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An important contributing factor to the evolutionary success of insects is nutritional association with microbial symbionts, which provide the host insects with nutrients lacking in their unbalanced diets. These symbionts are often compartmentalized in specialized cells of the host, the bacteriocytes. Even though bacteriocytes were first described more than a century ago, few studies have explored their dynamics throughout the insect life cycle and in response to environmental stressors. Here, we use the Buchnera aphidicola/pea aphid symbiotic system to study how bacteriocytes are regulated in response to nutritional stress throughout aphid development. Using artificial diets, we analyzed the effects of depletion or excess of phenylalanine or leucine, two amino acids essential for aphid growth and whose biosynthetic pathways are shared between the host and the symbiont. Bacteriocytes responded dynamically to those treatments, while other tissues showed no obvious morphological change. Amino acid depletion resulted in an increase in bacteriocyte numbers, with the extent of the increase depending on the amino acid, while excess either caused a decrease (for leucine) or an increase (for phenylalanine). Only a limited impact on survival and fecundity was observed, suggesting that the adjustment in bacteriocyte (and symbiont) numbers is sufficient to withstand these nutritional challenges. We also studied the impact of more extreme conditions by exposing aphids to a 24 h starvation period at the beginning of nymphal development. This led to a dramatic drop in aphid survival and fecundity and a significant developmental delay. Again, bacteriocytes responded dynamically, with a considerable decrease in number and size, correlated with a decrease in the number of symbionts, which were prematurely degraded by the lysosomal system. This study shows how bacteriocyte dynamics is integrated in the physiology of insects and highlights the high plasticity of these cells. ispartof: FRONTIERS IN PHYSIOLOGY vol:13 ispartof: location:Switzerland status: published

Insects are associated with multiple microbes that have been reported to influence various aspects of their biology. Most studies in insects, including pest species, focus on the bacterial communities of the microbiome even though the microbiome consists of members of many more kingdoms, which can also have large influence on the life history of insects. In this review, we present some key examples of how the different members of the microbiome, such as bacteria, fungi, viruses, archaea, and protozoa, affect the fitness and behavior of pest insects. Moreover, we argue that interactions within and among microbial groups are abundant and of great importance, necessitating the use of a community approach to study microbial-host interactions. We propose that the restricted focus on bacteria very likely hampers our understanding of the functioning and impact of the microbiome on the biology of pest insects. We close our review by highlighting a few open questions that can provide an in-depth understanding of how other components of the microbiome, in addition to bacteria, might influence host performance, thus contributing to pest insect ecology.

International audience; Transgenerational phenotypic plasticity is a fast non-genetic response to environmental modifications that can buffer the effects of environmental stresses on populations. However, little is known about the evolution of plasticity in the absence of standing genetic variation although several non-genetic inheritance mechanisms have now been identified. Here we monitored the pea aphid transgenerational phenotypic response to ladybird predators (production of winged offspring) during 27 generations of experimental evolution in the absence of initial genetic variation (clonal multiplication starting from a single individual). We found that the frequency of winged aphids first increased rapidly in response to predators and then remained stable over 25 generations, implying a stable phenotypic reconstruction at each generation. We also found that the high frequency of winged aphids persisted for one generation after removing predators. Winged aphid frequency then entered a refractory phase during which it dropped below the level of control lines for at least two generations before returning to it. Interestingly, the persistence of the winged phenotype decreased and the refractory phase lasted longer with the increasing number of generations of exposure to predators. Finally, we found that aphids continuously exposed to predators for 22 generations evolved a significantly weaker plastic response than aphids never exposed to predators, which, in turn, increased their fitness in presence of predators. Our findings therefore showcased an example of experimental evolution of plasticity in the absence of initial genetic variation and highlight the importance of integrating several components of non-genetic inheritance to detect evolutionary responses to environmental changes.

