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Trans-generational influences of sulfamethoxazole on lifespan, reproduction and population growth of Caenorhabditis elegans
Ecotoxicology and Environmental Safety 135 (2017) 312–318
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Trans-generational influences of sulfamethoxazole on lifespan, reproduction and population growth of Caenorhabditis elegans Zhenyang Yua, Guohua Sunc,d, Yanjun Liuc,e, Daqiang Yina, Jing Zhangb,
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a State Key Laboratory of Pollution Control and Resource Reuse, Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, PR China b Ecological Technique and Engineering College, Shanghai Institute of Technology, Shanghai 201418, PR China c College of Biological, Chemical Sciences and Engineering, Jiaxing University, Jiaxing, Zhejiang 314001, PR China d Department of Civil and Environmental Engineering, University of Ulsan, Nam Gu, Ulsan 680-748, South Korea e School of Civil Engineering and Environmental Science at Collage of Engineering, University of Oklahoma, Norman, OK 73019, USA
A R T I C L E I N F O
A BS T RAC T
Keywords: Trans-generational effect Sulfamethoxazole Lifespan Reproduction Population growth Caenorhabditis elegans
Trans-generational effects are increasingly used to indicate long-term influences of environmental pollutants. However, such studies can be complex and yield inconclusive results. In this study, the trans-generational effects of sulfamethoxazole (SMX) on Caenorhabditis elegans on lifespan, reproduction and population growth were tested for 7 consecutive generations, which included gestating generation (F0), embryo-exposed generation (F1), germline-exposed generation (F2), the first non-exposed generation (F3) and the three following generations (F4–F6). Results showed that lifespan was significantly affected by embryo exposure (F1) at 400 µm SMX with a value as low as 47% of the control. The reproduction (a total brood size as 49% of the control) and population growth (81% of the control) were significantly affected in germline exposure (F2). Lifespan and reproduction were severely inhibited in non-exposed generations, confirming the real transgenerational effects. Notably, initial reproduction and reproduction duration showed opposite generationrelated changes, indicating their interplay in the overall brood size. The population growth rate was well correlated with median lethal time, brood size and initial reproduction, which indicated that the population would increase when the nematodes lived longer and reproduced more offspring within shorter duration.
1. Introduction Environmental organisms are exposed to numerous pollutants at random life stages with various negative outcomes. When the exposure happens at early life stages (e.g., in gestation as a fetus), there would be long-term effects on the organisms in later life. Moreover, some residual consequences even occurred in progeny generations, i.e., causing trans-generational effects (Babenko et al., 2015; Van de Voort et al., 2015). Such effects provide developmental origins of behavior, health, and disease in the offspring (Swanson et al., 2009; Henrichs and Van den Bergh, 2015). Accordingly, the trans-generational effects are earning increasing attentions in demonstrating longterm outcomes of environmental pollutants (Skinner, 2008; Babenko et al., 2015; Van de Voort et al., 2015). Trans-generational tests are time consuming and laborious. Fortunately, such studies can be facilitated by model organisms. One example is Caenorhabditis elegans (C. elegans) whose lifespan and reproduction were employed to demonstrate effects of gold nanopar-
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ticles (Kim et al., 2013), cobalt (Wang et al., 2007) and nickel (Wang and Wang, 2008) in five, three and two generations, respectively. Another example is Daphnia magna whose growth and reproduction were used to show effects of antibacterials in two generations (Dalla Bona et al., 2015). Its metallothioneins were also used to indicate effects of cadmium in two generations (Li et al., 2016). Notably, the confusing generation arrangements in these studies can yield inconclusive results. Studies on the real trans-generational effects should consider the effects on the first non-exposed generation to demonstrate the actual long-term effects without the influence of the exposure. The choice of such generation depended on both the exposure duration and the life stages of organisms. The exposure in gestation results in exposure to the female adult (F0), the embryo (F1) and germline (F2) at the same time. Therefore, F3 is the first non-exposed generation (Skinner, 2008; Kim et al., 2013). Bearing this in mind, more studies are needed to reveal the real trans-generational effects of pollutants. Recently, sulfonamide antibiotics (SAs) became environmental
Corresponding author. E-mail address: [email protected] (J. Zhang).
http://dx.doi.org/10.1016/j.ecoenv.2016.10.017 Received 6 June 2016; Received in revised form 12 October 2016; Accepted 14 October 2016 0147-6513/ © 2016 Elsevier Inc. All rights reserved.
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Aladdin Industrial Co., China). After the bleach, the age-synchronized eggs were washed in sterile K-medium and transferred onto NGM plates with a bacterial lawn. After a 24 h incubation at 20 °C, the nematodes reached L2/L3 stage and were ready for chemical exposure. The choice of L2/L3 nematodes was to ensure a gestation exposure covering the whole formation of sperm, ovum and eggs (Hill et al., 2006). During the study, copper chloride was used as a reference toxicant (Dhawan et al., 2000). The L3 nematodes, age-synchronized from randomly chosen NGM plates, were exposed to copper chloride every month, and the median lethal concentrations (LC50) were not significantly different, verifying that the nematodes’ health and response to toxicants remained statistically constant throughout our study.
pollutants due to their ubiquitous existence in various environmental matrices and their ability to screen/sustain antibiotic resistance (Baran et al., 2011; Daniel et al., 2015). It was reported that SAs, e.g., sulfamethoxazole (SMX), at concentrations of 400 µm inhibited the behavior of C. elegans in F0 generation and the inhibitions transferred to F1 generation (Yu et al., 2011, 2013b). Moreover, SMX decreased the initial reproduction in F1 generation after an exposure in F0 generation (Liu et al., 2013). However, the potential of SMX to cause real trans-generational effects (i.e., on F3 and subsequent generations) needs more comprehensive studies. Furthermore, the changes of population growth provide essential information in judging effects of pollutants on the species maintenance and the ecosystem stability. Luckily, population growth rate (PGR) can be easily obtained by integrating the aforementioned lifespan and reproduction. Moreover, the PGR even showed potential superior sensitivity to toxicity than individual organisms (Alonzo et al., 2016). It was reported that the PGR of C. elegans was significantly decreased by tetracycline antibiotic at concentrations as low as 3 mg/L (Vangheel et al., 2014). Yet, population responses of C. elegans to SAs over generations remained unexplored. The present study aimed to explore the real trans-generational effects of SMX on C. elegans, combining indicators at both individual and population levels. To fulfill that purpose, we tested effects of SMX on the lifespan and reproduction, and calculated the PGR in six progeny generations (F1–F6) after an initial exposure in the parent generation (F0). Our findings demonstrated that the concerns of SAs should not only focus on the antibiotic resistance in pathogens, but also on the long-term influences of antibiotics themselves.
2.3. Exposure The toxicity experiments were performed according to previous studies with some modifications (Yu et al., 2013b). The results in a preliminary experiment showed that (1) there were no significant differences between nematodes in the solvent control (1% DMSO in Kmedium) and the absolute control (K-medium), and (2) the SMX concentration range of 0.04–400 µm was appropriate to ensure observable effects without acute lethality. In the formal experiments, five SMX concentrations and one solvent control (1% DMSO in Kmedium) were arranged in the 24-well culture plates with four wells for each concentration or control. Each well typically contained 500 μL SMX solutions or solvent control, and 500 μL K-medium containing approximately 100 L2/L3 nematodes. The exposure lasted 96 h in the absence of food at 20 °C. After the exposure, nematodes from each concentration or control were collected into centrifuge tubes. After a 30 min settlement, the nematodes in the bottom were transferred into new 1.5 mL centrifuge tubes, where they were washed in 1 mL sterile water. After another 30 min settlement, the nematodes in the bottom were transferred onto NGM plates with bacterial lawns to observe reproduction and lifespan.
