The evolution of antimicrobial peptide resistance in Pseudomonas aeruginosa is severely constrained by random peptide mixtures.

The prevalence of antibiotic-resistant pathogens has become a major threat to public health, requiring swift initiatives for discovering new strategies to control bacterial infections. Hence, antibiotic stewardship and rapid diagnostics, but also the development, and prudent use, of novel effective antimicrobial agents are paramount. Ideally, these agents should be less likely to select for resistance in pathogens than currently available conventional antimicrobials. The usage of antimicrobial peptides (AMPs), key components of the innate immune response, and combination therapies, have been proposed as strategies to diminish the emergence of resistance. Herein, we investigated whether newly developed random antimicrobial peptide mixtures (RPMs) can significantly reduce the risk of resistance evolution in vitro to that of single sequence AMPs, using the ESKAPE pathogen Pseudomonas aeruginosa (P. aeruginosa) as a model gram-negative bacterium. Infections of this pathogen are difficult to treat due the inherent resistance to many drug classes, enhanced by the capacity to form biofilms. P. aeruginosa was experimentally evolved in the presence of AMPs or RPMs, subsequentially assessing the extent of resistance evolution and cross-resistance/collateral sensitivity between treatments. Furthermore, the fitness costs of resistance on bacterial growth were studied and whole-genome sequencing used to investigate which mutations could be candidates for causing resistant phenotypes. Lastly, changes in the pharmacodynamics of the evolved bacterial strains were examined. Our findings suggest that using RPMs bears a much lower risk of resistance evolution compared to AMPs and mostly prevents cross-resistance development to other treatments, while maintaining (or even improving) drug sensitivity. This strengthens the case for using random cocktails of AMPs in favour of single AMPs, against which resistance evolved in vitro, providing an alternative to classic antibiotics worth pursuing.

Complete metamorphosis and microbiota turnover in insects.

The insects constitute the majority of animal diversity. Most insects are holometabolous: during complete metamorphosis their bodies are radically reorganized. This reorganization poses a significant challenge to the gut microbiota, as the gut is replaced during pupation, a process that does not occur in hemimetabolous insects. In holometabolous hosts, it offers the opportunity to decouple the gut microbiota between the larval and adult life stages resulting in high beta diversity whilst limiting alpha diversity. Here, we studied 18 different herbivorous insect species from five orders of holometabolous and three orders of hemimetabolous insects. Comparing larval and adult specimens, we find a much higher beta-diversity and hence microbiota turnover in holometabolous insects compared to hemimetabolous insects. Alpha diversity did not differ between holo- and hemimetabolous insects nor between developmental stages within these groups. Our results support the idea that pupation offers the opportunity to change the gut microbiota and hence might facilitate ecological niche shifts. This possible effect of niche shift facilitation could explain a selective advantage of the evolution of complete metamorphosis, which is a defining trait of the most speciose insect taxon, the holometabola.

Non-lethal exposure to H2O2 boosts bacterial survival and evolvability against oxidative stress.

Unicellular organisms have the prevalent challenge to survive under oxidative stress of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). ROS are present as by-products of photosynthesis and aerobic respiration. These reactive species are even employed by multicellular organisms as potent weapons against microbes. Although bacterial defences against lethal and sub-lethal oxidative stress have been studied in model bacteria, the role of fluctuating H2O2 concentrations remains unexplored. It is known that sub-lethal exposure of Escherichia coli to H2O2 results in enhanced survival upon subsequent exposure. Here we investigate the priming response to H2O2 at physiological concentrations. The basis and the duration of the response (memory) were also determined by time-lapse quantitative proteomics. We found that a low level of H2O2 induced several scavenging enzymes showing a long half-life, subsequently protecting cells from future exposure. We then asked if the phenotypic resistance against H2O2 alters the evolution of resistance against oxygen stress. Experimental evolution of H2O2 resistance revealed faster evolution and higher levels of resistance in primed cells. Several mutations were found to be associated with resistance in evolved populations affecting different loci but, counterintuitively, none of them was directly associated with scavenging systems. Our results have important implications for host colonisation and infections where microbes often encounter reactive oxygen species in gradients.