UMR BGPI équipe 6; UMR CBGP; Aphids are intimately linked with their host plants that constitute their only food resource and habitat, and thus impose considerable selective pressure on their evolution. It is therefore commonly assumed that host plants have greatly influenced the diversification of aphids. Here, we review what is known about the role of host plant association on aphid speciation by examining both macroevolutionary and population-level studies. Phylogenetic studies conducted at different taxonomic levels show that, as in many phytophagous insect groups, the radiation of angiosperms has probably favoured the major Tertiary diversification of aphids. These studies also highlight many aphid lineages constrained to sets of related host plants, suggesting strong evolutionary commitment in aphids' host plant choice, but they fail to document cospeciation events between aphid and host lineages. Instead, phylogenies of several aphid genera reveal that divergence events are often accompanied by host shifts, and suggest, without constituting a formal demonstration, that aphid speciation could be a consequence of adaptation to new hosts. Experimental and field studies below the species level support reproductive isolation between host races as partly due to divergent selection by their host plants. Selected traits are mainly feeding performances and life cycle adaptations to plant phenology. Combined with behavioural preference for favourable host species, these divergent adaptations can induce pre- and post-zygotic barriers between host-specialized aphid populations. However, the hypothesis of host-driven speciation is seldom tested formally and must be weighed against overlooked explanations involving geographic isolation and non-ecological reproductive barriers in the process of speciation.

1. Theoretical models predict that ovipositional decisions of parasitoid females should lead to the selection of the most profitable host for parasitoid development. Most parasitoid species have evolved specific adaptations to exploit a single host stage. However, females of the aphid hyperparasitoid Syrphophagous aphidivorus (Mayr) (Hymenoptera: Encyrtidae) display a unique and atypical oviposition behaviour by attacking either primary parasitoid larvae in live aphids, or parasitoid pupae in dead, mummified aphids. 2. In the laboratory, the correlation between host suitability and host preference of S. aphidivorus on the host Aphidius nigripes Ashmead parasitising the aphid Macrosiphum euphorbiae (Thomas) was investigated. 3. The relative suitability of the two host stages was determined by measuring hyperparasitoid fitness parameters (survival, development time, fecundity, sex ratio, and adult size of progeny), and calculating the intrinsic rate of population increase (rm). Host preference by S. aphidivorus females and the influence of aphid defence behaviour on host selection was also examined. 4. Hyperparasitoid offspring performance was highest when developing from hosts in aphid mummies and females consistently preferred this host to hosts in parasitised aphids. Although aphid defensive behaviour may influence host selection, it was not a determining factor. Ecological and evolutionary processes that might have led to dual oviposition behaviour in S. aphidivorus are discussed. [KEYWORDS: Host suitability ; offspring fitness ; oviposition ; preference ; parasitoid life history]

The mustard trypsin inhibitor, MTI-2, is a potent inhibitor of trypsin with no activity towards chymotrypsin. MTI-2 is toxic for lepidopteran insects, but has low activity against aphids. In an attempt to improve the activity of the inhibitor towards aphids, a library of inhibitor variants was constructed and cloned into the pRlac3 phagemid vector. The library of 9.3 107 independent colonies was created by randomisation of a stretch of five consecutive codons in the reactive site. Repeated selection rounds against bovine trypsin and chymotrypsin allowed the identification of novel, MTI-2 derived, antitrypsin and antichymotrypsin inhibitors. Chy8, the selected variant with highest affinity for bovine chymotrypsin (Ki = 32 nm versus >1000 nm for the wild-type) represents the strongest known recombinant chymotrypsin inhibitor of the MTI-2 family. It is highly toxic to nymphs of the aphid Acyrthosiphon pisum, and moderately toxic to nymphs of Aphis gossypii and Myzus persicae. The LC50 of 73 ?g ml1 towards A. pisum is the lowest value known among chymotrypsin inhibitors. The aphicidal activity of Chy8 was improved eightfold compared to the wild-type inhibitor. This demonstrates, for the first time, that bovine chymotrypsin provides a useful template to select engineered proteins highly toxic against these aphids. The selected gene will allow the development of transgenic crops that are protected against sucking insect pests.