2. Materials and methods 2.1. Test chemicals Sulfamethoxazole (SMX, CAS RN: 723-46-6, C10H11N3O3S) was prepared with K-medium (0.051 M NaCl and 0.032 M KCl) containing 1% dimethyl sulphoxide (DMSO, Amresco, USA) (Yu et al., 2013b). Five exposure concentrations were arranged as 0.04, 0.40, 4.0, 40.0 and 400.0 µm. The concentration arrangement was in accordance with the previous study on the effects of SMX on the nematode growth and behavior (Yu et al., 2011).
2.4. Reproduction and lifespan The reproduction was assayed according to an earlier report (Liu et al., 2013). The overall scheme for the experiments is shown in Fig. 1. Briefly, nematodes from the exposure (marked as F0) were picked individually onto new NGM plates with bacterial lawns (time marked as F0T0). Each NGM plate had two nematodes to facilitate the observation and operation. There were at least 48 nematodes (i.e., 24 NGM plates) for each concentration or solvent control. Then, F0 nematodes were transferred onto new NGM plates every 36 h, leaving the old NGM plates to count the newly hatched and easily visible offspring (F1) on the next day. The total offspring number within 72 h from F0T0 was referred as the initial reproduction of F0 generation (Yu et al., 2016). When the nematode reproduction stopped in four random NGM plates, the whole reproduction duration was noted and the transfer was stopped. Then, the living F0 nematodes were counted daily until they all died. Through this way, the brood size (accumulative fecundity), the initial reproduction, reproduction duration and lifespan in F0 generation were obtained. From the initial reproduction of F0, 48 nematodes (F1) were transferred individually onto 24 NGM plates with bacterial lawns (time marked as F1T0). Then, the F1 nematodes were transferred to new NGM plates every 36 h, leaving the old NGM plates to count the newly hatched and easily visible offspring (F2) on the next day. The total offspring number within 72 h from F1T0 was referred as the initial reproduction of F1 generation. When reproduction stopped in four NGM plates, the whole reproduction duration was noted and the transfer was stopped. Then, the living F1 nematodes were counted daily until their death. By this way, the brood size, the initial reproduction, reproduction duration and lifespan of nematodes in F1
2.2. Preparation of nematode The nematode was prepared according to earlier reports (Brenner, 1974; Yu et al., 2013b). Two kinds of culture medium were prepared beforehand to culture C. elegans, and their food, Escherichia coli OP50. The nematode growth medium (NGM) contained 3.0 g NaCl, 17.0 g agar, and 2.5 g peptone in 975 mL H2O. After an autoclave, the NGM at around 55 °C received 1.0 mL 1 M CaCl2, 1.0 mL 5 mg/mL cholesterol in ethanol, 1.0 mL 1 M MgSO4 and 25 mL 1 M potassium phosphate buffer (108.3 g KH2PO4 and 35.6 g K2HPO4 in 1.0 L H2O). Then, the NGM was dispensed into sterile petri plates (60 mm) to form agar (i.e., NGM plates). The lysogeny broth medium (LBM) to culture E. coli OP50 contained 10.0 g tryptone, 5.0 g yeast extract, 5.0 g NaCl and 1.0 L H2O. After the pH was adjusted to 7.0 with 1 M NaOH, the LBM was autoclaved and cooled down to room temperature. Both C. elegans and E. coli OP50 were provided by Department of Biochemistry and Molecular Biology, Southeast University Medical School, Nanjing, China. The bacteria were incubated in LBM at 120 rpm overnight at 37 °C. Next, 150 μL bacterial suspensions were spread across each NGM plate followed by an incubation overnight at 37 °C to form a bacterial lawn. Then, the NGM plates were used to culture the nematodes at 20 °C. Gravid nematodes were bleached for 5–10 min at room temperature in fresh clorox solutions containing 0.5 M NaOH and 1% NaOCl (diluted from sodium hypochlorite solution, AR 6% active chlorine, 313
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Fig. 1. The experimental scheme in testing effects of sulfamethoxazole (SMX) on lifespan, initial reproduction and total reproduction of C. elegans in the exposure generation (F0) and following six generations (F1–F6).
out independently for three times with similar results observed, and therefore the datasets in the present study were from one representative experiment.
generation were obtained. By repeating the same procedure, results of nematodes in F2–F6 generations were obtained. 2.5. Data presentation and statistical analysis
3. Results The lethality in the solvent control or each concentration was calculated as the dead nematodes on each day divided by the total nematodes on the first day. Then, the lethality was fitted by the Logistic model in Origin Pro 8.5 (Origin Lab Corp., USA). The median lethal time (LT50) in the solvent control or each concentration was calculated using the same model. Combining lethality and reproduction, the intrinsic growth rate (r) was calculated via POPULUS software (http://cbs.umn.edu/populus; by Don Alstad, Version 5.5) using the following equations:
3.1. Effects of SMX on the nematode lifespan in F0–F6 generations The nematode lifespan in each generation decreased with increasing SMX concentrations, and the lethality increased accordingly (Fig. S1 in Supplementary material). The lethality of SMX was well fitted by Logistic model according to the adjusted R2 ( > 0.88) and P values ( < 0.01) (Data not shown). The LT50 values showed a clear decrease over exposure concentrations in each generation (Fig. 2). Moreover, the LT50 values also showed generation-related changes. In nematodes from 400 µm SMX exposure, the LT50 values in F0–F6 generations were 62%, 37%, 49%, 47%, 72%, 78% and 66% of the concurrent control, respectively (Fig. 2). The results showed that nematodes in F1–F3 generations suffered more negative influences than those in F0 generation, and nematodes in F1 generation showed the shortest lifespan and strongest lethality (p < 0.05). Nematodes in F4–F6 generations showed recoveries in lifespan which showed similar inhibitions to those in F0 generation.
n
1=
∑ lx mx e−rx x=0
(1)
n
R0 =
∑ lx m x x =0
(2)
where lx is survivorship of nematodes at x day, and mx is offspring per individual (Lotka, 1913). All data were presented as a percentage of the mean value in the control (% of control) (Yu et al., 2013b). Through this way, the mean values in the control were presented as 100% of control. Values lower than 100% indicate inhibitions and those higher than 100% indicate stimulations. The two-way ANOVA were performed in Origin Pro 8.5 with Tukey's test to infer the differences within and between groups. The probability levels of 0.05 (p < 0.05) were considered statistically significant. The correlation among endpoints was analyzed through linear fitting, and Pearson cross-correlations (Pearson's r) were used to indicate the wellness of the correlation. The experiments were carried
3.2. Effects of SMX on the nematode reproduction in F0–F6 generations Effects of SMX on brood size, initial reproduction and reproduction duration of nematodes in F0–F6 generations are shown in Figs. 3–5. In F0, the nematode brood sizes at 0.04, 0.40 and 4.0 µm were 160%, 133% and 114% of the control, respectively, showing stimulatory effects. Meanwhile, the brood sizes at 40.0 and 400.0 µm were less 314
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LT50 (% of Control)
125 100
a
a aa b
b b
bb acb a d a
75
dd
bb d
b c b
b a
dc d
50
c
a ba b
25 0
0.04
0.4
4
40
a a
F0 F1 F2 F3 F4 F5 F6
400
Concentration (μM) Fig. 5. Reproduction duration of C. elegans in the exposure generation (F0) to sulfamethoxazole and the following generations (F1–F6). a: significantly different from the control by ANOVA (p < 0.05); b: significantly different from the control and from the earlier generation at the same concentration (p < 0.05); c: significantly different from the control and from the lower concentration in the same generation (p < 0.05); d: significantly different from the control and from the earlier generation at the same concentration and the lower concentration in the same generation (p < 0.05); e: significantly different from the earlier generation at the same concentration and the lower concentration in the same generation (p < 0.05); f: significantly different from the earlier generation at the same concentration (p < 0.05).
