Increased extinction probability and altered physiological characteristics in carbamate tolerant Daphnia magna

doi:10.21203/rs.3.rs-3632185/v1

Download PDF

Research Article

Increased extinction probability and altered physiological characteristics in carbamate tolerant Daphnia magna

https://doi.org/10.21203/rs.3.rs-3632185/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

We evaluated the physiological characteristics of chemical-tolerant cladocerans. Over the course of 26 generations (F25), D. 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 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 respiration efficiency may decrease in tolerant clones. 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.

Tolerance

Cladocera

Carbamates

Respiration rate

Extinction probability

Depuration rate

Artificial chemicals such as pesticides flow into rivers or lakes and affect the aquatic organisms inhabiting the area. However, several aquatic organisms struggle with the adverse effects of chemicals by acquiring chemical tolerance (Coors et al. 2009, Muggelberg et al. 2017). The chemical tolerance often accompanies fitness cost, such as longer development time, smaller eggs in several aquatic organisms, decreased population growth (fish, mosquitoes, and cladocerans) (Nusbaumer et al. 2021, Osoro et al. 2021, Tanaka and Tatsuta 2013). However, the fitness cost against chemical tolerance has rarely been determined (Jansen et al. 2010, Menger et al. 2022, Shephard et al. 2021), details of which still remain scarce. Further information on the fitness cost needs to be obtained to clarify the latency background of aquatic organisms. This study evaluated fitness cost using two parameters, extinction probability and respiration.

Daphnia sp. is commonly used in ecotoxicological studies (e.g., as guideline test for investigating chemical effects); this species was thus chosen as our test organism. Pirimicarb (an insecticide and acetylcholinesterase inhibitor) was used as the model chemical. As it has been previously reported that cladocerans exposed to lethal or sublethal levels of pirimicarb acquired chemical tolerance (Ishimota et al. 2022, Ishimota et al. 2020a), it is reasonable to consider this chemical as a model to investigate the chemical tolerance of insecticides that possess a major mode of action (acetylcholinesterase inhibitor).

Fitness costs are often detected only under stressful conditions such as high temperatures (Zhang et al. 2015). In a realistic environment, various factors, such as nutrient-poor foods (algae), may stress cladocerans. In particular, the abundance of cyanobacteria in lakes has increased due to recent warm temperatures and rising nutrient loads (Deng et al. 2023, Visser et al. 2016). Several scientists have reported that food quality affects chemical toxicity in cladocerans (Brown and Yan 2015, Ieromina et al. 2014). Increased cyanobacteria significantly delayed the maturation and reproduction of eggs and newborns in Daphnia (Cerbin et al. 2010). Because of their nutrient-poor composition, cyanobacteria depress the growth of cladocerans (Wacker and Martin-Creuzburg 2007). Thus, chemical-tolerant cladocerans in the field increasingly rely on nutrient-deficient food sources. Considering the chemical effects on cladocerans, fitness costs may be detected under such stressful conditions. To ascertain the population growth of chemical-tolerant cladocerans under stressful food conditions (expected nutrient-poor algae), we performed a life cycle test (Organisation for Economic Co-operation and Development, OECD 2012) using cyanobacteria (S. leopoliensis, OECD guideline species in the algal test) and green algae (C. vulgaris as a control, a recommended food for cladocerans in the OECD test guideline). Several types of cyanobacteria produce toxic substances, such as microcystins (Schaefer et al. 2020). However, since only the effects of nutrient-poor food on cladocerans were investigated in the life cycle test, non-toxic cyanobacteria (S. leopoliensis) were chosen.

Several studies have evaluated the respiratory effects in aquatic organisms exposed to pesticides, and found that chemical exposure altered parameters such as heart activity and oxygen consumption (Friberg-Jensen et al. 2010, Maki et al. 1973, Warming et al. 2009). Given that respiration is an essential parameter in the life cycle of organisms, these parameters may be useful for detecting the fitness cost of chemical tolerance.

Previous studies have demonstrated the various molecular mechanisms underlying pirimicarb tolerance in aquatic organisms (cladocerans and chironomids) (Ishimota et al. 2022, Ishimota et al. 2020b). Although pirimicarb is classified as an acetylcholinesterase inhibitor, it not only increases acetylcholinesterase activity but also peroxidase (PO) and superoxide dismutase activities, which are related to sensitivity changes (Ishimota et al. 2022). Because the biochemical approach was limited to further elucidating the mechanisms of chemical tolerance, we used a physiological approach in this research. High bioaccumulation and depuration rates have been linked to chemical tolerance in D. magna (Li et al. 2021); focus was laid on these parameters to ascertain the differences between chemical-sensitive and chemical-tolerant strains.

The primary objectives of this study were: (1) to evaluate the fitness cost for the life cycle parameters (extinction probability and respiration rates) and (2) to clarify the chemical tolerance mechanisms (changes in physiological characteristics, i.e., depuration rate) in the chemical-tolerant cladoceran (D. magna). If acquisition of chemical tolerance is accompanied by damage to population growth, the probability of extinction increases. If the respiratory system were altered in the chemical-tolerant population, the respiration rate would be altered. If the depuration rate in the chemical-tolerant clones was higher than that in the control, the altered metabolic rate in their bodies would be related to chemical tolerance.

