Diane Nacci

Detection of DNA damage in individual cells from marine organisms using the single cell gel assay

The single cell gel (SCG) or comet assay is a simple method by which DNA damage is expressed as relative nuclear ‘tail’ length of gel-embedded cells following alkaline electrophoresis. While potentially applicable to any cell type, laboratory experiments were conducted to examine the utility of the SCG method for the detection of genotoxicity in cells of marine fish and invertebrates. Selected cells included those from flounder (Pleuronectes americanus) and oysters (Crassostrea virginica). In vitro exposures were used to optimize parameters and evaluate sensitivity, reproducibility and dose-responsiveness of the SCG method. In vivo exposures were used to examine the effects of factors such as intra-animal variability on low level DNA damage detection. In one experimental series, individually identified oysters were repeatedly sampled to monitor DNA damage and recovery following in vivo exposure to genotoxicant-spiked sediment. Preliminary results suggest that the SCG may be a useful tool to monitor for genotoxic environmental exposures and investigate pollution-mediated health effects.

Population genetic diversity and fitness in multiple environments

BackgroundWhen a large number of alleles are lost from a population, increases in individual homozygosity may reduce individual fitness through inbreeding depression. Modest losses of allelic diversity may also negatively impact long-term population viability by reducing the capacity of populations to adapt to altered environments. However, it is not clear how much genetic diversity within populations may be lost before populations are put at significant risk. Development of tools to evaluate this relationship would be a valuable contribution to conservation biology. To address these issues, we have created an experimental system that uses laboratory populations of an estuarine crustacean, Americamysis bahia with experimentally manipulated levels of genetic diversity. We created replicate cultures with five distinct levels of genetic diversity and monitored them for 16 weeks in both permissive (ambient seawater) and stressful conditions (diluted seawater). The relationship between molecular genetic diversity at presumptive neutral loci and population vulnerability was assessed by AFLP analysis.ResultsPopulations with very low genetic diversity demonstrated reduced fitness relative to high diversity populations even under permissive conditions. Population performance decreased in the stressful environment for all levels of genetic diversity relative to performance in the permissive environment. Twenty percent of the lowest diversity populations went extinct before the end of the study in permissive conditions, whereas 73% of the low diversity lines went extinct in the stressful environment. All high genetic diversity populations persisted for the duration of the study, although population sizes and reproduction were reduced under stressful environmental conditions. Levels of fitness varied more among replicate low diversity populations than among replicate populations with high genetic diversity. There was a significant correlation between AFLP diversity and population fitness overall; however, AFLP markers performed poorly at detecting modest but consequential losses of genetic diversity. High diversity lines in the stressful environment showed some evidence of relative improvement as the experiment progressed while the low diversity lines did not.ConclusionsThe combined effects of reduced average fitness and increased variability contributed to increased extinction rates for very low diversity populations. More modest losses of genetic diversity resulted in measurable decreases in population fitness; AFLP markers did not always detect these losses. However when AFLP markers indicated lost genetic diversity, these losses were associated with reduced population fitness.

Methylmercury in Marine Ecosystems: Spatial Patterns and Processes of Production, Bioaccumulation, and Biomagnification

The spatial variation of MeHg production, bioaccumulation, and biomagnification in marine food webs is poorly characterized but critical to understanding the links between sources and higher trophic levels, such as fish that are ultimately vectors of human and wildlife exposure. This article discusses both large and local scale processes controlling Hg supply, methylation, bioaccumulation, and transfer in marine ecosystems. While global estimates of Hg supply suggest important open ocean reservoirs of MeHg, only coastal processes and food webs are known sources of MeHg production, bioaccumulation, and bioadvection. The patterns observed to date suggest that not all sources and biotic receptors are spatially linked, and that physical and ecological processes are important in transferring MeHg from source regions to bioaccumulation in marine food webs and from lower to higher trophic levels.

The genomic landscape of rapid repeated evolutionary adaptation to toxic pollution in wild fish

Mapping genetic adaptations to pollution Many organisms have evolved tolerance to natural and human-generated toxins. Reid et al. performed a genomic analysis of killifish, geographically separate and independent populations of which have adapted recently to severe pollution (see the Perspective by Tobler and Culumber). Sequencing multiple sensitive and resistant populations revealed signals of selective sweeps for variants that may confer tolerance to toxins, some of which were shared between resistant populations. Thus, high genetic diversity in killifish seems to allow selection to act on existing variation, driving rapid adaptation to selective forces such as pollution. Science, this issue p. 1305; see also p. 1232 Genetic diversity in Atlantic killifish has allowed for convergent evolution of pollution tolerance. Atlantic killifish populations have rapidly adapted to normally lethal levels of pollution in four urban estuaries. Through analysis of 384 whole killifish genome sequences and comparative transcriptomics in four pairs of sensitive and tolerant populations, we identify the aryl hydrocarbon receptor–based signaling pathway as a shared target of selection. This suggests evolutionary constraint on adaptive solutions to complex toxicant mixtures at each site. However, distinct molecular variants apparently contribute to adaptive pathway modification among tolerant populations. Selection also targets other toxicity-mediating genes and genes of connected signaling pathways; this indicates complex tolerance phenotypes and potentially compensatory adaptations. Molecular changes are consistent with selection on standing genetic variation. In killifish, high nucleotide diversity has likely been a crucial substrate for selective sweeps to propel rapid adaptation.