Fig. 2. Median lethality time (LT50, expressed in percentage of control) of C. elegans in the exposure generation (F0) to sulfamethoxazole and the following generations (F1–F6). a: significantly different from the control by ANOVA (p < 0.05); b: significantly different from the control and from the earlier generation at the same concentration (p < 0.05); c: significantly different from the control and from the lower concentration in the same generation (p < 0.05); d: significantly different from the control and from the earlier generation at the same concentration and the lower concentration in the same generation (p < 0.05).
than those in the control, showing inhibitory effects. The concentration-related results showed that SMX caused hormetic effects on the brood size, which was also observed in F1 and F5 generations. In the generation-related view, nematode brood sizes in F0–F6 generations at 0.04 µm SMX were 160%, 146%, 76%, 95%, 85%, 108% and 95% of the concurrent control, respectively (Fig. 3). The results indicated that the brood size was stimulated in F0 generation, and then it was less stimulated in F1 generation. The brood size changed from stimulation to inhibitions in F2 generation. The inhibitions decreased in F3 generation (less inhibitions than F2 generation) and increased in F4 generation (more inhibitions than F3 generation). At last, the effects changed to stimulations in F5 generation and to inhibitions in F6 generation. The effect fluctuations over generations indicated that nematodes attempted to recover from the initial damage from SMX exposure. Similarly in nematodes from 0.40 and 4.0 µm SMX exposure (Fig. 3), the earlier stimulations in F0 generation on the brood size decreased in F1 generation and changed to inhibitions in F2 generation. Then, the inhibitions showed fluctuated decreases from F3 to F6 generations. In nematodes from 40.0 and 400.0 µm SMX exposure, the brood size was inhibited in F0 generation. Then, the inhibitions first decreased in F1 generation and subsequently increased in F2 generation. At last, the inhibitions also showed fluctuated decreases from F3 to F6 generations. Notably, the brood size suffered the greatest inhibitions in F2 generation at all test concentrations (p < 0.05). The results of nematode initial reproduction in F0–F6 generations are shown in Fig. 4. At 0.04 µm SMX, the initial reproduction in F0–F6 generations was 77%, 78%, 97%, 121%, 118%, 106% and 99% of the control, respectively. The results indicated that the nematode initial reproduction was inhibited in F0 and F1 generations, and the effects changed from inhibition to stimulation in F3 generation. Then, the stimulation decreased gradually from F4 to F6 generations. In summary, the initial reproduction at 0.04 µm SMX showed inverse Ushaped changes over generations. At 0.40 µm, the initial reproduction in F0–F6 generations was 133%, 92%, 73%, 58%, 86%, 88% and 124% of the control, respectively (Fig. 4). The results indicated that the initial reproduction was stimulated in F0 generation. Then, it was inhibited in F1 generation, and the inhibitions first increased (F2 and F3 generations) and then decreased (F4 and F5 generations) over generations. Finally, it showed stimulation in F6 generation. In summary, the initial reproduction at 0.40 µm SMX showed U-shaped changes over generations. At 4.0 and 40.0 µm, the initial reproduction showed similar Ushaped changes to those from 0.40 µm SMX exposure (Fig. 4).
Fig. 3. Brood size (expressed in percentage of control) of C. elegans in the exposure generation (F0) to sulfamethoxazole and the following generations (F1–F6). a: significantly different from the control by ANOVA (p < 0.05); b: significantly different from the control and from the earlier generation at the same concentration (p < 0.05); c: significantly different from the control and from the lower concentration in the same generation (p < 0.05); d: significantly different from the control and from the earlier generation at the same concentration and the lower concentration in the same generation (p < 0.05); e: significantly different from the earlier generation at the same concentration and the lower concentration in the same generation (p < 0.05); f: significantly different from the earlier generation at the same concentration (p < 0.05).
Fig. 4. Initial reproduction of C. elegans in the exposure generation (F0) to sulfamethoxazole and the following generations (F1–F6). a: significantly different from the control by ANOVA (p < 0.05); b: significantly different from the control and from the earlier generation at the same concentration (p < 0.05); c: significantly different from the control and from the lower concentration in the same generation (p < 0.05); d: significantly different from the control and from the earlier generation at the same concentration and the lower concentration in the same generation (p < 0.05); e: significantly different from the earlier generation at the same concentration and the lower concentration in the same generation (p < 0.05); f: significantly different from the earlier generation at the same concentration (p < 0.05).
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4. Discussions
Meanwhile, in nematodes from 400.0 µm SMX exposure, the initial reproduction was inhibited in all generations ranging from 65–81% of the control without any generation-related changes. Notably, the initial reproduction was the lowest in F3 nematodes from 0.4 to 400 µm SMX exposure. The results of reproduction duration in F0–F6 generations are shown in Fig. 5. At 40.0 µm, the reproduction duration in F0–F6 generations was 82%, 89%, 100%, 110%, 91%, 89% and 80% of the control, respectively. In other words, reproduction duration showed an inverse U-shaped change over generations. From F0 to F3 generations, reproduction duration recovered gradually from inhibition to normal (control) level, and there was a significant stimulation in F3 generation. Then, the reproduction duration was inhibited in F4 and the inhibitions increased from F4 to F6 generations. Similarly at 400.0 µm, the reproduction duration also showed inverse U-shaped change. It was first inhibited (in F0 and F1 generations), and then stimulated (in F2 generation). Then, the stimulations decreased in F3 generation and changed to inhibitions in F4 generation. At last, the inhibitions increased from F5 to F6 generations. At 40.0 and 400.0 µm, the inverse U-shaped changes of reproduction duration over generations were opposite to the U-shaped changes of initial reproduction. At 0.04 µm, the U-shaped changes of reproduction duration over generations were opposite to the inverse U-shaped changes of initial reproduction.
4.1. Trans-generational toxicities of SMX and potential mechanisms In F0 generation, the lifespan was significantly shortened at all SMX concentrations in the present study which employed L2/L3 nematodes. However, the lifespan was lengthened at 100, 200 and 400 µm SMX in an earlier study which employed L4 nematodes (Liu et al., 2013). The differences caused by life stages were also observed in metal effects where L3 nematodes showed higher sensitivity than L4 ones (Yu et al., 2013a). Moreover, such differences were also reported in toxic effects across generations (Yu et al., 2013a). Summing up the present and previous studies, the life stage greatly influenced the toxic effects on nematodes, urging standard arrangement in future studies. The present study exposed L2/L3 nematodes for 96 h, which covered the whole formation of sperm, ovum and eggs (Hill et al., 2006). Such an arrangement ensured embryo and germline exposure in F1 and F2 nematodes, respectively. Among all generations, F1 nematodes suffered the greatest inhibition on the lifespan, and F2 nematodes showed the greatest inhibition on the brood size. The results showed different roles of embryo and germline in determining lifespan and reproduction in later generations which might be due to their different developmental stages and biological activities (Hanna et al., 2013). In the first non-exposed generation (F3) and the following F4–F6 generations, nematode lifespan and reproduction also showed significant inhibitions. The results confirmed the trans-generational effects of SMX on the offspring that was not involved in exposure. Such confirmation indicated long-term effects of sulfonamides on the behavior and health of the offspring (Henrichs and Van den Bergh, 2015). The results also demonstrated the influences of sulfonamides on the acclimation or adaptation of non-target organisms. Therefore, more attentions should be paid to the susceptibility of the host to negative effects of antibiotics, besides the concerns of antibiotic resistance in pathogens (Daniel et al., 2015). It was reported that the epigenetic mechanism (e.g., histone methylation) underlies the long-term effects on subsequent generations after an initial environmental disturbance in parent generation (Ho and Burggren, 2010; Burton and Metcalfe, 2014). In an earlier study, the histone methylation in Arabidopsis thaliana were significantly reduced by sulfonamides (e.g., sulfamethazine) (Zhang et al., 2012). Since the histone methylation in C. elegans played essential roles in determining the development of germ cells of the next generation to maintain an epigenetic memory (Furuhashi et al., 2010), the epigenetic effects could explain the observed trans-generational effects. Yet, such explanation still needs direct epigenetic evidence in future studies. The microbiota in the nematodes might also be involved in the trans-generational effects. The gut microbiota anticipated crucial physiological processes (e.g., development), the production of key elements (e.g., folic acid) and even co-evolution interactions with hosts (Bennett et al., 2015; Shapira, 2016). It was found that changes in microbiota were associated with negative effects even diseases (e.g., obesity which is well-known to influence reproduction) (Pietroiusti et al., 2016). Antibiotics had the abilities to modulate the gut microbiota (Zhang et al., 2014), and such modulation already showed potentials to transfer across generations (Mueller et al., 2015; Gérard, 2016). Yet, it still needs further studies to confirm the involvement of gut microbiota in the observed trans-generational effects.
