Biosensor Technologies for Early Detection and Quantification of Plant Pathogens.

Plant pathogens are a major reason of reduced crop productivity and may lead to a shortage of food for both human and animal consumption. Although chemical control remains the main method to reduce foliar fungal disease incidence, frequent use can lead to loss of susceptibility in the fungal population. Furthermore, over-spraying can cause environmental contamination and poses a heavy financial burden on growers. To prevent or control disease epidemics, it is important for growers to be able to detect causal pathogen accurately, sensitively, and rapidly, so that the best practice disease management strategies can be chosen and enacted. To reach this goal, many culture-dependent, biochemical, and molecular methods have been developed for plant pathogen detection. However, these methods lack accuracy, specificity, reliability, and rapidity, and they are generally not suitable for in-situ analysis. Accordingly, there is strong interest in developing biosensing systems for early and accurate pathogen detection. There is also great scope to translate innovative nanoparticle-based biosensor approaches developed initially for human disease diagnostics for early detection of plant disease-causing pathogens. In this review, we compare conventional methods used in plant disease diagnostics with new sensing technologies in particular with deeper focus on electrochemical and optical biosensors that may be applied for plant pathogen detection and management. In addition, we discuss challenges facing biosensors and new capability the technology provides to informing disease management strategies.

P2X7 receptor signaling during adult hippocampal neurogenesis.

Neurogenesis is a persistent and essential feature of the adult mammalian hippocampus. Granular neurons generated from resident pools of stem or progenitor cells provide a mechanism for the formation and consolidation of new memories. Regulation of hippocampal neurogenesis is complex and multifaceted, and numerous signaling pathways converge to modulate cell proliferation, apoptosis, and clearance of cellular debris, as well as synaptic integration of newborn immature neurons. The expression of functional P2X7 receptors in the central nervous system has attracted much interest and the regulatory role of this purinergic receptor during adult neurogenesis has only recently begun to be explored. P2X7 receptors are exceptionally versatile: in their canonical role they act as adenosine triphosphate-gated calcium channels and facilitate calcium-signaling cascades exerting control over the cell via calcium-encoded sensory proteins and transcription factor activation. P2X7 also mediates transmembrane pore formation to regulate cytokine release and facilitate extracellular communication, and when persistently stimulated by high extracellular adenosine triphosphate levels large P2X7 pores form, which induce apoptotic cell death through cytosolic ion dysregulation. Lastly, as a scavenger receptor P2X7 directly facilitates phagocytosis of the cellular debris that arises during neurogenesis, as well as during some disease states. Understanding how P2X7 receptors regulate the physiology of stem and progenitor cells in the adult hippocampus is an important step towards developing useful therapeutic models for regenerative medicine. This review considers the relevant aspects of adult hippocampal neurogenesis and explores how P2X7 receptor activity may influence the molecular physiology of the hippocampus, and neural stem and progenitor cells.

Wolbachia infection may improve learning and memory capacity of Drosophila by altering host gene expression through microRNA.

Wolbachia are endosymbiotic bacteria present in a wide range of invertebrates. Although their dramatic effects on host reproductive biology have been well studied, little is known about the effects of Wolbachia on the learning and memory capacity (LMC) of hosts, despite their distribution in the host nervous system, including brain. In this study, we found that Wolbachia infection significantly enhanced LMC in both Drosophila melanogaster and D. simulans. Expression of LMC-related genes was significantly increased in the head of D. melanogaster infected with the wMel strain, and among these genes, crebA was up-regulated the most. Knockdown of crebA in Wolbachia-infected flies significantly decreased LMC, while overexpression of crebA in Wolbachia-free flies significantly enhanced the LMC of flies. More importantly, a microRNA (miRNA), dme-miR-92b, was identified to be complementary to the 3'UTR of crebA. Wolbachia infection was correlated with reduced expression of dme-miR-92b in D. melanogaster, and dme-miR-92b negatively regulated crebA through binding to its 3'UTR region. Overexpression of dme-miR-92b in Wolbachia-infected flies by microinjection of agomirs caused a significant decrease in crebA expression and LMC, while inhibition of dme-miR-92b in Wolbachia-free flies by microinjection of antagomirs resulted in a significant increase in crebA expression and LMC. These results suggest that Wolbachia may improve LMC in Drosophila by altering host gene expression through a miRNA-target pathway. Our findings help better understand the host-endosymbiont interactions and, in particular, the impact of Wolbachia on cognitive processes in invertebrate hosts.

