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.


Introduction
The risk assessment of chemicals to aquatic organisms is primarily based on recommended tests on model species (Brock and Van Wijngaarden 2012;Taenzler et al. 2007). However, the chemical sensitivity of test species differs from that of their actual field clones. Because field clones may inhabit real environments with various stressors, they often acquire chemical tolerance (Haap and Köhler 2009;Jansen et al. 2011;Muyssen et al. 2002). For example, the LC 50 values of permethrin in Hyalella Azteca collected in the field were approximately 53 times higher than that of standard laboratory clones even after 22 months cultutre in permethrin-free water (Heim et al. 2018).
Several researchers have previously shown that aquatic organisms (i.e., cladocerans and chironomids) rapidly acquire chemical tolerance (Brausch and Smith 2009;Ward and Robinson 2005;Ishimota et al. 2020a, b). Gene mutation at the target site of the chemical in aquatic organisms is a major mechanism for the development of chemical tolerance (Perrier et al. 2021). Another factor that causes rapid tolerance to several stressors (e.g.,, pesticides and toxic algae) is maternal effect, which can rapidly enhance the rates of evolutionary response to selection (Ishimota et al. 2020b;Lyu et al. 2017;Mousseau et al. 2009). When the mother's environment affects her offspring's phenotype, in addition to the direct effect of transmitted genes, maternal effects are caused (Marshall and Uller 2007). The DNA methylation in the specific CpG sites is one of the factors for the maternal effects (Venney et al. 2020).
Recent studies have revealed that several factors could be related to the acquisition of chemical tolerance. Water temperature and food resource acquisition have been associated with chemical sensitivity in aquatic organisms (Tran et al. 2018;Wuerthner et al. 2019). Moreover, Venâncio et al. (2018) reported that Daphnia longispina, when exposed to lethal levels of sodium chloride, produced neonates that showed different chemical tolerance among clonal lineages. This finding suggests that aquatic organisms possess various genotypes to acquire chemical tolerance, even among the same species. Therefore, other contributory mechanisms need to be elucidated.
Despite their low sensitivity to chemicals, tolerant individuals encounter fitness costs for this adaptation, such as reduced population growth rate or shortened life span, compared with sensitive individuals (Heine-Fuster et al. 2017;Homem et al. 2020). Furthermore, insecticide-tolerant individuals require more energy and have greater fat body mass than sensitive individuals (Kliot and Ghanim 2012). Several characteristics of chemical-tolerant populations have recently been identified. When organisms adapt to one stressor, they often acquire high sensitivity to other stressors. For instance, the pyrethroid-resistant H. Azteca collected from the field is more sensitive to other stressors, such as thermal stress, 4, 4'-dichlorodiphenyltrichloroethane (DDT), and metal (copper) (Heim et al. 2018). However, several characteristics of fitness costs in chemical-tolerant populations remain unknown. No fitness cost was observed in the strain selected by continuous exposure to a chemical under laboratory conditions (Argentine et al. 1989;Kliot and Ghanim 2012). For instance, life-cycle parameters, such as life-span or offspring production, did not change in cadmium (Cd) tolerant D. magna selected by Cd, compared to the sensitive population (Ward and Robinson 2005); in other words, levels of each cycle parameter were almost the same between Cd exposure treatment and Cd sensitive population (i.e., control). Further research is warranted to elucidate the characteristics of chemical-tolerant populations, which will help understand the true effects of chemicals on field clones.
The adverse effects of pesticides on some non-target organisms in aquatic ecosystems need to be investigated (Hayasaka et al. 2013). Towards this goal, understanding the toxic mechanisms of pesticides will enable the protection of non-target aquatic organisms, such as cladocerans from pesticide-mediated toxicity. Acetylcholinesterase (AChE) inhibitors (organophosphorus and carbamate pesticides) are the most abundantly produced pesticides in the world (Zhang 2018). Although their main mode of action is AChE inhibition, researchers could not explain the chemical sensitivity in aquatic organisms only by the AChE activity, and other enzymes, such as glutathione-S-transferases activity, may also play a contributory role (Damásio et al. 2007;Ishimota and Tomiyama 2020). Carbamates (AChE inhibitors) are known to alter the expression of various genes in cladocera (Pereira et al. 2010). However, it is difficult to ascertain which enzymes are mainly related to chemical tolerance owing to the complexity of the regulatory mechanism (Silva et al. 2018;Wang et al. 2016). Therefore, understanding the toxic mechanisms, which are not considered primary, will provide further information on chemical tolerance in aquatic organisms.
