Shifts along the parasite-mutualist continuum are opposed by fundamental trade-offs.

Theory suggests that symbionts can readily evolve more parasitic or mutualistic strategies with respect to hosts. However, many symbionts have stable interactions with hosts that improve nutrient assimilation or confer protection from pathogens. We explored the potential for evolution of increased parasitism or decreased parasitism and mutualism in a natural gut symbiosis between larvae of Plutella xylostella and the microbe Enterobacter cloacae. We focused on interactions with the pathogen, Bacillus thuringiensis: selecting for parasitism in terms of facilitating pathogen infection, or increased mutualism in terms of host protection. Selection for parasitism led to symbionts increasing pathogen-induced mortality but reduced their competitive ability with pathogens and their in vitro growth rates. Symbionts did not evolve to confer protection from pathogens. However, several lineages evolved reduced parasitism, primarily in terms of moderating impacts on host growth, potentially because prudence pays dividends through increased host size. Overall, the evolution of increased parasitism was achievable but was opposed by trade-offs likely to reduce fitness. The evolution of protection may not have occurred because suppressing growth of B. thuringiensis in the gut might provide only weak protection or because evolution towards protective interactions was opposed by the loss of competitive fitness in symbionts.

Targeting antibiotic resistant bacteria with phage reduces bacterial density in an insect host.

Phage therapy is attracting growing interest among clinicians as antibiotic resistance continues becoming harder to control. However, clinical trials and animal model studies on bacteriophage treatment are still scarce and results on the efficacy vary. Recent research suggests that using traditional antimicrobials in concert with phage could have desirable synergistic effects that hinder the evolution of resistance. Here, we present a novel insect gut model to study phage-antibiotic interaction in a system where antibiotic resistance initially exists in very low frequency and phage specifically targets the resistance bearing cells. We demonstrate that while phage therapy could not reduce the frequency of target bacteria in the population during positive selection by antibiotics, it alleviated the antibiotic induced blooming by lowering the overall load of resistant cells. The highly structured gut environment had pharmacokinetic effects on both phage and antibiotic dynamics compared with in vitro: antibiotics did not reduce the overall amount of bacteria, demonstrating a simple turnover of gut microbiota from non-resistant to resistant population with little cost. The results imply moderate potential for using phage as an aid to target antibiotic resistant gut infections, and question the usefulness of in vitro inferences.

Lepidopteran species have a variety of defence strategies against bacterial infections.

The insect immune system has versatile ways of coping with microbial insults. Currently, innate immune priming has been described in several invertebrates, and the first insights into its mechanistic basis have been described. Here we studied infections with two different strains of Serratia marcescens bacteria in two different Lepidopteran hosts. The results reveal fundamental differences between the two hosts, a well-known model organism Galleria mellonella and a non-model species Arctia plantaginis. They differ in their strategies for resisting oral infections; priming their defences against a recurring sepsis; and upregulating immunity related genes as a response to the specific pathogen strains. The two bacterial strains (an environmental isolate and an entomopathogenic isolate) differ in their virulence, use of extracellular proteases, survival in the larval gut, and in the immune response they evoke in the hosts. This study explores the potential mechanistic explanations for both host and pathogen specific characters that significantly affect the outcome of Gram-negative bacterial infection in Lepidopteran larvae. The results highlight the need to pay greater attention to the differences between model and non-model hosts, and closely related pathogen strains, in immunological studies.

Within-host evolution decreases virulence in an opportunistic bacterial pathogen.

BACKGROUND: Pathogens evolve in a close antagonistic relationship with their hosts. The conventional theory proposes that evolution of virulence is highly dependent on the efficiency of direct host-to-host transmission. Many opportunistic pathogens, however, are not strictly dependent on the hosts due to their ability to reproduce in the free-living environment. Therefore it is likely that conflicting selection pressures for growth and survival outside versus within the host, rather than transmission potential, shape the evolution of virulence in opportunists. We tested the role of within-host selection in evolution of virulence by letting a pathogen Serratia marcescens db11 sequentially infect Drosophila melanogaster hosts and then compared the virulence to strains that evolved only in the outside-host environment. RESULTS: We found that the pathogen adapted to both Drosophila melanogaster host and novel outside-host environment, leading to rapid evolutionary changes in the bacterial life-history traits including motility, in vitro growth rate, biomass yield, and secretion of extracellular proteases. Most significantly, selection within the host led to decreased virulence without decreased bacterial load while the selection lines in the outside-host environment maintained the same level of virulence with ancestral bacteria. CONCLUSIONS: This experimental evidence supports the idea that increased virulence is not an inevitable consequence of within-host adaptation even when the epidemiological restrictions are removed. Evolution of attenuated virulence could occur because of immune evasion within the host. Alternatively, rapid fluctuation between outside-host and within-host environments, which is typical for the life cycle of opportunistic bacterial pathogens, could lead to trade-offs that lower pathogen virulence.

