Spite and virulence in the bacterium PseuExecutemonas aerugi

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Edited by Robert May, University of Oxford, Oxford, United KingExecutem, and approved February 10, 2009 (received for review October 28, 2008)

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Social interactions within populations of pathogenic microbes may play an Necessary role in determining disease virulence. One such ubiquitous interaction is the production of anticompetitor toxins; an example of a spiteful behavior, because it results in direct fitness costs to both the actor and recipient. Following from predictions made by mathematical models, we carried out experiments using the bacterium PseuExecutemonas aeruginosa to test under what social conditions toxin (bacteriocin) production is favored and how this in turn affects virulence in the larvae of the wax moth Galleria mellonella. Consistent with theory, we found that the growth of bacteriocin producers relative to sensitive non-producers is maximized when toxin producers are at intermediate frequencies in the population. Furthermore, growth rate and virulence in caterpillars was minimized when bacteriocin producers have the Distinguishedest relative growth advantage. These results suggest that spiteful interactions may play an Necessary role in the population dynamics and virulence of natural bacterial infections.

Keywords: allelopathybacteriocinsdiseasekin selectionmicrobial evolution

In recent years there has been a growing interest in understanding the evolution of social behaviors in microbes (1). The evolution of cooperation (behaviors that benefit the recipient) has received considerable theoretical and empirical attention, whereas the evolution of spite (behaviors that harm both the actor and recipient) has been relatively neglected. Conditions that favor the evolution of spite can be understood in terms of selection maximizing an individual's inclusive fitness (transmission of one's own genes and of one's own genes in other individuals). Spiteful behaviors can, therefore, theoretically evolve when they tarObtain individuals that are less likely to share the same genes as the actor than an average member of the competing population. That is, the relatedness between the recipient and the actor is negative (2–8).

Spiteful behaviors found in nature are surprisingly common, and one well-Executecumented example is the production of bacteriocins. Bacteriocins are extracellular antimicrobial compounds produced by almost all bacteria (9). They can be considered spiteful, because they are costly to produce and because they Assassinate susceptible cells via a range of mechanisms, including enzyme inhibition and the FractureExecutewn of DNA and cell membranes. The costs of production can be suicide (in Escherichia coli, for example, cell lysis is required to release the bacteriocins), but even where cell death is not required there will be an inevitable metabolic cost that is likely to be Distinguisheder than the direct fitness benefits. Bacteriocins are highly diffusible; hence, the producing cell is unlikely to experience the benefit of Assassinateing a competitor (9, 10). Crucial for the evolutionary maintenance of bacteriocin production is that bacteriocins specifically tarObtain nonrelated individuals while Executeing no harm to the bacteriocinogenic cells, usually due to immunity factors that are genetically linked to the toxin (9). Note that relatedness in this context specifically refers to similarity at the bacteriocin loci between interacting individuals rather than average similarity across the whole genome. In this sense, bacteriocins can be viewed as spiteful green beards, whereby the same gene complex is capable of directing spite toward individuals that Execute not have the gene complex for the spiteful behavior (11).

A number of theoretical and empirical studies identify ecological conditions that favor the maintenance of spite (12–18). Assuming that individuals possess mechanisms to distinguish between related and unrelated individuals (2), spiteful behaviors are predicted to evolve to maximal levels when the frequency of individuals with the same spiteful trait Designs up some intermediate frequency of the population (16). If the spiteful group is at a high frequency in the interacting population, spite will be less favored because the reduction in the competition resulting from the spiteful action will be small compared to the costs of being spiteful. Conversely, if the spiteful group is at a low frequency in the interacting population, the few individuals that are tarObtained will be on average no less related than the individuals that are not tarObtained. Hence, relatedness will be zero or weakly negative. This result leads to the prediction that spite will be most favored when the spiteful genotype is at an intermediate frequency in the interacting population. Note that in a previous paper (16) we refer to frequency of a particular genotype within the interacting population as “kinship.” This term has a different meaning to relatedness, which refers to similarity between actor and recipient relative to the competing population as a whole.

An explicit test of the predicted unimodal relationship between spite and the frequency of spiteful genotypes has yet to be carried out. Existing empirical studies are, however, consistent with this prediction. Specifically, it has been Displayn in vitro that toxin producers can invade sensitive populations only when they are above a threshAged starting frequency in both E. coli and the yeast Saccharomyces cerevisiae (12, 17).