Evidence for reduced immune gene diversity and activity during the evolution of termites.

The evolution of biological complexity is associated with the emergence of bespoke immune systems that maintain and protect organism integrity. Unlike the well-studied immune systems of cells and individuals, little is known about the origins of immunity during the transition to eusociality, a major evolutionary transition comparable to the evolution of multicellular organisms from single-celled ancestors. We aimed to tackle this by characterizing the immune gene repertoire of 18 cockroach and termite species, spanning the spectrum of solitary, subsocial and eusocial lifestyles. We find that key transitions in termite sociality are correlated with immune gene family contractions. In cross-species comparisons of immune gene expression, we find evidence for a caste-specific social defence system in termites, which appears to operate at the expense of individual immune protection. Our study indicates that a major transition in organismal complexity may have entailed a fundamental reshaping of the immune system optimized for group over individual defence.

Mating changes the genital microbiome in both sexes of the common bedbug Cimex lectularius across populations.

Many bacteria live on host surfaces, in cells and in specific organ systems. In comparison with gut microbiomes, the bacterial communities of reproductive organs (genital microbiomes) have received little attention. During mating, male and female genitalia interact and copulatory wounds occur, providing an entrance for sexually transmitted microbes. Besides being potentially harmful to the host, invading microbes might interact with resident genital microbes and affect immunity. Apart from the investigation of sexually transmitted symbionts, few studies have addressed how mating changes genital microbiomes. We dissected reproductive organs from virgin and mated common bedbugs, Cimex lectularius L., and sequenced their microbiomes to investigate composition and mating-induced changes. We show that mating changes the genital microbiomes, suggesting bacteria are sexually transmitted. Also, genital microbiomes varied between populations and the sexes. This provides evidence for local and sex-specific adaptation of bacteria and hosts, suggesting bacteria might play an important role in shaping the evolution of reproductive traits. Coadaptation of genital microbiomes and reproductive traits might further lead to reproductive isolation between populations, giving reproductive ecology an important role in speciation. Future studies should investigate the transmission dynamics between the sexes and populations to uncover potential reproductive barriers.

Legacy of a Butterfly's Parental Microbiome in Offspring Performance.

An insect's phenotype can be influenced by the experiences of the parental generation. However, the effects of the parental symbiotic microbiome and host plant use on the offspring are unclear. We addressed this gap of knowledge by studying Pieris brassicae, a multivoltine butterfly species feeding on different brassicaceous plants across generations. We investigated how disturbance of the parental bacterial community by antibiotic treatment affects F1 larval traits. We tested the effects depending on whether F1 larvae are feeding on the same plant species as their parents or on a different one. The parental treatment alone had no impact on the biomass of F1 larvae feeding on the parental plant species. However, the parental treatment had a detrimental effect on F1 larval biomass when F1 larvae had a different host plant than their parents. This effect was linked to higher larval prophenoloxidase activity and greater downregulation of the major allergen gene (MA), a glucosinolate detoxification gene of P. brassicae Bacterial abundance in untreated adult parents was high, while it was very low in F1 larvae from either parental type, and thus unlikely to directly influence larval traits. Our results suggest that transgenerational effects of the parental microbiome on the offspring's phenotype become evident when the offspring is exposed to a transgenerational host plant shift.IMPORTANCE Resident bacterial communities are almost absent in larvae of butterflies and thus are unlikely to affect their host. In contrast, adult butterflies contain conspicuous amounts of bacteria. While the host plant and immune state of adult parental butterflies are known to affect offspring traits, it has been unclear whether also the parental microbiome imposes direct effects on the offspring. Here, we show that disturbance of the bacterial community in parental butterflies by an antibiotic treatment has a detrimental effect on those offspring larvae feeding on a different host plant than their parents. Hence, the study indicates that disturbance of an insect's parental microbiome by an antibiotic treatment shapes how the offspring individuals can adjust themselves to a novel host plant.

