Pone.0003741 1.6

Rapid Experimental Evolution of Pesticide Resistance inC. elegans Entails No Costs and Affects the MatingSystem Patricia C. Lopes1,2, E´lio Sucena2, M. Emı´lia Santos2, Sara Magalha˜es2,3* 1 Programa Graduado em A´reas da Biologia Ba´sica e Aplicada (GABBA), Faculdade de Medicina da Universidade do Porto, Porto, Portugal, 2 Centro de Biologia do Desenvolvimento, Instituto Gulbenkian de Cieˆncia, Oeiras, Portugal, 3 Centro de Biologia Ambiental, Faculdade de Cieˆncias da Universidade de Lisboa, Campo Grande, Pesticide resistance is a major concern in natural populations and a model trait to study adaptation. Despite the importanceof this trait, the dynamics of its evolution and of its ecological consequences remain largely unstudied. To fill this gap, weperformed experimental evolution with replicated populations of Caenorhabditis elegans exposed to the pesticideLevamisole during 20 generations. Exposure to Levamisole resulted in decreased survival, fecundity and male frequency,which declined from 30% to zero. This was not due to differential susceptibility of males. Rather, the drug affected mobility,resulting in fewer encounters, probably leading to reduced outcrossing rates. Adaptation, i.e., increased survival andfecundity, occurred within 10 and 20 generations, respectively. Male frequency also increased by generation 20. Adaptationcosts were undetected in the ancestral environment and in presence of Ivermectin, another widely-used pesticide with anopposite physiological effect. Our results demonstrate that pesticide resistance can evolve at an extremely rapid pace.
Furthermore, we unravel the effects of behaviour on life-history traits and test the environmental dependence of adaptationcosts. This study establishes experimental evolution as a powerful tool to tackle pesticide resistance, and paves the way tofurther investigations manipulating environmental and/or genetic factors underlying adaptation to pesticides.
Citation: Lopes PC, Sucena E´, Santos ME, Magalha˜es S (2008) Rapid Experimental Evolution of Pesticide Resistance in C. elegans Entails No Costs and Affects theMating System. PLoS ONE 3(11): e3741. doi:10.1371/journal.pone.0003741 Editor: Robert Brooks, The University of New South Wales, Australia Received August 8, 2008; Accepted September 29, 2008; Published November 17, 2008 Copyright: ß 2008 Lopes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Marie Curie Reintegration grant (36589 - EVOL HET and ENV) and POCTI/BSE/48402/2002 grant from Fundac¸a˜o para a Cieˆncia e Tecnologia (Portugal) Competing Interests: The authors have declared that no competing interests exist.
of performance in other environments. Indeed, the presence of acost opens the possibility for managing resistance by creating areas Pesticides and antibiotics have been developed to induce high where the pesticide is not spread [14]. From a fundamental mortality rates on populations of parasites and pests. This imposes perspective, a cost of adaptation has often been evoked as the a strong selection pressure on these organisms, which may lead to mechanism underlying the evolution of specialization. Examples the evolution of resistance to such xenobiotics. Resistance has from the literature so far suggest that a cost of resistance is indeed indeed been observed in an impressive number of organisms common, but that its intensity is variable [15,16,17]. The presence [1,2,3]. Due to its ubiquity, pesticide resistance is also currently a of a cost of adapting to a pesticide, as well as its specific model trait for the study of adaptation to novel environments [4].
evolutionary dynamics will depend on the degree of resemblance Laboratory experiments with microorganisms and field studies among environments [18,19], on the genetic basis of adaptation with multicellular organisms have shown that resistance to [16], on the genetic background of the organism [20], and on the xenobiotics occurs within short time frames [5,6,7,8].
In addition to causing lethality, xenobiotics may also affect the Experimental evolution in replicate populations exposed to morphology, life history, or behaviour of organisms without killing pesticides can contribute to our understanding of the evolutionary them. For example, many pesticides reduce the fecundity and/or fate of lethal and sublethal effects caused by chemical stress, as well as longevity of organisms [9], whereas others cause paralysis, thereby to follow the building up of a cost of resistance. To date, few studies compromising the ability of organisms to find food or mates, or to have been carried out on the experimental evolution of pesticide escape from potential predators [10,11,12]. Despite being frequent- resistance in multicellular organisms using replicated evolving lines, ly overlooked, these sublethal effects can nonetheless affect the and none involve an androdioeceous organism. In this mating system, performance of organisms and significantly impact fitness [9,13].
males result from an outcrossing event between hermaphrodites and Hence, it is expected that natural selection will operate towards males, whereas hermaphrodites are also able to undergo selfing [24].
reducing these deleterious effects induced by pesticides.