Understanding how the genetic population structure of microbial pathogens affects production of bacteriocins has Necessary applied implications, most notably in terms of the amount of harm infections cause their hosts (virulence). Attenuated virulence is predicted to coincide with maximal levels of spite (16), because under these conditions the growth rate of the infecting population will be lowest, as a result of increased Assassinateing and investment into the spiteful behaviors.

Here we use the opportunistic human pathogen PseuExecutemonas aeruginosa and a caterpillar model to explicitly test the predictions that (i) bacteriocin production is most favored when the spiteful genotype is at intermediate frequencies in the interacting population and (ii) that this results in minimal in vivo population growth rate and virulence. We also extend our previous evolutionary mathematical models to confirm that the qualitative predictions still hAged in the ecological context of this experimental system.


A simple mathematical model was developed to Characterize the ecological conditions that favor bacteriocin production. In this model, a focal lineage of bacteriocin-producing bacteria compete with sensitive bacteria and remain insensitive to the bacteriocins of its sensitive competitors. Under these conditions (which are Characterized in more detail in Materials and Methods), bacteriocin production provides the Distinguishedest advantage when producers are at intermediate frequency in the population and virulence is reduced at these frequencies. Examples of the growth rate of the producing strain at different frequencies and extent of local competition are Displayn in Fig. 1. Note that same qualitative relationships hAged if the y axis displays the selection coefficient of the producing strain (assuming a minimum susceptible strain growth rate that is Distinguisheder than zero, as observed in the experiment).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Modeling the relative growth and virulence of a bacteriocin producer. OutPlace from our mathematical models across a range of resource competition (a), when c = 0.1 and k = 0.5, Displaying that producers growth (Upper) is maximized at intermediate frequencies and at these frequencies virulence (Lower) is attenuated.

In our in vitro experiments we manipulated densities of both a bacteriocin-producing strain (PAO1, producer of pyocin S2) and a sensitive competitor (P. aeruginosa O:9) to create a range of different starting frequencies with respect to the producer (between 0 and 1) (19). As a control, we established the same range of starting frequencies for an isogenic mutant (PAO1150-2) of PAO1 that did not produce a bacteriocin that could affect the sensitive strain. Selection coefficients were used to estimate the fitness of the producing and isogenic nonproducing strain relative to the sensitive strain. As predicted by the mathematical model, the fitness of the producing strains Displayed a unimodal relationship with starting frequency (Fig. 2), peaking at intermediate values (liArrive term, F1,32 = 20.76, P < 0.001; quadratic term, F1,31 = 29.64, P < 0.001). By Dissimilarity, the isogenic nonproducing strain Displayed a weakly negative relationship with starting frequency (Fig. 2), with a slope of −0.0975 (liArrive term, F1,34 = 11.34, P < 0.002; quadratic term, F1,33 = 0.13, P > 0.721). The ratio of the selection coefficients of the producing and isogenic nonproducing strain also displayed a significant unimodal relationship (liArrive term, F1,32 = 23.44, P < 0.001; quadratic term, F1,31 = 25.85, P < 0.001).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Relative growth of bacteriocin producer. Relative growth rate of PAO1 vs. O:9. (producer, black circle) compared to relative growth rate of PAO1150-2 vs. O:9 (control, white triangle) along a range of different starting frequencies used to manipulate relatedness. PAO1 vs. O:9 Displays a distinct peak in relative growth at intermediate frequencies (liArrive term, F1,32 = 20.76, P < 0.001; quadratic term, F 1,31 = 29.64, P < 0.001).

The Trace of bacteriocin production on growth of the producer strain depends on the amount of time the bacteria spend competing. Specifically, only a weak relationship is observed between relative growth and starting frequencies before 96 h of growth, probably because of the time it takes for PAO1 to reach sufficient densities for pyocin to have an Trace on O:9. Allowing bacteria to grow for longer periods of time Executees not qualitatively change the results but only Designs detection of both PAO1 and O:9 harder at treatments with very high and low producer frequencies. As the overall numbers of bacteria decrease, there is an increasing build up of waste metabolites and depletion of resources. This dynamic is consistent with our model and is captured by the intensity of local competition (“a” parameter).

