Historical exposure to chemicals reduces tolerance to novel chemical stress in Daphnia (waterflea)

Until the last few decades, anthropogenic chemicals used in most production processes didn't have a comprehensive assessment of their risk and impact on wildlife and humans. They are transported globally and usually end up in the environment as unintentional pollutants causing long-term adverse effects. Modern toxicology practises typically use acute toxicity tests of unrealistic concentrations of chemicals to determine their safe use, missing pathological effects arising from long-term exposures to environmentally relevant concentrations. Here, we study the transgenerational effect of environmentally relevant concentrations of five chemicals on the priority list of international regulatory frameworks on the keystone species Daphnia magna. We expose Daphnia genotypes resurrected from the sedimentary archive of a lake with a known history of chemical pollution to the five chemicals to understand how historical exposure to chemicals influences adaptive responses to novel chemical stress. We measure within and transgenerational plasticity in fitness-linked life history traits following exposure of 'experienced' and 'naive' genotypes to novel chemical stress. As the revived Daphnia originates from the same genetic pool sampled at different times in the past, we are able to quantify the long-term evolutionary impact of chemical pollution by studying genome-wide diversity and identifying functional pathways affected by historical chemical stress. Our results suggest that historical exposure to chemical stress causes reduced genome-wide diversity, leading to lower cross-generational tolerance to novel chemical stress. Lower tolerance is underpinned by reduced gene diversity at detoxification, catabolism and endocrine genes in experienced genotypes. We show that these genes sit within pathways that are conserved and potential chemical targets in other species, including humans.1 Abdullahi_etal_metadata_Atrazine: fitness-linked life history traits collected following exposure to Atrazine. GenotypeID; replicate; generation; treatment; mortality (including mortality event and day of mortality), size at maturity, age at maturity and fecundity measured after controlling for maternal effect across three generations are shown. If mortality occurred before sexual maturity, life history traits linked to later life stages were not recorded ('null'). 2Abdullahi_etal_metadata _PFOS: fitness-linked life history traits collected following exposure to Atrazine. GenotypeID; replicate; generation; treatment; mortality (including mortality event and day of mortality), size at maturity, age at maturity and fecundity measured after controlling for maternal effect across three generations are shown. If mortality occurred before sexual maturity, life history traits linked to later life stages were not recorded ('null'). 3Abdullahi_etal_metadata _Arsenic: fitness-linked life history traits collected following exposure to Atrazine. GenotypeID; replicate; generation; treatment; mortality (including mortality event and day of mortality), size at maturity, age at maturity and fecundity measured after controlling for maternal effect across three generations are shown. If mortality occurred before sexual maturity, life history traits linked to later life stages were not recorded ('null'). 4Abdullahi_etal_metadata_Trimethoprim: fitness-linked life history traits collected following exposure to Atrazine. GenotypeID; replicate; generation; treatment; mortality (including mortality event and day of mortality), size at maturity, age at maturity and fecundity measured after controlling for maternal effect across three generations are shown. If mortality occurred before sexual maturity, life history traits linked to later life stages were not recorded ('null'). 5Abdullahi_etal_metadata_Diclofenac: fitness-linked life history traits collected following exposure to Atrazine. GenotypeID; replicate; generation; treatment; mortality (including mortality event and day of mortality), size at maturity, age at maturity and fecundity measured after controlling for maternal effect across three generations are shown. If mortality occurred before sexual maturity, life history traits linked to later life stages were not recorded ('null').Funding provided by: Natural Environment Research CouncilCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100000270Award Number: NE/N016777/1Funding provided by: Alan Turing Institute (EPSRC)**Crossref Funder Registry ID: Award Number: EP/N510129/1Funding provided by: Petroleum Technology Development FundCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100009614Award Number: PTDF/ED/OSS/POF/1369/18Funding provided by: EU H2020 Marie Skłodowska-Curie Fellowship*Crossref Funder Registry ID: Award Number: 101028700For the current study, we used a genotype from each of the lake phases identified in Lake Ring 1 [Fig. 1; LRV0_1 (recovery), LRV8.5_3 (pesticides), LRV12_3 (eutrophication), LRII36_01 (semi-pristine)]. Following the process of resurrection, the genotypes were maintained in the laboratory for over a year as isoclonal lines in the following standard laboratory conditions: 16:8 hr light: dark photoperiod; 0.8 mg/L Chlorella vulgaris fed weekly; ambient temperature: 100C. Prior to the exposures, clonal lineages of the four genotypes were acclimated for at least two generations to the following conditions: 16:8 h light: dark photoperiod; 0.8 mg/L Chlorella vulgaris fed daily; ambient temperature: 200C, to reduce interference from maternal effect. After at least two generations in these conditions, 24h old individual juveniles from the second or following broods were randomly isolated and assigned to experimental conditions: PFOS (70 ng/L), Atrazine (0.2 mg/L), Trimethoprim (2 mg/L), Diclofenac (2 mg/L), and Arsenic (1,000 µg/L) (Fig. 1). The chemicals' concentrations were used to represent typical concentrations found in surface water (see Introduction). Where necessary, different broods from the same generation were used to ensure developmental synchrony among clonal lineages in the experiment. Exposures were conducted over three generations and five clonal replicates per genotype in individual jars of 100 ml, filled with filtered borehole water (growth medium), which was refreshed every second day. The experimental cultures were fed daily ad libitum with 0.8 mg Carbon/L of Chlorella vulgaris. To ensure constant concentrations of the chemicals throughout the experimental exposures, chemicals were introduced in the culture medium at each media change. The following fitness-linked life history traits were measured across three generations of four genotypes and 5 clonal replicates for exposed and non-exposed experimental animals: age at maturity (first time parthenogenetic eggs are released in the brood pouch), size at maturity (distance from the head to the base of the tail spine), fecundity (sum of juveniles across 2 broods), interval between broods and mortality. Size at maturity was measured after the release of the first brood in the brood pouch using image J software (https://imagej.nih.gov/ij/). The mortality rate per genotype was determined with the survival model fit using the psm function in the 'rms' package in R v.3.6.0. The day of mortality and mortality events were combined as a response variable while the term 'genotype' was treated as a fixed effect. The mortality curves per generation were plotted with the survplot function from the rms package in R v.3.6.0. Genotypes were fixed across all experimental conditions and generations, enabling us to control for confounding factors e.g. genetic changes occurring from one generation to the next and genetic variation among experimental exposures. This design permits the analysis of within (WGP) and transgenerational plasticity (TGP), as well as the analysis of evolutionary differences among genotypes