60S, and mitochondrial ribosomal proteins, suggests an impact of Microcystis on protein synthesis of D. pulex . In addition, differential regulation of the oxidative phosphorylation pathway (including the NADH:ubquinone oxidoreductase gene family) and the trypsin paralogous gene family (a major component of the digestive system in D. pulex ) could explain why fitness is reduced based on energy budget considerations.

Acute toxicity of arsenic to Daphnia pulex: Influence of organic functional groups and oxidation state

Investigations were conducted to determine the influence of organic functional groups (i.e., methyl, phenyl) and valence state (i.e., III, V) on acute (48-h) arsenic toxicity in Daphnia pulex. These included toxicity texts with a suite of inorganic (arsenite, arsenate) and organic arsenicals (trivalent and pentavalent methylated arsenicals, roxarsone, p-arsanilic acid). Toxicity, based on median lethal concentrations (LC50 values), clustered the arsenicals into three groups and followed the order (most toxic to least toxic) of monomethylarsonous acid (MMA(III)), 120 microg/L > inorganic arsenic, 2,500 to 3,900 microg/L > pentavalent methylated arsenicals and phenylarsonic compounds, 13,800 to 15,700 microg/L. Pentavalent organic arsenicals were less toxic than inorganic forms regardless of functional group. In contrast, the trivalent organic species (M MA(III)) was the most toxic arsenical studied. These findings, which are the first to include an aquatic organism, add to the growing body of evidence that find that MMA(III) is an extremely toxic intermediate of arsenic methylation and contradict theories of arsenic toxicity that regard methylation as a detoxication event.

Genome-wide transcription profiles reveal genotype-dependent responses of biological pathways and gene-families in Daphnia exposed to single and mixed stressors.

The present study investigated the possibilities and limitations of implementing a genome-wide transcription-based approach that takes into account genetic and environmental variation to better understand the response of natural populations to stressors. When exposing two different Daphnia pulex genotypes (a cadmium-sensitive and a cadmium-tolerant one) to cadmium, the toxic cyanobacteria Microcystis aeruginosa, and their mixture, we found that observations at the transcriptomic level do not always explain observations at a higher level (growth, reproduction). For example, although cadmium elicited an adverse effect at the organismal level, almost no genes were differentially expressed after cadmium exposure. In addition, we identified oxidative stress and polyunsaturated fatty acid metabolism-related pathways, as well as trypsin and neurexin IV gene-families as candidates for the underlying causes of genotypic differences in tolerance to Microcystis. Furthermore, the whole-genome transcriptomic data of a stressor mixture allowed a better understanding of mixture responses by evaluating interactions between two stressors at the gene-expression level against the independent action baseline model. This approach has indicated that ubiquinone pathway and the MAPK serine-threonine protein kinase and collagens gene-families were enriched with genes showing an interactive effect in expression response to exposure to the mixture of the stressors, while transcription and translation-related pathways and gene-families were mostly related with genotypic differences in interactive responses to this mixture. Collectively, our results indicate that the methods we employed may improve further characterization of the possibilities and limitations of transcriptomics approaches in the adverse outcome pathway framework and in predictions of multistressor effects on natural populations.

Natural Selection Canalizes Expression Variation of Environmentally Induced Plasticity-Enabling Genes

Many organisms survive fluctuating and extreme environmental conditions by manifesting multiple distinct phenotypes during adulthood by means of developmental processes that enable phenotypic plasticity. We report on the discovery of putative plasticity-enabling genes that are involved in transforming the gill of the euryhaline teleost fish, Fundulus heteroclitus, from its freshwater to its seawater gill-type, a process that alters both morphology and function. Gene expression that normally enables osmotic plasticity is inhibited by arsenic. Gene sets defined by antagonistic interactions between arsenic and salinity show reduced transcriptional variation among individual fish, suggesting unusually accurate and precise regulatory control of these genes, consistent with the hypothesis that they participate in a canalized developmental response. We observe that natural selection acts to preserve canalized gene expression in populations of killifish that are most tolerant to abrupt salinity change and that these populations show the least variability in their transcription of genes enabling plasticity of the gill. We found that genes participating in this highly canalized and conserved plasticity-enabling response had significantly fewer and less complex associations with transcriptional regulators than genes that respond only to arsenic or salinity. Collectively these findings, which are drawn from the relationships between environmental challenge, plasticity, and canalization among populations, suggest that the selective processes that facilitate phenotypic plasticity do so by targeting the regulatory networks that gives rise to the response. These findings also provide a generalized, conceptual framework of how genes might interact with the environment and evolve toward the development of plastic traits.