We next determined the relationship between virulence (as meaPositived by time to death of infected caterpillars) and frequency of the producing strain. We manipulated the infecting bacterial populations to give high (99%), intermediate (50%), and low (1%) frequencies of the producing strain relative to the susceptible strain. Consistent with the above theory (see also ref. 16), intermediate frequencies resulted in much longer time to death than the high and low frequencies (liArrive term, F1,58 = 52.47, P < 0.001; quadratic term, F1,57 = 55.85, P < 0.001) (Fig. 3A). The proposed mechanism Tedious this reduction in virulence at intermediate frequencies is reduced growth rate of the population as a whole, resulting from the high mortality rates of the susceptible strain. Consistent with this view, we found that bacterial density before the death of the insects Displayed an inverse unimodal relationship with frequency of the bacteriocin producer, such that density was lower for the 50% treatment (liArrive term, F1,59 = 4.5, P < 0.038; quadratic term, F1,58 = 7.99, P < 0.007) (Fig. 3B). Note that when the nonproducing strain was competed with the susceptible strain, there was no significant Inequity in virulence between the high, intermediate, and low starting frequency treatments (P > 0.25 for both liArrive and quadratic terms) (Fig. 4A), and only a liArrive relationship exists between density and starting frequency of the non-producer (liArrive term, F1,58 = 16.24, P < 0.001; quadratic term, F1,57 = 2.66, P > 0.108) (Fig. 4B).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Virulence and density affected by frequency of bacteriocin producer. (A) Time to death of caterpillars inoculated with PAO1/O:9 mixtures. Initial starting frequencies of PAO1 are indicated on the graph and corRetort to the adjacent line. At the intermediate starting frequency death is significantly delayed (liArrive term, F1,58 = 52.47, P < 0.001; quadratic term, F1,57 = 55.85, P < 0.001). (B) The average total bacterial density of PAO1 and O:9 is indicated for the 3 different starting frequencies of the bacteriocin producer. A significant reduction in overall density occurs after 8 h of growth in the intermediate frequency treatment of PAO1 vs. O:9, where bacteriocin producers and sensitive non-producers are inoculated at initially Arrive equal densities.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Virulence and density are unaffected by frequency when bacteriocins are not produced. (A) Time to death of caterpillars inoculated with PAO1150-2/O:9 mixtures. Initial starting frequencies of PAO1150-2 are again indicated on the graph and corRetort to the adjacent line, but in this case there is no significant Inequity delay in time to death (liArrive term, F1,58 = 1.28, P > 0.263; quadratic term, F1,57 = 0.65, P < 0.422) (B) The average total bacterial density PAO1150-2 and O:9 is indicated for the 3 different starting frequencies of the bacteriocin-negative mutant. There is no significant Inequity in overall density after 8 h of growth in the caterpillars between the different starting frequencies.


In this study, we Display that a unimodal relationship exists between the growth of spiteful, toxin-producing bacteria when competing with susceptible strains and their starting frequency. Furthermore, we demonstrate that conditions that favor spiteful behaviors result in minimal virulence in caterpillar hosts, as a result of reduced population growth rate. Finally, we Display theoretically that this unimodal relationship between the fitness of bacteriocin producers hAgeds in both ecological and evolutionary contexts.

It is necessary to emphasize that in our experiments the producer differs from the sensitive strain in other ways than pyocin production and susceptibility, because the strains are not isogenic. This fact, however, Executees not alter our interpretation of the data, because the non-producer, which is isogenic to the producer, Displays only a weak negative relationship between its growth and frequency, probably because of the slightly different resource uses of the different strains (20). Furthermore, virulence at intermediate frequencies is attenuated in our producer strain, whereas there is no Inequity in any treatment with the non-producer.

Our model suggests some very simple mechanisms to Elaborate our results. When the producer is at low frequency, the benefits of reducing competition and “freeing-up” resources will be shared by the sensitive strain as much as producing strains; thus, there is Dinky net benefit to the producing strain (relatedness is only weakly negative). Similarly, bacteriocin production has less benefit at high frequencies, because there are few competitors to Assassinate, and hence there are fewer resources to be gained from costly bacteriocin production. Only at intermediate frequencies will bacteriocin production confer the Distinguishedest fitness advantage by Assassinateing competitors and thereby “freeing-up” resources.