3.3. Effects of SMX on population growth of nematodes in F0–F6 generations The results of the population growth rate (r) of nematodes in F0–F6 generations are shown in Fig. 6. At 0.04 µm, the r values were similar to those in the control, except in F2 generation where the r was lower than 100% of the control showing inhibitions. At 0.40 µm, the r was 112% of the control in F0, and then it decreased to 87% of the control in F2 generation. In the following generations, it increased to approximately 100% of the control. That is to say, the population growth showed a U-shaped change over generations. In nematodes from 4.0 and 40.0 µm SMX exposure, the population growth showed similar Ushaped changes with the lowest values in F2 generation. At 400.0 µm, the population growth was inhibited without generation-related changes, but the lowest value was still in F2 generation. Collectively, the population growth in F2 generation suffered the greatest inhibition among generations in each concentration.
4.2. Interactions among endpoints over generations Fig. 6. Population growth of C. elegans in the exposure generation (F0) to sulfamethoxazole and the following generations (F1–F6). a: significantly different from POC in the control (p < 0.05); b: significantly different from the control and from the earlier generation at the same concentration (p < 0.05); c: significantly different from the control and from the lower concentration in the same generation (p < 0.05); d: significantly different from the earlier generation at the same concentration (p < 0.05).
In the present study, SMX caused hormetic effects on the brood size (Fig. 2) and initial reproduction (Fig. 3). It was reported that hormesis on the reproduction of C. elegans usually trades off decreases in lifespan (Tyne et al., 2015), or vice versa (Saul et al., 2013). Such trade-off among endpoints were widely reported in various organisms 316
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Appendix A. Supplementary material
Table 1 Pearson cross-correlations (Pearson's r) between median lethal time (LT50), brood size, initial reproduction, reproduction duration and population growth rate in C. elegans exposed to sulfamethaxozale. Brood size
LT50
Brood size Initial reproduction Reproduction duration Population growth rate * **
Initial reproduction
Reproduction duration
– 0.1548
–
−0.1297
0.2204
−0.0224
–
0.5494**
0.7410**
0.7219**
0.0102
0.4478 0.2659
*
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Multigenerational study of gold nanoparticles in Caenorhabditis elegans: transgenerational effect of maternal exposure. Environ. Sci. Technol. 47, 5393–5399. Li, S., Sheng, L., Xu, J., Tong, H., Jiang, H., 2016. The induction of metallothioneins during pulsed cadmium exposure to Daphnia magna: recovery and transgenerational effect. Ecotoxicol. Environ. Safe 126, 71–77. Liu, S., Saul, N., Pan, B., Menzel, R., Steinberg, C.E.W., 2013. The non-target organism Caenorhabditis elegans withstands the impact of sulfamethoxazole. Chemosphere 93, 2373–2380. Lotka, J., 1913. A natural population norm. J. Wash. Acad. Sci. 3, 241–248. Mueller, N.T., Whyatt, R., Hoepner, L., Oberfield, S., Dominguez-Bello, M.G., Widen, E.M., Hassoun, A., Perera, F., Rundle, A., 2015. Prenatal exposure to antibiotics, cesarean section and risk of childhood obesity. Int. J. Obes. 39, 665–670. Pietroiusti, A., Magrini, A., Campagnolo, L., 2016. New frontiers in nanotoxicology: gut microbiota/microbiome-mediated effects of engineered nanomaterials. Toxicol. Appl. Pharm. 299, 90–95. Roff, D.A., 2001. Age and Size at Maturity. Oxford University Press, New York. Saul, N., Pietsch, K., Stürzenbaum, S.R., Menzel, R., Steinberg, C.E.W., 2011. Diversity of polyphenol action in Caenorhabditis elegans: between toxicity and longevity. J. Nat. Prod. 74, 1713–1720. Saul, N., Pietsch, K., Stürzenbaum, S.R., Menzel, R., Steinberg, C.E.W., 2013. Hormesis and longevity with tannins: free of charge or cost-intensive? Chemosphere 93, 1005–1008. Shapira, M., 2016. Gut microbiotas and host evolution: scaling up symbiosis. Trends Ecol. Evol.. http://dx.doi.org/10.1016/j.tree.2016.03.006. Skinner, M.K., 2008. What is an epigenetic transgenerational phenotype? F3 or F2. Reprod. Toxicol. 25, 2–6. Swanson, J.M., Entringer, S., Buss, C., Wadhwa, P.D., 2009. Developmental origins of health and disease: environmental exposures. Semin Reprod. Med. 27, 391–402. Tyne, W., Little, S., Spurgeon, D.J., Svendsen, C., 2015. Hormesis depends upon the lifestage and duration of exposure: examples for a pesticide and a nanomaterial. Ecotoxicol. Environ. Safe 120, 117–123. Van de Voort, C.A., Grimsrud, K.N., Midic, U., Mtango, N., Latham, K.E., 2015. Transgenerational effects of binge drinking in a primate model: implications for human health. Fertil. Steril. 103, 560–569. Vangheel, M., Traunspurger, W., Spann, N., 2014. Effects of the antibiotic tetracycline on
p < 0.05. p < 0.01.
