Parasite host range and the evolution of host resistance.

doi: 10.1111/jeb.12639

Parasite host range and the evolution of host resistance F. A. GORTER*†, A. R. HALL*1, A. BUCKLING*2 & P. D. SCANLAN*3 *Department of Zoology, University of Oxford, Oxford, UK †Laboratory of Genetics, Department of Plant Sciences, Wageningen University, Wageningen, The Netherlands

Keywords:

Abstract

bacteria; bacteriophage; coevolution; experimental evolution; host range; host–parasite interactions.

Parasite host range plays a pivotal role in the evolution and ecology of hosts and the emergence of infectious disease. Although the factors that promote host range and the epidemiological consequences of variation in host range are relatively well characterized, the effect of parasite host range on host resistance evolution is less well understood. In this study, we tested the impact of parasite host range on host resistance evolution. To do so, we used the host bacterium Pseudomonas fluorescens SBW25 and a diverse suite of coevolved viral parasites (lytic bacteriophage Φ2) with variable host ranges (defined here as the number of host genotypes that can be infected) as our experimental model organisms. Our results show that resistance evolution to coevolved phages occurred at a much lower rate than to ancestral phage (approximately 50% vs. 100%), but the host range of coevolved phages did not influence the likelihood of resistance evolution. We also show that the host range of both single parasites and populations of parasites does not affect the breadth of the resulting resistance range in a na€ıve host but that hosts that evolve resistance to single parasites are more likely to resist other (genetically) more closely related parasites as a correlated response. These findings have important implications for our understanding of resistance evolution in natural populations of bacteria and viruses and other host–parasite combinations with similar underlying infection genetics, as well as the development of phage therapy.

Introduction Parasites are ubiquitous components of the natural world and play a fundamental role in both the ecology of communities and the evolution of the hosts they infect (Bohannan & Lenski, 2000; Decaestecker et al., 2005; Friman et al., 2009). The number of different host genotypes that a parasite can infect is a key determinant of coevolutionary interactions. Here, we refer to this property as host range. Parasites can be classified as either specialists, which infect only a narrow range of Correspondence: Pauline D. Scanlan, Department of Zoology, University of Oxford, Oxford OX1 3PS, UK. Tel.: +353 (25) 42300; fax: +353 (25) 42449; e-mails: pauline. [email protected]; [email protected] 1 Present address: Institute of Integrative Biology, ETH Z€ urich, 8092 Z€ urich, Switzerland 2 Present address: ESI, Biosciences, University of Exeter, Penryn Campus, Cornwall TR10 9FE, UK 3 Present address: Teagasc Food Research Centre, Moorepark, Fermoy, Co Cork, Ireland

closely related genotypes and/or species, or generalists, which infect a more diverse suite of hosts. Although in some fields, the term parasite host range is exclusively used to refer to the set of species a parasite can infect, in others, notably host–parasite coevolution, the term is also commonly used in relation to within-species or within-strain interactions (Parker, 1994; Sasaki, 2000; Holmfeldt et al., 2007; Best et al., 2010; Flores et al., 2011; Scanlan et al., 2013). Given that parasite host range has significant implications for economic and public health sectors including agriculture and medicine, the factors that promote host range are relatively well characterized, as are the epidemiological consequences of parasite host range (Woolhouse & Gowtage-Sequeria, 2005; Woolhouse et al., 2005; Parrish et al., 2008). Conversely, the evolutionary implications of variation in host range for host evolution are less clear. More specifically, the relative probability of resistance evolution of a na€ıve host (i.e. one that has not previously encountered the parasite) to specialist vs. generalist parasites is not well understood,

ª 2015 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY. J. EVOL. BIOL. 28 (2015) 1119–1130 JOURNAL OF EVOLUTIONARY BIOLOGY ª 2015 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY

1119

1120

F. A. GORTER ET AL.

nor is the effect of parasite host range on the resulting evolved host resistance range. However, the effect of parasite host range in driving host resistance evolution may have important epidemiological consequences: knowing whether it is harder to evolve resistance to generalist or specialist parasites could facilitate our understanding of the threats posed by pathogens with different host ranges and inform management strategies in both natural ecosystems, as well as clinical and agricultural settings. Here, we investigate the effects of variation in parasite host range on the likelihood of host resistance evolution in a na€ıve host and the breadth of resulting host resistance ranges. To this end, we used a collection of coevolved phages that varied greatly in their host range and were derived from an in vitro coevolutionary model system that exploits the interaction between the host bacterium Pseudomonas fluorescens and its viral parasite bacteriophage SBW25 Φ2 (Buckling & Rainey, 2002; Brockhurst et al., 2007; Scanlan et al., 2011). In this experimental system, both host and parasite rapidly coevolve as they alternately acquire and fix new beneficial alleles over the course of the coevolutionary process. This results in both host resistance range and parasite infectivity range expansion, that is both host and parasite evolve, respectively, to resist and infect an increasing number of genotypes. However, increases in range eventually decelerate due to pleiotropic costs in the form of reduced competitive fitness (Poullain et al., 2008; Hall et al., 2011b). This type of dynamic is broadly consistent with multilocus gene-for-gene (GFG) models of coevolution (Flor, 1956; Sasaki, 2000) and related phenotypic models (Best et al., 2010), under which fixation of universal generalists is only prevented by the pleiotropic costs associated with range expansion. The nested patterns typical of many bacteria– phage infectivity matrices (where generalist hosts can only be infected by generalist parasites) are also consistent with these models (Flores et al., 2011). Consequently, the GFG dynamics observed in the SBW-Φ2 model system are relevant not only to bacteria–phage infection studies but also to a much wider range of host–parasite interactions that display similar coevolutionary dynamics (such as some plant–pathogen interactions (Flor, 1956)). From a conceptual perspective and to facilitate our experimental design and hypothesis testing, we formulated a number of predictions based on the specific infection genetics and costs associated with host–parasite coevolution that are typically observed in this experimental system (Poullain et al., 2008; Hall et al., 2011a,b; Scanlan et al., 2011). On the one hand, we expected that it might be more difficult for a na€ıve bacterium to evolve resistance to highly coevolved generalist phages as these phages have acquired multiple infectivity substitutions that are associated with host range over the course of coevolution (Paterson et al.,