Contrasting Patterns of Virus Protection and Functional Incompatibility Genes in Two Conspecific Wolbachia Strains from Drosophila pandora.

Wolbachia infections can present different phenotypes in hosts, including different forms of reproductive manipulation and antiviral protection, which may influence infection dynamics within host populations. In populations of Drosophila pandora two distinct Wolbachia strains coexist, each manipulating host reproduction: strain wPanCI causes cytoplasmic incompatibility (CI), whereas strain wPanMK causes male killing (MK). CI occurs when a Wolbachia-infected male mates with a female not infected with a compatible type of Wolbachia, leading to nonviable offspring. wPanMK can rescue wPanCI-induced CI but is unable to induce CI. The antiviral protection phenotypes provided by the wPanCI and wPanMK infections were characterized; the strains showed differential protection phenotypes, whereby cricket paralysis virus (CrPV)-induced mortality was delayed in flies infected with wPanMK but enhanced in flies infected with wPanCI compared to their respective Wolbachia-cured counterparts. Homologs of the cifA and cifB genes involved in CI identified in wPanMK and wPanCI showed a high degree of conservation; however, the CifB protein in wPanMK is truncated and is likely nonfunctional. The presence of a likely functional CifA in wPanMK and wPanMK's ability to rescue wPanCI-induced CI are consistent with the recent confirmation of CifA's involvement in CI rescue, and the absence of a functional CifB protein further supports its involvement as a CI modification factor. Taken together, these findings indicate that wPanCI and wPanMK have different relationships with their hosts in terms of their protective and CI phenotypes. It is therefore likely that different factors influence the prevalence and dynamics of these coinfections in natural Drosophila pandora hosts.IMPORTANCEWolbachia strains are common endosymbionts in insects, with multiple strains often coexisting in the same species. The coexistence of multiple strains is poorly understood but may rely on Wolbachia organisms having diverse phenotypic effects on their hosts. As Wolbachia is increasingly being developed as a tool to control disease transmission and suppress pest populations, it is important to understand the ways in which multiple Wolbachia strains persist in natural populations and how these might then be manipulated. We have therefore investigated viral protection and the molecular basis of cytoplasmic incompatibility in two coexisting Wolbachia strains with contrasting effects on host reproduction.

P2X7 Receptors Regulate Phagocytosis and Proliferation in Adult Hippocampal and SVZ Neural Progenitor Cells: Implications for Inflammation in Neurogenesis.

Identifying the signaling mechanisms that regulate adult neurogenesis is essential to understanding how the brain may respond to neuro-inflammatory events. P2X7 receptors can regulate pro-inflammatory responses, and in addition to their role as cation channels they can trigger cell death and mediate phagocytosis. How P2X7 receptors may regulate adult neurogenesis is currently unclear. Here, neural progenitor cells (NPCs) derived from adult murine hippocampal subgranular (SGZ) and cerebral subventricular (SVZ) zones were utilized to characterize the roles of P2X7 in adult neurogenesis, and assess the effects of high extracellular ATP, characteristic of inflammation, on NPCs. Immunocytochemistry found NPCs in vivo and in vitro expressed P2X7, and the activity of P2X7 in culture was demonstrated using calcium influx and pore formation assays. Live cell and confocal microscopy, in conjunction with flow cytometry, revealed P2X7+ NPCs were able to phagocytose fluorescent beads, and this was inhibited by ATP, indicative of P2X7 involvement. Furthermore, P2X7 receptors were activated with ATP or BzATP, and 5-ethynyl-2'-deoxyuridine (EdU) used to observe a dose-dependent decrease in NPC proliferation. A role for P2X7 in decreased NPC proliferation was confirmed using chemical inhibition and NPCs from P2X7-/- mice. Together, these data present three distinct roles for P2X7 during adult neurogenesis, depending on extracellular ATP concentrations: (a) P2X7 receptors can form transmembrane pores leading to cell death, (b) P2X7 receptors can regulate rates of proliferation, likely via calcium signaling, and (c) P2X7 can function as scavenger receptors in the absence of ATP, allowing NPCs to phagocytose apoptotic NPCs during neurogenesis. Stem Cells 2018;36:1764-1777.