One possible sublethal effect of the pesticide is to affect this mating A crucial aspect for both resistance management and our system, and the build-up of resistance may also interact with it.
understanding of the evolutionary consequences of adaptation is to In this study, we followed the experimental evolution of evaluate whether the evolution of resistance entails a cost in terms resistance of the androdioecious free-living nematode Caenorhabditis November 2008 | Volume 3 | Issue 11 | e3741 elegans to the widely used pesticide Levamisole. This nematicide targets the nicotinergic acetylcholine receptor, resulting indepolarisation of neuronal and muscle cells [25,26]. Apart from Pesticides significantly affected the survival and fecundity of all inducing severe mortality, Levamisole modifies several life-history populations (GLM, effect of environment, F2,16.045 = 44.05; and behavioural traits of C. elegans, including egg laying and P,0.0001 and F2,16 = 98.34; P,0.0001). The interaction between mobility [27,28]. Hence, we measured adaptation not only as the environment and the selection regime was significant for changes in life-history traits such as fecundity and survival, but also survival, but not for fecundity (F2,16 = 12.34; P = 0.0006 and as behavioural modifications. To investigate whether resistance F2,16 = 2.64, P = 0.1, respectively). Subsequent analyses were entailed a cost, we measured the performance of resistant performed on each environment separately.
populations in the ancestral environment. As detecting a cost of Populations evolving in Levamisole had higher survival and resistance may depend on the environment where this cost is fecundity in this environment than populations evolving in a measured [16], we also measured this cost in the presence of Control environment (Fig. 1a and 1d; Table 1, effect of selection another nematicide, Ivermectin. Ivermectin acts by being an regime). Thus, exposure to Levamisole resulted in adaptation to this agonist of glutamate-mediated chloride channels, resulting in the environment within 20 generations. However, adaptation was very hyper-polarization of the membrane of neuronal and muscle cells heterogeneous among populations (Table 1, effect of population).
[29]. Since Levamisole and Ivermectin operate on excitatory and Differences in fecundity between selection regimes were observed at inhibitory networks, respectively, a strong trade-off in adaptation generation 20 only, whereas differences in survival were established to the two nematicides is expected. Indeed, negative cross- at generation 10 and remained constant thereafter (Figure 1; resistance between these pesticides has been shown [30].
Table 1, interaction generation*selection regime).
Therefore, we used Ivermectin as an environment where the In the Control environment, survival and fecundity of probability of detecting a cost of adapting to Levamisole is individuals from LE populations was not significantly different from that of individuals from C populations (Fig. 1b and 1e; Figure 1. Adaptation and its potential costs. Life history traits of populations in three different environments: Levamisole (a) and (d), Control (b)and (e), and Ivermectin (c) and (f). Survival (a, b, c) was measured as the proportion of individuals surviving from egg to adulthood (after 3 days).
Fecundity (d, e, f) was assessed by counting the number of eggs per hermaphrodite after individual bleaching at day 4. Black bars: Controlpopulations; white bars: LE populations. Vertical bars correspond to the standard error of the mean of the five populations in each selection regime.
doi:10.1371/journal.pone.0003741.g001 November 2008 | Volume 3 | Issue 11 | e3741 Table 1. Statistical analysis of life-history traits.
G: Generation; SR: Selection Regime; SR(P): Population nested within Selection Regime; F: F value; d.f.: degrees of freedom; P: significance. Survival: number ofindividuals reaching adulthood; Fecundity: number of eggs carried by hermaphrodites at day 4. Non-significant interactions (P,0.1, ‘‘NS’’) were removed from themodel. P,0.05 are highlighted in bold.
doi:10.1371/journal.pone.0003741.t001 Table 1, effect of selection regime). Thus, adaptation to t.,In populations naı¨ve to the Levamisole environment (the C Levamisole entailed no cost in the ancestral environment. These populations), the number of encounters in Levamisole is traits did not differ between generations (Table 1, effect of significantly lower than in the Control environment (Fig. 2c; generation), but fecundity in some populations changed between F1,4 = 23.28; P = 0.017). This is not the case for the LE1 generations, resulting in a significant interaction between gener- population, for which these variables do not differ across ation and population (Table 1). In Ivermectin, the survival and environments (Fig. 2c; t18 = 0.33; P = 0.74). The rate of encounter fecundity of the LE populations did not differ significantly from of LE1 individuals in the Control environment is comparable to that of C populations (Fig. 1c and 1f; Table 1, effect of selection that of C individuals (Fig. 2c, t11 = 2.2; P = 0.58), and so is the male regime). Therefore, resistance to Levamisole was not accompanied frequency of that population (Fig. 2a,b). However, compared to C by a cost in an environment with Ivermectin. As in the Control individuals, individuals of the LE1 population encounter mates environment, a significant interaction between generation and more often in the Levamisole environment (Fig. 2c; t10 = 2.23; P = 0.006). Therefore, resistance to Levamisole translated also into Male frequency did not differ significantly between the a behavioural change of the individuals, which allowed for an Levamisole and the Control environment (Fig. 2; GLM, effect of the environment, F1,8 = 0.8, P = 0.39). In addition, no significantinteraction was found between the environment and the population, selection regime nor generation (GLM, P.0.3 for allinteractions). Therefore, the environment where individuals Experimental evolution of C. elegans populations in a Levami- developed did not significantly affect the male frequency observed.