To investigate the characteristics of chemical tolerance, organisms with different degrees of chemical sensitivity must be produced, and multigenerational exposure to chemicals is a strategy to obtain such organisms. In a previous study, we investigated changes in the sensitivity of the cladoceran species Scapholeberis kingi to the carbamate insecticide pirimicarb, an acetylcholinesterase (AChE) inhibitor, which is one of the most common insecticides detected in surface water (Struger et al. 2016;Ishimota and Tomiyama 2020); we performed intergenerational experiments (two generations) by exposing neonates (< 24-h old) to pirimicarb for 48 h and observed that pirimicarb sensitivity differed between the pirimicarb-treated and non-treated individuals. Although we measured AChE activity in clones, we could not explain the intergenerational changes in pirimicarb sensitivity based on the activity of this enzyme. Pirimicarb has been reported to induce oxidative stress or produce genotoxic and cytotoxic effects in aquatic organisms, including fish, tadpoles, and snails (Natale et al. 2018;Raisi et al. 2018;Vera-Candioti et al. 2015), indicating the potential involvement of antioxidant enzymes in the chemical sensitivity of cladocerans.
S. kingi is a cladoceran commonly observed in paddy fields and ditches (Mano et al. 2010). The species is considered a suitable model species for evaluating chemical toxicity owing to its higher sensitivity to AChE inhibitors than that of other test species, such as Daphnia magna (Ishimota et al. 2020b).
To this end, the objectives of the present study were (1) to assess changes in the intrinsic growth rate (r) of field cladocerans (S. kingi) depending on their degree of pirimicarb tolerance; (2) to determine the association between r and feeding rate; and (3) to explore the involvement of antioxidant enzymes (PO and SOD) and AChE in pirimicarb sensitivity.
We collected several S. kingi clones and selected them via continuous exposure to lethal and sublethal pirimicarb concentrations for 15 generations (F0-F14). To investigate changes in the sensitivity of S. kingi, the half maximal effective concentration at 48 h (48 h-EC 50 ) for neonates (< 24-h old) from the F0, F4, and F14 generations was calculated. Additionally, the r and feeding rate of the chemical-tolerant population were determined. Finally, the association of the 48 h-EC 50 value with the activity of PO, SOD, and AChE was determined.

Chemical analysis
We obtained pirimicarb (99.9% purity) from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) and used it as the model AChE inhibitor. To determine its pirimicarb concentration, we diluted the test solution with acetonitrile in pure water (60:40 v/v). The pirimicarb concentration of each test solution was measured using liquid chromatography-mass spectrometry (LC-MS/MS) at a flow rate of 0.3 mL/min and column temperature of 40 °C (LC: 1290 HPLC; MS/MS: 6460 Triple Quad LC-MS/MS, Agilent Technologies, CA, USA; column: Acquity UPLC HSS T3, 1.8 µm × 2.1 mm × 100 mm, Waters, MA, USA). The analytical conditions were set as described previously (Ishimota et al. 2020a). The limit of quantification was set as 0.5 µg/L.
In a previous study, we confirmed that recoveries and their relative standard deviations (RSDs %, SD divided by mean) for pirimicarb were within the acceptable range using this method (mean recovery rate, 90-108%, RSD, 1-2%) (Ishimota and Tomiyama 2020). Furthermore, the target chemical was very stable during the exposure period (48 h). Thus, we measured pirimicarb concentration only at the beginning of exposure in each generation of the acute toxicity experiments (described in the Multigenerational experiment section). We set the acceptable recovery for pirimicarb concentrations as 70-120% (US EPA 2012; Stamatis et al. 2013).

Test organisms and culture methods
In a previous study, we confirmed that S. kingi clones exhibited different degrees of sensitivity to pirimicarb (the range of 48 h-EC 50 values 7-17 µg/L) (Ishimota and Tomiyama 2020). Thus, we used clones collected from four littoral sites, namely Lake Kasumigaura (36° 04′ 59″ N, 140° 13′ 06″ E), Lake Kitaura (36° 04′ 16″ N, 140° 31′ 44″ E), Tega pond (35° 51′ 40″ N, 140° 02′ 16″ E); and Moriya pond (35° 57′ 04″ N, 140° 00′ 19″ E), during the summer of 2015. These sites were located in a part of the Tone River drainage system with the agricultural land use (i.e., % of land area) ranging from 22 to 39% (Ishimota and Tomiyama 2020). Moreover, at these sites, several pesticides were previously found (Nohara and Iwakuma 1996;Tamura and Ogura 1994) and the water quality was very similar, e.g., electrical conductivity corrected at 25 °C (34-36 mS/m), pH value (7.9-8.8), and dissolved oxygen (8.1-9.1 mg/L) (Ishimota and Tomiyama 2020). However, the other water qualities differed from each other. For example, the total organic carbon values and chlorine ions ranged from 1.7 to 6.1 mg/L and 11.0 to 49.8 mg/L, respectively. Additionally, toxic ions, such as NO 3 − , were detected at several sites (0.6 mg/L in Lake Kasumigaura and Tega pond; 1.8 mg/L in Moriya pond) but not in Lake Kitaura. Although these values were lower than the 48 h EC 50 values in a cladoceran (23 mg N/L in D. magna) (Eytcheson and LeBlanc 2018), the field clones may have been constantly exposed to toxic chemicals in the field (Ishimota and Tomiyama 2020). Therefore, the differential water quality at these sites possibly changed their sensitivity to chemicals, thereby causing a genetic variability among these clones.
