A multigenerational study can detect the evolutionary response to BaP exposure in the non-biting freshwater midge Chironomus riparius.

The release of polycyclic aromatic hydrocarbons (PAHs) into the environment is posing a threat to ecosystems and human health. Benzo(a)pyrene (BaP) is considered a biomarker of PAH exposure and is classified as a Group 1 carcinogen. However, it was not known whether BaP is mutagenic, i.e. induces inherited germline mutations. In this study, we used a recently established method, which combines short-term mutation accumulation lines (MAL) with whole genome sequencing (WGS) to assess mutagenicity in the non-biting midge Chironomus riparius. The mutagenicity analysis was supplemented by an evaluation of the development of population fitness in three successive generations in the case of chronic exposure to BaP at a high concentration (100 µg/L). In addition, the level of ROS-induced oxidative stress was examined in vivo. Exposure to the higher BaP concentration led to an increase in germline mutations relative to the control, while the lower concentration showed no mentionable effect. Against expectations, BaP exposure decreased ROS-level compared to the control and is thus probably not responsible for the increased mutation rate. Likewise, the higher BaP concentration decreased fitness measured as population growth rate per day (PGR) significantly over all generations, without signs of rapid evolutionary adaptations. Our results thus highlighted that high BaP exposure may influence the evolutionary trajectory of organisms.

Spontaneous rate of clonal single nucleotide mutations in Daphnia galeata.

Mutations are the ultimate source of heritable variation and therefore the fuel for evolution, but direct estimates of mutation rates exist only for few species. We estimated the spontaneous single nucleotide mutation rate among clonal generations in the waterflea Daphnia galeata with a short-term mutation accumulation approach. Individuals from eighteen mutation accumulation lines over five generations were deep sequenced to count de novo mutations that were not present in a pool of F1 individuals, representing the parental genotype. We identified 12 new nucleotide mutations in 90 clonal generational passages. This resulted in an estimated single nucleotide mutation rate of 0.745 x 10-9 (95% c.f. 0.39 x 10-9-1.26 x 10-9), which is slightly lower than recent estimates for other Daphnia species. We discuss the implications for the population genetics of Cladocerans.

Polyamide microplastic exposure elicits rapid, strong and genome-wide evolutionary response in the freshwater non-biting midge Chironomus riparius.

Susceptibility to hazardous materials and contamination is largely determined by genetic make-up and evolutionary history of affected organisms. Yet evolutionary adaptation and microevolutionary processes triggered by contaminants are rarely considered in ecotoxicology. Using an evolve and resequencing approach, we investigated genome-wide responses of the midge C. riparius exposed to virgin polyamide microplastics (0-180 µm size range, at concentration 1 g kg-1) during seven consecutive generations. The results were integrated to a parallel life-cycle experiment ran under the same exposure conditions. Emergence, life-cycle trait, showed first a substantial reduction in larval survival, followed by a rapid recovery within three generations. On the genomic level, we observed substantial selectively driven allele frequency changes (mean 0.566 ± 0.0879) within seven generations, associated with a mean selection coefficient of 0.322, indicating very strong selection pressure. Putative selection targets were mainly connected to oxidative stress in the microplastics exposed C. riparius population. This is the first multigenerational study on chironomids to provide evidence that upon exposure to polyamide microplastic there are changes on the genomic level, providing basis to rapid adaptation of aquatic organisms to microplastics.

Whole genome sequencing and RNA-seq evaluation allowed to detect Cd adaptation footprint in Chironomus riparius.

Evolutionary adaptation and phenotypic plasticity are important processes on how organisms respond to pollutant exposure. We dissected here the contribution of both processes to increased tolerance in Chironomus riparius to cadmium (Cd) exposure in a multi-generation experiment and inferred the underlying genomic basis. We simulated environmentally realistic conditions by continuously increasing contaminant concentration in six replicates initiated with 1000 larvae each, three pre-exposed to Cd and three not exposed to Cd (no-Cd) over eight generations. We measured life-cycle traits, transcriptomic responses and genome-wide allele frequency changes from this evolve and resequencing (E&R) experiment. Overall, life cycle tests revealed little phenotypic adaptation to Cd exposure, but a slightly increase in survival in the first larval stage was observed. Population genomic analyses showed a strong genome-wide selective response in all replicates, highlighting two main biological functions involved in development and growth of the chironomids. Emphasizing that laboratory conditions continually exert selective pressure. However, the integration of the transcriptomic to the genomic data allowed to distinguish pathways specifically selected by the Cd exposure related to microtubules and organelles and cellular movement. Those pathways could be functionally related to an excretion of metals. Thus, our results indicate that genetic adaptation to Cd in C. riparius can happen within few generations under an environmentally relevant exposure scenario, but substantial phenotypic tolerance might take more time to arise. With our approach, we introduce an experimental setup to fill the existing gap in evolutionary ecotoxicology to investigate these early signs of genetic adaptation.

A multigenerational approach can detect early Cd pollution in Chironomus riparius.

Cadmium (Cd) is a non-essential highly toxic metal and its presence in the environment has been a concern over the years. On the present study we adopt the spiked water exposure scenario to study early Cd contamination across five generations of the model organism Chironomus riparius. Animals were, at the beginning of each generation, submitted to 0, 1, 3.2, 10, 32 and 100 µg/L of Cd. Classical endpoints like total emergence, EmT50, fertility and the integrative fitness measure, population growth rate (PGR), were calculated at each generation. Results could demonstrate that exposure to brief and low Cd concentrations can affect all the measured endpoints and, therefore, initial Cd pollution in previously unpolluted sites can be detected after just five consecutive generations. Importantly, at 100 µg/L of Cd fertility was greatly impaired after three generations. Also, PGR calculation is a sensitive tool for monitoring early pollution of Cd. Yet, no adaptation to Cd over five generations could be observed on the present experimental setup.