2010; Scanlan et al., 2011), and the effects of all of these need to be countered by the bacterium simultaneously. On the other hand, the pleiotropic costs that are associated with host range expansion for this parasite (Poullain et al., 2008) may in fact make it easier for the host to evolve resistance to ‘attenuated’ generalist phage than to uncompromised specialists, a principle that is commonly employed for vaccine development (Ebert, 1998; Duffy et al., 2006). As populations of viruses are typically not homogeneous (Boerlijst et al., 1996) and consist of phages with variable host ranges (Poullain et al., 2008), we also predicted that evolution of resistance to entire populations would be more difficult than evolving resistance to single phage genotypes. If so, this result would have important implications for the development of phage therapy (Loc-Carrillo & Abedon, 2011; Pirnay et al., 2011). Finally, we anticipated that resistance evolved in response to parasites with varying host ranges would entail several correlated responses. First, we predicted that hosts that evolve resistance to generalist parasites would have a broader resistance range than those that evolve resistance to specialists, as they should be able to resist the phage they evolved resistance to, as well as any other phages containing a subset of this parasite’s mutations. Second, such rapidly evolved resistant hosts may then have resistance ranges comparable to hosts that have coevolved with the same parasite lineage for many generations. Third, resistant hosts would be more likely to be resistant to parasites that are more genetically related to the parasite they evolved resistance to, than to parasites that are comparatively distantly related. Based on these predictions, we tested the following five hypotheses: (i) resistance is more likely to evolve to specialist than to generalist parasites, (ii) resistance is more likely to evolve to single parasites than to populations of parasites, (iii) hosts that evolve to resist generalist parasites can resist a broader set of other parasites than hosts that evolve to resist specialist parasites, (iv) hosts that evolve to resist a given parasite in a single step will have a similar resistance range as hosts that have become resistant to the same parasite following long-term coevolution, and (v) hosts that evolve to resist a given parasite are more likely to also resist genetically similar parasites than genetically dissimilar parasites. In our first experiment, we challenged the ancestral bacterium P. fluorescens SBW25 (na€ıve host) to evolve resistance to single phage genotypes (hereafter termed focal phage) that were isolated from different time points and replicate populations of a previously performed long-term coevolution experiment (Hall et al., 2011b). The resistance ranges of the resultant bacterial host mutants were then determined by screening them for resistance against a suite of both single phage genotypes and populations of phage. In our second and


Parasite host range and resistance evolution

similarly designed experiment, we challenged our ancestral bacterium (na€ıve host) to evolve resistance to mixed populations of phage (focal phage populations) that were isolated from the same coevolution experiment. Again, the resulting bacterial mutants were tested for resistance against the same suite of single phage genotypes and populations of phage. Finally, we tested whether evolution and long-term coevolution have the same effect on bacterial resistance range. To this end, we compared the resistance ranges of our evolved hosts to the resistance ranges of a large number of coevolved hosts isolated from the same coevolution experiment as the focal phage (Hall et al., 2011b). A graphical overview of our full experimental design and set-up is given in Fig. 1.

Materials and methods Phage genotypes The 50 phage genotypes (focal phages) and the 18 phage populations (focal phage populations) were

1121

isolated at three different time points from six independent populations of a previously performed long-term coevolution experiment (~400 bacterial generations) (Hall et al., 2011b), see Fig. 1a and also Tables 1 and 2. In brief, six independent populations (A-F) of Pseudomonas fluorescens SBW25 and bacteriophage SBW25Ф2 were established in static microcosms containing King’s medium B (KB) (Fig. 1a.1). Microcosms were maintained by batch culture serial transfer at 28 °C with 1% of the population transferred every 48 h for a total of 60 transfers. At different transfer time points (T10, T30 and T60), both whole populations of phages and sets of ten single phage genotypes were isolated (Fig. 1a.2). Each of the single phage genotypes was then characterized in terms of phenotype (host range calculated from the number of successful infections against a suite of bacterial genotypes isolated from each of the six replicate coevolving populations, at different time points, n = 60) and genotype (number of different amino acid substitutions at the tail fibre gene relative to each other and the ancestral sequence (Scanlan et al., 2011; Fig. 1a.3).

(a) Generating and isolating coevolved phages with variable host ranges for selection experiments and comparative analysis 1. Set up coevolution experiment. Transfer phage and bacteria every 48 h for 60 transfers (T)

3. Test each individual coevolved phage and phage population against a phenotypically diverse range of 60 coevolved hosts that were isolated from each of the six populations (A-F) and at each of the three time-points (T10, T30 and T60) to determine parasite host range. Sequence tail-fibre gene of each individual phage.

X 60 transfers

Host: Pseudomonas fluorescens SBW25 Parasite: phage SBW25Φ2

2. Isolate whole populations of phage (n = 18) and then 10 individual phage genotypes (n = 179*) at T10, T30 and T60 for each population.* see text.

A

A

Six coevolving populations (A to F) F

T0

T10

T30

T60

F

X T10, T30 and T60

4. Based on genetic and phenotypic analysis pick 50 individual phage genotypes each with a unique host range (focal phage) and also 18 populations of phage (focal phage populations) from T10, T30 and T60 of coevolution experiment for selection experiment

(b) Selection experiments (protocol is the same for both individual phage genotypes and populations of phage) Host: Ancestral Pseudomonas fluorescens SBW25 Parasite: focal phage and focal phage populations (derived from phage SBW25Φ2)

1. Co-inoculate each focal phage or focal phage population individually with ancestral host into a 96-well plate and incubate for 24 h (n = 50 + 18)

2. Plate out bacteria from each selection experiment and isolate individual colonies

3. Test 20 host colonies from each selection experiment (n = 50 + 18) for resistance to the focal phage or focal population they were co-inoculated with in the selection experiment

+

Focal phage: n = 50 Focal phage populations: n = 18

X

4. Isolate one resistant host from each selection experiment where resistance evolved (n = 27 and n = 8 for focal phage and focal phage populations, respectively), and test against panel of test phage (n = 179) and populations of test phage (n = 18) from earlier coevolution experiment (A.2) to determine resistance range of evolved host. Compare with resistance of coevolved hosts (n = 60) that were also isolated and characterised in earlier coevolution experiment (see A.1. and A.3.).