Transcription patterns of genes encoding four metallothionein homologs in Daphnia pulex exposed to copper and cadmium are time- and homolog-dependent

Metallothioneins are proteins that play an essential role in metal homeostasis and detoxification in nearly all organisms studied to date. Yet discrepancies between outcomes of chronic and acute exposure experiments hamper the understanding of the regulatory mechanisms of their isoforms following metal exposure. Here, we investigated transcriptional differences among four identified homologs (mt1-mt4) in Daphnia pulex exposed across time to copper and cadmium relative to a control. Transcriptional upregulation of mt1 and mt3 was detected on day four following exposure to cadmium, whereas that of mt2 and mt4 was detected on day two and day eight following exposure to copper. These results confirm temporal and metal-specific differences in the transcriptional induction of genes encoding metallothionein homologs upon metal exposure which should be considered in ecotoxicological monitoring programs of metal-contaminated water bodies. Indeed, the mRNA expression patterns observed here illustrate the complex regulatory system associated with metallothioneins, as these patterns are not only dependent on the metal, but also on exposure time and the homolog studied. Further phylogenetic analysis and analysis of regulatory elements in upstream promoter regions revealed a high degree of similarity between metallothionein genes of Daphnia pulex and Daphnia magna, a species belonging to the same genus. These findings, combined with a limited amount of available expression data for D. magna metallothionein genes, tentatively suggest a potential generalization of the metallothionein response system between these Daphnia species.

Will genetic adaptation of natural populations to chemical pollution result in lower or higher tolerance to future climate change

Evolved co-tolerance or cross-tolerance to chemical pollution and a future climate change stressor is observed

Adaptive iterative design (AID): a novel approach for evaluating the interactive effects of multiple stressors on aquatic organisms.

The study of stressor interactions by eco-toxicologists using nonlinear response variables is limited by required amounts of a priori knowledge, complexity of experimental designs, the use of linear models, and the lack of use of optimal designs of nonlinear models to characterize complex interactions. Therefore, we developed AID, an adaptive-iterative design for eco-toxicologist to more accurately and efficiently examine complex multiple stressor interactions. AID incorporates the power of the general linear model and A-optimal criteria with an iterative process that: 1) minimizes the required amount of a priori knowledge, 2) simplifies the experimental design, and 3) quantifies both individual and interactive effects. Once a stable model is determined, the best fit model is identified and the direction and magnitude of stressors, individually and all combinations (including complex interactions) are quantified. To validate AID, we selected five commonly co-occurring components of polluted aquatic systems, three metal stressors (Cd, Zn, As) and two water chemistry parameters (pH, hardness) to be tested using standard acute toxicity tests in which Daphnia mortality is the (nonlinear) response variable. We found after the initial data input of experimental data, although literature values (e.g. EC-values) may also be used, and after only two iterations of AID, our dose response model was stable. The model ln(Cd)*ln(Zn) was determined the best predictor of Daphnia mortality response to the combined effects of Cd, Zn, As, pH, and hardness. This model was then used to accurately identify and quantify the strength of both greater- (e.g. As*Cd) and less-than additive interactions (e.g. Cd*Zn). Interestingly, our study found only binary interactions significant, not higher order interactions. We conclude that AID is more efficient and effective at assessing multiple stressor interactions than current methods. Other applications, including life-history endpoints commonly used by regulators, could benefit from AIDs efficiency in assessing water quality criteria.

Single toxin dose-response models revisited

The goal of this paper is to offer a rigorous analysis of the sigmoid shape single toxin dose-response relationship. The toxin efficacy function is introduced and four special points, including maximum toxin efficacy and inflection points, on the dose-response curve are defined. The special points define three phases of the toxin effect on mortality: (1) toxin concentrations smaller than the first inflection point or (2) larger then the second inflection point imply low mortality rate, and (3) concentrations between the first and the second inflection points imply high mortality rate. Probabilistic interpretation and mathematical analysis for each of the four models, Hill, logit, probit, and Weibull is provided. Two general model extensions are introduced: (1) the multi-target hit model that accounts for the existence of several vital receptors affected by the toxin, and (2) model with a nonzero mortality at zero concentration to account for natural mortality. Special attention is given to statistical estimation in the framework of the generalized linear model with the binomial dependent variable as the mortality count in each experiment, contrary to the widespread nonlinear regression treating the mortality rate as continuous variable. The models are illustrated using standard EPA Daphnia acute (48h) toxicity tests with mortality as a function of NiCl or CuSO4 toxin.