RESUMEN
Acclimation increases tolerance to stress in individuals but is assumed to contribute fitness costs when the stressor is absent, though data supporting this widely held claim are sparse. Therefore, using clonal (i.e., genetically identical) cultures of Daphnia pulex, we isolated the contributions of acclimation to the regulation of the metal response gene, metallothionein 1 (MT1), and defined the reproductive benefits and costs of cadmium (Cd)-acclimation. Daphnia pulex were exposed for 50 parthenogenetic generations to environmentally realistic levels (1 µg Cd/L), and tolerance to Cd and other metals assessed during this period via standard toxicity tests. These tests revealed (1) increased tolerance to Cd compared to genetically identical nonacclimated cultures, (2) fitness costs in Cd-acclimated Daphnia when Cd was removed, and (3) cross-tolerance of Cd-acclimated Daphnia to zinc and silver, but not arsenic, thereby defining a functional role for metallothionein. Indeed, Cd-acclimated clones had significantly higher expression of MT1 mRNA than nonacclimated clones, when Cd exposed. Both the enhanced induction of MT1 and tolerant phenotype were rapidly lost when Cd was removed (1-2 generations), which is further evidence of acclimation costs. These findings provide evidence for the widely held view that acclimation is costly and are important for investigating evolutionary principles of genetic assimilation and the survival mechanisms of natural populations that face changing environments.
Asunto(s)
Daphnia , Contaminantes Químicos del Agua , Aclimatación , Animales , Cadmio , MetalotioneínaRESUMEN
Toxicogenomic approaches can detect and classify adverse interactions between environmental toxicants and other environmental stressors but require more complex experimental designs and analytical approaches. Here we use novel toxicogenomic techniques to analyze the effect of arsenic exposure in wild killifish populations acclimating to changing salinity. Fish from three populations were acclimated to full strength seawater and transferred to fresh water for 1 or 24 h. Linear models of gene expression in gill tissue identified 31 genes that responded to osmotic shock at 1 h and 178 genes that responded at 24 h. Arsenic exposure (100 µg/L) diminished the responses (reaction norms) of these genes by 22% at 1 h ( p = 1.0 × 10-6) and by 10% at 24 h ( p = 3.0 × 10-10). Arsenic also significantly reduced gene coregulation in gene regulatory networks ( p = 0.002, paired Levene's test), and interactions between arsenic and salinity acclimation were uniformly antagonistic at the biological pathway level ( p < 0.05, binomial test). Arsenic's systematic interference with gene expression reaction norms was validated in a mouse multistressor experiment, demonstrating the ability of these toxicogenomic approaches to identify biologically relevant adverse interactions between environmental toxicants and other environmental stressors.
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Arsénico , Fundulidae , Aclimatación , Animales , Expresión Génica , Branquias , Ratones , Salinidad , Agua de MarRESUMEN
The antipredator behavior diel vertical migration (DVM), common in aquatic keystone species Daphnia, involves daily migration from warmer surface waters before dawn to cooler deeper waters after dusk. Plasticity in Daphnia DVM behavior optimizes fitness via trade-offs between growth, reproduction, and predator avoidance. Migration behavior is affected by co-varying biotic and abiotic factors, including light, predator cues, and anthropogenic stressors making it difficult to determine each factor's individual contribution to the variation in this behavior. This study aims to better understand this ecologically significant behavior in Daphnia by: (1) determining how Daphnia pulicaria thermal preferences vary within and among natural populations; (2) distinguishing the role of temperature verses depth in Daphnia vertical migration; and (3) defining how two anthropogenic stressors (copper and nickel) impact Daphnia migratory behavior. Simulated natural lake stratification were constructed in 8L (0.5m tall, 14.5cm wide) water columns to monitor under controlled laboratory conditions the individual effects of temperature gradients, depth, and metal stressors on Daphnia vertical migration. Three major findings are reported. First, while no difference in thermal preference was found among the four populations studied, within lake populations variability among isolates was high. Second, decoupling temperature and depth revealed that depth was a better predictor of Daphnia migratory patterns over temperature. Third, exposure to environmentally relevant concentrations of copper or nickel inhibited classic DVM behavior. These findings revealed the high variability in thermal preference found within Daphnia populations, elucidated the individual roles that depth and temperature have on migratory behavior, and showed how copper and nickel can interfere with the natural response of Daphnia to fish predator cues. Thus contributing to the body of knowledge necessary to predict how natural populations of Daphnia will be affected by climate related changes in lake temperatures and increased presence of anthropogenic stressors.