Fig. 1 Graphical overview of experimental design and set-up. ª 2015 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY. J. EVOL. BIOL. 28 (2015) 1119–1130 JOURNAL OF EVOLUTIONARY BIOLOGY ª 2015 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY

X

1122

F. A. GORTER ET AL.

Table 1 Information on focal phage genotypes*.

Genotype

Population

Time

Phenotypic distance

Resistance

A1 A2 A3 A4 A5 B1 B2 B3 B4 B5 B6 B7 B8 B9 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 D1 D2 D3 D4 D5 D6 D7 D8 E1 E2 E3 E4 E5 E6 E7 E8 E9 F1 F2 F3 F4 F5 F6 F7 F8 F9

A A A A A B B B B B B B B B C C C C C C C C C C D D D D D D D D E E E E E E E E E F F F F F F F F F

10 10 10 30 60 10 10 10 30 30 30 60 60 60 10 10 10 10 10 10 30 30 60 60 10 10 10 10 30 30 30 60 10 10 10 30 30 30 60 60 60 10 10 10 30 30 30 60 60 60

13 18 10 16 14 11 13 13 16 30 58 34 11 58 50 50 46 48 46 45 27 28 27 27 3 3 3 3 42 40 41 48 3 8 8 15 15 14 12 12 13 25 25 9 53 51 52 50 51 4

0 0 1 1 1 0 0 0 0 0 0 1 1 1 1 0 0 0 1 1 1 1 1 1 0 0 0 0 1 1 1 1 1 1 1 0 1 1 1 1 1 0 0 0 0 0 0 0 1 1

Resistance range vs. single phage

Resistance range vs. populations of phage

0.50 0.50 0.75

0.56 0.56 0.39

0.60 0.63 0.52 0.60

0.22 0.22 0.17 0.34

0.60 0.55 0.53 0.55 0.46 0.58

0.45 0.39 0.28 0.28 0.28 0.33

0.49 0.66 0.69 0.58 0.64 0.62 0.63

0.16 0.22 0.22 0.39 0.39 0.39 0.39

0.57 0.56 0.48 0.88 0.42

0.39 0.39 0.22 0.61 0.61

0.22 0.20

0 0

*Columns from left to right display: (Genotype) single phage genotypes that were used in the selection experiment, (Population) coevolving populations from which phage genotypes were isolated, (Time) number of transfers after which phage genotypes were isolated, (Phenotypic distance) number (out of 60) of bacteria that phage genotypes could infect, (Resistance) outcome of selection experiment with ancestral bacteria (0 = no evolution of resistance, 1 = evolution of resistance), (Resistance range vs. single phage) evolved resistance range of bacterial resistance mutant (proportion data based on resistance against 179 of single phage genotypes), (Resistance range vs. populations of phage) evolved resistance range of bacterial resistance mutant against populations of phage (proportion data based on resistance against 18 of populations of phage).


1123


Table 2 Information on populations of phage**.

Genotype

Population

Time

Phenotypic distance

Resistance

A.T10 A.T30 A.T60 B.T10 B.T30 B.T60 C.T10 C.T30 C.T60 D.T10 D.T30 D.T60 E.T10 E.T30 E.T60 F.T10 F.T30 F.T60

A A A B B B C C C D D D E E E F F F

10 30 60 10 30 60 10 30 60 10 30 60 10 30 60 10 30 60

14 18 7 13 34 37 49 28 29 3 41 48 7 15 12 30 43 41

0 0 1 0 0 0 0 1 1 0 1 1 1 0 1 0 0 1


Resistance range vs. populations of phage

0.48

2

0.58 0.57

5 6

0.50 0.75 0.62

2 5 10

0.47

5

0.69

4

**Columns from left to right display: (Genotype) populations of phage that were used in the selection experiment, (Population) replicate coevolving populations from which populations of phage were isolated, (Time) number of transfers after which phage populations were isolated, (Phenotypic distance) average number (out of 60) of bacteria that the single phage genotypes within each population of phage could infect, (Resistance) outcome of selection experiment with ancestral bacteria (0 = no evolution of resistance, 1 = evolution of resistance), (Resistance range vs. single phage) evolved resistance range of bacterial resistance mutant (proportion data based on resistance against 179 of single phage genotypes), (Resistance range vs. populations of phage) evolved resistance range of bacterial resistance mutant against populations of phage (proportion data based on resistance against 18 of populations of phage).

Fifty unique phage genotypes that varied in both phenotypic and genotypic distance from each other and the ancestral genotype were selected for our experiment (Fig. 1a.4). These genotypes were representative of the diversity naturally present in different populations and at different stages of the coevolutionary process, see Table 1. Eighteen phage populations (focal phage populations) were isolated from the independent populations (a–f, n = 6) at three different time points (T10, T30 and T60, n = 3), see Table 2, and also included in our selection experiment (Fig. 1a.4). Selection experiments To test for the evolution of resistance in our host and select for bacterial resistance mutants, the ancestral bacterium SBW25 was independently challenged with either a single focal phage or with one of the focal phage populations (Fig. 1b). Microtitre plate wells containing 200 lL of liquid KB were inoculated with ~3.3 9 105 cells of wild-type (ancestral) P. fluorescens SBW25 and ~3.3 9 103 phage particles. Plates were

incubated at 28 °C (static) for 24 h and shaken for 5-s every 2 h (to minimize spatial refuges) (Fig. 1b.1). We also conducted a control experiment that was set up and run under identical conditions: 20 wells (i.e. 20 replicates) of a microtitre plate containing 200 lL of liquid KB were inoculated with ~3.3 9 105 cells of wild-type P. fluorescens SBW25 and ~3.3 9 103 wildtype phage particles. At the end of the selection experiments (24 h of incubation), twenty bacterial colonies from each individual selection experiment were isolated (Fig. 1b.2), tested for resistance to the same phage stock that had been used to select for bacterial resistance and scored for inhibition of growth after 24 h (Buckling & Rainey, 2002) (Fig. 1b.3). Resistance was further verified by spot plate assays conducted in triplicate using 5 lL of the associated phage stock. If no plaques were observed after 24-h incubation of the plates, the resistance phenotype was confirmed and the host bacterium was considered resistant to the focal phage or the focal phage population that had been used in the selection experiment.