Comparative transcriptomics implicates mechanisms of evolved pollution tolerance in a killifish population.

Wild populations of the killifish Fundulus heteroclitus resident in heavily contaminated North American Atlantic coast estuaries have recently and independently evolved dramatic, heritable, and adaptive pollution tolerance. We compared physiological and transcriptome responses to embryonic polychlorinated biphenyl (PCB) exposures between one tolerant population and a nearby sensitive population to gain insight into genomic, physiological and biochemical mechanisms of evolved tolerance in killifish, which are currently unknown. The PCB exposure concentrations at which developmental toxicity emerged, the range of developmental abnormalities exhibited, and global as well as specific gene expression patterns were profoundly different between populations. In the sensitive population, PCB exposures produced dramatic, dose‐dependent toxic effects, concurrent with the alterations in the expression of many genes. For example, PCB‐mediated cardiovascular system failure was associated with the altered expression of cardiomyocyte genes, consistent with sarcomere mis‐assembly. In contrast, genome‐wide expression was comparatively refractory to PCB induction in the tolerant population. Tolerance was associated with the global blockade of the aryl hydrocarbon receptor (AHR) signalling pathway, the key mediator of PCB toxicity, in contrast to the strong dose‐dependent up‐regulation of AHR pathway elements observed in the sensitive population. Altered regulation of signalling pathways that cross‐talk with AHR was implicated as one candidate mechanism for the adaptive AHR signalling repression and the pollution tolerance that it affords. In addition to revealing mechanisms of PCB toxicity and tolerance, this study demonstrates the value of comparative transcriptomics to explore molecular mechanisms of stress response and evolved adaptive differences among wild populations.

Effects of benzo[a]pyrene exposure on a fish population resistant to the toxic effects of dioxin-like compounds ☆

Effects of a model polycyclic aromatic hydrocarbon (PAH) were compared in populations of the estuarine fish Fundulus heteroclitus indigenous to a reference site and one highly contaminated with polychlorinated biphenyls (PCBs) and other compounds. The fish population resident to the PCB-contaminated site is genetically resistant to those PCB congeners categorized as dioxin-like compounds (DLCs) that act through the aryl hydrocarbon receptor (AHR). In response to DLC exposures, these DLC-resistant fish showed poor inducibility for enzymes known to be regulated by the AHR pathway and important for the metabolism of xenobiotics including some PAHs that also act as AHR agonists. Therefore, a laboratory study using the model PAH, benzo[a]pyrene (BaP), was conducted to evaluate how PAHs might affect these wild fish populations that differed in their inherent sensitivities to DLCs and in their tissue concentrations of contaminants. Following BaP treatment, the activities of two xenobiotic metabolizing enzymes and the concentrations of BaP-DNA adducts, as measured using the 32P-postlabeling method, were lower in the livers of DLC-resistant than reference fish. These results suggest that DLC-resistance could provide protection following chronic exposures to PAHs from the long-term consequences of DNA adduct formation, such as cancer. Alternatively, reduced metabolism and elimination of toxic or photo-activated PAHs could have acute consequences to the health and reproduction of these DLC-resistant fish and their progeny. These fish populations provide useful models to evaluate the potential costs and benefits of genetic adaptation in wildlife populations subject to anthropogenic stress.

Common mechanism underlies repeated evolution of extreme pollution tolerance

Human alterations to the environment can exert strong evolutionary pressures, yet contemporary adaptation to human-mediated stressors is rarely documented in wildlife populations. A common-garden experimental design was coupled with comparative transcriptomics to discover evolved mechanisms enabling three populations of killifish resident in urban estuaries to survive normally lethal pollution exposure during development, and to test whether mechanisms are unique or common across populations. We show that killifish populations from these polluted sites have independently converged on a common adaptive mechanism, despite variation in contaminant profiles among sites. These populations are united by a similarly profound desensitization of aryl-hydrocarbon receptor-mediated transcriptional activation, which is associated with extreme tolerance to the lethal effects of toxic dioxin-like pollutants. The rapid, repeated, heritable and convergent nature of evolved tolerance suggests that ancestral killifish populations harboured genotypes that enabled adaptation to twentieth-century industrial pollutants.