and also observed in trans-generational effects on C. elegans (Yu et al., 2016). However, the reproduction and lifespan did not show such trade-offs in the present study due to the lack of significant correlation (Table 1). Such absence might be attributed to the presence of food in the whole experiment which did not cause caloric restriction that was usually the prerequisite for trade-off effects (Adler et al., 2013). The population growth rate (PGR) was well correlated with LT50 and brood size, which indicated that the population would increase when the nematodes lived longer and reproduced more offspring. The PGR was also correlated with initial reproduction (Table 1). The results supported the natural observation that initial reproduction will determine the population survival against predation or adverse environmental conditions (Roff, 2001; Liu et al., 2013). In Figs. 4 and 5, initial reproduction and reproduction duration showed opposite changes over generations at 0.04, 40.0 and 400.0 µm. The results indicated an interplay between these two indicators in the overall reproduction. Such interplay was also implied in effects of tannic acid treatment which delayed the initial reproduction but did not change the overall reproduction (Saul et al., 2011). However, none of the initial reproduction, brood size and PGR showed any correlation with the reproduction duration (Table 1). Thus, effects of SMX on the population growth greatly relied on its effects on the initial reproduction, which could not be compensated with the responses in reproduction duration. Yet, the complex interactions among indicators still need further studies. 5. Conclusion Currently, the effects of sulfamethaxozale (SMX) on lifespan, reproduction and population growth in nematodes were studied for 7 consecutive generations after an exposure in F0 generation. Among all generations, the lifespan was severely affected following embryo exposure (F1) and reproduction was significantly affected following germline exposure (F2), indicating special roles of embryo and germline in lifespan and reproduction, respectively. In non-exposed F3–F6 generations, nematode lifespan and reproduction showed significant inhibitions, confirming the trans-generational effects. Moreover, the population growth was dependent on lifespan, brood size and initial reproduction, and there seemed an interplay between initial reproduction and reproduction duration. Further studies are needed to elucidate the mechanisms of the trans-generational effects. Acknowledgements The authors are grateful for the financial supports by the National Natural Science Foundation of China (Nos. 21307095, 21407061 and 51278353), the Collaborative Innovation Center for Regional Environmental Quality, and the Swedish Research Council (Contract Dnr. 639-2013-6913). 317
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Yu, Z.Y., Chen, X.X., Zhang, J., Wang, R., Yin, D.Q., 2013a. Transgenerational effects of heavy metals on L3 larva of Caenorhabditis elegans with greater behavior and growth inhibitions in the progeny. Ecotoxicol. Environ. Safe 88C, 178–184. Yu, Z.Y., Zhang, J., Chen, X.X., Yin, D.Q., Deng, H.P., 2013b. Inhibitions on the behavior and growth of the nematode progeny after prenatal exposure to sulfonamides at environmental concentrations. J. Hazard. Mater. 250–251, 198–203. Zhang, H.M., Deng, X.Y., Miki, D., Cutler, S., La, H.G., Hou, Y.-J., Oh, J., Zhu, J.-K., 2012. Sulfamethazine suppresses epigenetic silencing in Arabidopsis by impairing folate synthesis. Plant Cell 24, 1230–1241. Zhang, Y., Limaye, P.B., Renaud, H.J., Klaassen, C.D., 2014. Effect of various antibiotics on modulation of intestinal microbiota and bile acid profile in mice. Toxicol. Appl. Pharm. 277, 138–145.
the reproduction, growth and population growth rate of the nematode Caenorhabditis elegans. Nematology 16, 19–29. Wang, D., Wang, Y., 2008. Nickel sulfate induces numerous defects in Caenorhabditis elegans that can also be transferred to progeny. Environ. Pollut. 151, 585–592. Wang, Y., Xie, W., Wang, D.Y., 2007. Transferable properties of multi-biological toxicity caused by cobalt exposure in Caenorhabditis elegans. Environ. Toxicol. Chem. 26, 2405–2412. Yu, Z.Y., Jiang, L., Yin, D.Q., 2011. Behavior toxicity to Caenorhabditis elegans transferred to the progeny after exposure to sulfamethoxazole at environmentally relevant concentration. J. Environ. Sci. 23, 294–300. Yu, Z.Y., Zhang, J., Yin, D.Q., 2016. Multigenerational effects of heavy metals on feeding, growth, initial reproduction and antioxidants in Caenorhabditis elegans. PLoS ONE 11, e0154529.
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Trans-generational influences of sulfamethoxazole on lifespan, reproduction and population growth of Caenorhabditis elegans Zhenyang Yua, Guohua Sunc,d, Yanjun Liuc,e, Daqiang Yina, Jing Zhangb,
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a State Key Laboratory of Pollution Control and Resource Reuse, Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, PR China b Ecological Technique and Engineering College, Shanghai Institute of Technology, Shanghai 201418, PR China c College of Biological, Chemical Sciences and Engineering, Jiaxing University, Jiaxing, Zhejiang 314001, PR China d Department of Civil and Environmental Engineering, University of Ulsan, Nam Gu, Ulsan 680-748, South Korea e School of Civil Engineering and Environmental Science at Collage of Engineering, University of Oklahoma, Norman, OK 73019, USA
A R T I C L E I N F O
A BS T RAC T
Keywords: Trans-generational effect Sulfamethoxazole Lifespan Reproduction Population growth Caenorhabditis elegans
Trans-generational effects are increasingly used to indicate long-term influences of environmental pollutants. However, such studies can be complex and yield inconclusive results. In this study, the trans-generational effects of sulfamethoxazole (SMX) on Caenorhabditis elegans on lifespan, reproduction and population growth were tested for 7 consecutive generations, which included gestating generation (F0), embryo-exposed generation (F1), germline-exposed generation (F2), the first non-exposed generation (F3) and the three following generations (F4–F6). Results showed that lifespan was significantly affected by embryo exposure (F1) at 400 µm SMX with a value as low as 47% of the control. The reproduction (a total brood size as 49% of the control) and population growth (81% of the control) were significantly affected in germline exposure (F2). Lifespan and reproduction were severely inhibited in non-exposed generations, confirming the real transgenerational effects. Notably, initial reproduction and reproduction duration showed opposite generationrelated changes, indicating their interplay in the overall brood size. The population growth rate was well correlated with median lethal time, brood size and initial reproduction, which indicated that the population would increase when the nematodes lived longer and reproduced more offspring within shorter duration.
1. Introduction Environmental organisms are exposed to numerous pollutants at random life stages with various negative outcomes. When the exposure happens at early life stages (e.g., in gestation as a fetus), there would be long-term effects on the organisms in later life. Moreover, some residual consequences even occurred in progeny generations, i.e., causing trans-generational effects (Babenko et al., 2015; Van de Voort et al., 2015). Such effects provide developmental origins of behavior, health, and disease in the offspring (Swanson et al., 2009; Henrichs and Van den Bergh, 2015). Accordingly, the trans-generational effects are earning increasing attentions in demonstrating longterm outcomes of environmental pollutants (Skinner, 2008; Babenko et al., 2015; Van de Voort et al., 2015). Trans-generational tests are time consuming and laborious. Fortunately, such studies can be facilitated by model organisms. One example is Caenorhabditis elegans (C. elegans) whose lifespan and reproduction were employed to demonstrate effects of gold nanopar-
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ticles (Kim et al., 2013), cobalt (Wang et al., 2007) and nickel (Wang and Wang, 2008) in five, three and two generations, respectively. Another example is Daphnia magna whose growth and reproduction were used to show effects of antibacterials in two generations (Dalla Bona et al., 2015). Its metallothioneins were also used to indicate effects of cadmium in two generations (Li et al., 2016). Notably, the confusing generation arrangements in these studies can yield inconclusive results. Studies on the real trans-generational effects should consider the effects on the first non-exposed generation to demonstrate the actual long-term effects without the influence of the exposure. The choice of such generation depended on both the exposure duration and the life stages of organisms. The exposure in gestation results in exposure to the female adult (F0), the embryo (F1) and germline (F2) at the same time. Therefore, F3 is the first non-exposed generation (Skinner, 2008; Kim et al., 2013). Bearing this in mind, more studies are needed to reveal the real trans-generational effects of pollutants. Recently, sulfonamide antibiotics (SAs) became environmental
Corresponding author. E-mail address: [email protected] (J. Zhang).
http://dx.doi.org/10.1016/j.ecoenv.2016.10.017 Received 6 June 2016; Received in revised form 12 October 2016; Accepted 14 October 2016 0147-6513/ © 2016 Elsevier Inc. All rights reserved.