A single S. kingi clone from each site was individually maintained in the ISO medium (ISO 1996) with slight modification (36.8 mg/L CaCl 2 ·2H 2 O, 6.1 mg/L MgSO 4 ·7H 2 O; 64.8 mg/L NaHCO 3 , 8.60 mg/L KCl, 75.0 µg/L thiamine hydrochloride, 1.00 µg/L cyano-cobalamin, and 0.75 µg/L biotin) (Ishimota and Tomiyama 2020). For culture, including the acclimation period, 10 adults were incubated in 100-mL glass beakers containing 100 mL of the medium and fed at least five times per week with Chlorella vulgaris (Recenttec K. K, Japan, 5 × 10 5 cells/mL). The cultures were maintained under a 16:8 h light:dark photoperiod at 800 lx and 22 ± 1 °C. Neonates from the third or fourth brood were used for stock culture. The culture medium was changed three times per week.

Multigenerational experiment
To understand mechanisms underlying chemical tolerance (i.e., enzymes involved in the alteration of sensitivity to AChE inhibitors), we continuously exposed S. kingi to various concentrations of pirimicarb and established populations with varying degrees of sensitivity to this chemical. In a previous study, we noted changes in the chemical sensitivity of S. kingi following exposure to sublethal pirimicarb concentration (2.5 µg/L) for several clonal lineages (Ishimota and Tomiyama 2020). Thus, we expected clonal differences in the acquisition of chemical tolerance in the test species. Mano and Tanaka (2017) showed that variance in the genetic value of isofemale lines established from dormant eggs of Daphnia galeata and the heritability of organophosphate (fenitrothion) tolerance significantly differed between the sampling sites in Lake Kasumigaura (i.e., one of our sampling sites in this study). This finding suggests that the genetic variance in S. kingi from the four sampling sites also differed. If we combined the test clones from the differential sampling sites, the population might have representative genetic variation in this area. To normalize the ability to acquire pirimicarb tolerance, ten individuals (< 24-h old) collected from each sampling site were pooled and cultured together (40 individuals) in a 500-mL beaker containing 500 mL of the medium. To establish the combined population in the next generation, 40 neonates (< 24-h old) from the third or fourth brood were collected from the combined population and cultured in a 500-mL beaker containing 500 mL of the medium. We repeated this culturing for one month until we started the multigenerational study. The cultures were maintained under standardized culture conditions (described in the Test organisms and culture methods section).
We prepared a stock solution of 1.6 g/L pirimicarb with methanol: pure water solution (50:50, v/v). Next, we diluted this stock solution with the culture medium to prepare six concentrations of the test solution (0, 2.5, 5.0, 10.0, 20.0, and 40.0 µg/L) in methanol (12.5 µL/L). We then conducted acute toxicity experiments based on the OECD guideline no. 202 (OECD 2004). Five female neonates (< 24-h old), collected from the combined population (which we previously described) in the third or fourth brood, were exposed to 50 mL of test solution in a 50-mL glass beaker in four replicates without food under standard culture conditions (as previously described) for 48 h (total individuals, 20). Following exposure, we counted the neonates that could not swim during 15 s of gentle agitation of the beaker as immobilized individuals at each concentration. We determined the 48 h-EC 50 values and 95% confidence intervals for the first generation (F0) based on the immobilization data.
In the preliminary experiment, the 48 h-EC 50 values for pirimicarb for each clone ranged from 7 to 17 µg/L. Considering the lethal and sublethal concentration for each clone, neonates (< 24-h old) from the combined population in the third or fourth brood were continuously exposed to various pirimicarb concentrations (0, 2.5, 5.0, and 10.0 µg/L) until they reproduced third-or fourth-brood neonates. We exposed the test cladocerans (< 24-h old) to 100 mL of each test solution in a glass beaker containing 100 mL of the medium (n = 10) and cultured them according to the standard method (previously described). We collected the thirdor fourth-brood neonates to maintain the next generation and to determine the 48 h-EC 50 values and enzyme activity. Third-or fourth-brood neonates (< 24-h old) from the F0, F4, and F14 generations in each test group were exposed to various pirimicarb concentrations (0, 2.5, 5.0, 10.0, 20.0, and 40.0 µg/L) for 48 h to calculate the 48 h-EC 50 values and 95% confidence intervals.
To estimate the variation in 48 h-EC 50 values, we performed all experiments in duplicate.

Measurement of r
The r-value is a representative life cycle parameter providing information at the population level (growth and reproduction) (Buhl et al. 1993;Silva et al. 2017). According to the OECD test guideline, TG 211 (OECD 1997) reproduced 12 neonates (< 24-h old) from the third brood in each test group (0, 2.5, 5, and 10 µg/L); neonates in each test group were exposed to the same pirimicarb concentrations until 21 days after birth. From each test group, one neonate was placed in a 10-mL glass beaker containing 10 mL of each test solution; for each test concentration, four replicates were set, and the experiment was repeated in triplicate (total neonates, 12 per test group). The survival and reproductive rates were counted daily for 21 days in each generation (Ishimota and Tomiyama 2020). To avoid density effects, we removed the neonates immediately after counting. The neonates were cultured under standard culture conditions and fed with C. vulgaris (5 × 10 5 cells/mL) every day. The r-values were calculated using the daily age-specific survival and reproduction data. First, we calculated the age-specific fecundity during the experiment based on the Euler-Lotka equation and used it for the Leslie matrix (Lotka 1913).