Intensity of Mutualism Breakdown Is Determined by Temperature Not Amplification of Wolbachia Genes.

Wolbachia are maternally transmitted intracellular bacterial symbionts that infect approximately 40% of all insect species. Though several strains of Wolbachia naturally infect Drosophila melanogaster and provide resistance against viral pathogens, or provision metabolites during periods of nutritional stress, one virulent strain, wMelPop, reduces fly lifespan by half, possibly as a consequence of over-replication. While the mechanisms that allow wMelPop to over-replicate are still of debate, a unique tandem repeat locus in the wMelPop genome that contains eight genes, referred to as the "Octomom" locus has been identified and is thought to play an important regulatory role. Estimates of Octomom locus copy number correlated increasing copy number to both Wolbachia bacterial density and increased pathology. Here we demonstrate that infected fly pathology is not dependent on an increased Octomom copy number, but does strongly correlate with increasing temperature. When measured across developmental time, we also show Octomom copy number to be highly variable across developmental time within a single generation. Using a second pathogenic strain of Wolbachia, we further demonstrate reduced insect lifespan can occur independently of a high Octomom locus copy number. Taken together, this data demonstrates that the mechanism/s of wMelPop virulence is more complex than has been previously described.

Impact of ERK activation on fly survival and Wolbachia-mediated protection during virus infection.

Elevated levels of reactive oxygen species (ROS) provide protection against virus-induced mortality in Drosophila. In addition to contributing to oxidative stress, ROS are known to activate a number of signalling pathways including the extracellular signal-regulated kinases (ERK) signalling cascade. It was recently shown that ERK signalling is important for resistance against viral replication and invasion in cultured Drosophila cells and the gut epithelium of adult flies. Here, using a Drosophila loss-of-function ERK (rolled) mutant we demonstrated that ERK is important for fly survival during virus infection. ERK mutant flies subjected to Drosophila C virus (DCV) oral and systemic infection were more susceptible to virus-induced mortality as compared with wild-type flies. We have demonstrated experimentally that ERK activation is important for fly survival during oral and systemic virus infection. Given that elevated ROS correlates with Wolbachia-mediated antiviral protection, we also investigated the involvement of ERK in antiviral protection in flies infected by Wolbachia. The results indicate that ERK activation is increased in the presence of Wolbachia but this does not appear to influence Wolbachia-mediated antiviral protection, at least during systemic infection.

Hsp90 and Physiological Stress Are Linked to Autonomous Transposon Mobility and Heritable Genetic Change in Nematodes.

Transposable elements (TEs) have been recognized as potentially powerful drivers of genomic evolutionary change, but factors affecting their mobility and regulation remain poorly understood. Chaperones such as Hsp90 buffer environmental perturbations by regulating protein conformation, but are also part of the PIWI-interacting RNA pathway, which regulates genomic instability arising from mobile TEs in the germline. Stress-induced mutagenesis from TE movement could thus arise from functional trade-offs in the dual roles of Hsp90. We examined the functional constraints of Hsp90 and its role as a regulator of TE mobility by exposing nematodes (Caenorhabditis elegans and Caenorhabditis briggsae) to environmental stress, with and without RNAi-induced silencing of Hsp90. TE excision frequency increased with environmental stress intensity at multiple loci in several strains of each species. These effects were compounded by RNAi-induced knockdown of Hsp90. Mutation frequencies at the unc-22 marker gene in the offspring of animals exposed to environmental stress and Hsp90 RNAi mirrored excision frequency in response to these treatments. Our results support a role for Hsp90 in the suppression of TE mobility, and demonstrate that that the Hsp90 regulatory pathway can be overwhelmed with moderate environmental stress. By compromising genomic stability in germline cells, environmentally induced mutations arising from TE mobility and insertion can have permanent and heritable effects on both the phenotype and the genotype of subsequent generations.

Biotechnological production of hyaluronic acid: a mini review.

Hyaluronic acid (HA) is a polysaccharide found in the extracellular matrix of vertebrate epithelial, neural and connective tissues. Due to the high moisture retention, biocompatibility and viscoelasticity properties of this polymer, HA has become an important component of major pharmaceutical, biomedical and cosmetic products with high commercial value worldwide. Currently, large scale production of HA involves extraction from animal tissues as well as the use of bacterial expression systems in Streptococci. However, due to concerns over safety, alternative sources of HA have been pursued which include the use of endotoxin-free microorganisms such as Bacilli and Escherichia coli. In this review, we explore current knowledge of biosynthetic enzymes that produce HA, how these systems have been used commercially to produce HA and how the challenges of producing HA cheaply and safely are being addressed.