Culture methods for test cladocerans

D. magna was obtained from the National Institute for Environmental Studies (Ibaraki, Japan) and maintained with food (algae) and C. vulgaris (Recenttec K. K, Tokyo, Japan; cell concentration: 5 × 10⁵ cells/mL, fed more than five times per week). Elendt M4 medium was used for culture (Elendt and Bias 1990). One Daphnia was cultured in 100 mL of medium in a 100 mL glass beaker, and several other beakers were established in a similar manner. Neonates from the third or fourth brood were used for the subsequent stock cultures. The frequency of culture medium exchange was set to at least three times per week. The daphnia were kept under the water temperature: 20 ± 1°C, photoperiod: 16:8 h (light: dark duration), and light intensity: approximately 800 lux.

Constant exposure methods

Pirimicarb was purchased from the Fujifilm Wako Pure Chemical Corporation (Osaka, Japan). Referring to the 48 h EC₅₀ values (13–14 µg/L) in D. magna previously studied (Ishimota et al. 2020a), we established the continuous exposure concentrations of test chemical as 0, 3.8, 7.5, and 15 µg/L. We prepared a 1.6 g/L stock solution using a pure water: methanol solution (50:50, v/v), and the stock solution was diluted with the M4 medium to prepare the test water. Then, several sets of 100 mL beaker containing one Daphnia and 100 mL of test water were prepared for each group. Each water sample contained methanol (19 µL/L) and food (C. vulgaris). We confirmed that the 48 h EC₅₀ values of D. magna, cultured with the sub-lethal (7.5 and 15 µg/L) pirimicarb for 15 generations, was approximately two times higher than that in the control (unpublished work). Because we expected to obtain a higher pirimicarb-tolerant population, we maintained the continuous exposure methods until the 26 generations (from F0 to F25). It was expected that this population would become more tolerant to pirimicarb. We established third- or fourth-brood neonates for the next generation. All test conditions followed the Culture methods for test cladocerans.

Acute immobilization test

To confirm the sensitivity (48 h EC₅₀ value) of neonates from each exposure group in the F25 generations, acute immobilization tests were conducted according to the OECD guideline No. 202 (OECD 2004). We performed the test on neonates (< 24 h old) from the third and fourth broods. Immobilization of neonates was defined as neonates that were unable to swim for 15 s after gentle agitation of the beaker (OECD 2004). After we adjusted a stock solution of 1.6 g/L pirimicarb using a methanol: pure water solution (50:50, v/v), test solutions (0, 3.8, 7.5, 15, 30, and 60 µg/L) were prepared by diluting the stock solution with the culture medium. We added five neonates (< 24 h old) into a 100 mL glass beaker (n = 4, total number of neonates for this test: 20). The same experiment was performed for each test group in duplicate.

Life cycle test

To ascertain the extinction probability in the chemical-tolerant cladocerans, we conducted the life cycle tests by culturing Daphnia from each test group (0, 3.8, 7.5, and 15 µg/L) of F25, without pirimicarb exposure, for 21 days. Extinction probability is often used to determine the effects of chemicals on population size (Gergs et al. 2013, Tanaka 2003, Weir and Salice 2021). We prepared four sets of 100 mL glass beakers containing 100 mL of medium for each test group and added one neonate (< 24 h old) to each beaker. The triplicated experiments were performed with two foods, C. vulgaris and S. leopoliensis. We obtained C. vulgaris from Recenttec K. K (Tokyo, Japan) and S. leopoliensis from the National Institute for Environmental Studies (Ibaraki, Japan). The numbers of neonates born to the parents and dead parents were counted daily. To evaluate the changes in pirimicarb concentration with algae in the life cycle tests, we confirmed the recovery rates of pirimicarb concentrations in the test water at the beginning and after 48 h (because water exchange was performed at 48 h exposure).

Respiration experiment

The respiration rate was expressed as oxygen consumption, according to previous studies (Glazier 1991, Heisey and Porter 1977). We measured the respiration rates using the test neonates from F25 at 0, 3.8, 7.5, and 15 µg/L without any pollutants. We prepared several 150 mL glass bottles containing Elendt M4 medium and measured the dissolved oxygen (DO) concentration in each bottle using a DO meter (HQ 30D, Hach, Germany). After 10 neonates (< 24 h old) were washed with medium to remove any bacteria or particulate matter, they were added into one glass bottle with a lid in quadruplicate for each test group. Additionally, we used a bottle containing the culture medium without Daphnia as a blank sample in quadruplicate. Before being placed on the lid, the glass bottles were soaked in a bucket containing the culture medium to prevent air entry into the bottle. The glass bottle was covered with aluminum foil to prevent biological growth and respiration. The vessels were put into an incubator maintained at 20 ± 1°C (measured values: 20.1 to 20.3°C) under dark cycle and kept for 24 h. DO concentrations were assessed at the beginning and end of the exposure. Following this, we expressed the mean respiration rate as an individual/day in each group.

Chemical depuration test from daphnia body

To confirm the time-course depuration of the pirimicarb from daphnia body, we performed the chemical depuration test using the neonates collected from the 0 (control group) and 7.5 µg/L test group (which acquired pirimicarb tolerance) in the F25 generations. Thirty neonates (< 24 h old) from each test group were collected and exposed to 7.5 µg/L pirimicarb test solution with solvent (methanol; 19 µL/L) in the 1 L beaker in triplicate. Five sets of triplicate beakers were prepared (total number of beakers: 15; total number of individuals: 450). The neonates were then cultured for seven days, during which we exchanged the test water with 7.5 µg/L pirimicarb solution thrice a week to keep the nominal concentration. After seven days of exposure, we transferred the Daphnids into fresh medium without pirimicarb or solvent to start the depuration phase. Daphnids from each beaker were collected at each sampling point (0, 3, 6, 24, and 48 h after depuration), and 80 mg (wet weight) of which was weighed in triplicate; each sample was extracted with 1.5 mL of acetonitrile. After homogenization and sonication, the samples were certified. We collected the supernatant and evaporated them at 40°C water bath. Then, we added the acetonitrile: pure water (60:40, v/v) into the residue and filtered the solution with 0.20 µm membrane filter (Millex-LG hydrophilic PTFE membrane, Merck Millipore Corporation). We measured the pirimicarb concentrations in the solution by liquid chromatography-mass spectrometry (LC-MS/MS) using the following methods:

Pirimicarb concentrations in the test water were measured at the initiation and end of the exposure and at the initiation of the depuration phase.