where r is the intrinsic population growth rate, l x is the survival rate at a specific age (x), and m x is the reproduction rate at a specific age. Next, the r-values in the F0, F4, and F14 generations were estimated using the dominant eigenvalue (λ) of the Leslie matrix (i.e., daily time step, 21 age classes) for each treatment, using the following equation (Case 2000): where r is the intrinsic population growth rate, and λ is the dominant eigenvalue of the Leslie matrix. Mean and standard deviation of the r-values were calculated based on the rates for each replicate (n = 3).

Feeding experiment
To determine mechanisms underlying the generational changes in r, the feeding rate of mature S. kingi in each test group was calculated by comparing the food concentration at the beginning of the experiment with that at the end. This experiment was conducted using test individuals in the F0, F4, and F14 generations from each concentration group. Pirimicarb test solutions (0, 2.5, 5.0, and 10 µg/L) in methanol (12.5 µL/L), and C. vulgaris density was set at 5 × 10 5 cells/mL. One adult individual (7-day old) from each concentration group was placed in a 10-mL glass beaker containing 10 mL of each test solution, and five replicates were set for each test group. To confirm the decrease in algal cell density, a blank group (a 10-mL glass beaker containing 10 mL of the medium with the same density of algal cells but without S. kingi) was prepared. All experiments were performed in the dark at a controlled temperature (22 ± 1 °C) to minimize algal growth (Agra et al. 2010). Algal cell density was measured using flow cytometry (Guava EasyCyte Mini, Millipore, USA) at the beginning and after 48 h of exposure. The feeding rate (Fr) was calculated as follows: where Fr is feeding rate, Ct and Cb are the algal cell densities in the pirimicarb test (0, 2.5, 5.0, and 10 µg/L) and blank groups, respectively, and "0" indicates time at the beginning of pirimicarb exposure; "48" indicates time at 48 h after exposure (the end of the exposure).
Additionally, at the beginning of the test, the body sizes of S. kingi in each test group were measured (n = 5) and compared using ANOVA (p = 0.05). This experiment was conducted using test neonates in the F0, F4, and F14 generations from each concentration group.

Enzyme activity
We explored the involvement of antioxidant enzymes (PO and SOD) and AChE (target enzyme for pirimicarb) in pirimicarb tolerance of these population (Jeon et al. 2013). Initially, pirimicarb test solutions (0, 2.5, 5.0, and 10 µg/L) in methanol (12.5 µL/L) were prepared. Since the body size of S. kingi is very small (approximately 0.3 mm), 100 neonates (< 24-h old) were required to measure the activity in a single sample. Thus, three sets of 100 neonates (< 24-h old) in the third brood were exposed to 1 L of each test solution in a 1-L glass beaker for 48 h. Then, the F0 neonates in each beaker were homogenized according to the analytical method described previously (n = 3; total individuals, 300) (Ishimota and Tomiyama 2020). In addition, 100 F4 and F14 neonates (< 24-h old) in the third brood were exposed to the same pirimicarb concentrations and homogenized; three replicates were set for each test group. All samples were filtered using a cell strainer (70 µm, FALCON®) in 100 µL of 1% phosphate-buffered saline containing 0.25 mg/mL Pefa-bloc® SC (Sigma analytical standard, Sigma Aldrich, UK). The samples were sonicated for 15 min and centrifuged for 10 min at 4 °C and 15,000 × g. The supernatant of each sample was isolated, and the protein concentration was measured

Statistical analysis
All data were analyzed using R 3.6.1 (R Development Core Team 2019). Using the medrc package, the 48 h-EC 50 values with 95% confidence intervals were estimated by fitting the acute toxicity data to a two-parameter log-logistic model, considering duplicate testing of each test group as the random effect (Gerhard and Ritz 2017; Ishimota et al. 2020b). Significant differences in 48 h-EC 50 values among the test groups were analyzed with the ratio test using the EDcomp function in the drc package (with a significance level p = 0.05) (Ritz et al. 2006;Wheeler et al., 2006). The significance level for each pair was adjusted using Holm's method (Holm 1979), after we confirmed. After confirming the homoscedasticity by Levene's test in the lawstat package (Hui et al. 2008), each enzyme activity in all generations was compared using Dunnett's test using the multcomp package (Hothorn et al. 2008).
If we observed the negative effects of pirimicarb on r in the F0 group, we investigated the correlation between 48 h-EC 50 and mean r for all generations using Pearson's correlation analysis with the Rcmdr package (Fox 2005). Before the analysis, we confirmed that the variable was normally distributed using the Shapiro-Wilk normality test (p = 0.05) with the Rcmdr package (Fox 2005). Pearson's coefficients (cor) were calculated with a significance level of 0.05, with a coefficient of 1 (p < 0.05), indicating a perfect positive correlation, and a coefficient of − 1 (p < 0.05), indicating a perfect negative correlation.