Plant responses to insect eggs are not induced by egg-associated microbes, but by a secretion attached to the eggs.

Plants can enhance their defence against herbivorous insects by responding to insect egg depositions preceding larval feeding. The similarity of plant responses to insect eggs with those to phytopathogens gave rise to the hypothesis that egg-associated microbes might act as elicitors. We tested this hypothesis by investigating first if elimination of microbes in the butterfly Pieris brassicae changes the responses of Brassica nigra and Arabidopsis thaliana to eggs and larvae of this insect species. An antibiotic treatment of butterflies mitigated the plant transcriptional response to the eggs and the egg-mediated enhancement of the plant's defence against larvae. However, application of cultivated microbial isolates from the eggs onto Arabidopsis thaliana did not enhance the plant's anti-herbivore defence. Instead, application of an egg-associated glandular secretion, which is attaching the eggs to the leaves, elicited the enhancing effect on the plant's defence against larvae. However, this effect was only achieved when the secretion was applied in similar quantities as released by control butterflies, but not when applied in the reduced quantity as released by antibiotic-treated butterflies. We conclude that glandular secretions rather than egg-associated microbes act in a dose-dependent manner as elicitor of the egg-mediated enhancement of the plant's defence against insect larvae.

Daphnia galeata responds to the exposure to an ichthyosporean gut parasite by down-regulation of immunity and lipid metabolism.

BACKGROUND: Regulatory circuits of infection in the emerging experimental model system, water flea Daphnia and their microparasites, remain largely unknown. Here we provide the first molecular insights into the response of Daphnia galeata to its highly virulent and common parasite Caullerya mesnili, an ichthyosporean that infects the gut epithelium. We generated a transcriptomic dataset using RNAseq from parasite-exposed (vs. control) Daphnia, at two time points (4 and 48 h) after parasite exposure. RESULTS: We found a down-regulation of metabolism and immunity-related genes, at 48 h (but not 4 h) after parasite exposure. These genes are involved in lipid metabolism and fatty acid biosynthesis, as well as microbe recognition (e.g. c-type lectins) and pathogen attack (e.g. gut chitin). CONCLUSIONS: General metabolic suppression implies host energy shift from reproduction to survival, which is in agreement with the known drastic reduction in Daphnia fecundity after Caullerya infection. The down-regulation of gut chitin indicates a possible interaction between the peritrophic matrix and the evading host immune system. Our study provides the first description of host transcriptional responses in this very promising host-parasite experimental system.

Host and Symbiont Jointly Control Gut Microbiota during Complete Metamorphosis.

Holometabolous insects undergo a radical anatomical re-organisation during metamorphosis. This poses a developmental challenge: the host must replace the larval gut but at the same time retain symbiotic gut microbes and avoid infection by opportunistic pathogens. By manipulating host immunity and bacterial competitive ability, we study how the host Galleria mellonella and the symbiotic bacterium Enterococcus mundtii interact to manage the composition of the microbiota during metamorphosis. Disenabling one or both symbiotic partners alters the composition of the gut microbiota, which incurs fitness costs: adult hosts with a gut microbiota dominated by pathogens such as Serratia and Staphylococcus die early. Our results reveal an interaction that guarantees the safe passage of the symbiont through metamorphosis and benefits the resulting adult host. Host-symbiont "conspiracies" as described here are almost certainly widespread in holometobolous insects including many disease vectors.

Evolution of defence cocktails: Antimicrobial peptide combinations reduce mortality and persistent infection.