sole-enriched environment resulted in adaptation to this environ- Male frequency differed significantly between LE and C ment within 20 generations. This adaptation to a novel populations when exposed to Levamisole (Fig. 2a; GLM, effect environment entailed no cost in the ancestral environment or in of the selection regime: F1,8 = 34.79, P,0.0001). Indeed, the male Ivermectin, another pesticide with an opposite physiological mode frequency of LE populations at generation 10 was near 0%, of action. Levamisole paralyzed the nematodes. This resulted in whereas that of C populations varied between 14 and 35%. By fewer encounter rates between males and hermaphrodites and led generation 20, male frequency increased in 3 of the 5 LE to the disappearance of males from the populations. A build-up of populations (Fig. 2a, GLM. interaction generation*population(se- resistance has re-established the mobility of the worms, and lection regime), F9,80 = 3.51, P = 0.0005).
concomitantly the male frequency increased.
To understand the disappearance of males after 10 generations Resistance in our outbred populations accumulated within very in Levamisole, we tested the effect of this drug on the survival of few generations. Therefore, adaptation was most likely due to the each sex separately. Significant differences in susceptibility were standing genetic variation of populations. The fact that pesticide found between sexes (F1,40 = 68.48; P = 0.001). However, males resistance is a trait that is relatively easy to select for under artificial were less sensitive to Levamisole than hermaphrodites. Indeed, on selection [31] is in agreement with the prediction that genes average 43.963.42% of the hermaphrodites of each population conferring pesticide resistance may be present in populations at survived to Levamisole, while this proportion was of 69.361.4% low frequencies. Even though data from natural populations and for males (on average 97.761.4% of the hermaphrodites and from artificial selection suggest that resistance can indeed rapidly 100% of the males survived in the control). Hence, differences in accumulate, this is the first study providing a direct demonstration susceptibility to the pesticide between sexes do not explain the of the speed of this process. The speed of adaptation varied with disappearance of males in the LE populations. Subsequently, we the trait measured. Indeed, survival increased within 10 generation tested if outcrossing was impaired in the Levamisole environmen- and had reached a plateau at 20 generations, whereas fecundity November 2008 | Volume 3 | Issue 11 | e3741 Figure 2. Evolution of the mating system. Male frequency (number of males/total number of individuals) of all populations in the environmentwith Levamisole (a) and in the Control environment (b), measured at day 3. (c): behavioural observations of the populations C1, C3, C5 and L1 in theControl or in the Levamisole environment during 20 minutes: encounter rates between males and hermaphrodites: Black bars: Control populations(C1–C5); white bars: LE populations (L1–L5). Vertical bars correspond to the standard error of the mean.
doi:10.1371/journal.pone.0003741.g002 increased mostly at generation 20. This difference suggests that environment where costs were tested. It is possible that the period these traits evolve independently, at least to a certain extent.
of experimental evolution was too short to create a measurable The rapid evolution of resistance to Levamisole was not cost of adaptation. However, the fact that adaptation was detected accompanied by a cost in the ancestral environment. This result during the experimental period, and that it was not accompanied differs from most studies of pesticide resistance, where a cost was by a cost indicates that adaptation to each environment is, at least detected [16,32,33,but see 34]. This discrepancy may be due to to a certain extent, determined by independent loci [35,36].
