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

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 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.

Increased extinction probability and altered physiological characteristics in pirimicarb-tolerant Daphnia magna.

We evaluated the physiological characteristics of chemical-tolerant cladocerans. Over the course of 26 generations (F25), Daphnia magna was continuously exposed to pirimicarb (carbamate) solutions (0, 3.8, 7.5, and 15 µg/L) in sub-lethal or lethal levels. The 48 h EC50 values (29.2-29.9 µg/L) for 7.5 and 15 µg/L exposure groups were found to be nearly two times higher than that in the control (17.2 µg/L). Subsequently, we investigated whether the extinction probability changed when the chemical-tolerant daphnids were fed two different types of food, Chlorella vulgaris and Synechococcus leopoliensis. Furthermore, we ascertained how chemical tolerance influences respiration and depuration rates. The 48 h EC50 value was positively related to the extinction probability when the daphnids were fed S. leopoliensis. Because the measured lipid content of S. leopoliensis was three times lower than that of C. vulgaris, the tolerant daphnids struggled under nutrient-poor conditions. Respiration rates across all pirimicarb treatment groups were higher than those in the control group, suggesting that they may produce large amounts of energy through respiration to maintain the chemical tolerance. Since the pirimicarb depuration rate for 7.5 µg/L exposure groups was higher than that in the control, the altered metabolic/excretion rate may be one factor for acquiring chemical tolerance. These altered physiological characteristics are crucial parameters for evaluating the mechanisms of chemical tolerance and associated fitness costs.

Possible enzymatic mechanism underlying chemical tolerance and characteristics of tolerant population in Scapholeberis kingi.

To determine the potential effects of pesticides on aquatic organisms inhabiting a realistic environment, we explored the characteristics and mechanisms of chemical tolerance in Scapholeberis kingi(Cladocera). We established a chemical-tolerant population via continuous exposure to pirimicarb, an acetylcholinesterase (AChE) inhibitor, and examined the effects of pirimicarb concentration on the intrinsic growth rates (r) of tolerant cladocerans. We also explored the association between r and feeding rate and tested the involvement of antioxidant enzymes [peroxidase (PO) and superoxide dismutase] and AChE in pirimicarb sensitivity. S. kingi was continuously exposed to lethal and sublethal pirimicarb concentrations (0, 2.5, 5, and 10 µg/L) for 15 generations, and changes (half maximal effective concentration at 48 h, 48 h-EC50) in chemical sensitivity were investigated. In the F14 generation, the sensitivity of the 10 µg/L group was three times lower than that of the control group, suggesting the acquisition of chemical tolerance. Moreover, r was significantly and negatively correlated with 48 h-EC50, suggesting a fitness cost for tolerance. Surprisingly, there was no significant correlation between r and feeding rate. There was a weak but significant positive correlation between each enzyme activity and the 48 h-EC50 value (p < 0.05). Thus, oxidative stress regulation and enhanced AChE may be involved in the acquisition of chemical tolerance in cladocerans. These findings will help elucidate the characteristics and mechanisms of chemical tolerance in aquatic organisms.