1124

F. A. GORTER ET AL.

Measuring resistance range Next, we tested the effect of the host range of a parasite on the resistance range of the hosts that evolved resistance to it. That is, if a bacterium evolved to resist a particular (specialist or generalist) phage, how many other phages could this bacterium resist as a correlated response? To address this, we randomly picked a single resistant bacterium from each (focal phage or focal phage population) selection experiment in which resistance evolved (n = 27 for the focal phage experiments; n = 8 for the focal phage population experiments) (Fig. 1b.4). Each of these resistant bacteria, as well as the ancestral host bacterium SBW25, was separately grown up overnight in liquid culture and used to make soft agar plates, that is plates covered with a lawn of growing bacteria. Different phage stocks were then applied to these lawns in order to determine whether each bacterium was resistant to these stocks, that is whether the stocks were unable to produce visible plaques on that particular bacterial lawn. We first determined the resistance range of each bacterium against whole populations of phage, where 5 lL of viral stock from different populations of phage from the coevolution experiment (n = 18; populations A–F, T10, T30 and T60) was spotted onto each bacterial lawn in triplicate. The resistance range of each bacterium against single phage genotypes was then assayed against a set of single genotypes from the coevolution experiment (n = 179; 10 phage isolates from each population A-F, T10, T30 and T60, 9 phage isolates from population C, T60) in triplicate using a pin replicator to apply phage to bacterial lawns. Bacteria were scored as resistant if no plaques were observed in any of the three replicates and as sensitive if plaques were observed in at least one replicate. Resistance range was calculated as the proportion of phage populations to which a bacterium was resistant, or the proportion of single phage populations to which a bacterium was resistant (Fig. 1b.4). Data analysis

Evolution of resistance Resistance data from experiments with focal phage genotypes and focal phage populations were analysed separately. To determine the effect of phage host range on whether resistance evolved during our selection experiment (yes/no), we used generalized linear models with resistance evolution as the binary response variable, phage host range and time point from which phages were isolated (T10, T30 or T60) as fixed effects and population (A–F in the coevolution experiment) as a random effect. We assumed a binomial error structure and used a logit link function. For both analyses, we started with full models including the interaction between the fixed effects and compared different models using likelihood ratio tests to find the minimal model.

Next, we tested whether the same explanatory variables (host range, time, population) affected evolved resistance range using general linear models, where resistance range is the arcsine-transformed proportion of populations to which the evolved bacterium was resistant (assessed against either 18 populations of phage or 179 single phage genotypes). To analyse the resistance ranges evolved in response to focal phage genotypes, we used the model selection strategy described above. To analyse resistance ranges evolved in response to focal phage populations, host range and time spent coevolving were each tested independently in combination with population because of the limited number of observations/degrees of freedom. To compare resistance evolved against single phage genotypes and whole populations, we first tested whether resistance was more likely to evolve against one or the other using a v2-test. We then tested whether the resistance range of mutants that evolved resistance to focal phage genotypes differed on average from that of mutants that evolved resistance to focal phage populations. We did this by calculating the average resistance range of bacteria that evolved resistance to focal phage genotypes at each time point and for each population, before fitting a linear model with arcsine-transformed resistance range as the response variable, whether resistance was evolved against focal phage or focal phage populations and time as fixed effects, and population as a random effect.

Resistance range of evolved vs. coevolved hosts We used the previously determined resistance range (assayed against the same suite of single phage as above, see Table S1) of 36 bacteria isolated from the same coevolution experiment as the focal phages used in this study to compare evolved and coevolved resistance ranges. We then used general linear models to determine the effect of treatment (evolved or coevolved), time point of isolation (T10, T30 and T60) and the interaction between these variables on arcsinetransformed resistance range, including population as a random effect to account for nonindependence among bacteria from the same population at different time points. We also accounted for heteroscedasticity by modelling different variances for each treatment 9 time combination (Pinheiro & Bates, 2000). For the evolved treatment, we combined information from bacteria that evolved against focal phage genotypes and bacteria that evolved against focal phage populations, as these bacteria had very similar resistance ranges at each time point.

Genetic distance analysis The tail fibre sequence of each of the single phage genotypes (Scanlan et al., 2011) was used to calculate the genetic distance between the focal phage and the phage genotypes that were used in the resistance assay



(test phage). Genetic distance was calculated as the pairwise difference in amino acid sequence of the tail fibre gene. We then tested whether genetic distance could predict whether a resistance mutant was resistant to the test phage, where we anticipated that smaller genetic distances (and thus higher relatedness of focal and test phage) would increase the likelihood of resistance. We implemented this as a generalized linear model with resistance score against each test phage as the binary response variable, genetic distance and time point of isolation of the test phage as fixed effects and both focal phage and test phage as random effects. Again, we assumed a binomial error structure and used a logit link function. All statistical analyses were performed in R version 2.15.2 (R company for Statistical Computing, Vienna, Austria).

Results Evolution of resistance against single phage genotypes

the ability of ancestral bacteria to evolve resistance, bacteria were more likely to evolve resistance against phages from the final time point (T60) than to phages from T10 and T30 (host range: v2 = 0.63, d.f. = 1, P = 0.43; time: v2 = 20.23, d.f. = 2, P < 0.0001; pairwise differences assessed with a Tukey post hoc test), see Fig. 2a,b. However, there was a strong relationship between host range and time point of isolation (oneway ANOVA with host range as response variable, F2,47 = 5.43, P = 0.008). To investigate the effect of parasite host range on evolved host resistance range, we tested those bacteria that evolved resistance to focal phage for resistance against both a suite of single phage genotypes and phage populations. Although there were no main effects of focal phage host range or time point of isolation on evolved resistance ranges against single phage genotypes (see Fig. 2c) (host range: F1,16 = 1.13, P = 0.30; time: F2,16 = 0.46, P = 0.64), resistance ranges did depend on the interaction between these variables (host range 9 time: F2,16 = 7.74, P = 0.004):bacteria that evolved resistance to phages from T60 with a broader host range had a narrower resistance range. Note that phages from one of the six replicate coevolving populations (population F) consistently resulted in evolved bacteria with much narrower resistance ranges than all others, which largely drove this relationship. The same results were obtained for evolved resistance