Asunto(s)
Migración Animal , Daphnia/fisiología , Animales , Cambio Climático , Cobre/metabolismo , Níquel/metabolismo , Conducta Predatoria , TemperaturaRESUMEN
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.
Asunto(s)
Adaptación Fisiológica/genética , Proteínas de Peces/genética , Fundulidae/genética , Redes Reguladoras de Genes , Genoma , Animales , Arsénico/toxicidad , Evolución Biológica , Proteínas de Peces/metabolismo , Agua Dulce/química , Fundulidae/metabolismo , Regulación de la Expresión Génica , Interacción Gen-Ambiente , Branquias/efectos de los fármacos , Branquias/metabolismo , Masculino , Fenotipo , Salinidad , Sales (Química)/farmacología , Agua de Mar/química , Selección GenéticaRESUMEN
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.
Asunto(s)
Daphnia/efectos de los fármacos , Daphnia/genética , Microcystis/patogenicidad , Contaminantes Químicos del Agua/toxicidad , Animales , Cadmio/toxicidad , Daphnia/metabolismo , Daphnia/microbiología , Perfilación de la Expresión Génica , Genotipo , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Transcriptoma/efectos de los fármacos , Transcriptoma/fisiologíaRESUMEN
Certain individuals have a disproportionate effect on group responses. Characteristics may include susceptibility to pollutants, such as cadmium (Cd), a potent trace metal. Here, we show how a pair of Cd-exposed individuals can impact the behavior of unexposed groups. We used behavioral assessments to characterize the extent of the effects of the Cd-exposed individuals on group boldness, cohesion, foraging, activity, and responses to plants. We found that groups with a pair of Cd-exposed fish remained closer to novel stimuli and plants than did groups with untreated (control) fish. The presence of plants reduced Cd-induced differences in shoal cohesion and delays feeding in male shoals. Shoals with Cd- and water-treated fish were equally active. The results suggest that fish acutely exposed to environmentally relevant Cd concentrations can have profound effects on the un-exposed majority. However, the presence of plants may mitigate the effects of contaminants on some aspects of social behavior.
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Perciformes , Contaminantes Químicos del Agua , Animales , Pez Cebra/fisiología , Cadmio/toxicidad , Conducta Social , Contaminantes Químicos del Agua/toxicidadRESUMEN
Some individuals have a disproportionate effect on group responses. These individuals may possess distinct attributes that differentiate them from others. These characteristics may include susceptibility to contaminant exposure such as cadmium, a potent trace metal present in water and food. Here, we tested whether a pair of cadmium-exposed individuals could exert an impact on the behavior of the unexposed majority. We used behavioral assessments to characterize the extent of the effects of the cadmium-exposed pair on group boldness, cohesion, activity and responses to landmarks. We found that groups with a pair of cadmium-exposed fish approached and remained closer to novel stimuli and landmarks than did groups with pairs of fish treated with uncontaminated water (control). Shoals with cadmium and water treated fish exhibited similar levels of cohesion and activity. The results suggest that fish acutely exposed to environmentally-relevant cadmium concentrations can have profound effects on the un-exposed majority.
RESUMEN
To account for global contamination events, we must identify direct and indirect pollutant effects. Although pollutants can have direct effects on individuals, it is unknown how a few contaminated individuals affect groups, a widespread social organization. We show environmentally relevant levels of cadmium (Cd) can have indirect social effects revealed in the social context of a larger group. Cd-contaminated individuals had poor vision and more aggressive responses, but no other behavioral effects. The presence of experienced Cd-exposed pairs in the groups had an indirect effect on the un-exposed individual's social interactions leading to the shoal becoming bolder and moving closer to a novel object than control groups. Because a few directly affected individuals could indirectly affect social behavior of the un-exposed majority, we believe that such acute but potentially important heavy metal toxicity could inform reliable predictions about the consequences of their use in a changing world.
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Contaminantes Ambientales , Contaminantes Químicos del Agua , Animales , Cadmio/toxicidad , Pez Cebra/fisiología , Contaminantes Químicos del Agua/toxicidad , Conducta SocialRESUMEN
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.