Chemical analysis

We followed our prior analytical procedure for determining the pirimicarb concentration in the test water (Ishimota et al. 2020b). First, we diluted the test solution with acetonitrile: pure water (6:4, v/v) and measured the chemical concentrations using LC-MS/MS. The limit of quantification was set to 0.5 µg/L. A flow rate was set as 0.3 mL/min using a column at 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 monitored precursor and product ions of pirimicarb were 239.1/72.1 m/z. The peak area of the calibration standard was plotted against the corresponding amount to construct a calibration curve for pirimicarb using the least-squares method.

Although we validated the pirimicarb analysis in the test water and confirmed pirimicarb stability (48 h) in our previous study (Ishimota et al. 2020b), the analytical validation for the Daphnia body was unclear. Therefore, we performed the following analytical validation of the Daphnia body: Pirimicarb solution was added to homogenized Daphnia samples to obtain 0.5 (limit of quantification) and 10 µg/kg samples. After determining these concentrations using the aforementioned methods, we determined the mean recovery rate and relative standard deviation (RSD (%), calculated by dividing the standard deviation by the mean concentration) in triplicate.

Statistical analysis

We used R version 3.6.1 and 4.1.0 (R Core Team 2020; 2021) for all statistical analyses. We estimated the 48 h EC₅₀ values and confidence intervals using a two-parameter log-logistic model. The lower range was set to 0, and the upper range was set to 1, calculated using Eq. 1 as follows:

$$f\left(x\right)=\frac{1}{(1+exp(a\left({log}\left(x\right)-{log}\left(e\right)\right))}$$

where f(x) denotes the immobility probability, x is the nominal concentration of pirimicarb, e is the inflection point of the fitted line (equivalent to the concentration required to cause 50% immobility (48 h EC₅₀)), and a is the relative slope around e. Because we considered duplicate experiments as random effects, we estimated EC₅₀ values using the medrc package (Gerhard and Ritz 2017). We statistically compared EC₅₀ values among the test groups using the EDcomp function (drc package) (Ishimota et al. 2022, Ritz et al. 2006). This function was tested to determine whether the ratios of the EC₅₀ values were comparable to those of 1. Additionally, we adapted the significance level for each pair using Holm’s method (Holm 1979).

In the life cycle test, the extinction probability was estimated using the countCDFxt function in the R package “popbio” with 95% confidence intervals using bootstrapped estimation (Stubben and Milligan 2007, William and Daniel 2002). The calculation was performed using Eq. 2 below:

$G\left(T\right|d, \mu ,$ σ ²) = Φ$\left(\frac{-d-\mu T}{\sqrt{{\sigma }^{2}T}}\right)$ + $\text{e}\text{x}\text{p}(-2\mu d/$σ²) Φ$\left(\frac{-d+\mu T}{\sqrt{{\sigma }^{2}T}}\right)$ (2)

where, $G\left(T\right|d, \mu ,$σ²) denotes the extinction probability, Φ is the area of the extinction probability under a normal distribution curve, T is a future time of interest. Additionally, the equation includes log mean (µ) and log variation (σ²) of population growth rate during the life-cycle experiments (21 d). The population growth rate was calculated from the daily age-specific survival, parents, and reproduction numbers. In the countCDFxt function, increasing σ² increases extinction probability. When µ value is negative or zero, the ultimate extinction is certain. We ran the simulation for 21 d (exposure time) for 500 replicates using bootstrapping methods. The quasi-extinction threshold was defined as that of an individual.

For the data analysis of extinction probability and respiration rate, variances in data among test groups were checked using Levene’s test in the car package (Fox and Weisberg 2019). Since the homogeneity of variance was confirmed using Levene’s test, we performed Tukey's HSD method in the multcomp package for each data point (Hothorn et al. 2022).

In the depuration test, we estimated the half-life of pirimicarb in Daphnia using the following equation:

$${t}_{1∕2}=\frac{-ln\left(0.5\right)}{k}$$

where t_1/2 is the half-life (50% depuration) of pirimicarb in the Daphnia body; ln is the natural logarithm; k is the depuration rate constant, which is the slope of the linear regression curve (natural logarithms of the pirimicarb concentration at multiple sampling points vs. depuration time) (Castro et al. 2019).

To estimate (1) whether chemical sensitivity (48 h EC₅₀) affects the extinction probability under the two algal food conditions and (2) whether the sensitivity would be related to the respiration rate, a generalized liner model (GLM) analysis was performed. Each distribution was assumed to be gamma (log link) distribution. In these GLM analysis, we used Wald statistic test to calculate the statistical significance of each variable with the χ² test (p < 0.05) (Hosmer and Lemeshow 2000).