Frequency of resistance evolution

Our host bacterium, ancestral SBW25, evolved resistance against 27 of the 50 focal phage genotypes used in our selection experiment. By contrast, resistance against the ancestral phage evolved in all 20 replicates within 24 h. Although phage host range did not predict

No

Yes


0

5 10 15 20 Focal phage host range

(c) 160

120

80

40 0


1.00

(b)

0.75

0.50

0.25

0.00

25

25

10 30 60 Focal phage time point Resistance range vs. phage populations

Resistance evolution

(a)

Fig. 2 Evolution of resistance against focal phage genotypes. (a) Effect of focal phage host range on whether resistance evolved (yes/no) in our experiments. (b) Effect of coevolutionary time point (T10, T30 and T60) at which focal phage was isolated on whether resistance evolved (yes/no), expressed as the frequency of selection experiments in which at least one bacterial isolate evolved resistance per time point (error bars indicate binomial 95% confidence intervals). (c) Effect of focal phage host range and coevolutionary time point (T10: open circles, T30: squares, T60: crosses) on evolved resistance range, as assayed against a suite of single phage genotypes. (d) As in C), assayed against populations of phage.

1125

(d) 10

5

0


0


25

1126

F. A. GORTER ET AL.

populations used in the selection experiment, see Fig. 3a. Again, parasite host range did not affect resistance evolution, whereas time did (host range: v2 = 0.14, d.f. = 1, P = 0.71; time: v2 = 6.33, d.f. = 2, P = 0.04). Of the eight populations to which bacteria evolved resistance, one population was from T10, two were from T30, and five were from T60, see Fig. 3b. A Tukey post hoc test indicated that none of the time points was significantly different from any of the others, but this is likely due to the small number of observations. Contrary to results from single genotypes, we found no significant relationship between parasite host range and time (one-way ANOVA with host range as response variable, F2,15 = 1.48, P = 0.26). Although resistance mutants varied in their resistance range to both single phage genotypes and phage populations, see Fig. 3c,d, we did not detect any relationship between evolved resistance range and either parasite host range or time (resistance to single phage: host range F1,2 = 0.40, P = 0.59, time F2,1 = 0.20, P = 0.84; resistance to phage populations: host range F1,2 = 1.45, P = 0.35, time F2,1 = 6.00, P = 0.28).

Evolution of resistance against populations of phage

Comparison of resistance evolved against single phage and populations of phage

In our second selection experiment, the ancestral host bacterium evolved resistance to 8 of the 18 focal phage

Contrary to our expectations, the likelihood of the ancestral bacterium evolving resistance to focal phage

Frequency of resistance evolution

ranges of hosts generated against focal phage genotypes when assayed against populations of phages (see Fig. 2d) (host range F1,11 = 0.27, P = 0.62; time: F2,5 = 1.48, P = 0.31; host range 9 time: F2,11 = 6.17, P = 0.02); in contrast to all other resistance mutants, resistance mutants generated against focal phage from population F were not resistant to any of the phage populations they were tested against. Evolved bacteria were more likely to be resistant to phages that were genetically closely related to the focal phage they evolved resistance to (effect of genetic distance: v2 = 23.75, d.f. = 1, P < 0.0001), although this effect was weak (r2 = 0.02; average distance between focal phage and other phages to which bacteria were resistant: 12.99; average distance between focal phage and other phages to which bacteria were sensitive: 14.03. See Fig. S1). Additionally, evolved bacteria were more likely to be resistant to test phages from later time points (effect of test phage time point: v2 = 75.90, d.f. = 2, P < 0.0001; a Tukey post hoc test indicated that all three time points were significantly different at P < 0.01).

Resistance evolution

(a) No

Yes

0

5

10

15

20

1.00

(b)

0.75

0.50

0.25

0.00

25

10

Resistance range vs. phage populations


(c) 160

120

80

40 0

5

10

15

20

30

60

Focal phage population time point

Focal phage population host range

25


(d) 10

5

0 0

5

10

15

20

25


Fig. 3 Evolution of resistance against focal phage populations. (a) Effect of focal phage host range on whether resistance evolved (yes/no) in our experiments. (b) Effect of coevolutionary time point (T10, T30 and T60) at which focal phage was isolated on whether resistance evolved (yes/no), expressed as the frequency of selection experiments in which at least one bacterial isolate evolved resistance per time point (error bars indicate binomial 95% confidence intervals). (c) Effect of focal phage host range and coevolutionary time point (T10: open circles, T30: squares, T60: crosses) on evolved resistance range, as assayed against a suite of single phage genotypes. (d) As in C), assayed against populations of phage.



populations was not different than that of the bacterium evolving resistance to focal phage genotypes (v2 = 0.18, d.f. = 1, P = 0.67). Similarly, there was no difference in the resistance range of focal phage resistance mutants and focal phage population resistance mutants, when defined as either the proportion of single phage genotypes to which they were resistant or the proportion of populations of phage to which they were resistant (resistance to single genotypes: F1,13 = 0.30, P = 0.59; resistance to populations: F1,13 = 0.04, P = 0.84). Evolved vs. coevolved resistance ranges To assess whether evolution and coevolution give rise to similar bacterial resistance ranges, we compared the resistance ranges that evolved in our selection experiments to the resistance ranges of coevolved bacteria that were isolated from the same time points and replicate populations as the phage used in our selection experiments. Although bacteria that evolved resistance to phage from different time points had similar resistance ranges, coevolved bacteria from later time points had larger resistance ranges than coevolved bacteria from the initial time point, such that evolved bacteria had larger resistance ranges than coevolved bacteria from T10, but smaller resistance ranges than coevolved bacteria from T30 and T60 (treatment F1,60 = 32.54, P < 0.0001; time: F2,60 = 0.06, P = 0.94; treatment 9 time: F2,60 = 16.34, P < 0.0001. T10: P = 0.0328; T30:

Resistance range

160

120

80

40

10

20

30

40

50

60

Time Fig. 4 Comparison of evolved resistance ranges generated against phage from time points T10, T30 and T60 (open circles) and coevolved resistance ranges of bacteria isolated from time points T10, T30 and T60 (squares).