the fact that we used a selection pressure that allowed the survival Exposure to Levamisole resulted in fewer encounters between of 25% of the initial population, whereas most studies deal with males and hermaphrodites. Since males are produced mainly as natural populations, where pesticide doses aim at eradicating all the result of an outcrossing event, which involves an encounter individuals of a pest population. In those cases, probably only the between a male and a hermaphrodite, males in populations most effective mutation conferring resistance is selected. Indeed, exposed to the pesticide disappeared within 10 generations. This most resistance mutations described are a one-base-pair change result supports the hypothesis that encounter rates are an that modifies the binding site of the pesticide in the corresponding important factor in determining male frequency in C. elegans neuroreceptor, which is likely to be costly, as other molecules also populations [37,38], and may underlie the frequencies in the base bind to that site [2]. As the size of populations surviving pesticide population. However, in other studies of experimental evolution use increases, several gene combinations may build up and be in the laboratory, where encounter rates were probably similar as selected, hence reducing the probability of a costly resistance.
those of our base population, male frequencies were extremely Detecting a cost of resistance may depend on the environment low [39,40]. Hence, additional factors need to be invoked to where such cost is measured [18,19]. With the aim of maximizing explain the male frequency observed in the base population used the possibility of detecting a cost, we selected an environment in this study. Had we used a non-selfing species, Levamisole expected to have an opposite physiological effect on the worms to would probably have impaired nearly all mating events, leading that imposed by Levamisole. As Levamisole and Ivermectin to severe reduction in population growth. This suggests that operate on excitatory and inhibitory circuits respectively, resis- sublethal pesticide effects can have dramatic consequences on tance to one of these drugs may well increase the susceptibility to populations. As C. elegans is capable of both selfing and the other, entailing a cost of adaptation. However, even in such an outcrossing, the action of the pesticide resulted in a remarkable environment, resistance to Levamisole did not entail any cost.
reduction in outcrossing rates, but populations were maintained Therefore, the lack of cost is probably not contingent on the November 2008 | Volume 3 | Issue 11 | e3741 If the speed at which males disappeared from the populations Levamisole (hereafter the Levamisole environment), while the exposed to Levamisole was striking, the same is true for the pace at control (drug-free) environment and the environment containing which male frequency increased in the populations that became Ivermectin 0.04 mM served to measure potential costs of resistant to the drug. The latter suggests that outcrossing is indeed adaptation. Prior to testing performance, all populations (C1–C5 advantageous in these populations; otherwise male frequency and LE1–LE5) spent three generations in a drug-free environ- would be expected to remain near 0% [40]. 30% of males is a ment, to ensure that the responses observed were due to genetic frequency that corresponds to the locally-stable equilibrium differences among populations. Subsequently, 100 eggs from each predicted by Stewart and Phillips (2002) [40]. This male frequency population were placed onto fresh petri dishes of each environ- may be expected because outcrossing produces two to four times ment (N = 5 plates/environment) and incubated for 3 days at 20uC the offspring obtained through selfing [41]. Therefore, the increase and 80% RH. When individuals reached adulthood (4th day of in male frequency observed, as a result of restored mobility, may culture), 30 gravid hermaphrodites from each plate were collected be seen as yet another expression of the evolution of pesticide and individually submitted to a hypochloride/sodium hydroxide solution. The surviving eggs were counted, yielding the fecundity Pesticide resistance has been used as a ‘model trait’ to study measure. This method mimics the conditions used in the adaptation to novel environments for the past 20 years [4,31]. Our experimental evolution setup, but at an individual level. The study underscores the potential use of this model trait in plates with the remaining individuals were placed at 4uC for two experimental evolution. By using pesticide resistance in a days to immobilize the individuals to be counted. Survival was controlled setting, we were able to shed light on the reciprocal obtained by counting the number of individuals per plate interactions between behavior and evolution, as well as to test the (accounting for the 30 removed to measure fecundity) and dividing multidimensionality of adaptation costs. However, the potential- it by the initial number of eggs plated (100). Male frequency was ities of this system are not restricted to the results obtained in the estimated as the ratio between the number of males and the total current study. Using experimental evolution to tackle pesticide resistance allows for the manipulation of a variety of environmen- Next, we aimed at understanding the male frequencies observed tal and genetic factors. Indeed, manipulating selection intensity, (cf. Results). We first tested whether males were more susceptible environmental stability, population size and genetic background, to Levamisole than hermaphrodites. 20 adult males and 20 provide direct tests of the effects such factors may have on the hermaphrodites from each C population at generation 20 were placed separately in Levamisole and in Control plates (5 plates perpopulation per environment). After one day, the number of individuals surviving was counted. Subsequently, we measured theencounter rate between males and hermaphrodites. Four her- The base population of Caenorhabditis elegans used in this maphrodites from one population were placed on a small drop of study was composed of a mixture of the strains used in Teoto´nio et bacteria (10 mL) that had grown overnight in a 5-cm diameter al. 2006 [42]. It was kept in the experimental conditions described plate containing either 0.15 mM Levamisole or no drugs.
in Manoel et al. 2007 [43], for over 80 generations prior to our Subsequently, a male was introduced and this group was observed study. Levamisole (Levamisol hydrochloride, C11H12N2S ? HCl), for 20 minutes. We registered the number of male-hermaphrodite an imidazothiazole and Ivermectin (22,23-Dihydroavermectin B1), encounters. This was done ten times for C1, C3, C5 and LE1 at a macrocyclic lactone, were purchased from Sigma-Aldrich.