Climate change can alter the phenology of organisms. It may thus lead seasonal organisms to face different day lengths than in the past, and the fitness consequences of these changes are as yet unclear. To study such effects, we used the pea aphid Acyrthosiphon pisum as a model organism, as it has obligately asexual clones which can be used to study day length effects without eliciting a seasonal response. We recorded life-history traits under short and long days, both with two realistic temperature cycles with means differing by 2 degrees C. In addition, we measured the population growth of aphids on their host plant Pisum sativum. We show that short days reduce fecundity and the length of the reproductive period of aphids. Nevertheless, this does not translate into differences at the population level because the observed fitness costs only become apparent late in the individual's life. As expected, warm temperature shortens the development time by 0.7 days/degrees C, leading to faster generation times. We found no interaction of temperature and day length. We conclude that day length changes cause only relatively mild costs, which may not decelerate the increase in pest status due to climate change.

Aphids maintain an obligate, endosymbiotic association with Buchnera sp., a bacterium closely related to Escherichia coli. Bacteria are housed in specialized cells of organ-like structures called bacteriomes in the hemocoel of the aphid and are maternally transmitted. Phylogenetic studies have shown that the association had a single origin, dated about 200-250 million years ago, and that host and endosymbiont lineages have evolved in parallel since then. However, the pattern of deepest branching within the aphid family remains unsolved, which thereby hampers tin appraisal of, for example, the role played by horizontal gene transfer in the early evolution of Buchnera. The main role of Buchnera in this association is the biosynthesis and provisioning of essential amino acids to its aphid host. Physiological and metabolic studies have recently substantiated such nutritional role. In addition, genetic studies of Buchnera from several aphids have shown additional modifications, such as strong genome reduction, high A+T content compared to free-living bacteria, differential evolutionary rates, a relative increase in the number of non-synonymous substitutions, and gene amplification mediated by plasmids. Symbiosis is an active process in insect evolution cis revealed by the intermediate values of the previous characteristics showed by secondary symbionts compared to free-living bacteria and Buchnera.

The biochemical profile and metabolism of Acyrthosiphon pisum (Harris) (Homoptera, Aphididae) are markedly altered and redirected in response to parasitization by the endophagous braconid Aphidius ervi Haliday (Hymenoptera, Braconidae). In the present study, the role played in the host regulation process by teratocytes, cells deriving from the dissociation of the embryonic membrane of the parasitoid, is taken into consideration. The protein synthesis activity of these cells of embryonic origin is analysed in vitro and an essential characterization of those proteins de novo synthesized and released in the incubation medium is provided. Teratocytes, obtained by dissecting parasitized host aphids, 3, 4 and 5 days after parasitoid oviposition, were incubated in vitro and, at the end of the incubation period, were separated from the medium for SDS-PAGE analysis of both cellular and secreted proteins. Various cellular proteins were more abundant as the time between parasitization and teratocyte collection increased. Furthermore, two proteins, showing an approximate molecular mass of 15 kD (p15) and 45 kD (p45) respectively, were abundantly secreted in the incubation medium by 5 day-old teratocytes. Incubations in presence of S-35 radiolabelled amino acids indicated that p15 and p45 are both synthesized by A. ervi teratocytes. The amino acid composition of these two proteins was similar to that reported for other insect proteins with a demonstrated nutritional function. The p45 protein was found to be glycosylated. A tentative physiological model describing the host regulation role played by different parasitoid-derived factors is proposed.

Background The widespread use of genome sequencing provided evidences for the high degree of conservation in innate immunity signalling pathways across animal phyla. However, the functioning and evolutionary history of immune-related genes remains unknown for most invertebrate species. A striking observation coming from the analysis of the pea aphid Acyrthosiphon pisum genome is the absence of important conserved genes known to be involved in the antimicrobial responses of other insects. This reduction in antibacterial immune defences is thought to be related to their long-term association with beneficial symbiotic bacteria and to facilitate symbiont maintenance. An additional possibility to avoid elimination of mutualistic symbionts is a fine-tuning of the host immune response. To explore this hypothesis we investigated the existence and potential involvement of immune regulators in aphid agonistic and antagonistic interactions. Results In contrast to the limited antibacterial arsenal, we showed that the pea aphid Acyrthosiphon pisum expresses 5 members of Macrophage Migration Inhibitory Factors (ApMIF), known to be key regulators of the innate immune response. In silico searches for MIF members in insect genomes followed by phylogenetic reconstruction suggest that evolution of MIF genes in hemipteran species has been shaped both by differential losses and serial duplications, raising the question of the functional importance of these genes in aphid immune responses. Expression analyses of ApMIFs revealed reduced expression levels in the presence, or during the establishment of secondary symbionts. By contrast, ApMIFs expression levels significantly increased upon challenge with a parasitoid or a Gram-negative bacteria. This increased expression in the presence of a pathogen/parasitoid was reduced or missing, in the presence of facultative symbiotic bacteria. Conclusions This work provides evidence that while aphid’s antibacterial arsenal is reduced, other immune genes widely absent from insect genomes are present, diversified and differentially regulated during antagonistic or agonistic interactions. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-762) contains supplementary material, which is available to authorized users.