Acute immobilization test for pirimicarb

In the acute immobilization test, the ratios of the measured pirimicarb concentrations to the nominal concentrations ranged from 93 to 102% at initial exposure. In the acute immobilization tests of F25, the water quality was appropriate for culturing Daphnia (the water temperature: 20.1–20.3°C; pH: 7.95–8.15; DO concentration: 7.52–8.20 mg/L). These results met the criteria of OECD test guideline No. 202 (OECD 2004).

The 48 h EC₅₀ values (29.2–29.9 µg/L) at 7.5 and 15 µg/L pirimicarb treatment groups in F25 were significantly (two times) higher than that in the control (17.2 µg/L), whereas that (18.5 µg/L) at 3.8 µg/L treatment was comparable to that in the control (Fig. 1). No immobilized neonates were observed at 0 µg/L level during the acute toxicity test.

Life cycle data

For the pirimicarb concentrations in the test water with C. vulgaris, the ratios of the measured pirimicarb concentrations to the nominal concentrations at the initiation and end of exposure (48 h) were 92–111% and those of S. leopoliensis were 92–114%. These results confirmed that pirimicarb concentrations were stable even in the presence of algae, suggesting that the absorption of pirimicarb into the algae was negligible. These findings suggested that the chemical was stable during the life cycle test.

The mean extinction probability of the S. leopoliensis control (0.40) was higher than that with C. vulgaris control (0.005) (Fig. 2). For the C. vulgaris treatment, the extinction probability in all the continuous exposure groups did not change significantly among the test groups (Fig. 2a), whereas the probability in all the continuous exposure groups of S. leopoliensis treatment (0.70–0.79) was higher than that in the control (0.40) with statistical significance (p < 0.05) (Fig. 2b). The GLM approach showed that extinction probability in S. leopoliensis treatment alone was positively related to the 48 h EC₅₀ (p < 0.05) (Table 1).

Table 1

Generalized linear model (GLM) estimation for the relationship between 48 h EC₅₀ for pirimicarb and extinction probability; 48 h EC₅₀ and extinction probability
	Parameter coefficients
Variable	Slope	Standard error	Pr(>\|t\|)
C. vulgaris treatment (48 h EC₅₀ vs extinction probability)
Intercept	-5.48	0.70	1.41 × 10^− 5***
48 h EC₅₀	-0.01	0.03	0.681
S. leopoliensis treatment (48 h EC₅₀ vs extinction probability)
Intercept	-1.08	0.29	4.0 × 10^− 3**
48 h EC₅₀	0.03	0.01	0.049*
Respiration test (48 h EC₅₀ vs respiration rate)
Intercept	-3.17	0.30	5.14 × 10^− 8***
48 h EC₅₀	0.03	0.01	0.037*
Asterisks represent explanatory variables with Wald statistics (p < 0.05, * p < 0.01, and *** p < 0.001).

Respiration rates

Mean respiration rate (RR) in the blank (without any neonates and pirimicarb) (0.01 mg/L/individual/day) significantly differed from that in the control (0 µg/L) (with neonates without pirimicarb) (0.05 mg/L/individual/day) (p < 0.05) (Fig. 3). The results thus confirm that our experimental methods detect the changes in Daphnid respiration. The RRs in the neonates from all the continuous exposure groups were significantly higher than that in the control (p < 0.05) and the highest RR (0.11 mg/L/individual/day) was observed in 15 µg/L continuous exposure group. The GLM approach showed that the RRs were positively related to the 48 h EC₅₀ (p < 0.05) (Table 1).

Depuration rates

During the analytical validation of Daphnia body, the mean recovery rates for spiked 0.5 and 10 µg/kg pirimicarb solutions were 111 and 112%, respectively, and the RSDs at those concentrations were 1.4 and 0.9%, respectively, which met the criteria (60–120%) in the guidance document (SANTE/2020/12830, Rev.2) (European Commission 2023).

The monitored mean water temperature was kept at 20.2°C during the depuration test. LC/MS/MS analysis detected no peaks in the blank samples of the test water and Daphnia body. During the exposure (uptake phase), the ratios of the measured pirimicarb concentration to the nominal concentration in the test water for the control and treatment at the initiation of exposure were 93 and 101%, respectively; those in the final concentrations (after seven days of exposure) were 88 and 91%, respectively. After pirimicarb was removed, that in the initiation of the depuration phase became < 0.5 µg/L in each experimental group.

In the depuration phase of the control group, pirimicarb concentration in daphnia sample at the initiation was 4.0 µg/kg; it was decreased to 0.8 µg/kg at 24 h (Fig. 4). Finally, the concentration became less than LOQ level (< 0.5 µg/kg) at 48 h depuration phase. In the continuous exposure group (from the daphnia at 7.5 µg/L), the initial pirimicarb concentration was 8.3 µg/kg and it was rapidly decreased to < 0.5 µg/kg at 24 h depuration phase. The estimated half-life times of the pirimicarb in the daphnia body in the control and 7.5 µg/L group were 11.6 and 1.7 h, respectively. Therefore, the pirimicarb accumulation in the continuous-exposure group was higher than that in the control group, and the depuration rate in the continuous-exposure group was higher than that in the control group.