To determine whether the enzyme activity and 48 h-EC 50 were altered depending on the generations (F0, F4, and F14), we built a linear mixed model (LMM) using the lmer function with the lme4 package, and the correlation between the generation alteration and other parameters (enzyme activity and 48 h-EC 50 ) was investigated (Bates et al. 2015). This model estimated the slope values of generation as a variable by adding a random effect for pirimicarb concentrations as the subject. Additionally, we calculated the correlations between enzyme activity and the 48 h-EC 50 value in multiple generations. Since we have only one EC 50 value against several enzyme activity results (i.e., exposure to 0, 2.5, 5.0, and 10 µg/L pirimicarb solutions) for the F0 generation, it was difficult to show the relationship between the enzyme activity and EC 50 value . Thus, we used the data from the F4 and F14 generations for the correlation analysis. After confirming that the variable of each parameter was not normally distributed, using the Shapiro-Wilk normality test (p = 0.05), Spearman's coefficients (rho) were calculated with a significance level of 0.05, using the Rcmdr package (Fox 2005). A coefficient of 1 (p < 0.05) indicates a perfect positive correlation, and a coefficient of − 1 (p < 0.05) indicates a perfect negative correlation.

Insecticide concentration and water quality in test solutions
In the intergenerational experiment, the ratio of the measured concentration to the nominal concentration in the water sample at the start of pirimicarb exposure ranged from 80 to 104%, which was within the acceptable range (70-120%).
Temperature, pH, and dissolved oxygen concentration in the water ranged from 22.1 to 22.6 °C, 8.11 to 8.44, and 8.10 to 9.36 mg/L, respectively, and variations in these parameters remained within the OECD test guideline no. 202 throughout the experiment (OECD 2004).

Multigenerational experiments
The 48 h-EC 50 values in the control group (0 µg/L) were comparable during the test generations (Fig. 1), and no immobilized neonates were not found in any generations of controls (0 µg/L).
In the F4 and F14 generations, the 48 h-EC 50 values in the 2.5 µg/L concentration group were comparable to those in the control group. In contrast, in the F4 generation, the 48 h-EC 50 value (3.9 µg/L) in the 5.0 µg/L concentration group was significantly decreased and almost 2.5 times lower than that of the control group (9.7 µg/L) (p < 0.05). Likewise, in the F4 generation, the 48 h-EC 50 value in the 10 µg/L concentration group (5.5 µg/L) was decreased compared with those in the control group, albeit non-significantly. In the F14 generation, the 48 h-EC 50 values in the 5.0 and 10 µg/L concentration groups (15.2-25.8 µg/L) were increased compared with those in the control group (7.7-9.7 µg/L) (p < 0.05). In particular, the 48 h-EC 50 value in the 10 µg/L concentration group was approximately three times higher than that in the control group (0 µg/L) (p < 0.05), which was the highest among the exposure groups. These results suggest that S. kingi was likely to acquire pirimicarb tolerance in the F14 generation.
Additionally, the LMM showed that the 48 h-EC 50 values were positively correlated with generational alteration (Table 1).

r and feeding rate
The r-values in the control group (0 µg/L) were comparable throughout the experimental period (Fig. 2). The r-values in the 10 µg/L concentration group were significantly lower than those in the control group in the F0 and F14 generations but comparable to those in the control group in the F4 generation (p < 0.05). Additionally, the 48 h-EC 50 value was significantly and negatively correlated with the r-values (cor = − 0.82, p < 0.01). The feeding rates in the control group (0 µg/L) were comparable throughout the experimental period. In the F0 generation, the feeding rates in all test groups were decreased compared with those in the control group, albeit non-significantly (Fig. 3). In contrast, in the F4 generation, the feeding rates were comparable among the test and control groups. However, in the F14 generation, Fig. 1 Half maximal effective concentration at 48 h (48 h-EC 50 ) and 95% confidence intervals (error bars) for Scapholeberis kingi exposed to pirimicarb (0, 2.5, 5.0, and 10 µg/L) for 48 h in F0, F4, and F14 generations. Different letters indicate significant differences (p < 0.05, corrected by Holm's method) the feeding rates in the test groups were significantly higher than those in the control group (p < 0.05). Contrary to our expectations, the feeding rate was not related to the r-value (cor = − 0.39, p ≥ 0.05). Furthermore, the body size of the test species (0.60-0.65 mm) did not significantly differ among the test groups, suggesting that body size hardly affected the feeding rate.