1127

P < 0.0001; T60 P < 0.0001; all significant after sequential Bonferroni correction; Fig. 4).

Discussion We investigated how variation in the host range and coevolutionary history of both individual parasite genotypes and populations of parasites influences the evolution of host resistance. We tested five hypotheses and our results show that (i) parasite host range did not affect the likelihood of resistance evolution, (ii) our na€ıve host was as likely to evolve resistance to individual parasites as to diverse populations of parasites, (iii) there was no correlation between the host range of the parasite (focal phage) that the host evolved resistance to and the breadth of its resulting resistance range, (iv) hosts that evolved resistance in a single step to parasites from different time points had similar resistance ranges, whereas hosts that had coevolved with parasites over longer timescales had relatively small resistance ranges at early time points and relatively broad ranges at later time points, and (v) evolved hosts were relatively likely to be resistant to other parasites that were genetically closely related to the parasite they were directly selected to evolve resistance against. A key question this study raises is why there is no correlation between host range and the probability of resistance evolution. Although it is clearly more difficult for bacteria to evolve resistance to previously coevolved phages than to ancestral phage (~50% vs. 100% resistance evolution), it is remarkable that in half of our 24-h selection experiments, bacteria evolved resistance ranges that in many cases were equivalent to the outcome of hundreds of generations of coevolution. These results are likely explained by the molecular mechanisms of infection and resistance: resistance in this system can be achieved by altering the receptor used by the phage [which in this and other systems is associated with the lipopolysaccharide component of the cell wall (Labrie et al., 2010; Scanlan et al., 2015)], whereas phages have to specifically match this receptor to infect a bacterium. Crucially, single mutations can alter the receptor, potentially explaining the lack of relationship between range and resistance probability. Our results also show that evolved resistance confers resistance to a broad set of other parasite genotypes and populations as a correlated response, indicating that host and phage are not specifically matched and resistance evolution is to some degree aspecific. Similar effects have been reported in other bacteria–phage systems (Stoddard et al., 2007; Hall et al., 2012; Betts et al., 2013, 2014) and different host–parasite combinations such as Daphnia dentifera and its natural yeast parasite Metschnikowia (Duffy & Sivars-Becker, 2007) and Drosophila melanogaster and its viral parasite Drosophila C virus (Martins et al., 2014). However, phages to which evolved bacteria were resistant to as a correlated


1128

F. A. GORTER ET AL.

response were on average more closely related to the focal phage than phages to which they were not resistant to, even if this effect was small (Fig. S1). Although each evolved host had a unique resistance profile (see Fig. S2a–d), the evolved resistance ranges were relatively uniform in breadth (Fig. 4). Such similarity in breadth of resistance range is not surprising given the short time span and thus potential for divergence in our experiment; we would expect our resistance mutants to contain a single, or at the most a few, mutations conferring resistance. Surprisingly, the amount of time a given phage has previously spent coevolving significantly increased the probability of host resistance evolution, and this cannot be explained by host range. There was no significant difference in breadth of the host range of parasites between T30 and T60 in this system, but we observed that some phages from T60, in contrast to phages with similar host ranges from T30, had a greatly reduced plaque size, a phenomenon that is commonly used as an indicator of impaired phage fitness (Burch & Chao, 2004). The exact nature of these costs is not clear from our results, but one possibility is that they are conferred by deleterious mutations that were able to hitchhike along with infectivity mutations and accumulated over time even when host range expansion (but not shift) ended. Alternatively, mutations at either the tail fibre gene or elsewhere on the viral genome that accumulated between T30 and T60 may have conferred infectivity to newly encountered bacteria, or increased virulence on predominant genotypes, but simultaneously impaired virulence on the ancestral host. Whatever the underlying mechanism, it is interesting to note the parallel between the relationship between time and resistance evolution that we found for phage, and the process of attenuation that is commonly employed in vaccine development. We anticipated that the diversity of phages within populations would reduce the likelihood of resistance evolution compared to that observed against single phage genotypes, with the rationale that more mutations would be required to resist the former. However, we found no difference in the likelihood of resistance evolution to genotypes or populations. It is possible that bacteria evolved independent resistance mechanisms to all phage within a population, but this seems unlikely given the short duration of resistance evolution. A more likely explanation is that phage populations were not particularly diverse and resistance to one phage in a population correlated with resistance to other phage genotypes in the same population, in accordance with the high degree of correlated resistance noted above. As indicated in other recent studies, our results highlight the asymmetry in coevolution between this bacterium and phage (Hall et al., 2011b; Scanlan et al., 2011, 2015). Recent genetic analysis shows that host resistance can result from a wide range of mutations at dif-

ferent loci and that relatively broad resistance ranges do not necessarily require a large number of mutations (Scanlan et al., 2015), a finding consistent with the ease of resistance evolution in the present study. This is in contrast to phages, where mutations required for host range expansion are acquired in a contingency-like manner and are restricted to one or two loci (Scanlan et al., 2011). This may also explain why bacteria are typically ahead in the coevolutionary arms race and are locally adapted as evidenced by high levels of sympatric resistance (Buckling & Rainey, 2002). Such extreme asymmetries are not typically incorporated in coevolutionary models. Given the parallels of the coevolutionary dynamics between this and other bacteria–phage systems (Flores et al., 2011), our findings are likely to be broadly relevant to bacteria–phage interactions where resistance is mediated by modification of receptors, as is this case here and in some natural systems (Avrani et al., 2011). An important implication of this is that bacteria should be able to evolve resistance to highly coevolved phages as readily as to less coevolved ones, which may also explain why the massive impact phages can have on bacterial populations is fairly short lived (Faruque et al., 2005). Our results also have important implications for the therapeutic use of phages: coevolution may be costly to some phages, making it easier for bacteria to evolve resistance. Finally, although we detected no difference in the capacity for resistance evolution against single phages and against whole populations, this does not exclude the possibility that populations containing multiple phage species that use different receptors will be relatively difficult to overcome (Chan et al., 2013). Note that it is unclear whether these findings will hold in the context of the other common phage resistance mechanisms such as CRISPR–Cas-based post-infection resistance, where hosts evolve resistance by incorporating phage DNA spacers into the CRISPR–Cas locus (Westra et al., 2012). CRISPR–Cas-imposed selection can favour CRISPR–Cas interference mechanisms, suggesting that in this context, it might be much harder for bacteria to evolve resistance against coevolved phages (Bondy-Denomy et al., 2013). Our findings may also be relevant to the evolutionary epidemiology of any host–parasite system where hosts and parasites can evolve broad ranges (i.e. GFG-like specificities). Outside bacteria–phage interactions, the best-documented cases of such range coevolution are plant–pathogen interactions (Dodds et al., 2006). However, in this case, resistance often appears to require successful recognition of parasite Avr elements by host R genes, and consequently, it is not the host but the parasite that benefits from loss of recognition (Chisholm et al., 2006; Kanzaki et al., 2012). The requirement for a specific match by the host could seriously reduce host evolutionary potential, implying that in