Continuous exposure to sub-lethal or lethal levels (7.5 and 15 µg/L) of pirimicarb solutions induce chemical tolerance (based on the acute toxicity test) (Fig. 1). In a previous study (unpublished work), D. magna was exposed to pirimicarb for shorter periods (15 generations) and sensitivity was maintained at the same level at F25. Shaw et al. (2019) also reported the similar findings. Daphnia pulex exposed to cadmium for five generations induced twice higher chemical tolerance; however, the levels of tolerance remained the same for five generations on exposure to the chemical for 50 generations. Thus, cladocerans potentially has a threshold for the acquisition of chemical tolerance and cannot exceed this despite sufficient exposure duration. However, considering the long-term exposure of aquatic organisms to several chemicals in the field, laboratory exposure studies may underestimate their ability to adapt to chemicals.

The cumulative extinction probability in cyanobacteria (S. leopoliensis) control group was approximately 100 times higher than that in the green algal (C. vulgaris) control despite the same amounts of foods being provided in the two algal treatments (Fig. 2). Since feeding rates in D. magna did not change in almost all continuous exposure groups, compared to the control in each algal treatment (Supplementary Information, Fig. S1), the different extinction probabilities could not be explained by feeding rates. We found that the lipid content in the S. leopoliensis treatment was lower than that in the C. vulgaris treatment (Supplementary Information 2.1). This finding is consistent with previous reports, where cyanobacteria (S. leopoliensis) that contained poor nutrients (lipids) depressed the growth of cladocerans (Wacker and Martin-Creuzburg 2007, Wang et al. 2016). Thus, poor nutrient availability may induce a high probability of extinction in the control group of S. leopoliensis treatment. Considering that the extinction probability in all continuous exposure groups of the S. leopoliensis treatment was significantly higher than that in the control (Fig. 2b), the fitness cost ( increased extinction probability) for chemical tolerance may become more visible under poor nutrient conditions (cyanobacteria treatment). Because the extinction probability was at the same level between the continuous exposure groups, concentration dependency was unclear in our study. However, because the extinction probability in the S. leopoliensis treatment was positively related to pirimicarb sensitivity in the GLM analysis (Table 1), the pirimicarb-tolerant groups tended to become extinct. Since significantly fewer reproductions were observed in the cyanobacteria treatment than in the control (Supplementary Information, Table S1), the decreased reproduction may have contributed to the increased extinction probability.

Fitness costs are often only represented under stressful conditions. For example, bacteria (Salmonella Typhimurium) (which are tolerant to multiple drugs) showed a decrease in growth rates under stressful conditions at low pH and high salt concentrations, but not under non-stressful conditions (Petersen et al. 2009). Considering these findings, D. magna may also show fitness costs for their growth under nutrient-poor food conditions (cyanobacteria, i.e., S. leopoliensis). Because various kinds of algae exist as food for cladocerans in the field, they may not always consume nutrient-rich algae. Cyanobacteria tend to dominate with increasing temperatures (Kosten et al. 2012, Markensten et al. 2010). Gophen (2021) reported that the dinoflagellate (Peridinium Spp) population was replaced by cyanobacteria owing to the recent increase in field temperatures. Considering the domination of cyanobacteria in recent years, our findings contribute to determining the potential chemical risks to aquatic organisms in realistic environments.

Owing to the lack of experiments regarding chemical effects at higher temperatures, our study may be limited in evaluating the fitness cost. In addition, because natural stressors, such as predators, have been reported to affect the population size of D. magna (Gergs et al. 2013), a realistic population may have further costs against their life cycle.

Moreover, the respiration rate of D. magna in the continuous pirimicarb exposure group was higher than that in the control group (Fig. 3). Thus, they may consume more energy for respiration to repair their body systems. The oxygen consumption of several aquatic organisms (e.g., cladocerans and fish) is a useful parameter for evaluating chemical toxicity or stressors as a biological early warning system (Alkimin et al. 2020, Bansal et al. 1979, Waiwood and Johansen 1974). For example, pyrethroid (organochlorine) exposure significantly increases oxygen consumption in D. magna (Martins et al. 2007). The GLM approach showed that the increased respiration rates in D. magna were positively related to the 48 h EC₅₀ despite the weak relationship (Table 1). Thus, pirimicarb-tolerant populations may exhibit high respiration rates as a fitness cost for chemical tolerance. As this respiration experiment was performed without any contaminants, maternal exposure to parent Daphnia was likely to be associated with altered respiration.

Finally, chemical tolerance mechanisms were assessed (Fig. 4). After the uptake phase, the initial pirimicarb concentration in the pirimicarb-tolerant D. magna body was twice higher than that in the control, suggesting that they could accumulate more chemicals in their body. The pyrethroid-resistant Hyalella azteca (Cladocera) possessed greater bioaccumulation than the sensitive strains (Castro et al. 2019, Muggelberg et al. 2017), which is in line with our study. Because the chemical depuration rate in the tolerant population was higher than that in the control group, they were thought to first excrete the pesticide from their bodies. These two findings may be related to the high pirimicarb tolerance. However, other possible causes of chemical tolerance must be considered. The altered bacterial composition in D. magna exposed to chlorpyrifos (organophosphate) was related to its sensitivity to the same chemical (Janssens et al. 2022); the authors suggested that the bacteria in D. magna may possess enzymes that can transform or degrade xenobiotics. Considering these findings, our study was limited to checking whether pirimicarb tolerance was induced by changes in bacterial enzymes or physiological changes in Daphnia. Therefore, further research is required to clarify the detailed metabolic mechanisms in chemical-tolerant organisms.

