Use of pesticides can have substantial nonlethal impacts on nontarget species, including driving evolutionary change, often with unknown consequences for species, ecosystems, and society. Hyalella ...azteca , a species complex of North American freshwater amphipods, is widely used for toxicity testing of water and sediment and has frequently shown toxicity due to pyrethroid pesticides. We demonstrate that 10 populations, 3 from laboratory cultures and 7 from California water bodies, differed by at least 550-fold in sensitivity to pyrethroids. The populations sorted into four phylogenetic groups consistent with species-level divergence. By sequencing the primary pyrethroid target site, the voltage-gated sodium channel, we show that point mutations and their spread in natural populations were responsible for differences in pyrethroid sensitivity. At least one population had both mutant and WT alleles, suggesting ongoing evolution of resistance. Although nonresistant H. azteca were susceptible to the typical neurotoxic effects of pyrethroids, gene expression analysis suggests the mode of action in resistant H. azteca was not neurotoxicity but was oxidative stress sustained only at considerably higher pyrethroid concentrations. The finding that a nontarget aquatic species has acquired resistance to pesticides used only on terrestrial pests is troubling evidence of the impact of chronic pesticide transport from land-based applications into aquatic systems. Our findings have far-reaching implications for continued uncritical use of H. azteca as a principal species for monitoring and environmental policy decisions.
Coastal harbor areas are subjected to a myriad of contamination sources with largely unknown effects. Such complex chemical mixtures are difficult to monitor but transcriptomics is a promising ...approach for such biomonitoring. The present study was designed to verify the use of the Coastal Biosensor for Endocrine Disruption (C-BED) assay, previously developed to detect emerging contaminants and their effects on Mytilus edulis, on another mussel species, Mytilus galloprovincialis. Mussels were caged on St-Florent harbor (contaminated) and on Revellata Bay (reference) for three months. A classical multibiomarkers approach was coupled to the C-BED assay. The results of both approaches were analysed using the Integrated Biomarkers Responses (IBR) and compared to each other. Both approaches demonstrated a higher contamination and probable endocrine disruption of mussels in St-Florent, compared to the reference station. These results confirm that the C-BED assay provides an innovative method to expand our ability to detect emerging contaminants.
•C-BED assay provides an innovative method to monitor for chemicals.•There is a probable endocrine disruption of mussels in St. Florent.•Both classical multibiomarkers approach and transcriptomic approach demonstrate a higher contamination in St. Florent.•The use of the C-BED assay is validated on a new specie, Mytilus galloprovincialis.
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•Daphnia magna neonates were exposed to genotoxic levels of the anticancer drugs: imatinib mesylate, cisplatinum, etoposide.•RNA-seq analysis was performed to uncover insights into ...the mechanism of action of these compounds.•Differentially expressed transcripts were related to cell cycle regulation and G-protein coupled receptor (GPCR) signaling.•Angiotensin converting enzyme and DNA topoisomerase II are potential biomarkers of anticancer drug exposure.
The use of anticancer drugs in chemotherapy is increasing, leading to growing environmental concentrations of imatinib mesylate (IMA), cisplatinum (CDDP), and etoposide (ETP) in aquatic systems. Previous studies have shown that these anticancer drugs cause DNA damage in the crustacean Daphnia magna at low, environmentally relevant concentrations. To explore the mechanism of action of these compounds and the downstream effects of DNA damage on D. magna growth and development at a sensitive life stage, we exposed neonates to low level concentrations equivalent to those that elicit DNA damage (IMA: 2000 ng/L, ETP: 300 ng/L, CDDP: 10 ng/L) and performed transcriptomic analysis using an RNA-seq approach. RNA sequencing generated 14 million reads per sample, which were aligned to the D. magna genome and assembled, producing approximately 23,000 transcripts per sample. Over 90% of the transcripts showed homology to proteins in GenBank, revealing a high quality transcriptome assembly, although functional annotation was much lower. RT-qPCR was used to identify robust biomarkers and confirmed the downregulation of an angiotensin converting enzyme-like gene (ance) involved in neuropeptide regulation across all three anticancer drugs and the down-regulation of DNA topoisomerase II by ETP. RNA-seq analysis also allowed for an in depth exploration of the differential splicing of transcripts revealing that regulation of different gene isoforms predicts potential impacts on translation and protein expression, providing a more meaningful assessment of transcriptomic data. Enrichment analysis and investigation of affected biological processes suggested that the DNA damage caused by ETP and IMA influences cell cycle regulation and GPCR signaling. This dysregulation is likely responsible for effects to neurological system processes and development, and overall growth and development. Our transcriptomic approach provided insight into the mechanisms that respond to DNA damage caused by anticancer drug exposure and generated novel hypotheses on how these chemicals may impact the growth and survival of this ecologically important zooplankton species.
