Evaluation of gene expression of different molecular biomarkers of stress response as an effect of copper exposure on the earthworm EIsenia Andrei

Abstract

The paper reports the results of a laboratory test on the bioaccumulation and toxicological effects of sub-lethal soil concentration of copper, a widely used fungicide in organic farming, on DNA damage, a critical marker increasingly used in ecotoxicology in the earthworm Eisenia andrei. In the same experimental setting we evaluated gene expression of classical biomarker of stress induced by xenobiotic. [Heat Shock Protein 70 (HSP70) and Metallothionein (MET)], as well as genes coding for enzymes involved in detoxification of reactive oxygen species [Superoxide dismutase (SOD) and catalase (CAT)]. Additionally, expression of genes involved in the immune response were investigated: a Toll-like receptor (TLR), a receptor with cytolytic activity named Cytolytic Factor (CCF) and two antimicrobial peptides, fetidin (FET) and lysenin (LYS). Results showed significant time-dependent bioaccumulation of Cu and DNA damage at concentrations remarkably lower than those found in most agricultural soils worldwide. MET was increased as was FET and TLR. The present work gives new insights into the mechanisms of sub-lethal toxicity of copper as an environmental pollutant and in the identification of novel sub-lethal biomarkers of cellular response to the stressor such as immune response genes.

The Effects of In Vivo Exposure to Copper Oxide Nanoparticles on the Gut Microbiome, Host Immunity, and Susceptibility to a Bacterial Infection in Earthworms

“…The effects of NP were compared to those of metal salts, to test whether any effects were attributed to particles or ions. We hypothesized that (i) earthworms that have their microbiome changed through exposure to CuO-NP and copper salts are more susceptible to a bacterial infection; and (ii) exposure to CuO-NP, copper salts, and the proceeding bacterial challenge will have an effect on the gene expression of tested immune markers, in line with previous studies [ 44 , 62 ]. The holistic methodological approach applied in this paper provides a useful framework for improved assessment of immuno-safety of NMs.…”

“…This cellular complexity means that it is possible for different cell subpopulations to have different sensitivities to pollutant exposure [ 82 , 97 ]. Exposure to metals and xenobiotics, but also immunostimulants like LPS [ 98 ], has been demonstrated to change the ratio between the different coelomocyte cell subpopulations [ 82 , 97 , 99 ] and to alter the expression of earthworm immune markers [ 62 ]. In this study, coelomic fluid was extruded and sampled without separation of different cell subpopulations.…”

Sub-Lethal Effects of Pesticides on the DNA of Soil Organisms as Early Ecotoxicological Biomarkers

“…With the exception of this paper, all other screened articles were conducted in standardized laboratory conditions through the application of OECD guidelines. Moreover, these studies did not use wild earthworms; on the contrary, they mainly used as a reference organism E. fetida, with the exception of three articles (Fouché et al, 2016;Chevillot et al, 2017;Mincarelli et al, 2019) that analyzed the effects of pesticides on the other commonly used earthworm reference species Eisenia andrei. As reported in Table 3, most of the studies used three organisms as an experimental unit for experimental conditions, and exceptionally few studies increased the observational sample to up to 10 or 18 samples (Wang et al, 2015;Chen et al, 2018;Espinosa-Reyes et al, 2019).…”

“…In all studies, DNA damage assessment was conducted on coelomocytes – hemocytes of the annelids that can be recovered from the coelomic fluid with non-invasive techniques by submerging the organism in ethanol 5% saline solution that might be integrated with the chelating agent EDTA and the mucolytic agent guaiacol or similar. The studies explored the potential genotoxic effect of a broad set of chemicals including as insecticides Cyantraniliprole ( Qiao et al, 2019 ), Acetamiprid ( Li B. et al, 2018 ), Neonicotionoids ( Chevillot et al, 2017 ), Imidacloprid ( Wang et al, 2016 ), Spirotetramat ( Zhang et al, 2015 ), Thiacloprid ( Feng et al, 2015 ), and Guadipyr ( Wang et al, 2015 ) that represented the most numerous, followed by fungicides Pyraclostrobin ( Ma et al, 2019 ), Fluoxastrobin ( Zhang et al, 2018 ), Tebuconazole ( Chen et al, 2018 ), and a smaller set referred to herbicides Tribenuron ( Chen et al, 2018 ) and Mesotrione ( Li X. et al, 2018 ) or general pesticides including Polychlorinated biphenyls ( Duan et al, 2017 ) and natural toxins used as biofumigants ( Fouché et al, 2016 ) and copper sulfide ( Mincarelli et al, 2019 ) that is one of the few chemical treatments allowed in organic farming. In all studies, synthetic compounds were studies at increasing doses of exposure normally below 10 mg/kg of soil, with the exception of the insecticide Guadipyr ( Wang et al, 2015 ) that was used between 10 and 100 mg/kg of soil.…”

“…with a dose-dependent response to the chemical at each time point analyzed; however, when they are observed on a longitudinal time scale during the typical month of exposure (28 days) at the lowest dose of chemicals used, often a decrease in DNA damage is observed from the second week of exposure highlighting an adaptive response; at intermediate dosage, DNA damage tends to stabilize during the last 2 weeks of exposure while at the highest concentration DNA damage is able to increase in comparison to the previous time points even after 1 month of exposure ( Li X. et al, 2018 ; Zhang et al, 2018 ; Ma et al, 2019 ). Mincarelli et al (2019) also showed an adaptive response at the level of gene expression of metallothionein as well as proteins involved in the immunological response (antimicrobial peptide fetidin and toll-like receptors).…”