such systems it might actually, as we anticipated, be harder for the host to evolve resistance to highly coevolved parasites and diverse sets of parasites. This has potentially serious consequences for pest management in agricultural crops.

Acknowledgments We are grateful to the European Research Council and NERC (UK) for funding and to Dr Ville Friman for useful comments on the manuscript. AB also gratefully acknowledges support from the Royal Society. None of the authors declare any conflict of interests.

References Avrani, S., Wurtzel, O., Sharon, I., Sorek, R. & Lindell, D. 2011. Genomic island variability facilitates Prochlorococcusvirus coexistence. Nature 474: 604–608. Best, A., White, A., Kisdi, E., Antonovics, J., Brockhurst, M.A. & Boots, M. 2010. The evolution of host-parasite range. Am. Nat. 176: 63–71. Betts, A., Vasse, M., Kaltz, O. & Hochberg, M.E. 2013. Back to the future: evolving bacteriophages to increase their effectiveness against the pathogen Pseudomonas aeruginosa PAO1. Evol. Appl. 6: 1054–1063. Betts, A., Kaltz, O. & Hochberg, M.E. 2014. Contrasted coevolutionary dynamics between a bacterial pathogen and its bacteriophages. Proc. Natl. Acad. Sci. USA 111: 11109– 11114. Boerlijst, M.C., Bonhoeffer, S. & Nowak, M.A. 1996. Viral quasi-species and recombination. Proc. R. Soc. B Biol. Sci. 263: 1577–1584. Bohannan, B.J.M. & Lenski, R.E. 2000. Linking genetic change to community evolution: insights from studies of bacteria and bacteriophage. Ecol. Lett. 3: 362–377. Bondy-Denomy, J., Pawluk, A., Maxwell, K.L. & Davidson, A.R. 2013. Bacteriophage genes that inactivate the CRISPR/ Cas bacterial immune system. Nature 493: 429–U181. Brockhurst, M.A., Morgan, A.D., Fenton, A. & Buckling, A. 2007. Experimental coevolution with bacteria and phage. The Pseudomonas fluorescens–Phi2 model system. Infect. Genet. Evol. 7: 547–552. Buckling, A. & Rainey, P.B. 2002. Antagonistic coevolution between a bacterium and a bacteriophage. Proc. Biol. Sci. 269: 931–936. Burch, C.L. & Chao, L. 2004. Epistasis and its relationship to canalization in the RNA virus phi 6. Genetics 167: 559–567. Chan, B.K., Abedon, S.T. & Loc-Carrillo, C. 2013. Phage cocktails and the future of phage therapy. Future Microbiol. 8: 769–783. Chisholm, S.T., Coaker, G., Day, B. & Staskawicz, B.J. 2006. Host-microbe interactions: shaping the evolution of the plant immune response. Cell 124: 803–814. Decaestecker, E., Declerck, S., De Meester, L. & Ebert, D. 2005. Ecological implications of parasites in natural Daphnia populations. Oecologia 144: 382–390. Dodds, P.N., Lawrence, G.J., Catanzariti, A.M., Teh, T., Wang, C.I.A., Ayliffe, M.A. et al. 2006. Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust avirulence genes. Proc. Natl. Acad. Sci. USA 103: 8888–8893.

1129

Duffy, M.A. & Sivars-Becker, L. 2007. Rapid evolution and ecological host-parasite dynamics. Ecol. Lett. 10: 44–53. Duffy, S., Turner, P.E. & Burch, C.L. 2006. Pleiotropic costs of niche expansion in the RNA bacteriophage phi 6. Genetics 172: 751–757. Ebert, D. 1998. Experimental evolution of parasites. Science 282: 1432–1435. Faruque, S.M., Bin Naser, I., Islam, M.J., Faruque, A.S.G., Ghosh, A.N., Nair, G.B. et al. 2005. Seasonal epidemics of cholera inversely correlate with the prevalence of environmental cholera phages. Proc. Natl. Acad. Sci. USA 102: 1702–1707. Flor, H.H. 1956. The complementary genic systems in flax and flax rust. Adv. Genet. Incorp. Mol. Genet. Med. 8: 29–54. Flores, C.O., Meyer, J.R., Valverde, S., Farr, L. & Weitz, J.S. 2011. Statistical structure of host-phage interactions. Proc. Natl. Acad. Sci. USA 108: E288–E297. Friman, V.P., Lindstedt, C., Hiltunen, T., Laakso, J. & Mappes, J. 2009. Predation on multiple trophic levels shapes the evolution of pathogen virulence. PLoS ONE 4: e6761. Hall, A.R., Scanlan, P.D. & Buckling, A. 2011a. Bacteria-phage coevolution and the emergence of generalist pathogens. Am. Nat. 177: 44–53. Hall, A.R., Scanlan, P.D., Morgan, A.D. & Buckling, A. 2011b. Host-parasite coevolutionary arms races give way to fluctuating selection. Ecol. Lett. 14: 635–642. Hall, A.R., De Vos, D., Friman, V.P., Pirnay, J.P. & Buckling, A. 2012. Effects of sequential and simultaneous applications of bacteriophages on populations of Pseudomonas aeruginosa in vitro and in wax moth larvae. Appl. Environ. Microbiol. 78: 5646–5652. Holmfeldt, K., Middelboe, M., Nybroe, O. & Riemann, L. 2007. Large variabilities in host strain susceptibility and phage host range govern interactions between lytic marine phages and their Flavobacterium hosts. Appl. Environ. Microbiol. 73: 6730–6739. Kanzaki, H., Yoshida, K., Saitoh, H., Fujisaki, K., Hirabuchi, A., Alaux, L. et al. 2012. Arms race co-evolution of Magnaporthe oryzae AVR-Pik and rice Pik genes driven by their physical interactions. Plant J. 72: 894–907. Labrie, S.J., Samson, J.E. & Moineau, S. 2010. Bacteriophage resistance mechanisms. Nat. Rev. Microbiol. 8: 317–327. Loc-Carrillo, C. & Abedon, S.T. 2011. Pros and cons of phage therapy. Bacteriophage 1: 111–114. Martins, N.E., Faria, V.G., Nolte, V., Scholtterer, C., Teixeira, L., Sucena, E. et al. 2014. Host adaptation to viruses relies on few genes with different cross-resistance properties. Proc. Natl. Acad. Sci. USA 111: 5938–5943. Parker, M.A. 1994. Pathogens and sex in plants. Evol. Ecol. 8: 560–584. Parrish, C.R., Holmes, E.C., Morens, D.M., Park, E.C., Burke, D.S., Calisher, C.H. et al. 2008. Cross-species virus transmission and the emergence of new epidemic diseases. Microbiol. Mol. Biol. Rev. 72: 457–470. Paterson, S., Vogwill, T., Buckling, A., Benmayor, R., Spiers, A.J., Thomson, N.R. et al. 2010. Antagonistic coevolution accelerates molecular evolution. Nature 464: 275–278. Pinheiro, J.C. & Bates, D.M. 2000. Mixed-Effects Models in S and S-PLUS. Springer Science & Business Media, New York. Pirnay, J.P., De Vos, D., Verbeken, G., Merabishvili, M., Chanishvili, N., Vaneechoutte, M. et al. 2011. The phage therapy paradigm: pret-a-porter or sur-mesure? Pharm. Res. 28: 934–937. Poullain, V., Gandon, S., Brockhurst, M.A., Buckling, A. & Hochberg, M.E. 2008. The evolution of specificity in