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Daphnia/genética , Redes Reguladoras de Genes , Microcystis/patogenicidad , Familia de Multigenes , Animales , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
BACKGROUND: Germline mutations provide the raw material for all evolutionary processes and contribute to the occurrence of spontaneous human diseases and disorders. Yet despite the daily interaction of humans and other organisms with an increasing number of chemicals that are potentially mutagenic, precise measurements of chemically induced changes to the genome-wide rate and spectrum of germline mutation are lacking. OBJECTIVES: A large-scale Daphnia pulex mutation-accumulation experiment was propagated in the presence and absence of an environmentally relevant cadmium concentration to quantify the influence of cadmium on germline mutation rates and spectra. RESULTS: Cadmium exposure dramatically changed the genome-wide rates and regional spectra of germline mutations. In comparison with those in control conditions, Daphnia exposed to cadmium had a higher overall A:TâG:C mutation rates and a lower overall C:GâG:C mutation rate. Daphnia exposed to cadmium had a higher intergenic mutation rate and a lower exonic mutation rate. The higher intergenic mutation rate under cadmium exposure was the result of an elevated intergenic A:TâG:C rate, whereas the lower exon mutation rate in cadmium was the result of a complete loss of exonic C:GâG:C mutations-mutations that are known to be enriched at 5-hydroxymethylcytosine. We experimentally show that cadmium exposure significantly reduced 5-hydroxymethylcytosine levels. DISCUSSION: These results provide evidence that cadmium changes regional mutation rates and can influence regional rates by interfering with an epigenetic process in the Daphnia pulex germline. We further suggest these observed cadmium-induced changes to the Daphnia germline mutation rate may be explained by cadmium's inhibition of zinc-containing domains. The cadmium-induced changes to germline mutation rates and spectra we report provide a comprehensive view of the mutagenic perils of cadmium and give insight into its potential impact on human population health. https://doi.org/10.1289/EHP8932.
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Cadmio , Daphnia , Animales , Cadmio/toxicidad , Daphnia/genética , Mutación de Línea Germinal , Tasa de MutaciónRESUMEN
In toxicology, standard sigmoidal concentration-response curves are used to predict effects concentrations and set chemical regulations. However, current literature also establishes the existence of complex, bimodal concentration-response curves, as is the case for arsenic toxicity. This bimodal response has been observed at the molecular level, but not characterized at the whole organism level. This study investigated the effect of arsenic (sodium arsenite) on post-gastrulated zebrafish embryos and elucidated effects of bimodal concentration-responses on different phenotypic perturbations. Six hour post fertilized (hpf) zebrafish embryos were exposed to arsenic to 96 hpf. Hatching success, mortality, and morphometric endpoints were evaluated both in embryos with chorions and dechorionated embryos. Zebrafish embryos exhibited a bimodal response to arsenic exposure. Concentration-response curves for exposed embryos with intact chorions had an initial peak in mortality (88%) at 1.33 mM arsenic, followed by a decrease in toxicity (~20% mortality) at 1.75 mM, and subsequently peaked to 100% mortality at higher concentrations. To account for the bimodal response, two distinct concentration-response curves were generated with estimated LC10 values (and 95% CI) of 0.462 (0.415, 0.508) mM and 1.69 (1.58, 1.78) mM for the 'low concentration' and 'high concentration' peaks, respectively. Other phenotypic analyses, including embryo length, yolk and pericardial edema all produced similar concentration-response patterns. Tests with dechorionated embryos also resulted in a bimodal toxicity response but with lower LC10 values of 0.170 (0.120, 0.220) mM and 0.800 (0.60, 0842) mM, respectively. Similarities in bimodal concentration-responses between with-chorion and dechorionated embryos indicate that the observed effect was not caused by the chorion limiting arsenic availability, thus lending support to other studies such as those that hypothesized a conserved bimodal mechanism of arsenic interference with nuclear receptor activation.
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Arsénico , Pez Cebra , Animales , Arsénico/toxicidad , Corion , Embrión no MamíferoRESUMEN
BACKGROUND: Genomic 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. RESULTS: Our 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. CONCLUSION: The 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.
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Cadmio/toxicidad , Daphnia/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Metalotioneína/genética , Contaminantes Químicos del Agua/toxicidad , Animales , Biomarcadores , ADN Complementario , Daphnia/metabolismo , Bases de Datos Genéticas , Relación Dosis-Respuesta a Droga , Monitoreo del Ambiente , Genoma/efectos de los fármacos , Metalotioneína/metabolismo , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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.
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Arsénico/toxicidad , Daphnia/efectos de los fármacos , Animales , Metilación , Oxidación-ReducciónRESUMEN
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.
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Cadmio/toxicidad , Cobre/toxicidad , Daphnia/genética , Regulación de la Expresión Génica/efectos de los fármacos , Metalotioneína/genética , Contaminantes Químicos del Agua/toxicidad , Animales , Daphnia/clasificación , Perfilación de la Expresión Génica , Datos de Secuencia Molecular , FilogeniaRESUMEN
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 AID's efficiency in assessing water quality criteria.