We confirmed acquired pirimicarb tolerance in D. magna on exposure to the chemicals for 26 generations. When D. magna clones were fed S. leopoliensis instead of C. vulgaris, the extinction probability of the chemical-tolerant population was higher than that of the control. Considering the low lipid content of S. leopoliensis, the risk of extinction under poor nutrient conditions may be high in the cyanobacterial treatments. The GLM approach showed that respiration rates were related to the 48 h EC₅₀; respiration in pirimicarb treatment was likely altered as a fitness cost for chemical tolerance. Moreover, the depuration rate in the pirimicarb-tolerant population was higher than that in the control population, suggesting that high depuration velocity is an essential mechanism for acquiring chemical tolerance. Our findings contribute to the understanding of the potential characteristics of chemical-tolerant populations in the field.

Acknowledgements

We thank our technical staff, Aya Kitahara, for supporting this study and Editage (https://www.editage.com/) for editing and reviewing this manuscript for English.

Compliance with ethical standards

Disclosure of potential conflicts of interest

The authors declare that no potential conflict of interest when preparing this manuscript.

Research involving human participants and/or animals

Although the test organism (invertebrate) was not the target animal for the animal care guidelines, we performed this experiment according to the ethical standards of animal care at the Institute of Environmental Toxicology (Japan). This study did not involve any studies with human participants or animals performed by any of the authors.

Informed consent

Not applicable.

Funding

The authors declare that no funds, grants, or other support was received during the preparation of this manuscript.

Competing interests

The authors declare no competing interests.

Data availability

Data related to the findings of this study are available in the article and supplemental data. Additional documentation is available upon reasonable request.

Author contributions

All authors discussed the results and contributed to the final manuscript. Makoto Ishimota performed and designed the experiments and analyzed the data. Makoto Ishimota wrote the first draft of this manuscript. Naruto Tomiyama, Mebuki Kodama and Kazutoshi Ohyama discussed the results and commented on the manuscript. All the authors have read and approved the final version of the manuscript.