International audience; The host-associated differentiation (HAD) hypothesis states that higher trophic levels in parasitic associations should exhibit similar divergence in case of host sympatric speciation. We tested HAD on populations of Aphidius ervi the main parasitoid of the pea aphid Acyrthosiphon pisum, emerging from host populations specialized on either alfalfa or red clover. Host and parasitoid populations were assessed for genetic variation and structure, while considering geography, host plant and host aphid protective symbionts Regiella insecticola and Hamiltonella defensa as potential covariables. Cluster and hierarchical analyses were used to assess the contribution of these variables to population structure, based on genotyping pea aphids and associated A. ervi with microsatellites, and host aphid facultative symbionts with 16S rDNA markers. Pea aphid genotypes were clearly distributed in two groups closely corresponding with their plant origins, confirming strong plant associated differentiation of this aphid in North America. Overall parasitism by A. ervi averaged 21.5 % across samples, and many parasitized aphids producing a wasp hosted defensive bacteria, indicating partial or ineffective protective efficacy of these symbionts in the field. The A. ervi population genetic data failed to support differentiation according to the host plant association of their pea aphid host. Potential for parasitoid specialization was also explored in experiments where wasps from alfalfa and clover aphids were reciprocally transplanted on alternate hosts, the hypothesis being that wasp behaviour and parasitic stages should be most adapted to their host of origin. Results revealed higher probability of oviposition on the alfalfa aphids, but higher adult emergence success on red clover aphids, with no interaction as expected under HAD. We conclude that our study provides no support for the HAD in this system. We discuss factors that might impair A. ervi specialization on its divergent aphid hosts on alfalfa and clover.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Understanding the drivers of speciation is critical to interpreting patterns of biodiversity. The identification of the genetic changes underlying adaptation and reproductive isolation is necessary to link barriers to gene flow to the causal origins of divergence. Here, we present a novel approach to the genetics of speciation, which should complement the commonly used approaches of quantitative trait locus mapping and genome-wide scans for selection. We present a large-scale candidate gene approach by means of sequence capture, applied to identifying the genetic changes underlying reproductive isolation in the pea aphid, a model system for the study of ecological speciation. Targeted resequencing enabled us to scale up the candidate gene approach, specifically testing for the role of chemosensory gene families in host plant specialization. Screening for the signature of divergence under selection at 172 candidate and noncandidate loci, we revealed a handful of loci that show high levels of differentiation among host races, which almost all correspond to odorant and gustatory receptor genes. This study offers the first indication that some chemoreceptor genes, often tightly linked together in the genome, could play a key role in local adaptation and reproductive isolation in the pea aphid and potentially other phytophagous insects. Our approach opens a new route toward the functional genomics of ecological speciation.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Most aphids show reproductive polyphenism, i.e. they alternate their reproductive modes from parthenogenesis to sexual reproduction in response to short photoperiods. Although juvenile hormone (JH) has been considered a likely candidate for regulating the transition from asexual to sexual reproduction after photoperiod sensing, there are few studies investigating the direct relationship between JH titres and the reproductive-mode change. In addition, the sequencing of the pea aphid genome has allowed identification of the genes involved in the JH pathway, which in turn allows us to examine their expression levels in relation to the reproductive-mode change. Using liquid chromatography-mass spectrometry in the pea aphid, JHIII titre was shown to be lower in aphids producing sexual morphs under short-day conditions than in aphids producing parthenogenetic morphs under long-day conditions. The expression levels of genes upstream and downstream of JH action were quantified by real-time quantitative reverse-transcription-PCR across the reproductive-mode change. The expression level of JH esterase, which is responsible for JH degradation, was significantly higher in aphids reared under short-day conditions. This suggests that the upregulation of the JH degradation pathway may be responsible for the lower JHIII titre in aphids exposed to short-days, leading to the production of sexual morphs.