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Aladdin Industrial Co., China). After the bleach, the age-synchronized eggs were washed in sterile K-medium and transferred onto NGM plates with a bacterial lawn. After a 24 h incubation at 20 °C, the nematodes reached L2/L3 stage and were ready for chemical exposure. The choice of L2/L3 nematodes was to ensure a gestation exposure covering the whole formation of sperm, ovum and eggs (Hill et al., 2006). During the study, copper chloride was used as a reference toxicant (Dhawan et al., 2000). The L3 nematodes, age-synchronized from randomly chosen NGM plates, were exposed to copper chloride every month, and the median lethal concentrations (LC50) were not significantly different, verifying that the nematodes’ health and response to toxicants remained statistically constant throughout our study.
pollutants due to their ubiquitous existence in various environmental matrices and their ability to screen/sustain antibiotic resistance (Baran et al., 2011; Daniel et al., 2015). It was reported that SAs, e.g., sulfamethoxazole (SMX), at concentrations of 400 µm inhibited the behavior of C. elegans in F0 generation and the inhibitions transferred to F1 generation (Yu et al., 2011, 2013b). Moreover, SMX decreased the initial reproduction in F1 generation after an exposure in F0 generation (Liu et al., 2013). However, the potential of SMX to cause real trans-generational effects (i.e., on F3 and subsequent generations) needs more comprehensive studies. Furthermore, the changes of population growth provide essential information in judging effects of pollutants on the species maintenance and the ecosystem stability. Luckily, population growth rate (PGR) can be easily obtained by integrating the aforementioned lifespan and reproduction. Moreover, the PGR even showed potential superior sensitivity to toxicity than individual organisms (Alonzo et al., 2016). It was reported that the PGR of C. elegans was significantly decreased by tetracycline antibiotic at concentrations as low as 3 mg/L (Vangheel et al., 2014). Yet, population responses of C. elegans to SAs over generations remained unexplored. The present study aimed to explore the real trans-generational effects of SMX on C. elegans, combining indicators at both individual and population levels. To fulfill that purpose, we tested effects of SMX on the lifespan and reproduction, and calculated the PGR in six progeny generations (F1–F6) after an initial exposure in the parent generation (F0). Our findings demonstrated that the concerns of SAs should not only focus on the antibiotic resistance in pathogens, but also on the long-term influences of antibiotics themselves.
2.3. Exposure The toxicity experiments were performed according to previous studies with some modifications (Yu et al., 2013b). The results in a preliminary experiment showed that (1) there were no significant differences between nematodes in the solvent control (1% DMSO in Kmedium) and the absolute control (K-medium), and (2) the SMX concentration range of 0.04–400 µm was appropriate to ensure observable effects without acute lethality. In the formal experiments, five SMX concentrations and one solvent control (1% DMSO in Kmedium) were arranged in the 24-well culture plates with four wells for each concentration or control. Each well typically contained 500 μL SMX solutions or solvent control, and 500 μL K-medium containing approximately 100 L2/L3 nematodes. The exposure lasted 96 h in the absence of food at 20 °C. After the exposure, nematodes from each concentration or control were collected into centrifuge tubes. After a 30 min settlement, the nematodes in the bottom were transferred into new 1.5 mL centrifuge tubes, where they were washed in 1 mL sterile water. After another 30 min settlement, the nematodes in the bottom were transferred onto NGM plates with bacterial lawns to observe reproduction and lifespan.
2. Materials and methods 2.1. Test chemicals Sulfamethoxazole (SMX, CAS RN: 723-46-6, C10H11N3O3S) was prepared with K-medium (0.051 M NaCl and 0.032 M KCl) containing 1% dimethyl sulphoxide (DMSO, Amresco, USA) (Yu et al., 2013b). Five exposure concentrations were arranged as 0.04, 0.40, 4.0, 40.0 and 400.0 µm. The concentration arrangement was in accordance with the previous study on the effects of SMX on the nematode growth and behavior (Yu et al., 2011).
2.4. Reproduction and lifespan The reproduction was assayed according to an earlier report (Liu et al., 2013). The overall scheme for the experiments is shown in Fig. 1. Briefly, nematodes from the exposure (marked as F0) were picked individually onto new NGM plates with bacterial lawns (time marked as F0T0). Each NGM plate had two nematodes to facilitate the observation and operation. There were at least 48 nematodes (i.e., 24 NGM plates) for each concentration or solvent control. Then, F0 nematodes were transferred onto new NGM plates every 36 h, leaving the old NGM plates to count the newly hatched and easily visible offspring (F1) on the next day. The total offspring number within 72 h from F0T0 was referred as the initial reproduction of F0 generation (Yu et al., 2016). When the nematode reproduction stopped in four random NGM plates, the whole reproduction duration was noted and the transfer was stopped. Then, the living F0 nematodes were counted daily until they all died. Through this way, the brood size (accumulative fecundity), the initial reproduction, reproduction duration and lifespan in F0 generation were obtained. From the initial reproduction of F0, 48 nematodes (F1) were transferred individually onto 24 NGM plates with bacterial lawns (time marked as F1T0). Then, the F1 nematodes were transferred to new NGM plates every 36 h, leaving the old NGM plates to count the newly hatched and easily visible offspring (F2) on the next day. The total offspring number within 72 h from F1T0 was referred as the initial reproduction of F1 generation. When reproduction stopped in four NGM plates, the whole reproduction duration was noted and the transfer was stopped. Then, the living F1 nematodes were counted daily until their death. By this way, the brood size, the initial reproduction, reproduction duration and lifespan of nematodes in F1
2.2. Preparation of nematode The nematode was prepared according to earlier reports (Brenner, 1974; Yu et al., 2013b). Two kinds of culture medium were prepared beforehand to culture C. elegans, and their food, Escherichia coli OP50. The nematode growth medium (NGM) contained 3.0 g NaCl, 17.0 g agar, and 2.5 g peptone in 975 mL H2O. After an autoclave, the NGM at around 55 °C received 1.0 mL 1 M CaCl2, 1.0 mL 5 mg/mL cholesterol in ethanol, 1.0 mL 1 M MgSO4 and 25 mL 1 M potassium phosphate buffer (108.3 g KH2PO4 and 35.6 g K2HPO4 in 1.0 L H2O). Then, the NGM was dispensed into sterile petri plates (60 mm) to form agar (i.e., NGM plates). The lysogeny broth medium (LBM) to culture E. coli OP50 contained 10.0 g tryptone, 5.0 g yeast extract, 5.0 g NaCl and 1.0 L H2O. After the pH was adjusted to 7.0 with 1 M NaOH, the LBM was autoclaved and cooled down to room temperature. Both C. elegans and E. coli OP50 were provided by Department of Biochemistry and Molecular Biology, Southeast University Medical School, Nanjing, China. The bacteria were incubated in LBM at 120 rpm overnight at 37 °C. Next, 150 μL bacterial suspensions were spread across each NGM plate followed by an incubation overnight at 37 °C to form a bacterial lawn. Then, the NGM plates were used to culture the nematodes at 20 °C. Gravid nematodes were bleached for 5–10 min at room temperature in fresh clorox solutions containing 0.5 M NaOH and 1% NaOCl (diluted from sodium hypochlorite solution, AR 6% active chlorine, 313
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Fig. 1. The experimental scheme in testing effects of sulfamethoxazole (SMX) on lifespan, initial reproduction and total reproduction of C. elegans in the exposure generation (F0) and following six generations (F1–F6).
out independently for three times with similar results observed, and therefore the datasets in the present study were from one representative experiment.
generation were obtained. By repeating the same procedure, results of nematodes in F2–F6 generations were obtained. 2.5. Data presentation and statistical analysis
3. Results The lethality in the solvent control or each concentration was calculated as the dead nematodes on each day divided by the total nematodes on the first day. Then, the lethality was fitted by the Logistic model in Origin Pro 8.5 (Origin Lab Corp., USA). The median lethal time (LT50) in the solvent control or each concentration was calculated using the same model. Combining lethality and reproduction, the intrinsic growth rate (r) was calculated via POPULUS software (http://cbs.umn.edu/populus; by Don Alstad, Version 5.5) using the following equations:
3.1. Effects of SMX on the nematode lifespan in F0–F6 generations The nematode lifespan in each generation decreased with increasing SMX concentrations, and the lethality increased accordingly (Fig. S1 in Supplementary material). The lethality of SMX was well fitted by Logistic model according to the adjusted R2 ( > 0.88) and P values ( < 0.01) (Data not shown). The LT50 values showed a clear decrease over exposure concentrations in each generation (Fig. 2). Moreover, the LT50 values also showed generation-related changes. In nematodes from 400 µm SMX exposure, the LT50 values in F0–F6 generations were 62%, 37%, 49%, 47%, 72%, 78% and 66% of the concurrent control, respectively (Fig. 2). The results showed that nematodes in F1–F3 generations suffered more negative influences than those in F0 generation, and nematodes in F1 generation showed the shortest lifespan and strongest lethality (p < 0.05). Nematodes in F4–F6 generations showed recoveries in lifespan which showed similar inhibitions to those in F0 generation.