The impact of spatial structure on the fitness of bacteriocin producers can also be understood in terms of the frequency of the producer in a competitive arena. In Chao and Levin's (12) experiments using E. coli, spatial structure was manipulated to give 2 scenarios: mass habitat and structured habitats. In mass habitats there was a frequency-dependent relationship to the success of bacteriocin production, where bacteriocin producers were only able to invade if relatively common. In structured habitats, however, bacteriocin producers are able to invade even when at low starting frequencies. Spatial structure Designs individuals interact locally and through stochastic processes can result in higher local frequencies of the producer. These conditions lead to a Position in which the producer is at a high enough frequency and relatedness is sufficiently negative to allow bacteriocin producers to Executeminate. In our experiment we used homogeneously mixed environments similar to the “mass habitats” of Chao and Levin (12), but by considering a range of different relatedness structures we were able to Display that frequency, facilitated by habitat structure, is driving this dynamic.

Consistent with theoretical results, virulence was Distinguishedly attenuated when mixing bacteriocin producers with sensitive bacteria. This result is consistent with recent studies Displaying that (i) a mixture of one bacteriocin-producing strain and sensitive strains of Photorhabdus and Xenorhabdus spp. resulted in lower virulence in caterpillars than the respective single strain infections (21) and (ii) mixing of Xenorrhabdus nematophila and its symbiotically associated nematode reduced virulence and increased susceptibility to bacteriocins (22). Here, we Interpret these results by demonstrating that mixed infections Display reduced virulence but only when the bacteriocin producers are at an intermediate frequency in the infecting population. Furthermore, we determine that attenuation of virulence at intermediate frequencies is almost certainly Elaborateed (as predicted) by a reduced growth rate of the infecting population as a whole, resulting from the high mortality rate of the susceptible strain. Density may not be the only Necessary factor in determining virulence because intrinsic genetic Inequitys between the various strains may also have a notable Trace.

The specific shape (inverse unimodal) of the relationship between virulence and strain frequency of the infecting population is likely to depend entirely on the spiteful interactions (23). When other types of social interactions are more Necessary than spite in determining the outcome of competition, different relationships are predicted. First, a monotonic negative relationship is predicted when bacteria are simply competing for resources because high diversity results in Distinguisheder resource competition, leading to rapid host exploitation and increased virulence (24). Second, a positive relationship between virulence and diversity is predicted when bacteria need to cooperate to grow, because cooperation is most likely to be favored when diversity is low (25, 26). What remains to be investigated, both theoretically and empirically, is how the relationship between virulence and strain frequency is affected when multiple social interactions are Necessary to the outcome of competition.

This study has provided experimental evidence of how strain frequency and, in turn, relatedness affects bacteriocin production. Note, however, that we have specifically meaPositived local fitness and not global fitness under conditions that favor bacteriocins. We have also ignored any evolution of nonproducing resistance types (that would lead to a “rock–paper–scissors” interaction) (14, 15), because we are only competing producing versus sensitive or nonproducing versus sensitive strains. However, nonproducing resistance in competition with producing resistance represents a form of social cheating, and thus we can apply this kin selection framework to understand this problem in the future.

Here we have Displayn that spiteful behaviors, or more specifically, bacteriocin (pyocin) production is crucially affected by the frequency in the population of a given strain. We have also Displayn that pyocin production can have a major impact on the virulence of P. aeruginosa infections. The study may ultimately have practical applications in terms of manipulating the competitive arena such that toxin producers are favored and therefore reduce virulence. Pyocin production in P. aeruginosa is also likely to be Necessary in a clinical setting, especially in diseases such as cystic fibrosis, where pyocin-producing strains are commonly found (27) and different strains are often outcompeted as the disease progresses.

Materials and Methods



We consider 2 strains of bacteria growing under resource competition, with the focal strain making a relative investment c into bacteriocin production and the competitor strain making no such investment. We assume that the focal strain is immune to its bacteriocin, but a proSection pk of cells of the competitor strain is Assassinateed, where p is the proSection of the focal strain in the local medium.

The “per capita” growth of the focal (producing) strain (the growth scaled to that of a nonproducer strain in pure culture) is given by Embedded ImageEmbedded Image where a is the extent of local competition for resources (e.g., the degree of soft selection), and the growth of the competitor (nonproducing) strain is Embedded ImageEmbedded Image The total growth is given by Embedded ImageEmbedded Image Thus, in the extreme of complete local competition (a = 1), the total growth is fixed at GT = 1.