Enzyme activity
The PO activity of the F0 generation in the 2.5 and 5.0 µg/L concentration groups and of the F4 generation in the 5.0 and 10.0 µg/L concentration groups was decreased compared with that in the control group (0 µg/L) (p < 0.05) (Fig. 4). Although the PO activity of the F0 generation in the 10.0 µg/L concentration group did not decrease, this Fig. 2 Mean intrinsic population growth rate (r) of Scapholeberis kingi continuously exposed to pirimicarb (0, 2.5, 5.0, and 10 µg/L) in F0, F4, and F14 generations. Error bars indicate standard deviation (n = 3). Different letters indicate significant differences (p < 0.05, corrected by Holm's method) Fig. 3 Mean feeding rate of adult Scapholeberis kingi (7-day old) continuously exposed to pirimicarb (0, 2.5, 5.0, and 10 µg/L) in F0, F4, and F14 generations. S. kingi individuals in each group (0, 2.5, 5.0, and 10 µg/L) were exposed to the same concentration of pirimicarb for 48 h in the presence of food (Chlorella vulgaris, 5 × 10 5 cells/ mL), and the feeding rate was calculated based on the loss of food. Error bars indicate standard deviation (n = 5). Different letters indicate significant differences (p < 0.05, corrected by Holm's method) Fig. 4 Peroxidase (PO) activity in third-brood neonates (< 24-h old) exposed to pirimicarb (0, 2.5, 5.0, and 10 µg/L) for 48 h in F0, F4, and F14 generations. Neonates were continuously exposed pirimicarb (0, 2.5, 5.0, and 10 µg/L) until 21 days after birth. The reproduced neonates (< 24-h old) from each test group were exposed to the same concentration of pirimicarb (0, 2.5, 5.0, and 10 µg/L) for 48 h and used for the estimation of PO activity. Data represent the mean ± standard deviation (ratio to control, 0 µg/L, n = 3). Asterisks indicate significant differences compared with the control in each generation (p < 0.05) may be attributed to the large variation in the activity of this enzyme. The PO activity of the F14 generations in all test groups was comparable to that in the control group (0 µg/L).
The SOD activity of the F0 generation in all test groups was decreased compared with that in the control group (0 µg/L) (p < 0.05) (Fig. 5). In the F4 generation, although the SOD activity in the 5.0 and 10.0 µg/L concentration groups was decreased compared with that in the control group, the activity in the 2.5 µg/L concentration group was comparable to the control level (p < 0.05). Finally, in the F14 generation, the SOD activity in all test groups was comparable to that in the control group.
The AChE activity in the test groups was significantly decreased compared with that in the control group in the F0 and F4 generations but remained comparable to the control level in the F14 generation (Fig. 6).
Overall, a significant decrease in the activity of all enzymes was confirmed in the F0 and F4 generations, but the activity recovered to the control level in the F14 generation. The LMM showed that PO, SOD, and AChE activity was positively correlated with generational alterations (Table 1). In the correlation analysis, the elevated enzyme activity was significantly but slightly correlated with 48 h-EC 50 ( rho values for PO, SOD, and AChE activity were 0.37, 0.45, and 0.35, respectively, p < 0.05) ( Table 2).

Discussion
In F4 generation, individuals exposed to higher pirimicarb concentrations (5.0 and 10 µg/L) were more sensitive than the controls (Fig. 1), suggesting that they could not recover from the lethal and sublethal effects of the chemical even after a few generations. However, at the end of the experimental period (in the F14 generation), individuals exposed to the highest pirimicarb concentration (10 µg/L) were the most tolerant. These findings indicate that the chemical sensitivity of the test populations was strongly affected at the initiation of pirimicarb exposure, but they could adapt to the effects of this pollutant over generations.
The longevity (14.5-19.1 days) at 10.0 µg/L group throughout the generations was significantly shorter compared to that of the controls (20-21 days), which may suggest that the test cladocerans at the 10.0 µg/L were selected by the exposure (Supplementary Information Fig. S2). The Superoxide dismutase (SOD) activity in third-brood neonates (< 24-h old) exposed to pirimicarb (0, 2.5, 5.0, and 10 µg/L) for 48 h in F0, F4, and F14 generations. Neonates were continuously exposed pirimicarb (0, 2.5, 5.0, and 10 µg/L) until 21 days after birth. The reproduced neonates (< 24-h old) from each test group were exposed to the same concentration of pirimicarb (0, 2.5, 5.0, and 10 µg/L) for 48 h and used for the estimation of SOD activity. Data represent the mean ± standard deviation (ratio to control, 0 µg/L, n = 3). Asterisks indicate significant differences compared with the control in each generation (p < 0.05) Fig. 6 Acetylcholinesterase (AChE) activity in third-brood neonates (< 24-h old) exposed to pirimicarb (0, 2.5, 5.0, and 10 µg/L) for 48 h in F0, F4, and F14 generations. Neonates were continuously exposed pirimicarb (0, 2.5, 5.0, and 10 µg/L) until 21 days after birth. The reproduced neonates (< 24-h old) from each test group were exposed to the same concentration of pirimicarb (0, 2.5, 5.0, and 10 µg/L) for 48 h and used for the estimation of AChE activity. Data represent the mean ± standard deviation (ratio to control, 0 µg/L, n = 3). Asterisks indicate significant differences compared with the control in each generation (p < 0.05)  (18.8-20.6 days) at 5 µg/L in F4 and F14 generations was slightly shoter than that of the controls; however, the group could acquire chemical tolerance in F14. Exposure to sub-lethal levels of insecticide induced chemical tolerance in an insect (Leptinotarsa decemlineata), which coincided with our study (Margus et al. 2019). These findings suggest that chemical tolerance may be developed not only by the selection of individuals. Genetic diversity, for example, heterozygosity, is an effective parameter for investigating chemical tolerance (Bendis and Relyea 2014; Pedrosa et al. 2017). For instance, mercury tolerance was caused in the chironomid population collected from mercury-contaminated sites with high genetic diversity (Pedrosa et al. 2017). Although our study is limited to ascertaining the chemical tolerance caused without this parameter, combining clones from the four sites may increase the genetic diversity. In any case, we need to confirm the genetic diversity of the test clones in future studies.