n
1=
∑ lx mx e−rx x=0
(1)
n
R0 =
∑ lx m x x =0
(2)
where lx is survivorship of nematodes at x day, and mx is offspring per individual (Lotka, 1913). All data were presented as a percentage of the mean value in the control (% of control) (Yu et al., 2013b). Through this way, the mean values in the control were presented as 100% of control. Values lower than 100% indicate inhibitions and those higher than 100% indicate stimulations. The two-way ANOVA were performed in Origin Pro 8.5 with Tukey's test to infer the differences within and between groups. The probability levels of 0.05 (p < 0.05) were considered statistically significant. The correlation among endpoints was analyzed through linear fitting, and Pearson cross-correlations (Pearson's r) were used to indicate the wellness of the correlation. The experiments were carried
3.2. Effects of SMX on the nematode reproduction in F0–F6 generations Effects of SMX on brood size, initial reproduction and reproduction duration of nematodes in F0–F6 generations are shown in Figs. 3–5. In F0, the nematode brood sizes at 0.04, 0.40 and 4.0 µm were 160%, 133% and 114% of the control, respectively, showing stimulatory effects. Meanwhile, the brood sizes at 40.0 and 400.0 µm were less 314
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LT50 (% of Control)
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b a
dc d
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Concentration (μM) Fig. 5. Reproduction duration of C. elegans in the exposure generation (F0) to sulfamethoxazole and the following generations (F1–F6). a: significantly different from the control by ANOVA (p < 0.05); b: significantly different from the control and from the earlier generation at the same concentration (p < 0.05); c: significantly different from the control and from the lower concentration in the same generation (p < 0.05); d: significantly different from the control and from the earlier generation at the same concentration and the lower concentration in the same generation (p < 0.05); e: significantly different from the earlier generation at the same concentration and the lower concentration in the same generation (p < 0.05); f: significantly different from the earlier generation at the same concentration (p < 0.05).
Fig. 2. Median lethality time (LT50, expressed in percentage of control) of C. elegans in the exposure generation (F0) to sulfamethoxazole and the following generations (F1–F6). a: significantly different from the control by ANOVA (p < 0.05); b: significantly different from the control and from the earlier generation at the same concentration (p < 0.05); c: significantly different from the control and from the lower concentration in the same generation (p < 0.05); d: significantly different from the control and from the earlier generation at the same concentration and the lower concentration in the same generation (p < 0.05).
than those in the control, showing inhibitory effects. The concentration-related results showed that SMX caused hormetic effects on the brood size, which was also observed in F1 and F5 generations. In the generation-related view, nematode brood sizes in F0–F6 generations at 0.04 µm SMX were 160%, 146%, 76%, 95%, 85%, 108% and 95% of the concurrent control, respectively (Fig. 3). The results indicated that the brood size was stimulated in F0 generation, and then it was less stimulated in F1 generation. The brood size changed from stimulation to inhibitions in F2 generation. The inhibitions decreased in F3 generation (less inhibitions than F2 generation) and increased in F4 generation (more inhibitions than F3 generation). At last, the effects changed to stimulations in F5 generation and to inhibitions in F6 generation. The effect fluctuations over generations indicated that nematodes attempted to recover from the initial damage from SMX exposure. Similarly in nematodes from 0.40 and 4.0 µm SMX exposure (Fig. 3), the earlier stimulations in F0 generation on the brood size decreased in F1 generation and changed to inhibitions in F2 generation. Then, the inhibitions showed fluctuated decreases from F3 to F6 generations. In nematodes from 40.0 and 400.0 µm SMX exposure, the brood size was inhibited in F0 generation. Then, the inhibitions first decreased in F1 generation and subsequently increased in F2 generation. At last, the inhibitions also showed fluctuated decreases from F3 to F6 generations. Notably, the brood size suffered the greatest inhibitions in F2 generation at all test concentrations (p < 0.05). The results of nematode initial reproduction in F0–F6 generations are shown in Fig. 4. At 0.04 µm SMX, the initial reproduction in F0–F6 generations was 77%, 78%, 97%, 121%, 118%, 106% and 99% of the control, respectively. The results indicated that the nematode initial reproduction was inhibited in F0 and F1 generations, and the effects changed from inhibition to stimulation in F3 generation. Then, the stimulation decreased gradually from F4 to F6 generations. In summary, the initial reproduction at 0.04 µm SMX showed inverse Ushaped changes over generations. At 0.40 µm, the initial reproduction in F0–F6 generations was 133%, 92%, 73%, 58%, 86%, 88% and 124% of the control, respectively (Fig. 4). The results indicated that the initial reproduction was stimulated in F0 generation. Then, it was inhibited in F1 generation, and the inhibitions first increased (F2 and F3 generations) and then decreased (F4 and F5 generations) over generations. Finally, it showed stimulation in F6 generation. In summary, the initial reproduction at 0.40 µm SMX showed U-shaped changes over generations. At 4.0 and 40.0 µm, the initial reproduction showed similar Ushaped changes to those from 0.40 µm SMX exposure (Fig. 4).
Fig. 3. Brood size (expressed in percentage of control) of C. elegans in the exposure generation (F0) to sulfamethoxazole and the following generations (F1–F6). a: significantly different from the control by ANOVA (p < 0.05); b: significantly different from the control and from the earlier generation at the same concentration (p < 0.05); c: significantly different from the control and from the lower concentration in the same generation (p < 0.05); d: significantly different from the control and from the earlier generation at the same concentration and the lower concentration in the same generation (p < 0.05); e: significantly different from the earlier generation at the same concentration and the lower concentration in the same generation (p < 0.05); f: significantly different from the earlier generation at the same concentration (p < 0.05).
Fig. 4. Initial reproduction of C. elegans in the exposure generation (F0) to sulfamethoxazole and the following generations (F1–F6). a: significantly different from the control by ANOVA (p < 0.05); b: significantly different from the control and from the earlier generation at the same concentration (p < 0.05); c: significantly different from the control and from the lower concentration in the same generation (p < 0.05); d: significantly different from the control and from the earlier generation at the same concentration and the lower concentration in the same generation (p < 0.05); e: significantly different from the earlier generation at the same concentration and the lower concentration in the same generation (p < 0.05); f: significantly different from the earlier generation at the same concentration (p < 0.05).
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4. Discussions
Meanwhile, in nematodes from 400.0 µm SMX exposure, the initial reproduction was inhibited in all generations ranging from 65–81% of the control without any generation-related changes. Notably, the initial reproduction was the lowest in F3 nematodes from 0.4 to 400 µm SMX exposure. The results of reproduction duration in F0–F6 generations are shown in Fig. 5. At 40.0 µm, the reproduction duration in F0–F6 generations was 82%, 89%, 100%, 110%, 91%, 89% and 80% of the control, respectively. In other words, reproduction duration showed an inverse U-shaped change over generations. From F0 to F3 generations, reproduction duration recovered gradually from inhibition to normal (control) level, and there was a significant stimulation in F3 generation. Then, the reproduction duration was inhibited in F4 and the inhibitions increased from F4 to F6 generations. Similarly at 400.0 µm, the reproduction duration also showed inverse U-shaped change. It was first inhibited (in F0 and F1 generations), and then stimulated (in F2 generation). Then, the stimulations decreased in F3 generation and changed to inhibitions in F4 generation. At last, the inhibitions increased from F5 to F6 generations. At 40.0 and 400.0 µm, the inverse U-shaped changes of reproduction duration over generations were opposite to the U-shaped changes of initial reproduction. At 0.04 µm, the U-shaped changes of reproduction duration over generations were opposite to the inverse U-shaped changes of initial reproduction.