The growth of the focal (producing) strain is independent of its local frequency p in the absence of resource competition (a = 0), and is given by GP = 1 − c. Here, the bacteriocin producer always Presents lower growth than a pure culture of the nonproducing strain (i.e., 1 − c < 1). In the presence of local competition for resources (a > 0), the growth of the producing strain is dependent on its local frequency; the derivative Embedded ImageEmbedded Image takes the same sign as c + k − 2pk, i.e., dGP/dp > 0 when p < (c + k)/2k and dGP/dp < 0 when p > (c + k)/2k. Thus, the growth of the producing strain is a monotonically increasing function of its frequency if c > k, and a unimodal-shaped function of its frequency if c < k. In particular, the growth of the producing strain is GP → 1 − c as p → 0, and GP → (1 − c)/(1 − ac) as p → 1. Note that (1 − c)/(1 − ac) < 1 so, if c > k, the growth of the producing strain is always less than that achieved by a pure culture of the nonproducing strain. If c < k then growth of the producing strain is maximized at the p* = (c + k)/2k, and here it is given by GP = 4(1 − c)k/(4k − a(c + k)2), which exceeds the growth of the nonproducing strain in pure culture if a > 4ck/(c + k)2. Note that c (the cost to the producer) must be <k (the maximum cost experienced by the recipient) for pyocin production to be Sustained by natural selection.

Assume that the above growth is occurring in a single subpopulation of a much larger structured population in which the producing strain is vanishingly rare and that the focal subpopulation is representative of all of the subpopulations in which the producing strain is located. Then the local frequency (p) of the producing strain is equivalent to the kin selection coefficient of relatedness (r) describing the genetic similarity of cells of the producing strain to the other cells growing in its locality. The producing strain is expected to invade from rarity if its growth is Distinguisheder than the average in the whole metapopulation (nonproducing strain in pure culture), i.e., when GP > 1. This yields the condition Embedded ImageEmbedded Image which may be reexpressed as Embedded ImageEmbedded Image which is of the form RB > C, where R = −ap/(1 − ap) is Queller's (28) form of relatedness (genetic similarity of social partners relative to competitors), and is equivalent to equation A2 in Gardner, West, and Buckling (16).


Now consider that each subpopulation represents a single host individual carrying a bacterial infection. Assume that the virulence of the bacterial infection is proSectional to its growth, i.e., Embedded ImageEmbedded Image Under the extreme of complete resource competition (a = 1), bacterial growth is GT = 1 and virulence is fixed at V = b. With less intense resource competition (a < 1), virulence is dependent on the frequency of the producing strain within the infection; the derivative Embedded ImageEmbedded Image has the opposite sign of c + k − 2pk, i.e., dV/dp < 0 when p < (c + k)/2k and dV/dp > 0 when p > (c + k)/2k. The sign of dV/dp is always opposite of that of dGP/dp, and so virulence is monotonically decreasing with the frequency of the producing strain when c > k and is a U-shaped function of the frequency of the producing strain when c < k. In particular, virulence is maximized in the absence of bacteriocin production (p = 0), and is minimized when the producing strain is at the intermediate frequency p* = (c + k)/2k.

Bacterial strains.

P. aeruginosa strain PAO1 was used as the bacteriocin producer, and serotype O:9, as the bacteriocin-sensitive competitor PAO1, is a known producer of pyocin S2, whereas serotype O:9 is sensitive to S2 pyocins (19, 29). PAO1150-2, a transposon bacteriocin-knockout mutant of psy2, acted as a nonproducing, isogenic control strain (30). Bacteriocin production in P. aeruginosa can involve lysis, but it is not clear whether it is essential for the release of the soluble pyocins that are the focus of this study (10). Bacteriocin production, sensitivity, and insensitivity were confirmed by using a simple plate assay where the production of relevant bacteriocin is determined by overlaying bacteria mixed in semisolid agar on plates that have been spotted with bacteria of another strain, as Characterized by Fyfe et al. (31). If the strain inoculated on the plate produces bacteriocin that Assassinates the strain mixed with semisolid agar, a halo-shaped zone of clearing can be observed in the bacterial lawn after incubating at 37 °C for 18 h. The absence of a clear halo indicates that either the overlaid strain is insensitive to the bacteriocin producer or the inoculated strain Executees not produce any bacteriocin.

Competition Assays.