International audience; Sex chromosomes play a role in many important biological processes, including sex determination, genomic conflicts, imprinting and speciation. In particular, they exhibit several unusual properties such as inheritance pattern, hemizygosity and reduced recombination, which influence their response to evolutionary factors (e.g. drift, selection, demography). Here, we examine the evolutionary forces driving X chromosome evolution in aphids, an XO system where females are homozygous (XX) and males hemizygous (X0) at sex chromosomes. We show by simulations that the unusual mode of transmission of the X chromosome in aphids coupled to cyclical parthenogenesis results in similar effective population sizes and predicted levels of genetic diversity for X chromosomes and autosomes under neutral evolution. These results contrast with expectations from standard XX/XY or XX/X0 systems (where the effective population size of the X is 3/4 of that of autosomes) and have deep consequences for aphid X chromosome evolution. We then localized 52 microsatellites markers on the X and 351 on autosomes. We genotyped 167 individuals with 356 of these loci and found similar levels of allelic richness on the X and on the autosomes, as predicted by our simulations. In contrast, we detected higher dN and dN/dS ratio for X-linked genes compared to autosomal genes, a pattern compatible with either positive or relaxed selection. Given that both types of chromosomes have similar effective population sizes and that the single copy of the X chromosome of male aphids exposes its recessive genes to selection, some degree of positive selection seems to best explain the higher rates of evolution of X-linked genes. Overall, this study highlights the particular relevance of aphids to study the evolutionary factors driving sex chromosomes and genome evolution.

International audience; Insects harbour a wild diversity of symbionts that can spread and persist within populations by providing benefits to their host. The pea aphid Acyrthosiphon pisum maintains a facultative symbiosis with the bacterium Hamiltonella defensa, which provides enhanced resistance against the aphid parasitoid Aphidius ervi. Although the mechanisms associated with this symbiotic-mediated protection have been investigated thoroughly, little is known about its evolutionary effects on parasitoid populations. We used an experimental evolution procedure in which parasitoids were exposed either to highly resistant aphids harbouring the symbiont or to low innate resistant hosts free of H. defensa. Parasitoids exposed to H. defensa gained virulence over time, reaching the same parasitism rate as those exposed to low aphid innate resistance only. A fitness reduction was associated with this adaptation as the size of parasitoids exposed to H. defensa decreased through generations. This study highlighted the considerable role of symbionts in host-parasite co-evolutionary dynamics.

Most terrestrial prey species are assumed to assess predation risk by detecting predators directly rather than using cues of previous attacks on conspecifics. However, such cues might represent valuable information, and prey can be expected to respond to the presence of congeners killed by enemies. Such cues are available in aphid colonies attacked by parasitic wasps because they do not remove parasitized hosts from the colony. Colonies are thus often a mixture of healthy, parasitized, and killed aphids, which corpses ("mummies") stay attached to the plant and can be encountered by live aphids. Aphids exhibit a dispersal polyphenism. Recent studies show that they produce more winged offspring when directly exposed to natural enemies or to alarm pheromone emitted by conspecifics. We hypothesized that aphids perceive the corpses of congeners killed by parasitoids and respond by increasing the production of winged morphs, but we surprisingly found the opposite. We determined the adaptive value of this response by analyzing the foraging behavior of parasitoids in aphid colonies with killed aphids ("mummies"). Parasitoid females responded to the presence of mummies by reducing both their time allocation and parasitism activity in the patch. The strategy of aphids to reduce emigration (i.e., they produce more wingless morphs when mummies are present) is adaptive because the presence of killed congeners reduces parasitoid pressure on colonies. This demonstrates that the remains of individuals killed by natural enemies can still have an ecological relevance in prey populations and that enemy-induced phenotypic plasticity depends on the type of predation cues. Copyright 2009, Oxford University Press.