The simultaneous expression of costly immune effectors such as multiple antimicrobial peptides is a hallmark of innate immunity of multicellular organisms, yet the adaptive advantage remains unresolved. Here, we test current hypotheses on the evolution of such defence cocktails. We use RNAi gene knock-down to explore, the effects of three highly expressed antimicrobial peptides, displaying different degrees of activity in vitro against Staphylococcus aureus, during an infection in the beetle Tenebrio molitor. We find that a defensin confers no survival benefit but reduces bacterial loads. A coleoptericin contributes to host survival without affecting bacterial loads. An attacin has no individual effect. Simultaneous knock-down of the defensin with the other AMPs results in increased mortality and elevated bacterial loads. Contrary to common expectations, the effects on host survival and bacterial load can be independent. The expression of multiple AMPs increases host survival and contributes to the control of persisting infections and tolerance. This is an emerging property that explains the adaptive benefit of defence cocktails.

Sex-specific gene expression in the mosquito Culex pipiens f. molestus in response to artificial light at night.

BACKGROUND: Artificial light at night (ALAN) is a typical feature of urban areas and most organisms living in urban or suburban habitats are exposed to low levels of ALAN. Light is one of the most important environmental cues that organisms use to regulate their activities. Studies have begun to quantify the influence of ALAN on the behavior and ecology of organisms, but research on the effects at the molecular level remains limited. Mosquitoes in the Culex pipiens complex (Diptera, Culicidae) are widespread and abundant in urban areas where they are potential disease vectors. It is thus of particular interest to understand how ALAN may influence biologically and ecologically relevant traits. RESULTS: We used RNAseq to evaluate the transcriptome response in a Cx. pipiens f. molestus laboratory population that was exposed to near-natural light conditions (light:dark L16:D8 hours, "control") and ALAN conditions with 3 h of constant low-level light at night (L16 + Llow3:D5 hours, "low-light"). The resulting transcripts were mapped to the reference genome of the closely related Culex quinquefasciatus. Female expression patterns differed significantly between control and treatment conditions at five genes although none showed an absolute fold change greater than two (FC > 2). In contrast, male expression differed at 230 genes (74 with FC > 2). Of these, 216 genes (72 with FC > 2) showed reduced expression in the low-light treatment, most of which were related to gametogenesis, lipid metabolism, and immunity. Of the 14 genes (two with FC > 2) with increased expression, only five had any functional annotation. There was a pronounced sex-bias in gene expression regardless of treatment, with 11,660 genes (51 % of annotated genes; 8694 with FC > 2; 48 % of annotated genes) differentially expressed between males and females, including 14 genes of the circadian clock. CONCLUSION: Our data suggest a stronger response to artificial light by males of Cx. pipiens f. molestus than by females, and that a wide range of physiological pathways may be affected by ALAN at the molecular level. The fact that differences in gene expression appear to be sex-specific may have a strong influence at the population level.

Eating in a losing cause: limited benefit of modified macronutrient consumption following infection in the oriental cockroach Blatta orientalis.

BACKGROUND: Host-pathogen interactions can lead to dramatic changes in host feeding behaviour. One aspect of this includes self-medication, where infected individuals consume substances such as toxins or alter their macronutrient consumption to enhance immune competence. Another widely adopted animal response to infection is illness-induced anorexia, which is thought to assist host immunity directly or by limiting the nutritional resources available to pathogens. Here, we recorded macronutrient preferences of the global pest cockroach, Blatta orientalis to investigate how shifts in host macronutrient dietary preference and quantity of carbohydrate (C) and protein (P) interact with immunity following bacterial infection. RESULTS: We find that B. orientalis avoids diets enriched for P under normal conditions, and that high P diets reduce cockroach survival in the long term. However, following bacterial challenge, cockroaches significantly reduced their overall nutrient intake, particularly of carbohydrates, and increased the relative ratio of protein (P:C) consumed. Surprisingly, these behavioural shifts had a limited effect on cockroach immunity and survival, with minor changes to immune protein abundance and antimicrobial activity between individuals placed on different diets, regardless of infection status. CONCLUSIONS: We show that cockroach feeding behaviour can be modulated by a pathogen, resulting in an illness-induced anorexia-like feeding response and a shift from a C-enriched to a more P:C equal diet. However, our results also indicate that such responses do not provide significant immune protection in B. orientalis, suggesting that the host's dietary shift might also result from random rather than directed behaviour. The lack of an apparent benefit of the shift in feeding behaviour highlights a possible reduced importance of diet in immune regulation in these invasive animals, although further investigations employing pathogens with alternative infection strategies are warranted.