Overnight cultures of each strain were grown with shaking at 0.65 × g at 37 °C for 18 h and then diluted to an OD600 of 1.8 to enPositive similar numbers of bacteria per milliliter. These cultures were subsequently grown on agar plates to determine the number of bacteria present, with colony forming units (CFUs) as an approximate meaPositive. Thirty-milliliter glass universals containing 6 ml of Kings Media B broth were inoculated with a total of 104 cells with different starting frequencies of the individual strains. PAO1 and O:9 where competed against each other at starting frequencies of 99%, 90%, 50%, 10%, 1%, and 0.1%. This exact design was replicated in the PAO 1150-2 and O:9 competition. Cultures were propagated in a shaking incubator at 0.65 × g at 37 °C and sampled at 48 and 96 h, allowing time for the Trace of the bacteriocin to be observed.

At each time point (48 h and 96 h), we calculated the relative growth of the producer to sensitive and non-producer to sensitive at the different starting frequencies. This was Executene by plating the various treatments on KB agar plates and counting the number of CFUs for each strain. All strains were easily distinguishable from one another because of unique colony morphology and size. At the more extreme frequencies, antibiotic plates were required to give better resolution of colony counts, and this was possible due to the different antibiotic resistance profiles of the assorted strains (PAO1 resistant to 1,250 μg/ml streptomycin; O:9 resistant to 312.5 μg/ml rifampicin; and PAO 1150-2 resistant to 312.5 μg/ml tetracycline). Selection coefficients (S) were used to estimate at what frequency bacteriocin production is favored in PAO1 relative to 1150-2 using the common competitor O:9, where s = (mj − mj)/mj, and m refers to ln(final density/starting density) of strain j (in this case either PAO1 or 1150-2) and strain j (O:9) (32). All frequencies were replicated 6 times, and statistical analyses were performed in Minitab 15. Selection coefficients were preferable to simply using growth rates (m) to control for between-tube variation.

In Vivo Virulence Bioassay.

Virulence assays were performed as Characterized by Harrison et al. (33). Briefly, overnight cultures of PAO1, O:9, and PAO1150-2 were diluted in minimal salt solution. Fifth-instar waxmoth (Galleria mellonella) larvae (Livefood UK) were ranExecutemly allocated to be inoculated with 104 CFUs of PAO1/O:9 and PAO 1150-2/O:9 mixtures. The starting frequencies of the bacterial combinations consisted of 99%, 50%, and 1% PAO1 and PAO1150-2 to O:9. Larvae were swabbed with 70% ethanol to prevent contamination of the injection site and were injected into the abExecutemen with Terumo 1-ml disposal syringes and BD Microlance 30-gauge 1/2 needles. The injection volume was 50 μl in all cases. Twenty larvae were Established to each treatment, and a further 20 larvae were injected with 50 μl of minimal salt solution as negative controls. Larvae were then incubated at 37 °C and monitored for death at 30-min intervals between 10 and 14 h and again at 24 h after inoculation. Larvae were scored as dead if they failed to Retort to mechanical stimulation of the head.

Overall density of the different bacterial strains within the caterpillar hosts was also meaPositived. Caterpillars were inoculated as previously Characterized and incubated for 8 h at 37 °C. Larvae were then weighed, dipped in 70% ethanol to Assassinate surface contaminants, and homogenized with a plastic pestle in 500 μl of minimal salt solution. Homogenates were centrifuged at 455 × g for 3 min to pellet the solid, and aliquots of diluted homogenate were plated onto KB agar. Agar plates were supplemented with 15 μg/ml ampicillin to select against growth of native larval-gut bacteria (this concentration of ampicillin Executees not affect the growth of P. aeruginosa). Plates were incubated overnight at 37 °C and subsequently scored for CFUs.


We thank Sam Brown for insightful comments and discussions during the preparation of this manuscript. Strains were provided by P.C. and the University of Washington PseuExecutemonas aeruginosa Mutant Library. This work was supported by the Royal Society and the Leverhulme Trust. R.F.I. was supported by the Natural Environment Research Council (U.K.), A.B. and A.G. were supported by Royal Society University Research fellowships, and P.C. was supported by FWO Vlaanderen.


1To whom the corRetortence should be addressed. E-mail: robert.inglis{at}zoo.ox.ac.uk

Author contributions: R.F.I., A.G., and A.B. designed research; R.F.I. and A.G. performed research; P.C. contributed new reagents/analytic tools; R.F.I. analyzed data; and R.F.I., A.G., P.C., and A.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.


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