In previous studies, when cladocerans were continuously exposed to various chemicals (nano-scaled titanium dioxide and organophosphate) over several generations, they produced more sensitive clones than controls (Jacobasch et al. 2014;Zalizniak and Nugegoda 2006). For instance, following continuous exposure to chlorpyrifos (organophosphate) over three successive generations, D. carinata produced almost two times more sensitive neonates than the original clones (Zalizniak and Nugegoda 2006), which supports our findings. In contrast, several reports have suggested that cladocerans could rapidly acquire chemical tolerance following exposure to synthetic chemicals (mercury and carbamates (pirimicarb and carbaryl)) (Tsui and Wang 2005;Ishimota et al. 2020b;Jansen et al. 2010). Given these contradictory findings regarding the changes in chemical sensitivity, we cannot definitively establish the number of generations required to obtain a chemical-tolerant population via exposure to chemicals. In a study by Wuerthner et al. (2019), D. pulex acquired higher pesticide tolerance when exposed to sublethal carbaryl concentrations at early life stages (carbaryl) than when exposed at later life stages. Thus, exposure at early life stages might be an important factor in the acquisition of chemical tolerance. In this study, we continuously exposed S. kingi to various chemical concentrations at early life stages, which possibly induced chemical tolerance of varying degrees in the test cladocerans.
The r-value is a sensitive life cycle parameter that combines the number of broods, survival rate, and time to the first brood release (Silva et al. 2017;Zalizniak and Nugegoda 2006). Since the total number of neonates (reproduction) was significantly and positively correlated with the r-value (Table S1 in Supplementary Information), changes in r-values may be explained by reproduction. Although the r-value of the F0 generation in the 10 µg/L concentration group was decreased compared with that in the control group, it recovered to the control level in the F4 generation (Fig. 2). In several studies, the decreased r-value of D. magna exposed to various chemicals (nickel and molinate) was increased following exposure over several subsequent generations (three to six generations) (Münzinger 1990;Sánchez et al. 2004), which supports our findings in the F4 generation. However, the recovered r-values in the F4 generation decreased again in the F14 generation, suggesting that the potential effects of chemicals are underestimated by short-term (up to only five generations) multigenerational studies. Thus, longer exposure to chemicals may, in fact, negatively affect the growth rate of cladocerans. When the population is already exposed to chemicals, there is a chance for genetic erosion due to the further impact of the chemical (Bendis and Relyea 2014; Ribeiro and Lopes, 2013). After the initial impact removed the most sensitive genotypes, the surviving tolerant genotypes showed slower physical recovery from the damage (Ribeiro and Lopes 2013). Therefore, the low r-value in the F14 at 10 µg/L concentration group may suggest that they were genetically eroded.
Additionally, the 48 h-EC 50 value was significantly correlated with the r-value, indicating a potential fitness cost for tolerance. Under field conditions, three months of continuous exposure to low levels of several pesticides (i.e., commonly observed in agricultural streams) induced chemical tolerance in Gammarus pulex (Siddique et al. 2020). They had fitness costs, such as reduced survival and mating. As we have described in the introduction section, fitness cost was not reported in the laboratory conditions because the conditions are not similar to those in the environmental conditions (where there may be several stressors) or because the short-term generational study may not cause the genes to evolve (Kilot and Ghanim 2012). Considering the observed fitness cost in our laboratory, the cladoceran could rapidly alter their energy balance to adapt to chemical stressors despite short-term generations. Thus, our findings provide valuable information for understanding the characteristics of a chemical-tolerant population.
We hypothesized that the r-value would be positively correlated to the feeding rate in S. kingi. In other words, decreased food intake may decrease the growth rate. Feeding behavior is an important index to explore the effects of pollutants on ecosystems (Liu et al. 2019). Several studies have evaluated chemical toxicity using feeding rate as the endpoint and reported decreased rates following exposure to chemicals (Liu et al. 2019;Ogonowski et al. 2016;Villarroel et al. 1999). In D. longispina, the feeding rate of metal-tolerant individuals was lower than that of sensitive controls (Agra et al. 2010). In this study, contrary to our expectations, there was no significant correlation between these two parameters.