Asexuality confers demographic advantages to invasive taxa, but generally limits adaptive potential for colonizing of new habitats. Therefore, pre-existing adaptations and habitat tolerance are essential in the success of asexual invaders. We investigated these key factors of invasiveness by assessing reproductive modes and host-plant adaptations in the pea aphid, Acyrthosiphon pisum, a pest recently introduced into Chile. The pea aphid encompasses lineages differing in their reproductive mode, ranging from obligatory cyclical parthenogenesis to fully asexual reproduction. This species also shows variation in host use, with distinct biotypes specialized on different species of legumes as well as more polyphagous populations. In central Chile, microsatellite genotyping of pea aphids sampled on five crops and wild legumes revealed three main clonal genotypes, which showed striking associations with particular host plants rather than sampling locations. Phenotypic analyses confirmed their strong host specialization and demonstrated parthenogenesis as their sole reproductive mode. The genetic relatedness of these clonal genotypes with corresponding host-specialized populations from the Old World indicated that each clone descended from a particular Eurasian biotype, which involved at least three successful introduction events followed by spread on different crops. This study illustrates that multiple introductions of highly specialized clones, rather than local evolution in resource use and/or selection of generalist genotypes, can explain the demographic success of a strictly asexual invader.

Organisms often perceive risk to be attacked by natural enemies through cues that accompany or persist after attacks on conspecifics. Under the risk of attack, preys may prioritize anti-enemy behaviours, sacrificing their feeding activities. These enemy-induced changes can affect the prey population growth. The presence of killed congeners is a past attack events cue but, in terrestrial prey-enemy systems, its effect on prey population biology is an open question. This was addressed here by studying both the space use and reproductive effort of aphids exposed to congeners killed by parasitoids. To test whether preys adjust their responses with the threat intensity, aphids' responses in presence of killed conspecifics were compared to those measured when directly exposed to non-consumptive parasitoids. The results showed that killed congeners are perceived by aphids and led to both changes in their space use and a strong decline in their population size. This experiment is the first to demonstrate that the presence of congeners killed by an enemy can contribute to the suppressive effects of natural enemies on prey populations.

Seasonal timing is assumed to involve the circadian clock, an endogenous mechanism to track time and measure day length. Some debate persists, however, and aphids were among the first organisms for which circadian clock involvement was questioned. Inferences about links to phenology are problematic, as the clock itself is little investigated in aphids. For instance, it is unknown whether aphids possess diurnal rhythms at all. Possibly, the close interaction with host plants prevents independent measurements of rhythmicity. We reared the pea aphid Acyrthosiphon pisum (Harris) on an artificial diet, and recorded survival, moulting, and honeydew excretion. Despite their plant-dependent life style, aphids were independently rhythmic under light-dark conditions. This first demonstration of diurnal aphid rhythms shows that aphids do not simply track the host plant's rhythmicity.

The mustard trypsin inhibitor, MTI-2, is a potent inhibitor of trypsin with no activity towards chymotrypsin. MTI-2 is toxic for lepidopteran insects, but has low activity against aphids. In an attempt to improve the activity of the inhibitor towards aphids, a library of inhibitor variants was constructed and cloned into the pRlac3 phagemid vector. The library of 9.3 107 independent colonies was created by randomisation of a stretch of five consecutive codons in the reactive site. Repeated selection rounds against bovine trypsin and chymotrypsin allowed the identification of novel, MTI-2 derived, antitrypsin and antichymotrypsin inhibitors. Chy8, the selected variant with highest affinity for bovine chymotrypsin (Ki = 32 nm versus >1000 nm for the wild-type) represents the strongest known recombinant chymotrypsin inhibitor of the MTI-2 family. It is highly toxic to nymphs of the aphid Acyrthosiphon pisum, and moderately toxic to nymphs of Aphis gossypii and Myzus persicae. The LC50 of 73 ?g ml1 towards A. pisum is the lowest value known among chymotrypsin inhibitors. The aphicidal activity of Chy8 was improved eightfold compared to the wild-type inhibitor. This demonstrates, for the first time, that bovine chymotrypsin provides a useful template to select engineered proteins highly toxic against these aphids. The selected gene will allow the development of transgenic crops that are protected against sucking insect pests.