1130

F. A. GORTER ET AL.

evolving and coevolving antagonistic interactions between a bacteria and its phage. Evolution 62: 1–11. Sasaki, A. 2000. Host-parasite coevolution in a multilocus genefor-gene system. Proc. R. Soc. B Biol. Sci. 267: 2183–2188. Scanlan, P.D., Hall, A.R., Lopez-Pascua, L.D. & Buckling, A. 2011. Genetic basis of infectivity evolution in a bacteriophage. Mol. Ecol. 20: 981–989. Scanlan, P.D., Hall, A.R., Burlinson, P., Preston, G. & Buckling, A. 2013. No effect of host-parasite co-evolution on host range expansion. J. Evol. Biol. 26: 205–209. Scanlan, P.D., Hall, A.R., Blackshields, G., Friman, V.P., Davis, M.R. Jr, Goldberg, J.B. et al. 2015. Coevolution with bacteriophages drives genome-wide host evolution and constrains the acquisition of abiotic-beneficial mutations. Mol. Biol. Evol. (in press). Stoddard, L.I., Martiny, J.B.H. & Marston, M.F. 2007. Selection and characterization of cyanophage resistance in marine Synechococcus strains. Appl. Environ. Microbiol. 73: 5516–5522. Westra, E.R., Swarts, D.C., Staals, R.H.J., Jore, M.M., Brouns, S.J.J. & van der Oost, J. 2012. The CRISPRs, they are Achangin’: how prokaryotes generate adaptive immunity. Annu. Rev. Genet. 46: 311–339. Woolhouse, M.E. & Gowtage-Sequeria, S. 2005. Host range and emerging and reemerging pathogens. Emerg. Infect. Dis. 11: 1842–1847.

Woolhouse, M.E., Haydon, D.T. & Antia, R. 2005. Emerging pathogens: the epidemiology and evolution of species jumps. Trends Ecol. Evol. 20: 238–244.

Supporting information Additional Supporting Information may be found in the online version of this article: Figure S1 Genetic distance (pairwise difference in protein sequence of the tail fibre gene) between focal phage, to which bacteria evolved resistance, and test phage, to which bacteria were resistant (resistance: yes) or not (resistance: no) as a correlated response. Figure S2 Similarity in resistance ranges of resistance mutants evolved against single phage genotypes ((a) and (b)) or phage populations ((c) and (d)). Table S1 Overview of coevolved bacterial hosts used in this study. Received 14 October 2014; revised 31 March 2015; accepted 1 April 2015


Gene Expression Contributes to the Recent Evolution of Host Resistance in a Model Host Parasite System.

Host-parasite interactions and the evolution of nonrandom mating.

Insect host-parasite coevolution in the light of experimental evolution.

Effects of epistasis on infectivity range during host-parasite coevolution.

Parasite evolution in response to sex-based host heterogeneity in resistance and tolerance.

Rapid evolution of virulence leading to host extinction under host-parasite coevolution.

Host age modulates within-host parasite competition.

Heritable variation in host tolerance and resistance inferred from a wild host-parasite system.

Comparative aspects of host-parasite and host-tumor relationships.

Combining experimental evolution and field population assays to study the evolution of host range breadth.

Parasite-host coevolution.

Virulence evolution in a host-parasite system in the absence of viral evolution.

Do-or-die life cycles and diverse post-infection resistance mechanisms limit the evolution of parasite host ranges.

Parasite and host elemental content and parasite effects on host nutrient excretion and metabolic rate.

Conflict RNA modification, host-parasite co-evolution, and the origins of DNA and DNA-binding proteins1.

Understanding the ecology and evolution of host-parasite interactions across scales.

Parasite infection drives the evolution of state-dependent dispersal of the host.

Schistosoma mansoni compatibility polymorphism.

The role of models in translating within-host dynamics to parasite evolution.

Evolution of Caenorhabditis elegans host defense under selection by the bacterial parasite Serratia marcescens.

Invasion success of a scarab beetle within its native range: host range expansion versus host-shift.

Vaccinia virus host range genes.

Accelerated evolution of schistosome genes coding for proteins located at the host-parasite interface.

Host density and competency determine the effects of host diversity on trematode parasite infection.