Alkimin GD, Nunes B, Soares AM, Bellot M, Gómez-Canela C, Barata C (2020) Daphnia magna responses to fish kairomones and chlorpromazine exposures. Chem Biol Interact 325: 109123. https://doi.org/10.1016/j.cbi.2020.109123
Bansal SK, Verma SR, Gupta AK, Rani S, Dalela RC (1979) Pesticide-induced alterations in the oxygen uptake rate of a freshwater major carp Labeo rohita. Ecotoxicol Environ Saf 3: 374–382. https://doi.org/10.1016/0147-6513(79)90027-7
Brown AH, Yan ND (2015) Food quantity affects the sensitivity of Daphnia to road salt. Environ Sci Technol 49: 4673–4680. https://doi.org/10.1021/es5061534
Castro M, Sobek A, Yuan B, Breitholtz M (2019) Bioaccumulation potential of CPs in aquatic organisms: uptake and depuration in Daphnia magna. Environ Sci Technol 53: 9533–9541. https://doi.org/10.1021/acs.est.9b01751
Cerbin S, Kraak MHS, de Voogt P, Visser PM, Van Donk E (2010) Combined and single effects of pesticide carbaryl and toxic Microcystis aeruginosa on the life history of Daphnia pulicaria. Hydrobiologia 643: 129–138. https://doi.org/10.1007/s10750-010-0130-1
Coors A, Vanoverbeke J, De Bie T, De Meester L (2009) Land use, genetic diversity and toxicant tolerance in natural populations of Daphnia magna. Aquat Toxicol 95: 71–79. https://doi.org/10.1016/j.aquatox.2009.08.004
Deng J, Shan K, Shi K, Qian SS, Zhang Y, Qin B, Zhu G (2023) Nutrient reduction mitigated the expansion of cyanobacterial blooms caused by climate change in Lake Taihu according to Bayesian network models. Water Res 236: 119946. https://doi.org/10.1016/j.watres.2023.119946
Elendt B-P, Bias W-R (1990) Trace nutrient deficiency in Daphnia magna cultured in standard medium for toxicity testing. Effects of the optimization of culture conditions on life history parameters of D. magna. Water Res 24: 1157–1167. https://doi.org/10.1016/0043-1354(90)90180-E
European Commission (2023) Guidance document on pesticide analytical methods for risk assessment and post-approval control and monitoring purposes, Ref: SANTE/2020/12830, rev.2
Fox J, Weisberg S (2019) An R Companion to Applied Regressions, 3rd ed. https://www.john-fox.ca/Companion/index.html
Friberg-Jensen U, Nachman G, Christoffersen KS (2010) Early signs of lethal effects in Daphnia magna (Branchiopoda, Cladocera) exposed to the insecticide cypermethrin and the fungicide azoxystrobin. Environ Toxicol Chem 29: 2371–2378. https://doi.org/10.1002/etc.290
Gergs A, Zenker A, Grimm V, Preuss TG (2013) Chemical and natural stressors combined: from cryptic effects to population extinction. Scientific reports 3: 2036. https://doi.org/10.1038/srep02036
Gerhard D, Ritz C (2017) Marginalization in nonlinear mixed-effects models with an application to dose-response analysis
Glazier DS (1991) Separating the respiration rates of embryos and brooding females of Daphnia magna: Implications for the cost of brooding and the allometry of metabolic rate. Limnology and Oceanography 36: 354–361. https://doi.org/10.4319/lo.1991.36.2.0354
Gophen M (2021) Climate change-enhanced cyanobacteria domination in Lake Kinneret: a retrospective overview. Water 13: 163
Heisey D, Porter KG (1977) The effect of ambient oxygen concentration on filtering and respiration rates of Daphnia galeata mendotae and Daphnia magna. Limnology and Oceanography 22: 839–845. https://doi.org/10.4319/lo.1977.22.5.0839
Holm S (1979) A simple sequentially rejective multiple test procedure. Scandinavian Journal of Statistics 6: 65-70
Hosmer DW, Lemeshow S (2000) Applied logistic regression, 2nd edn. https://doi.org/10.1002/0471722146
Hothorn T, Bretz F, Westfall P, Heiberger RM, Schuetzenmeister A, Scheibe S (2022) Multcomp: Simultaneous Inference in General Parametric Models, Accessed 28 June 2023
Ieromina O, Peijnenburg WJGM, de Snoo G, Müller J, Knepper TP, Vijver MG (2014) Impact of Imidacloprid on Daphnia magna under different food quality regimes. Environ Toxicol Chem 33: 621–631. https://doi.org/10.1002/etc.2472
Ishimota M, RisakoTajiki-Nishino, Fukuyama T, Tomiyama N, Sakamoto M, Ohyama K (2020a) Long-term tolerance acquisition and changes in acetylcholinesterase activity in three cladoceran species after a 48-h pulsed exposure to pirimicarb. Water Air Soil Pollut 231: 287. https://doi.org/10.1007/s11270-020-04670-3
Ishimota M, Tajiki-Nishino R, Fukuyama T, Tomiyama N (2020b) Rapid adaptation of Chironomus yoshimatsui to acetylcholinesterase inhibitors (pyraclofos and pirimicarb) in a multi-generation study. J Environ Sci Health B 55: 429–437. https://doi.org/10.1080/03601234.2019.1708165
Ishimota M, Kodama M, Tomiyama N (2022) Possible enzymatic mechanism underlying chemical tolerance and characteristics of tolerant population in Scapholeberis kingi. Environmental Science and Pollution Research 29: 18989–19002. https://doi.org/10.1007/s11356-021-17071-8
Jansen M, Stoks R, Coors A, De Meester L (2010) No evidence for a cost of selection by carbaryl exposure in terms of vulnerability to fish predation in Daphnia magna. Hydrobiologia 643: 123–128. https://doi.org/10.1007/s10750-010-0129-7
Janssens L, Van de Maele M, Delnat V, Theys C, Mukherjee S, De Meester L, Stoks R (2022) Evolution of pesticide tolerance and associated changes in the microbiome in the water flea Daphnia magna. Ecotoxicol Environ Saf 240: 113697. https://doi.org/10.1016/j.ecoenv.2022.113697
Kosten S, Huszar VLM, Bécares E, Costa LS, van Donk E, Hansson L-A, Jeppesen E, Kruk C, Lacerot G, Mazzeo N, De Meester L, Moss B, Lürling M, Nõges T, Romo S, Scheffer M (2012) Warmer climates boost cyanobacterial dominance in shallow lakes. Glob Chang Biol 18: 118–126. https://doi.org/10.1111/j.1365-2486.2011.02488.x
Li H, Zhang Q, Su H, You J, Wang W-X (2021) High tolerance and delayed responses of Daphnia magna to neonicotinoid insecticide imidacloprid: Toxicokinetic and toxicodynamic modeling. Environ Sci Technol 55: 458-467. https://doi.org/10.1021/acs.est.0c05664
Maki AW, Stewart KW, Silvey JKG (1973) The Effects of Dibrom on Respiratory Activity of the Stonefly, Hydroperla crosbyi, Hellgrammite, Corydalus cornutus and the Golden Shiner, Notemigonus crysoleucas. Transactions of the American Fisheries Society 102: 806–815. https://doi.org/10.1577/1548-8659(1973)102<806:TEODOR>2.0.CO;2
Markensten H, Moore K, Persson I (2010) Simulated lake phytoplankton composition shifts toward cyanobacteria dominance in a future warmer climate. Ecol Appl 20: 752–767. https://doi.org/10.1890/08-2109.1