Signalling pathways and mechanistic cues highlighted by transcriptomic analysis of primordial, primary, and secondary ovarian follicles in domestic cat.

In vitro growth (IVG) of dormant primordial ovarian follicles aims to produce mature competent oocytes for assisted reproduction. Success is dependent on optimal in vitro conditions complemented with an understanding of oocyte and ovarian follicle development in vivo. Complete IVG has not been achieved in any other mammalian species besides mice. Furthermore, ovarian folliculogenesis remains sparsely understood overall. Here, gene expression patterns were characterised by RNA-sequencing in primordial (PrF), primary (PF), and secondary (SF) ovarian follicles from Felis catus (domestic cat) ovaries. Two major transitions were investigated: PrF-PF and PF-SF. Transcriptional analysis revealed a higher proportion in gene expression changes during the PrF-PF transition. Key influencing factors during this transition included the interaction between the extracellular matrix (ECM) and matrix metalloproteinase (MMPs) along with nuclear components such as, histone HIST1H1T (H1.6). Conserved signalling factors and expression patterns previously described during mammalian ovarian folliculogenesis were observed. Species-specific features during domestic cat ovarian folliculogenesis were also found. The signalling pathway terms "PI3K-Akt", "transforming growth factor-ß receptor", "ErbB", and "HIF-1" from the functional annotation analysis were studied. Some results highlighted mechanistic cues potentially involved in PrF development in the domestic cat. Overall, this study provides an insight into regulatory factors and pathways during preantral ovarian folliculogenesis in domestic cat.

Resistance Evolution Against Antimicrobial Peptides in Staphylococcus aureus Alters Pharmacodynamics Beyond the MIC.

Antimicrobial peptides (AMPs) have been proposed as a promising class of new antimicrobials partly because they are less susceptible to bacterial resistance evolution. This is possibly caused by their mode of action but also by their pharmacodynamic characteristics, which differ significantly from conventional antibiotics. Although pharmacodynamics of antibiotic resistant strains have been studied, such data are lacking for AMP resistant strains. Here, we investigated if the pharmacodynamics of the Gram-positive human pathogen Staphylococcous aureus evolve under antimicrobial peptide selection. Interestingly, the Hill coefficient (kappa κ) evolves together with the minimum inhibition concentration (MIC). Except for one genotype, strains harboring mutations in menF and atl, all mutants had higher kappa than the non-selected sensitive controls. Higher κ results in steeper pharmacodynamic curve and, importantly, in a narrower mutant selection window. S. aureus selected for resistance to melittin displayed cross resistant against pexiganan and had as steep pharmacodynamic curves (high κ) as pexiganan-selected lines. By contrast, the pexiganan-sensitive tenecin-selected lines displayed lower κ. Taken together, our data demonstrate that pharmacodynamic parameters are not fixed traits of particular drug/strain interactions but actually evolve under drug treatment. The contribution of factors such as κ and the maximum and minimum growth rates on the dynamics and probability of resistance evolution are open questions that require urgent attention.

A mid-gut microbiota is not required for the pathogenicity of Bacillus thuringiensis to diamondback moth larvae.