Quantitative Proteomic Analyses of Molecular Mechanisms Associated with Cytoplasmic Incompatibility in Drosophila melanogaster Induced by Wolbachia.

To investigate the molecular mechanisms of cytoplasmic incompatibility (CI) induced by Wolbachia bacteria in Drosophila melanogaster, we applied an isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic assay to identify differentially expressed proteins extracted from spermathecae and seminal receptacles (SSR) of uninfected females mated with either 1-day-old Wolbachia-uninfected (1T) or infected males (1W) or 5-day-old infected males (5W). In total, 1317 proteins were quantified; 83 proteins were identified as having at least a 1.5-fold change in expression when 1W was compared with 1T. Differentially expressed proteins were related to metabolism, immunity, and reproduction. Wolbachia changed the expression of seminal fluid proteins (Sfps). Wolbachia may disrupt the abundance of proteins in SSR by affecting ubiquitin-proteasome-mediated proteolysis. Knocking down two Sfp genes (CG9334 and CG2668) in Wolbachia-free males resulted in significantly lower embryonic hatch rates with a phenotype of chromatin bridges. Wolbachia-infected females may rescue the hatch rates. This suggests that the changed expression of some Sfps may be one of the mechanisms of CI induced by Wolbachia. This study provides a panel of candidate proteins that may be involved in the interaction between Wolbachia and their insect hosts and, through future functional studies, may help to elucidate the underlying mechanisms of Wolbachia-induced CI.

Wolbachia Influences the Production of Octopamine and Affects Drosophila Male Aggression.

Wolbachia bacteria are endosymbionts that infect approximately 40% of all insect species and are best known for their ability to manipulate host reproductive systems. Though the effect Wolbachia infection has on somatic tissues is less well understood, when present in cells of the adult Drosophila melanogaster brain, Wolbachia exerts an influence over behaviors related to olfaction. Here, we show that a strain of Wolbachia influences male aggression in flies, which is critically important in mate competition. A specific strain of Wolbachia was observed to reduce the initiation of aggressive encounters in Drosophila males compared to the behavior of their uninfected controls. To determine how Wolbachia was able to alter aggressive behavior, we investigated the role of octopamine, a neurotransmitter known to influence male aggressive behavior in many insect species. Transcriptional analysis of the octopamine biosynthesis pathway revealed that two essential genes, the tyrosine decarboxylase and tyramine ß-hydroxylase genes, were significantly downregulated in Wolbachia-infected flies. Quantitative chemical analysis also showed that total octopamine levels were significantly reduced in the adult heads.

Oxidative stress correlates with Wolbachia-mediated antiviral protection in Wolbachia-Drosophila associations.

Wolbachia mediates antiviral protection in insect hosts and is being developed as a potential biocontrol agent to reduce the spread of insect-vectored viruses. Definition of the molecular mechanism that generates protection is important for understanding the tripartite interaction between host insect, Wolbachia, and virus. Elevated oxidative stress was previously reported for a mosquito line experimentally infected with Wolbachia, suggesting that oxidative stress is important for Wolbachia-mediated antiviral protection. However, Wolbachia experimentally introduced into mosquitoes impacts a range of host fitness traits, some of which are unrelated to antiviral protection. To explore whether elevated oxidative stress is associated with antiviral protection in Wolbachia-infected insects, we analyzed oxidative stress of five Wolbachia-infected Drosophila lines. In flies infected with protective Wolbachia strains, hydrogen peroxide concentrations were 1.25- to 2-fold higher than those in paired fly lines cured of Wolbachia infection. In contrast, there was no difference in the hydrogen peroxide concentrations in flies infected with nonprotective Wolbachia strains compared to flies cured of Wolbachia infection. Using a Drosophila mutant that produces increased levels of hydrogen peroxide, we investigated whether flies with high levels of endogenous reactive oxygen species had altered responses to virus infection and found that flies with high levels of endogenous hydrogen peroxide were less susceptible to virus-induced mortality. Taken together, these results suggest that elevated oxidative stress correlates with Wolbachia-mediated antiviral protection in natural Drosophila hosts.