Applications for silver nanomaterials in consumer products are rapidly expanding, creating an urgent need for toxicological examination of the exposure potential and ecological effects of silver ...nanoparticles (AgNPs). The integration of genomic techniques into environmental toxicology has presented new avenues to develop exposure biomarkers and investigate the mode of toxicity of novel chemicals. In the present study we used a 15k oligonucleotide microarray for Daphnia magna, a freshwater crustacean and common indicator species for toxicity, to differentiate between particle specific and ionic silver toxicity and to develop exposure biomarkers for citrate-coated and PVP-coated AgNPs. Gene expression profiles revealed that AgNO3 and AgNPs have distinct expression profiles suggesting different modes of toxicity. Major biological processes disrupted by the AgNPs include protein metabolism and signal transduction. In contrast, AgNO3 caused a downregulation of developmental processes, particularly in sensory development. Metal responsive and DNA damage repair genes were induced by the PVP AgNPs, but not the other treatments. In addition, two specific biomarkers were developed for the environmental detection of PVP AgNPs; although further verification under different environmental conditions is needed.
Transcriptomics, high-throughput assays, and adverse outcome pathways (AOP) are promising approaches applied to toxicity monitoring in the 21st century, but development of these methods is ...challenging for nonmodel organisms and emerging contaminants. For example, Endocrine Disrupting Compounds (EDCs) may cause reproductive impairments and feminization of male bivalves; however, the mechanism linked to this adverse outcome is unknown. To develop mechanism-based biomarkers that may be linked through an AOP, we exposed Mytilus edulis to 17-alpha-ethinylestradiol (5 and 50 ng/L) and 4-nonylphenol (1 and 100 μg/L) for 32 and 39 days. When mussels were exposed to these EDCs, we found elevated female specific transcripts and significant female-skewed sex ratios using a RT-qPCR assay. We performed gene expression analysis on digestive gland tissue using an M. edulis microarray and through network and targeted analyses identified the nongenomic estrogen signaling pathway and steroidogenesis pathway as the likely mechanisms of action for a putative AOP. We also identified several homologues to genes within the vertebrate steroidogenesis pathway including the cholesterol side chain cleavage complex. From this AOP, we designed the Coastal Biosensor for Endocrine Disruption (C-BED) assay which was confirmed in the laboratory and tested in the field.
•Blue mussels Mytilus edulis were deployed at four locations in Boston Harbor.•We identified transcriptomic biomarkers that correlated with PCBs and PAHs.•We targeted endocrine disrupting compounds ...(EDCs) with the C-BED assay.•An Integrated Biomarker Response (IBR) approach identified sites impacted by EDCs.
Within monitoring frameworks, biomarkers provide several benefits because they serve as intermediates between pollutant exposure and effects, and integrate the responses of contaminants that operate through the same mechanism of action. This study was designed to verify the use of transcriptomic biomarkers developed in our prior work (i.e., Coastal Biosensor of Endocrine Disruption; C-BED assay) on Mytilus edulis and identify additional biomarkers for legacy pollutants. M. edulis were collected from a reference site in Pemaquid, ME, USA and deployed by the Massachusetts Water Resources Authority (MWRA) at locations in and outside Boston Harbor, MA, USA: including (1) Boston Inner Harbor (IH), (2) the current outfall (OS), (3) 1 km away from the current outfall (LNB), and (4) Deer Island (DI), the site where untreated wastewater was formerly discharged into the bay. Differential gene expression was quantified with a high density microarray. Seven genes significantly correlated with whole tissue concentration of PAHs, and six genes significantly correlated with whole body concentrations of PCBs, two groups of legacy contaminants that were elevated at stations IH, OS, and DI. Enrichment analysis indicated that IH mussels had the highest induction of stress response genes, which correlated with the higher levels of contaminants measured at this site. Based on the C-BED assay gene analysis, stations IH and OS exhibited signs of endocrine disruption, which were further confirmed by incorporating the results for the C-BED assay within the Integrated Biomarker Response (IBR) approach. This study successfully demonstrated the potential use of transcriptomic biomarkers within a monitoring program to identify the presence and organismal responses to endocrine disrupting and legacy contaminant classes.