The mode of action of the entomopathogenic bacterium Bacillus thuringiensis (Bt) remains a matter of debate. Recent reports have claimed that aseptic lepidopteran hosts were not susceptible to Bt and that inoculation with mid-gut bacteria restores pathogenicity. These claims are controversial because larvae were rendered aseptic by consuming antibiotics, although the effect of these antibiotics on Bt was not examined. We tested the generality of the mid-gut bacteria hypothesis in the diamondback moth, Plutella xylostella using properly controlled experiments that investigated the effect of antibiotic consumption and absence of gut microbiota separately. We found that purified Bt toxin and spore/toxin mixtures were fully pathogenic to larvae reared aseptically. Persistence of antibiotics in larval tissues was implicated in reducing host mortality because larval consumption of the antibiotic rifampicin reduced the pathogenicity of rifampicin-sensitive Bt strains but not rifampicin-resistant strains. Inoculating larvae with Enterobacter sp. Mn2 reduced the mortality of larvae feeding on Bt HD-1 and the presence of a culturable gut microbiota also reduced the pathogenicity of the Bt toxin Cry1Ac, in agreement with other studies indicating that an intestinal microbiota can protect taxonomically diverse hosts from pathogen attack. As ingestion of antibiotics suppresses host mortality the vegetative growth of Bt in the host must be important for its pathogenicity. Furthermore, claims that aseptic larvae are not susceptible to Bt must be supported by experiments that control for the effect of administering antibiotics.

Gut bacteria are not required for the insecticidal activity of Bacillus thuringiensis toward the tobacco hornworm, Manduca sexta.

It was recently proposed that gut bacteria are required for the insecticidal activity of the Bacillus thuringiensis-based insecticide, DiPel, toward the lepidopterans Manduca sexta, Pieris rapae, Vanessa cardui, and Lymantria dispar. Using a similar methodology, it was found that gut bacteria were not required for the toxicity of DiPel or Cry1Ac or for the synergism of an otherwise sublethal concentration of Cry1Ac toward M. sexta. The toxicities of DiPel and of B. thuringiensis HD73 Cry(-) spore/Cry1Ac synergism were attenuated by continuously exposing larvae to antibiotics before bioassays. Attenuation could be eliminated by exposing larvae to antibiotics only during the first instar without altering larval sterility. Prior antibiotic exposure did not attenuate Cry1Ac toxicity. The presence of enterococci in larval guts slowed mortality resulting from DiPel exposure and halved Cry1Ac toxicity but had little effect on B. thuringiensis HD73 Cry(-) spore/Cry1Ac synergism. B. thuringiensis Cry(-) cells killed larvae after intrahemocoelic inoculation of M. sexta, Galleria mellonella, and Spodoptera litura and grew rapidly in plasma from M. sexta, S. litura, and Tenebrio molitor. These findings suggest that gut bacteria are not required for B. thuringiensis insecticidal activity toward M. sexta but that B. thuringiensis lethality is reduced in larvae that are continuously exposed to antibiotics before bioassay.

Complete metamorphosis of insects.

The majority of described hexapod species are holometabolous insects, undergoing an extreme form of metamorphosis with an intercalated pupal stage between the larva and adult, in which organs and tissues are extensively remodelled and in some cases completely rebuilt. Here, we review how and why this developmental strategy has evolved. While there are many theories explaining the evolution of metamorphosis, many of which fit under the hypothesis of decoupling of life stages, there are few clear adaptive hypotheses on why complete metamorphosis evolved. We propose that the main adaptive benefit of complete metamorphosis is decoupling between growth and differentiation. This facilitates the exploitation of ephemeral resources and enhances the probability of the metamorphic transition escaping developmental size thresholds. The evolution of complete metamorphosis comes at the cost of exposure to predators, parasites and pathogens during pupal life and requires specific adaptations of the immune system at this time. Moreover, metamorphosis poses a challenge for the maintenance of symbionts and the gut microbiota, although it may also offer the benefit of allowing an extensive change in microbiota between the larval and adult stages. The regulation of metamorphosis by two main players, ecdysone and juvenile hormone, and the related signalling cascades are now relatively well understood. The mechanics of metamorphosis have recently been studied in detail because of the advent of micro-CT and research into the role of cell death in remodelling tissues and organs. We support the argument that the adult stage must necessarily have preceded the larval form of the insect. We do not resolve the still contentious question of whether the larva of insects in general originated through the modification of existing preadult forms or through heterochrony as a modified embryonic stage (pronymph), nor whether the holometabolous pupa arose as a modified hemimetabolous final stage larva. This article is part of the theme issue 'The evolution of complete metamorphosis'.