Genomic evolution of the pathogenic Wolbachia strain, wMelPop.

Most strains of the widespread endosymbiotic bacterium Wolbachia pipientis are benign or behave as reproductive parasites. The pathogenic strain wMelPop is a striking exception, however: it overreplicates in its insect hosts and causes severe life shortening. The mechanism of this pathogenesis is currently unknown. We have sequenced the genomes of three variants of wMelPop and of the closely related nonpathogenic strain wMelCS. We show that the genomes of wMelCS and wMelPop appear to be identical in the nonrepeat regions of the genome and differ detectably only by the triplication of a 19-kb region that is unlikely to be associated with life shortening, demonstrating that dramatic differences in the host phenotype caused by this endosymbiont may be the result of only minor genetic changes. We also compare the genomes of the original wMelPop strain from Drosophila melanogaster and two sequential derivatives, wMelPop-CLA and wMelPop-PGYP. To develop wMelPop as a novel biocontrol agent, it was first transinfected into and passaged in mosquito cell lines for approximately 3.5 years, generating wMelPop-CLA. This cell line-passaged strain was then transinfected into Aedes aegypti mosquitoes, creating wMelPop-PGYP, which was sequenced after 4 years in the insect host. We observe a rapid burst of genomic changes during cell line passaging, but no further mutations were detected after transinfection into mosquitoes, indicating either that host preadaptation had occurred in cell lines, that cell lines are a more selectively permissive environment than animal hosts, or both. Our results provide valuable data on the rates of genomic and phenotypic change in Wolbachia associated with host shifts over short time scales.

Draft genome sequence of the male-killing Wolbachia strain wBol1 reveals recent horizontal gene transfers from diverse sources.

BACKGROUND: The endosymbiont Wolbachia pipientis causes diverse and sometimes dramatic phenotypes in its invertebrate hosts. Four Wolbachia strains sequenced to date indicate that the constitution of the genome is dynamic, but these strains are quite divergent and do not allow resolution of genome diversification over shorter time periods. We have sequenced the genome of the strain wBol1-b, found in the butterfly Hypolimnas bolina, which kills the male offspring of infected hosts during embyronic development and is closely related to the non-male-killing strain wPip from Culex pipiens. RESULTS: The genomes of wBol1-b and wPip are similar in genomic organisation, sequence and gene content, but show substantial differences at some rapidly evolving regions of the genome, primarily associated with prophage and repetitive elements. We identified 44 genes in wBol1-b that do not have homologs in any previously sequenced strains, indicating that Wolbachia's non-core genome diversifies rapidly. These wBol1-b specific genes include a number that have been recently horizontally transferred from phylogenetically distant bacterial taxa. We further report a second possible case of horizontal gene transfer from a eukaryote into Wolbachia. CONCLUSIONS: Our analyses support the developing view that many endosymbiotic genomes are highly dynamic, and are exposed and receptive to exogenous genetic material from a wide range of sources. These data also suggest either that this bacterial species is particularly permissive for eukaryote-to-prokaryote gene transfers, or that these transfers may be more common than previously believed. The wBol1-b-specific genes we have identified provide candidates for further investigations of the genomic bases of phenotypic differences between closely-related Wolbachia strains.

Antiviral protection and the importance of Wolbachia density and tissue tropism in Drosophila simulans.

Wolbachia, a maternally transmitted endosymbiont of insects, is increasingly being seen as an effective biological control agent that can interfere with transmission of pathogens, including dengue virus. However, the mechanism of antiviral protection is not well understood. The density and distribution of Wolbachia in host tissues have been implicated as contributing factors by previous studies with both mosquitoes and flies. Drosophila flies infected with five diverse strains of Wolbachia were screened for the ability to mediate antiviral protection. The three protective Wolbachia strains were more closely related and occurred at a higher density within whole flies than the two nonprotective Wolbachia strains. In this study, to further investigate the relationship between whole-fly Wolbachia density and the ability to mediate antiviral protection, tetracycline was used to decrease the abundance of the high-density, protective Wolbachia strain wAu prior to viral challenge. Antiviral protection was lost when the density of the protective Wolbachia strain was decreased to an abundance similar to that of nonprotective Wolbachia strains. We determined the Wolbachia density and distribution in tissues of the same five fly-Wolbachia combinations as used previously. The Wolbachia density within the head, gut, and Malpighian tubules correlated with the ability to mediate antiviral protection. These findings may facilitate the development of Wolbachia biological control strategies and help to predict host-Wolbachia pairings that may interfere with virus-induced pathology.