Measuring mutagenicity in ecotoxicology: A case study of Cd exposure in Chironomus riparius.

Existing mutagenicity tests for metazoans lack the direct observation of enhanced germline mutation rates after exposure to anthropogenic substances, therefore being inefficient. Cadmium (Cd) is a metal described as a mutagen in mammalian cells and listed as a group 1 carcinogenic and mutagenic substance. But Cd mutagenesis mechanism is not yet clear. Therefore, in the present study, we propose a method coupling short-term mutation accumulation (MA) lines with subsequent whole genome sequencing (WGS) and a dedicated data analysis pipeline to investigate if chronic Cd exposure on Chironomus riparius can alter the rate at which de novo point mutations appear. Results show that Cd exposure did not affect the basal germline mutation rate nor the mutational spectrum in C. riparius, thereby arguing that exposed organisms might experience a range of other toxic effects before any mutagenic effect may occur. We show that it is possible to establish a practical and easily implemented pipeline to rapidly detect germ cell mutagens in a metazoan test organism. Furthermore, our data implicate that it is questionable to transfer mutagenicity assessments based on in vitro methods to complex metazoans.

Time does matter! Acute copper exposure abolishes rhythmicity of clock gene in Danio rerio.

The circadian clock is a key cellular timing system that coordinates physiology and behavior. Light is a key regulator of the clock mechanism via its activation of Per and Cry clock gene expression. Evidence points to a key role of reactive oxygen species (ROS) in resetting this process. In this context, the aim of the present study was to explore copper as a ROS generator, using an innovative approach investigating its effects on circadian timing. Liver and brain from Danio rerio specimens exposed to 0, 5, 25 and 45 µg/L copper concentrations were obtained. Daily oscillations of superoxide dismutase (SOD) and catalase (CAT) enzymatic activity and their correlations both with clock genes (per1, per2, and cry1a) and with organism energy cost were determined. CAT expression correlates with per2 and cry1a and, thus, provides data to support the hypothesis of hydrogen peroxide production by a phototransducing flavin-containing oxidase. Higher SOD activity is correlated with higher intracellular ATP levels. Copper disturbed the daily oscillation of antioxidant enzymes and clock genes, with disturbed per1 rhythmicity in both the brain and liver, while cry1a rhythmicity was abolished in the liver at 25 µg/L copper. Coordination between the SOD and the CAT enzymes was lost when copper concentrations exceeded the limits established by international laws. These results indicate that organism synchronization with the environment may be impaired due to acute copper exposure.

How and where to perform biomonitoring studies: different levels of toxic metal pollution are detected in the Alagados Reservoir in Southern Brazil.

The Alagados Reservoir located in the Brazilian city of Ponta Grossa is used to supply water for human consumption but is impacted by toxic metals. The current study combined chemical, biochemical, and multivariate analyses to determine the bioavailability of toxic metals at three sites (AL-A, AL-B, and AL-C) within the Alagados Reservoir. Metal bioaccumulation was analyzed in the liver, gills, and muscle tissue of a native fish species (Geophagus brasiliensis), and neurotoxicity, xenobiotic metabolism, and oxidative stress were evaluated using biochemical biomarkers. Additionally, histopathological studies were performed on the gills and the liver using scanning electron microscopy (SEM) and conventional light microscopy (LM), respectively. Overall, the bioaccumulation of metals, biomarkers of oxidative stress, and melanomacrophage counts indicate that the AL-C and the AL-A sites are the most and least affected by metals, respectively. The AL-B site presented the lowest acetylcholinesterase enzyme activity, a finding which was probably associated with the agricultural activities around this area of the reservoir. The biomarkers clearly revealed that toxic metals negatively affect all three sites studied herein and that human activity is the major source of pollutants. Despite the existence of different pollution levels within the Alagados Reservoir, it is still used as a human water supply.

Bioconcentration and bioaccumulation of metal in freshwater Neotropical fish Geophagus brasiliensis.

From the concentration in water and sediments, bioconcentration and bioaccumulation of copper (Cu), manganese (Mn), zinc (Zn), iron (Fe), cobalt (Co), cadmium (Cd), chrome (Cr), silver (Ag), lead (Pb), nickel (Ni), aluminum (Al), and arsenic (As) were determined in the gills, liver, and muscles of Geophagus brasiliensis in the Alagados Reservoir, Ponta Grossa, Paraná, Brazil. Metals were quantified through AAS, and a study was carried out on the existing relations between metal and body weight, size, and genre of this species. The level of metal in the water of the reservoir was lower than the maximum set forth in the legislation, except for that of Cd and Fe. In sediments, Cu, Cd, Cr, and Ni presented concentrations above the threshold effect level (TEL). Pb and Cr were above the limits for the G. brasiliensis. The tendency of metals present in the muscles of G. brasiliensis was Al > Cu > Zn > Fe > Co > Mn > Cr > Ag > Ni > Pb > Cd > As. In the gills, it was Al > Fe > Zn > Mn > Co > Ag > Cr > Ni > Cu > As > Pb > Cd, and the liver presented Al > Cu > Zn > Co > Fe > Mn > Pb > Ag > Ni > Cr > As > Cd. The bioconcentration and bioaccumulation of metal in the tissues follow the global tendency liver > gills > muscle. The statistical analysis did not point to significant differences in the metal concentration and body weight, size, and gender of the species in the three tissues under analysis.