Chemical tolerance related to the ABC transporter gene and DNA methylation in cladocera (Daphnia magna)

Abstract

AbstractWe 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 μg/L) for 15 generations (F0‐F14). We then determined the 48 h‐EC50 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 μg/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‐EC50 values in neonates (<24 h old) from the treated parents. The EC50 values (30.3–31.6 μg/L) in F14 of the 7.5 and 15 μg/L groups were approximately two times higher than that in the control (16.0 μg/L). A linear mixed model analysis showed that EC50 and ABCt mRNA levels were significantly increased with generational alterations; further analysis showed that the ABCt mRNA level was positively related to the EC50. Therefore, ABCt may be associated with altered pirimicarb sensitivity. In addition, the EC50 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.