Wolbachia-mediated antibacterial protection and immune gene regulation in Drosophila.

The outcome of microbial infection of insects is dependent not only on interactions between the host and pathogen, but also on the interactions between microbes that co-infect the host. Recently the maternally inherited endosymbiotic bacteria Wolbachia has been shown to protect insects from a range of microbial and eukaryotic pathogens. Mosquitoes experimentally infected with Wolbachia have upregulated immune responses and are protected from a number of pathogens including viruses, bacteria, Plasmodium and filarial nematodes. It has been hypothesised that immune upregulation underpins Wolbachia-mediated protection. Drosophila is a strong model for understanding host-Wolbachia-pathogen interactions. Wolbachia-mediated antiviral protection in Drosophila has been demonstrated for a number of different Wolbachia strains. In this study we investigate whether Wolbachia-infected flies are also protected against pathogenic bacteria. Drosophila simulans lines infected with five different Wolbachia strains were challenged with the pathogenic bacteria Pseudomonas aeruginosa PA01, Serratia marcescens and Erwinia carotovora and mortality compared to paired lines without Wolbachia. No difference in mortality was observed in the flies with or without Wolbachia. Similarly no antibacterial protection was observed for D. melanogaster infected with Wolbachia. Interestingly, D. melanogaster Oregon RC flies which are naturally infected with Wolbachia showed no upregulation of the antibacterial immune genes TepIV, Defensin, Diptericin B, PGRP-SD, Cecropin A1 and Attacin D compared to paired flies without Wolbachia. Taken together these results indicate that Wolbachia-mediated antibacterial protection is not ubiquitous in insects and furthermore that the mechanisms of antibacterial and antiviral protection are independent. We suggest that the immune priming and antibacterial protection observed in Wolbachia-infected mosquitoes may be a consequence of the recent artificial introduction of the symbiont into insects that normally do not carry Wolbachia and that antibacterial protection is unlikely to be found in insects carrying long-term Wolbachia infections.

Solving the Wolbachia paradox: modeling the tripartite interaction between host, Wolbachia, and a natural enemy.

Wolbachia is one of the most common symbionts of arthropods. Its establishment requires lateral transfer to and successful transmission within novel host species. However, Wolbachia performs poorly when introduced into new host species, and models predict that Wolbachia should seldom be able to establish from low initial frequencies. Recently, various symbionts, including Wolbachia, have been shown to protect their hosts from natural enemies. Hence, Wolbachia invasion may be facilitated by the dynamic interaction between it, its host, and a natural enemy. We model such an interaction whereby Wolbachia induces either complete resistance, partial resistance, or tolerance to a host-specific pathogen and also induces the common manipulation phenotype of cytoplasmic incompatibility (CI). We show that the presence of the pathogen greatly facilitates Wolbachia invasion from rare and widens the parameter space in which "imperfect" Wolbachia strains can invade. Furthermore, positive frequency-dependent selection through CI can drive Wolbachia to very high frequencies, potentially excluding the pathogen. These results may explain a poorly understood aspect of Wolbachia biology: it is widespread, despite performing poorly after transfer to new host species. They also support the intriguing possibility that Wolbachia strains that encode both CI and natural-enemy resistance could potentially rid insects, including human disease vectors, of important pathogens.

Symbiont-mediated protection in insect hosts.

Microbes influence the ecology and evolution of their hosts in a variety of ways, including the formation of life-long beneficial or detrimental parasitic infections. Understanding the molecular and biochemical events that underpin symbiosis - beneficial or parasitic - has been a long-term goal of molecular symbiosis research. In addition to beneficial symbionts provisioning scarce resources to their hosts, a growing body of evidence shows that bacterial symbionts can protect their hosts from parasitic symbionts and predators. Here, we review recent theoretical predictions and experimental observations of symbiont-mediated protection in insects. We discuss the implications that protection has for the ecology and evolution of host, symbiont and pathogen and describe what is known about the molecular mechanisms that underpin symbiont protection.