Estrogen responses in killifish (Fundulus heteroclitus) from polluted and unpolluted environments are site- and gene-specific

Epidemiological, ecological, and laboratory-based studies support the hypothesis that endocrine disrupting chemicals (EDCs) in the environment are responsible for developmental and reproductive abnormalities. We have previously described a killifish population resident in a highly polluted Superfund site (New Bedford Harbor, NBH) that shows evidence of exposure to an estrogenic environment and endocrine disruption. Here, we compare NBH with a local reference population (Scorton Creek, SC) for developmental patterns and direct effects of exogenous estradiol on the estrogenic markers, brain cytochrome P450 aromatase (CYP19A2 or AroB), hepatic vitellogenin (Vtg), and hepatic estrogen receptor alpha (ER alpha). In contrast to our previous observation of elevated ER alpha in NBH embryos, developmental levels of AroB and Vtg mRNAs did not differ between the two sites, demonstrating that not all estrogen-responsive genes are upregulated in NBH embryos. A dose-response experiment showed that NBH larvae are less responsive (lower maximum induction, as measured by ER alpha) and less sensitive (higher EC(50) for induction, as measured by AroB) to estradiol than SC larvae, changes that would be adaptive in an estrogenic environment. In contrast, induction of Vtg mRNA is similar in the two populations, indicating that the adaptive mechanism is target gene-specific. Based on the lower basal levels of ER alpha mRNA in several tissues from adult NBH fish vs SC fish (Greytak and Callard, 2007), we predicted estrogen hyporesponsiveness; however, induction of ER alpha by estradiol exposure in reproductively inactive males did not differ between the two sites. Moreover, AroB was more responsive and Vtg induction was greater (2d) or similar (5d) in NBH as compared to SC males. Worth noting is the high inter-individual variability in estrogen responses of gene targets, especially in NBH killifish, which may indicate evolving preadaptive or adaptive mechanisms. In conclusion, although multi-generational exposure to a highly polluted environment is associated with changes in basal levels of ER alpha mRNA, this is not a simple predictor of estrogen responsiveness. We hypothesize that adaptation of killifish to the estrogenic and polluted environment may be occurring through diverse mechanisms that are gene-, tissue type- and life-stage-specific.

Targeted approach to identify genetic loci associated with evolved dioxin tolerance in Atlantic Killifish (Fundulus heteroclitus)

BackgroundThe most toxic aromatic hydrocarbon pollutants are categorized as dioxin-like compounds (DLCs) to which extreme tolerance has evolved independently and contemporaneously in (at least) four populations of Atlantic killifish (Fundulus heteroclitus). Surprisingly, the magnitude and phenotype of DLC tolerance is similar among these killifish populations that have adapted to varied, but highly aromatic hydrocarbon-contaminated urban/industrialized estuaries of the US Atlantic coast. Multiple tolerant and neighboring sensitive killifish populations were compared with the expectation that genetic loci associated with DLC tolerance would be revealed.ResultsSince the aryl hydrocarbon receptor (AHR) pathway partly or fully mediates DLC toxicity in vertebrates, single nucleotide polymorphisms (SNPs) from 42 genes associated with the AHR pathway were identified to serve as targeted markers. Wild fish (N = 36/37) from four highly tolerant killifish populations and four nearby sensitive populations were genotyped using 59 SNP markers. Similar to other killifish population genetic analyses, strong genetic differentiation among populations was detected, consistent with isolation by distance models. When DLC-sensitive populations were pooled and compared to pooled DLC-tolerant populations, multi-locus analyses did not distinguish the two groups. However, pairwise comparisons of nearby tolerant and sensitive populations revealed high differentiation among sensitive and tolerant populations at these specific loci: AHR 1 and 2, cathepsin Z, the cytochrome P450s (CYP1A and 3A30), and the NADH dehydrogenase subunits. In addition, significant shifts in minor allele frequency were observed at AHR2 and CYP1A loci across most sensitive/tolerant pairs, but only AHR2 exhibited shifts in the same direction across all pairs.ConclusionsThe observed differences in allelic composition at the AHR2 and CYP1A SNP loci were identified as significant among paired sensitive/tolerant populations of Atlantic killifish with multiple statistical tests. The genetic patterns reported here lend support to the argument that AHR2 and CYP1A play a role in the adaptive response to extreme DLC contamination. Additional functional assays are required to isolate the exact mechanism of DLC tolerance.

Population Growth and Demography of Common Loons in the Northern United States

Abstract We used recent developments in theoretical population ecology to construct basic models of common loon (Gavia immer) demography and population dynamics. We parameterized these models using existing survival estimates and data from long-term monitoring of loon productivity and abundance. Our models include deterministic, 2-stage, density-independent matrix models, yielding population growth-rate estimates (λ) of 0.99 and 1.01 for intensively studied populations in our Wisconsin, USA, and New Hampshire, USA, study areas, respectively. Perturbation analysis of these models indicated that estimated growth rate is extremely sensitive to adult survival, as expected for this long-lived species. Also, we examined 20 years of count data for the 2 areas and evaluated support for a set of count-based models of population growth. We detected no temporal trend in Wisconsin, which would be consistent with fluctuation around an average equilibrium state but could also result from data limitations. For New Hampshire, the model set included varying formulations of density dependence and partitioning of stochasticity that were enabled by the annual sampling resolution. The best model for New Hampshire included density regulation of population growth and, along with the demographic analyses for both areas, provided insight into the possible importance of breeding habitat availability and the abundance of nonbreeding adults. Based on these results, we recommend that conservation organizations include nonbreeder abundance in common loon monitoring efforts and that additional emphasis be placed on identifying and managing human influences on adult loon survival.