In the F0 generation, the feeding rates in all test groups were significantly lower than those in the control group; however, in the F14 generation (chemical tolerant generation), the feeding rates in all test groups were higher than those in the control group (Fig. 3). Similarly, in previous studies, higher algal consumption in daphnids continuously exposed to lead or fungicides (carbendazim) over several generations has been observed (Araujo et al. 2019;Silva et al. 2017;Skjolding et al. 2014), suggesting that increased food consumption accelerates gut clearance and helps detoxification. Thus, our findings provide evidence that chemicaltolerant clones consume more energy to eliminate pollutants from their body. Energy and resource allocation for adaptation and survival is essential when organisms cope with the toxicity of insecticides (Kliot and Ghanim 2012). In fact, the total carbohydrate, protein, and lipid content in D. magna exposed to several chemicals, such as tributyltin chloride and linear alkylbenzene sulfonic acid, was related to survival, growth, and reproduction (De Coen and Janssen 2003). Therefore, these parameters should be investigated to determine the characteristics of chemical-tolerant clones in detail.
Finally, we discuss the enzymatic mechanism underlying the differential chemical sensitivity of various clones. Decreased AChE activity of the F0 and F4 generations in the 5.0 and 10.0 µg/L concentration groups suggests that pirimicarb inhibited AChE (Fig. 6). These observations are consistent with our previous findings of decreased AChE activity in S. kingi exposed to pirimicarb (Ishimota and Tomiyama 2020). In addition, pirimicarb acts via other toxic mechanisms, such as oxidative stress, genotoxicity, and cytotoxicity (Natale et al. 2018;Raisi et al. 2018;Vera-Candioti et al. 2015). Raisi et al. (2018) reported that the oxidative biomarker levels (catalase activity) in snails were altered following pirimicarb exposure. Similarly, the glutathione PO (a type of PO) and SOD activity was decreased in D. magna exposed to various insecticides (chlorantraniliprole, cyantraniliprole, and flubendiamide) (Cui et al. 2017). In the present study, in the F0 and F4 generations, the PO and SOD activity in the test groups was significantly lower than that in the control groups (Figs. 4 and 5), suggesting that pirimicarb induced oxidative stress in S. kingi.
In all test groups, the decreased activity of all enzymes in the F0 generation was recovered to the control level in the F14 generation (when the individuals had acquired pirimicarb tolerance) (Figs. 4, 5, and 6). The SOD, PO, and AChE activity was significantly but weakly correlated with the pirimicarb sensitivity of S. kingi (Table 2). Considering our study's short exposure period (i.e., only 15 generations), chemical tolerance may not be induced by genetic mutations. While the relationship between chemical tolerance and the three enzyme activity is controversial, the three enzyme activity may be enhanced in the pirimicarb-tolerant population. Moreover, epigenetic changes may cause a chemical-tolerant population (Field et al. 2004;Vandegehuchte et al. 2009) and DNA methylation of the amplified genes of each enzyme may be related to chemical tolerance.
We previously reported that increased AChE activity in other types of cladocerans (Ceriodaphnia cornuta) might be related to changes in pirimicarb sensitivity (Ishimota et al. 2020b). Thus, the recovery of this enzyme may also be a factor in acquiring chemical tolerance in S. kingi. SOD and PO activity is related to chemical tolerance in many plants (Kim et al. 2008;Liu et al. 2015;Sreenivasulu et al. 1999;Tseng et al. 2007;Wu et al. 2017); however, only a few researchers have reported an association between SOD and PO activity and chemical tolerance in animals, including insects (e.g., bed bugs, butterfly) (Li et al. 2021;Mamidala et al. 2012). Moreover, we did not find any report on these associations in cladocerans. Thus, our novel findings will contribute to elucidating the enzymatic mechanism underlying chemical tolerance in animals. The measurement of peroxidation levels in S. kingi (based on the levels of H 2 O 2 and lipid peroxidation byproduct 4-hydroxynonenal) (Terhzaz et al. 2015) will provide useful insights to determine the oxidative mechanism of action of pirimicarb.
Overall, our findings shed light on the mechanism of insecticide tolerance in cladocerans (S. kingi) and the characteristics of chemical-tolerant population, including decreased r and increased feeding rate. In particular, the elevated activity of the three enzymes may be related to chemical tolerance, suggesting that the regulation of oxidative stress and enhanced AChE enzyme are involved in the acquisition of chemical tolerance.

Conclusions
Following continuous exposure to lethal and sublethal concentrations of pirimicarb for 15 generations, the test S. kingi individuals became three times more tolerant than the controls at the highest exposure concentration (10 µg/L). The r-value was significantly but negatively correlated with the 48 h-EC 50 value, indicating the fitness cost for pirimicarb tolerance; however, we could not explain changes in the r-value based on the feeding rates, and additional experiments are warranted to clarify these effects. Moreover, we explored the involvement of antioxidant enzymes (PO and SOD) and AChE in changes in the sensitivity of cladocerans to pirimicarb. Since there was a weak but significant positive correlation between each enzyme activity and 48 h-EC 50 value, the elevated activity of the three enzymes may be related to chemical tolerance. While the relationship between chemical tolerance and the three enzyme activity was controversial, the regulation of oxidative stress and enhanced AChE may contribute to the acquisition of