The genome of the spider mite Tetranychus urticae, a herbivore, is missing important elements of the canonical Drosophila immune pathways necessary to fight bacterial infections. However, it is not known whether spider mites can mount an immune response and survive bacterial infection. In other chelicerates, bacterial infection elicits a response mediated by immune effectors leading to the survival of infected organisms. In T. urticae, infection by either Escherichia coli or Bacillus megaterium did not elicit a response as assessed through genome-wide transcriptomic analysis. In line with this, spider mites died within days even upon injection with low doses of bacteria that are non-pathogenic to Drosophila. Moreover, bacterial populations grew exponentially inside the infected spider mites. By contrast, Sancassania berlesei, a litter-dwelling mite, controlled bacterial proliferation and resisted infections with both Gram-negative and Gram-positive bacteria lethal to T. urticae. This differential mortality between mite species was absent when mites were infected with heat-killed bacteria. Also, we found that spider mites harbour in their gut 1000-fold less bacteria than S. berlesei. We show that T. urticae has lost the capacity to mount an induced immune response against bacteria, in contrast to other mites and chelicerates but similarly to the phloem feeding aphid Acyrthosiphon pisum. Hence, our results reinforce the putative evolutionary link between ecological conditions regarding exposure to bacteria and the architecture of the immune response., N.W. was supported by a Marie Sklodowska-Curie Action (MSCA) Individual fellowship (658795-DOGMITE) of Horizon 2020. T.V.L. acknowledges funding from the Fund for Scientific Research Flanders (FWO) (grant nos. G009312N and G053815N), the European Commission (EC contract 618105) via FACCE ERA-NET Plus and FACCE-JP (Genomite, project ID 137 via NWO). This work was supported by Instituto Gulbenkian de Ciencia/Fundacao Calouste Gulbenkian to E.S. and by Fundacao para a Ciencia e a Tecnologia (FCT, Portugal) grant nos. ANR/BIA-EVF/0013/2012 to S.M. and Isabelle Olivieri and FCT-TUBITAK/0001/2014 to S.M. and Ibrahim Cakmak. M.G. acknowledges funding from the Government ofCanada through Genome Canada and the Ontario Genomics Institute (OGI-046), Ontario Research Fund – Global Leadership in Genomics and Life Sciences GL2-01-035, NSERC Strategic Grant STPGP322206-05 and JGI Community Sequencing Program grant no. 777506.

Aphids maintain an obligate, endosymbiotic association with Buchnera sp., a bacterium closely related to Escherichia coli. Bacteria are housed in specialized cells of organ-like structures called bacteriomes in the hemocoel of the aphid and are maternally transmitted. Phylogenetic studies have shown that the association had a single origin, dated about 200-250 million years ago, and that host and endosymbiont lineages have evolved in parallel since then. However, the pattern of deepest branching within the aphid family remains unsolved, which thereby hampers an appraisal of, for example, the role played by horizontal gene transfer in the early evolution of Buchnera. The main role of Buchnera in this association is the biosynthesis and provisioning of essential amino acids to its aphid host. Physiological and metabolic studies have recently substantiated such nutritional role. In addition, genetic studies of Buchnera from several aphids have shown additional modifications, such as strong genome reduction, high A+T content compared to free-living bacteria, differential evolutionary rates, a relative increase in the number of non-synonymous substitutions, and gene amplification mediated by plasmids. Symbiosis is an active process in insect evolution as revealed by the intermediate values of the previous characteristics showed by secondary symbionts compared to free-living bacteria and Buchnera., The authors thank the Servicio Central de Soporte a la Investigación Científica (Universitat de València) for bioinformatics and sequencing facilities. The work has been supported by grants GV-3216/95 from Generalitat Valenciana (Spain), BFM2000-1383 from MEC (Spain) and FEDER 2FD1997-1006 (Spain).