Immune gene regulation in the gut during metamorphosis in a holo- versus a hemimetabolous insect.

During metamorphosis, holometabolous insects completely replace the larval gut and must control the microbiota to avoid septicaemia. Rapid induction of bactericidal activity in the insect gut at the onset of pupation has been described in numerous orders of the Holometabola and is best-studied in the Lepidoptera where it is under control of the 20-hydroxyecdysone (20E) moulting pathway. Here, using RNAseq, we compare the expression of immune effector genes in the gut during metamorphosis in a holometabolous (Galleria mellonella) and a hemimetabolous insect (Gryllus bimaculatus). We find that in G. mellonella, the expression of numerous immune effectors and the transcription factor GmEts are upregulated, with peak expression of three antimicrobial peptides (AMPs) and a lysozyme coinciding with delamination of the larval gut. By contrast, no such upregulation was detectable in the hemimetabolous Gr. bimaculatus. These findings support the idea that the upregulation of immune effectors at the onset of complete metamorphosis is an adaptive response, which controls the microbiota during gut replacement. This article is part of the theme issue 'The evolution of complete metamorphosis'.

In vivo exposure of insect AMP resistant Staphylococcus aureus to an insect immune system.

Antimicrobial peptides (AMPs) are important immune effectors in insects. Bacteria have a limited number of ways to resist AMPs, and AMP-resistance is often costly. Recently, it has become clear that AMP activities in vitro and in vivo differ. Although some studies have followed the in vivo survival of AMP resistant pathogens, studying a pathogen resistant to the AMPs of that particular host has never been reported. Here, we infected the mealworm beetle Tenebrio molitor with Staphylococcus aureus strains that were evolved in vitro in the presence of one or two antimicrobial peptides from T. molitor. We found that the Tenebrio immune system could clear mutant Tenecin resistant strains at least as efficiently as sensitive controls. The bacterial load of Tenecin resistant S. aureus segregated by mutation. Strains with mutations in both the pmt and rpo operons showed the highest in vivo survival and therefore showed the lowest fitness cost amongst the evolved resistance mutations. In contrast, Tenecin resistant strains with mutations in the nsa and rpo operons showed much lower survival within the hosts. Our study shows that Tenecin resistant strains are phagocytosed at a lower rate. The nsa/rpo mutants were phagocytosed at a higher rate than other Tenecin resistant S. aureus strains. The differences in resistance against AMPs and phagocytosis did not translate into changes in virulence. AMP resistance, while a prerequisite for an infection in vertebrates, does not provide a survival advantage to S. aureus in a host environment that is dominated by AMPs.

Gene expression response to a nematode parasite in novel and native eel hosts.

Invasive parasites are involved in population declines of new host species worldwide. The high susceptibilities observed in many novel hosts have been attributed to the lack of protective immunity to the parasites which native hosts acquired during their shared evolution. We experimentally infected Japanese eels (Anguilla japonica) and European eels (Anguilla anguilla) with Anguillicola crassus, a nematode parasite that is native to the Japanese eel and invasive in the European eel. We inferred gene expression changes in head kidney tissue from both species, using RNA-seq data to determine the responses at two time points during the early stages of infection (3 and 23 days postinfection). At both time points, the novel host modified the expression of a larger and functionally more diverse set of genes than the native host. Strikingly, the native host regulated immune gene expression only at the earlier time point and to a small extent while the novel host regulated these genes at both time points. A low number of differentially expressed immune genes, especially in the native host, suggest that a systemic immune response was of minor importance during the early stages of infection. Transcript abundance of genes involved in cell respiration was reduced in the novel host which may affect its ability to cope with harsh conditions and energetically demanding activities. The observed gene expression changes in response to a novel parasite that we observed in a fish follow a general pattern observed in amphibians and mammals, and suggest that the disruption of physiological processes, rather than the absence of an immediate immune response, is responsible for the higher susceptibility of the novel host.