Insect immunity: oral exposure to a bacterial pathogen elicits free radical response and protects from a recurring infection.

BACKGROUND: Previous exposure to a pathogen can help organisms cope with recurring infection. This is widely recognised in vertebrates, but increasing occasions are also being reported in invertebrates where this phenomenon is referred to as immune priming. However, the mechanisms that allow acquired pathogen resistance in insects remain largely unknown. RESULTS: We studied the priming of bacterial resistance in the larvae of the tiger moth, Parasemia plantaginis using two gram-negative bacteria, a pathogenic Serratia marcescens and a non-pathogenic control, Escherichia coli. A sublethal oral dose of S. marcescens provided the larvae with effective protection against an otherwise lethal septic infection with the same pathogen five days later. At the same time, we assessed three anti-bacterial defence mechanisms from the larvae that had been primarily exposed to the bacteria via contaminated host plant. Results showed that S. marcescens had induced a higher amount of reactive oxygen species (ROS) in the larval haemolymph, possibly protecting the host from the recurring infection. CONCLUSIONS: Our study supports the growing evidence of immune priming in insects. It shows that activation of the protective mechanism requires a specific induction, rather than a sheer exposure to any gram-negative bacteria. The findings indicate that systemic pathogen recognition happens via the gut, and suggest that persistent loitering of immune elicitors or anti-microbial molecules are a possible mechanism for the observed prophylaxis. The self-harming effects of ROS molecules are well known, which indicates a potential cost of increased resistance. Together these findings could have important implications on the ecological and epidemiological processes affecting insect and pathogen populations.

Evolution of bacterial life-history traits is sensitive to community structure.

Very few studies have experimentally assessed the evolutionary effects of species interactions within the same trophic level. Here we show that when Serratia marcescens evolve in multispecies communities, their growth rate exceeds the growth rate of the bacteria that evolved alone, whereas the biomass yield gets lower. In addition to the community effects per se, we found that few species in the communities caused strong effects on S. marcescens evolution. The results indicate that evolutionary responses (of a focal species) are different in communities, compared to species evolving alone. Moreover, selection can lead to very different outcomes depending on the community structure. Such context dependencies cast doubt on our ability to predict the course of evolution in the wild, where species often inhabit very different kinds of communities.

Fluctuating temperature leads to evolution of thermal generalism and preadaptation to novel environments.

Environmental fluctuations can select for generalism, which is also hypothesized to increase organisms' ability to invade novel environments. Here, we show that across a range of temperatures, opportunistic bacterial pathogen Serratia marcescens that evolved in fluctuating temperature (daily variation between 24°C and 38°C, mean 31°C) outperforms the strains that evolved in constant temperature (31°C). The growth advantage was also evident in novel environments in the presence of parasitic viruses and predatory protozoans, but less clear in the presence of stressful chemicals. Adaptation to fluctuating temperature also led to reduced virulence in Drosophila melanogaster host, which suggests that generalism can still be costly in terms of reduced fitness in other ecological contexts. While supporting the hypothesis that evolution of generalism is coupled with tolerance to several novel environments, our results also suggest that thermal fluctuations driven by the climate change could affect both species' invasiveness and virulence.

Life history trade-offs and relaxed selection can decrease bacterial virulence in environmental reservoirs.

Pathogen virulence is usually thought to evolve in reciprocal selection with the host. While this might be true for obligate pathogens, the life histories of opportunistic pathogens typically alternate between within-host and outside-host environments during the infection-transmission cycle. As a result, opportunistic pathogens are likely to experience conflicting selection pressures across different environments, and this could affect their virulence through life-history trait correlations. We studied these correlations experimentally by exposing an opportunistic bacterial pathogen Serratia marcescens to its natural protist predator Tetrahymena thermophila for 13 weeks, after which we measured changes in bacterial traits related to both anti-predator defence and virulence. We found that anti-predator adaptation (producing predator-resistant biofilm) caused a correlative attenuation in virulence. Even though the direct mechanism was not found, reduction in virulence was most clearly connected to a predator-driven loss of a red bacterial pigment, prodigiosin. Moreover, life-history trait evolution was more divergent among replicate populations in the absence of predation, leading also to lowered virulence in some of the 'predator absent' selection lines. Together these findings suggest that the virulence of non-obligatory, opportunistic bacterial pathogens can decrease in environmental reservoirs through life history trade-offs, or random accumulation of mutations that impair virulence traits under relaxed selection.