From the initial population, we derived 10 experimental lines: Differences in survival and fecundity were first analyzed with five maintained in standard conditions [C1–C5] and five kept in General Lineal Models using the GLM procedure in SAS. The plates containing the nematicide Levamisole (LE1–LE5). The factors of the model were ‘‘environment’’ (levamisole, ivermectin populations were cultured for 20 generations at 20uC and 80% or control), ‘‘generation’’ (10 or 20), ‘‘selection regime’’ (LE or C RH and frozen at generation 10 (G10) and 20 (G20) for later use lines), a factor ‘‘selection line’’ (C1–C5 and LE1–LE5) nested to in the assays. Our standard experimental evolution protocol the factor ‘‘selection regime’’, and the interactions ‘‘environment*- followed that of Manoel et al. 2007 [43]. Each generation lasted 4 selection regime’’, ‘‘generation’’ * ‘‘selection regime’’, ‘‘environ- days. At day 1, 1000 individuals at the first larval stage (L1) were ment*selection line’’ and ‘‘generation’’ * ‘‘selection line’’. The placed onto Petri dishes (9 cm diameter) containing Nematode factor ‘‘selection line’’ and its interactions with other factors were Growth Media-light agar (NGM) (US Biological) with a lawn of considered random factors. The interaction terms with P-values HT115 Escherichia coli as food source, then incubated for 3 days. At larger than 0.1), were sequentially dropped from the analysis and day 4, individuals were washed off the plates and exposed to a included in the error term [44]. Subsequently, we performed hypochloride/sodium hydroxide solution, which kills all life stages statistical tests within each environment to answer specific except the eggs inside the hermaphrodites. These eggs were questions. Adaptation was tested by comparing survival and subsequently kept in a M9 buffer solution in 15mL falcon tubes in fecundity of LE and C populations in the Levamisole environ- an incubator at 20uC and 120 rpm overnight. The next day, the ment. The analysis and the factors used were the same as before, number of larvae on each tube was estimated with five sample except for the factor environment and its interactions with the drops of 5 mL from each tube and the volume corresponding to other factors. A cost of adaptation was tested with the same model, 1000 of individuals was placed in fresh Petri dishes. Each but with the data collected in the other two environments.
population was composed of 10 Petri dishes, hence N = 10 000, Differences in male frequencies were tested with a GLM individuals per population. The NGM-light agar in which LE procedure in SAS, with the same model as for fecundity and populations were kept contained Levamisole 0.15 mM. This survival, but excluding the Ivermectin environment. Comparisons concentration was lethal for 75% of the individuals in the base between the control and the levamisole environment aimed at population, but had no effect on bacterial growth (T-test, N = 10 testing whether an immediate physiological effect of the environ- petri dishes per environment, t = 1.26, P = 0.23).
ment could affect the male frequencies observed; comparisons Adaptation was assessed by comparing the performance of LE among selection regimes tested the effect of the pesticide on male populations to those of C populations in petri dishes containing frequency, while comparisons between generations of the November 2008 | Volume 3 | Issue 11 | e3741 levamisole lines tested recovery due to the evolution of resistance.
population varied between environments, we performed a T-test To test differences in survival between males and hermaphrodites, only C populations were used. The sex of the individuals wasintroduced as a fixed factor and population as a random factor. To test the effect of the Levamisole environment on the ability to finda mate, we compared the number of encounters of individuals We thank Rute Viera for statistical advice, Henrique Teoto´nio for the C.
from C populations in the Levamisole versus the Control elegans base population, Marta Moita and Isabel Gordo for inspiringdiscussions and Margarida Matos, Pierrick Labbe´ and Lilia Perfeito for environment. Environment was introduced as a fixed factor and population as a random factor. As there were no significantdifferences among populations, these were grouped in the subsequent analysis. To test whether resistant individuals hadrecovered their ability to find a mate, we used individuals from the Conceived and designed the experiments: ES SM. Performed the most resistant population at generation 20, LE1, and compared experiments: PCL ES ES SM. Analyzed the data: PCL SM. Wrote the their behavior to that of individuals from the C selection regime.
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