Resistance alleles within the voltage-gated sodium channel (vgsc) have been correlated with pyrethroid resistance in wild populations of the nontarget amphipod, Hyalella azteca from California (CA), ...U.S.A. In the present study, we expand upon the relationship between land use and the evolution of pesticide resistance in H. azteca to develop a quantitative methodology to target and screen novel populations for resistance allele genotypes in a previously uninvestigated region of the U.S. (New England: NE). By incorporating urban land development and toxicity-normalized agricultural pesticide use indices into our site selection, we successfully identified three amino acid substitutions associated with pyrethroid resistance. One of the resistance mutations has been described in H. azteca from CA (L925I). We present the remaining two (vgsc I936F and I936V) as novel pyrethroid-resistance alleles in H. azteca based on previous work in insects and elevated cyfluthrin resistance in one NE population. Our results suggest that urban pesticide use is a strong driver in the evolution of resistance alleles in H. azteca. Furthermore, our method for resistance allele screening provides an applied framework for detecting ecosystem impairment on a nationwide scale that can be incorporated into ecological risk assessment decisions.
Hyalella azteca are epibenthic amphipods that have developed resistance to pyrethroid and organophosphate insecticides due to single amino acid substitutions in the voltage-gated sodium channel and ...the acetylcholinesterase-1 gene, respectively. Aquatic systems are often contaminated with several different types of insecticides, therefore there is a possibility that H. azteca have also developed resistance to other classes of insecticides. The aims of the current study were to verify that pyrethroid- and organophosphate-resistant H. azteca have retained their resistance after being cultured in the absence of selective pressure for 5 years (Escondido Creek population) and 9 years (Mosher Slough population), to determine if these populations have cross-resistance to carbaryl (carbamate) and 4,4′-dichlorodiphenyltrichloroethane (DDT; organochlorine), and determine whether previous field exposure to fipronil (phenylpyrazole) and imidacloprid (neonicotinoid) caused resistance in cultured pyrethroid- and organophosphate-resistant H. azteca populations. Escondido Creek and Mosher Slough H. azteca populations both maintained high tolerances for bifenthrin due to L925I and I936F amino acid substitutions. Resistance was also found for chlorpyrifos in the Escondido Creek and Mosher Slough populations with lower genotype frequencies of the G119S substitution, indicating that additional factors may be responsible for organophosphate resistance in this study. Mosher Slough H. azteca were moderately resistant to DDT, and Escondido Creek and Mosher Slough H. azteca were moderately resistant to carbaryl, suggesting cross-resistance. No differences were observed in acute toxicity values across the three populations of H. azteca for fipronil and imidacloprid, and this is possibly due to the lack of exposure to toxic concentrations of these insecticides in the field and lack of similar modes of action to pyrethroids and organophosphates. Resistance is known to be associated with fitness costs that can place insecticide-resistant populations at risk for decline through decreased survival and reduced fecundity.
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•The over usage of pesticides has resulted in the development of resistance in H. azteca.•Resistance to bifenthrin is due to the L925I and I936F amino acid substitutions.•Pesticide-resistant H. azteca are capable of developing cross-resistance to other pesticides.•No resistance was observed for pesticide-resistant H. azteca for fipronil and imidacloprid.•Development of insecticide resistance can have unwanted fitness costs.
Global climate change continues to cause alterations in environmental conditions which can be detrimental to aquatic ecosystem health. The development of pesticide resistance in organisms such as ...Hyalella azteca can lead to increased susceptibility to environmental change. This research provides a robust assessment of the effects of alterations in salinity on the fitness of H. azteca. Full-life cycle bioassays were conducted with non-resistant and pyrethroid-resistant H. azteca cultured under two salinity conditions representing a rise from freshwater control (0.2 psu) to increased salinity due to salt-water intrusion, reduced snowpack and evaporative enrichment (6.0 psu). Additionally, the upper thermal tolerance was defined for each population at each salinity. Pyrethroid-resistant H. azteca exhibited reduced thermal tolerance; however, they produced more offspring per female than non-resistant animals. Compared to the low salinity water, both non-resistant and pyrethroid-resistant H. azteca produced more offspring, grew larger (based on dry mass), and produced larger offspring in elevated salinity, although pyrethroid-resistant animals had lower survival and lipid levels. This study provides fundamental information about the fitness potential of H. azteca in a changing climate, suggesting advantages for non-resistant animals under future climate scenarios. In addition, this research further supports the need to consider the effects of global climate change when conducting risk assessment of contaminants of concern, as well as the contribution of contaminants when investigating climate change impacts on populations, as exposure may contribute to niche contraction.
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•Studied interaction of pesticide resistance and salinity on fitness in H. azteca•Thermal tolerance was reduced at higher salinities and in resistant H. azteca.•Pyrethroid-resistant H. azteca produced more offspring regardless of salinity.•Non-resistant H. azteca reproduced more and grew larger at higher salinity.•Lower survival at 6.0 psu in resistant H. azteca suggests reduced adaptive capacity.
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