Martins JC, Saker ML, Teles LFO, Vasconcelos VM (2007) Oxygen consumption by Daphnia magna straus as a marker of chemical stress in the aquatic environment. Environ Toxicol Chem 26: 1987–1991. https://doi.org/10.1897/07-051R.1
Menger JP, Ribeiro AV, Potter BD, Valmorbida I, Hodgson EW, Knodel JJ, Koch RL (2022) Lack of evidence for fitness costs in soybean aphid (Hemiptera: Aphididae) with resistance to pyrethroid insecticides in the upper midwest region of the United States. J Econ Entomol 115: 1191–1202. https://doi.org/10.1093/jee/toac096
Muggelberg LL, Huff Hartz KE, Nutile SA, Harwood AD, Heim JR, Derby AP, Weston DP, Lydy MJ (2017) Do pyrethroid-resistant Hyalella azteca have greater bioaccumulation potential compared to non-resistant populations? Implications for bioaccumulation in fish. Environmental Pollution 220: 375–382. https://doi.org/10.1016/j.envpol.2016.09.073
Nusbaumer D, Marques da Cunha L, Wedekind C (2021) Testing for population differences in evolutionary responses to pesticide pollution in brown trout (Salmo trutta). Evol Appl 14: 462–475. https://doi.org/10.1111/eva.13132
OECD (2004): Guidelines for the Testing of Chemicals, Section 2, Test No.202: Daphnia sp., Acute Immobilization Test. https://doi.org/10.1787/20745761. Accessed 28 June 2023
OECD (2012): Guidelines for the Testing of Chemicals, Section 2, Test No.211: Daphnia magna Reproduction Test. https://doi.org/10.1787/9789264185203-en. Accessed 28 June 2023
Osoro JK, Machani MG, Ochomo E, Wanjala C, Omukunda E, Munga S, Githeko AK, Yan G, Afrane YA (2021) Insecticide resistance exerts significant fitness costs in immature stages of Anopheles gambiae in western Kenya. Malar J 20: 259. https://doi.org/10.1186/s12936-021-03798-9
Petersen A, Aarestrup FM, Olsen JE (2009) The in vitro fitness cost of antimicrobial resistance in Escherichia coli varies with the growth conditions. FEMS Microbiol Lett 299: 53–59. https://doi.org/10.1111/j.1574-6968.2009.01734.x
R Core Team (2020; 2021) R: A language and environment for statistical. Computing, Ver 3.6.1, 4.1.0. MSOR connections, 1. R Foundation for Statistical Computing, Vienna, Austria
Ritz C, Cedergreen N, Jensen J, Streibig J (2006) Relative potency in nonsimilar dose–response curves. Weed Sci 54: 407–412. https://doi:10.1614/WS-05-185R.1
Schaefer AM, Yrastorza L, Stockley N, Harvey K, Harris N, Grady R, Sullivan J, McFarland M, Reif JS (2020) Exposure to microcystin among coastal residents during a cyanobacteria bloom in Florida. Harmful algae 92: 101769. https://doi.org/10.1016/j.hal.2020.101769
Shaw JR, Colbourne JK, Glaholt SP, Turner E, Folt CL, Chen CY (2019) Dynamics of Cadmium Acclimation in Daphnia pulex: Linking Fitness Costs, Cross-Tolerance, and Hyper-Induction of metallothionein. Environ Sci Technol 53: 14670–14678. https://doi.org/10.1021/acs.est.9b05006
Shephard AM, Zambre AM, Snell-Rood EC (2021) Evaluating costs of heavy metal tolerance in a widely distributed, invasive butterfly. Evol Appl 14: 1390–1402. https://doi.org/10.1111/eva.13208
Stubben C, Milligan B (2007) Estimating and Analyzing Demographic Models Using the popbio Package in R. J Stat Softw 22: 1–23. https://doi.org/10.18637/jss.v022.i11
Tanaka Y (2003) Ecological risk assessment of pollutant chemicals: extinction risk based on population-level effects. Chemosphere 53: 421–425. https://doi.org/10.1016/S0045-6535(03)00016-X
Tanaka Y, Tatsuta H (2013) Retrospective estimation of population-level effect of pollutants based on local adaptation and fitness cost of tolerance. Ecotoxicology (London, England) 22: 795–802. https://doi.org/10.1007/s10646-013-1081-x
Visser PM, Verspagen JMH, Sandrini G, Stal LJ, Matthijs HCP, Davis TW, Paerl HW, Huisman J (2016) How rising CO₂ and global warming may stimulate harmful cyanobacterial blooms. Harmful algae 54: 145–159. https://doi.org/10.1016/j.hal.2015.12.006
Wacker A, Martin-Creuzburg D (2007) Allocation of essential lipids in Daphnia magna during exposure to poor food quality. Funct Ecol 21: 738–747. https://doi.org/10.1111/j.1365-2435.2007.01274.x
Waiwood KG, Johansen PH (1974) Oxygen consumption and activity of the white sucker (Catostomus commersoni), in lethal and nonlethal levels of the organochlorine insecticide, methoxychlor. Water Res 8: 401–406. https://doi.org/10.1016/0043-1354(74)90070-0
Wang X, Shen Z, Miao X (2016) Nitrogen and hydrophosphate affects glycolipids composition in microalgae. Scientific reports 6: 30145. https://doi.org/10.1038/srep30145
Warming TP, Mulderij G, Christoffersen KS (2009) Clonal variation in physiological responses of Daphnia magna to the strobilurin fungicide azoxystrobin. Environ Toxicol Chem 28: 374–380. https://doi.org/10.1897/08-279.1
Weir SM, Salice CJ (2021) Investigating potential toxic effects of pollutants on population growth rates and probability of extinction for a representative squamate. Ecotoxicology (London, England) 30: 175-186. https://doi.org/10.1007/s10646-020-02289-y
William F, Morris; , Daniel F, Doak (2002) Quantitative conservation biology: Theory and practice of population viability analysis. Sinauer Associates, Sunderland, MA
Zhang LJ, Jing YP, Li XH, Li CW, Bourguet D, Wu G (2015) Temperature-sensitive fitness cost of insecticide resistance in Chinese populations of the diamondback moth Plutella xylostella. Mol Ecol 24: 1611–1627. https://doi.org/10.1111/mec.13133

SupplementalInformation231118.docx

Download PDF

Editorial decision: Major Revision
21 Mar, 2024
Reviewers agreed at journal
28 Dec, 2023
Reviewers invited by journal
22 Dec, 2023
Editor assigned by journal
05 Dec, 2023
First submitted to journal
28 Nov, 2023

You are reading this latest preprint version

Increased extinction probability and altered physiological characteristics in carbamate tolerant Daphnia magna

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Culture methods for test cladocerans

Constant exposure methods

Acute immobilization test

Life cycle test

Respiration experiment

Chemical depuration test from daphnia body

Chemical analysis

Statistical analysis

Results

Acute immobilization test for pirimicarb

Life cycle data

Respiration rates

Depuration rates

Discussion

Conclusion

Declarations

References

Supplementary Files

Status:

Version 1