Ishimota_2023_Environ.Toxicol__

We performed multigenerational tests to clarify the chemical tolerance mechanisms of a nontarget aquatic organism, Daphnia magna. We continuously exposed D. magna to a carbamate insecticide (pirimicarb) at lethal or sublethal concentrations (0, 3.8, 7.5, and 15 microg/L) for 15 generations (F0-F14). We then determined the 48 h-EC(50) values and mRNA expression levels of acetylcholinesterase, glutathione S-transferase, and ATP (Adenosine triphosphate)-binding cassette transporter (ABCt) in neonates (<24 h old) from F0, F4, F9, and F14. To ascertain the effects of DNA methylation on pirimicarb sensitivity, we measured 5-methylcytosine levels (DNA methylation levels) in neonates of parents in the last generation (F14). In addition, we cultured groups exposed to 0 and 7.5 microg/L (the latter of which acquired chemical tolerance to pirimicarb) with or without 5-azacytidine (de-methylating agent) and determined methylation levels and 48 h-EC(50) values in neonates (<24 h old) from the treated parents. The EC(50) values (30.3-31.6 microg/L) in F14 of the 7.5 and 15 microg/L groups were approximately two times higher than that in the control (16.0 microg/L). A linear mixed model analysis showed that EC(50) and ABCt mRNA levels were significantly increased with generational alterations; further analysis showed that the ABCt mRNA level was positively related to the EC(50) . Therefore, ABCt may be associated with altered pirimicarb sensitivity. In addition, the EC(50) value and DNA methylation levels in pirimicarb-tolerant clones decreased after exposure to 5-azacytidine, suggesting that DNA methylation contributes to chemical tolerance. These findings improved our knowledge regarding the acquisition of chemical tolerance in aquatic organisms.