4.1. Trans-generational toxicities of SMX and potential mechanisms In F0 generation, the lifespan was significantly shortened at all SMX concentrations in the present study which employed L2/L3 nematodes. However, the lifespan was lengthened at 100, 200 and 400 µm SMX in an earlier study which employed L4 nematodes (Liu et al., 2013). The differences caused by life stages were also observed in metal effects where L3 nematodes showed higher sensitivity than L4 ones (Yu et al., 2013a). Moreover, such differences were also reported in toxic effects across generations (Yu et al., 2013a). Summing up the present and previous studies, the life stage greatly influenced the toxic effects on nematodes, urging standard arrangement in future studies. The present study exposed L2/L3 nematodes for 96 h, which covered the whole formation of sperm, ovum and eggs (Hill et al., 2006). Such an arrangement ensured embryo and germline exposure in F1 and F2 nematodes, respectively. Among all generations, F1 nematodes suffered the greatest inhibition on the lifespan, and F2 nematodes showed the greatest inhibition on the brood size. The results showed different roles of embryo and germline in determining lifespan and reproduction in later generations which might be due to their different developmental stages and biological activities (Hanna et al., 2013). In the first non-exposed generation (F3) and the following F4–F6 generations, nematode lifespan and reproduction also showed significant inhibitions. The results confirmed the trans-generational effects of SMX on the offspring that was not involved in exposure. Such confirmation indicated long-term effects of sulfonamides on the behavior and health of the offspring (Henrichs and Van den Bergh, 2015). The results also demonstrated the influences of sulfonamides on the acclimation or adaptation of non-target organisms. Therefore, more attentions should be paid to the susceptibility of the host to negative effects of antibiotics, besides the concerns of antibiotic resistance in pathogens (Daniel et al., 2015). It was reported that the epigenetic mechanism (e.g., histone methylation) underlies the long-term effects on subsequent generations after an initial environmental disturbance in parent generation (Ho and Burggren, 2010; Burton and Metcalfe, 2014). In an earlier study, the histone methylation in Arabidopsis thaliana were significantly reduced by sulfonamides (e.g., sulfamethazine) (Zhang et al., 2012). Since the histone methylation in C. elegans played essential roles in determining the development of germ cells of the next generation to maintain an epigenetic memory (Furuhashi et al., 2010), the epigenetic effects could explain the observed trans-generational effects. Yet, such explanation still needs direct epigenetic evidence in future studies. The microbiota in the nematodes might also be involved in the trans-generational effects. The gut microbiota anticipated crucial physiological processes (e.g., development), the production of key elements (e.g., folic acid) and even co-evolution interactions with hosts (Bennett et al., 2015; Shapira, 2016). It was found that changes in microbiota were associated with negative effects even diseases (e.g., obesity which is well-known to influence reproduction) (Pietroiusti et al., 2016). Antibiotics had the abilities to modulate the gut microbiota (Zhang et al., 2014), and such modulation already showed potentials to transfer across generations (Mueller et al., 2015; Gérard, 2016). Yet, it still needs further studies to confirm the involvement of gut microbiota in the observed trans-generational effects.
3.3. Effects of SMX on population growth of nematodes in F0–F6 generations The results of the population growth rate (r) of nematodes in F0–F6 generations are shown in Fig. 6. At 0.04 µm, the r values were similar to those in the control, except in F2 generation where the r was lower than 100% of the control showing inhibitions. At 0.40 µm, the r was 112% of the control in F0, and then it decreased to 87% of the control in F2 generation. In the following generations, it increased to approximately 100% of the control. That is to say, the population growth showed a U-shaped change over generations. In nematodes from 4.0 and 40.0 µm SMX exposure, the population growth showed similar Ushaped changes with the lowest values in F2 generation. At 400.0 µm, the population growth was inhibited without generation-related changes, but the lowest value was still in F2 generation. Collectively, the population growth in F2 generation suffered the greatest inhibition among generations in each concentration.
4.2. Interactions among endpoints over generations Fig. 6. Population growth of C. elegans in the exposure generation (F0) to sulfamethoxazole and the following generations (F1–F6). a: significantly different from POC in the control (p < 0.05); b: significantly different from the control and from the earlier generation at the same concentration (p < 0.05); c: significantly different from the control and from the lower concentration in the same generation (p < 0.05); d: significantly different from the earlier generation at the same concentration (p < 0.05).
In the present study, SMX caused hormetic effects on the brood size (Fig. 2) and initial reproduction (Fig. 3). It was reported that hormesis on the reproduction of C. elegans usually trades off decreases in lifespan (Tyne et al., 2015), or vice versa (Saul et al., 2013). Such trade-off among endpoints were widely reported in various organisms 316
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Appendix A. Supplementary material
Table 1 Pearson cross-correlations (Pearson's r) between median lethal time (LT50), brood size, initial reproduction, reproduction duration and population growth rate in C. elegans exposed to sulfamethaxozale. Brood size
LT50
Brood size Initial reproduction Reproduction duration Population growth rate * **
Initial reproduction
Reproduction duration
– 0.1548
–
−0.1297
0.2204
−0.0224
–
0.5494**
0.7410**
0.7219**
0.0102
0.4478 0.2659
*
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p < 0.05. p < 0.01.
and also observed in trans-generational effects on C. elegans (Yu et al., 2016). However, the reproduction and lifespan did not show such trade-offs in the present study due to the lack of significant correlation (Table 1). Such absence might be attributed to the presence of food in the whole experiment which did not cause caloric restriction that was usually the prerequisite for trade-off effects (Adler et al., 2013). The population growth rate (PGR) was well correlated with LT50 and brood size, which indicated that the population would increase when the nematodes lived longer and reproduced more offspring. The PGR was also correlated with initial reproduction (Table 1). The results supported the natural observation that initial reproduction will determine the population survival against predation or adverse environmental conditions (Roff, 2001; Liu et al., 2013). In Figs. 4 and 5, initial reproduction and reproduction duration showed opposite changes over generations at 0.04, 40.0 and 400.0 µm. The results indicated an interplay between these two indicators in the overall reproduction. Such interplay was also implied in effects of tannic acid treatment which delayed the initial reproduction but did not change the overall reproduction (Saul et al., 2011). However, none of the initial reproduction, brood size and PGR showed any correlation with the reproduction duration (Table 1). Thus, effects of SMX on the population growth greatly relied on its effects on the initial reproduction, which could not be compensated with the responses in reproduction duration. Yet, the complex interactions among indicators still need further studies. 5. Conclusion Currently, the effects of sulfamethaxozale (SMX) on lifespan, reproduction and population growth in nematodes were studied for 7 consecutive generations after an exposure in F0 generation. Among all generations, the lifespan was severely affected following embryo exposure (F1) and reproduction was significantly affected following germline exposure (F2), indicating special roles of embryo and germline in lifespan and reproduction, respectively. In non-exposed F3–F6 generations, nematode lifespan and reproduction showed significant inhibitions, confirming the trans-generational effects. Moreover, the population growth was dependent on lifespan, brood size and initial reproduction, and there seemed an interplay between initial reproduction and reproduction duration. Further studies are needed to elucidate the mechanisms of the trans-generational effects. Acknowledgements The authors are grateful for the financial supports by the National Natural Science Foundation of China (Nos. 21307095, 21407061 and 51278353), the Collaborative Innovation Center for Regional Environmental Quality, and the Swedish Research Council (Contract Dnr. 639-2013-6913). 317
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