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Evolutionary plasticity of insect immunity
Journal of Insect Physiology xxx (2012) xxx–xxx
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Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Evolutionary plasticity of insect immunity Andreas Vilcinskas ⇑ Institute of Phytopathology and Applied Zoology, Justus-Liebig-University of Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany
a r t i c l e
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Article history: Available online xxxx Keywords: Insects Immunity Evolutionary ecology Antimicrobial peptides Fitness costs Genomics
a b s t r a c t Many insect genomes have been sequenced and the innate immune responses of several species have been studied by transcriptomics, inviting the comparative analysis of immunity-related genes. Such studies have demonstrated significant evolutionary plasticity, with the emergence of novel proteins and protein domains correlated with insects adapting to both abiotic and biotic environmental stresses. This review article focuses on effector molecules such as antimicrobial peptides (AMPs) and proteinase inhibitors, which display greater evolutionary dynamism than conserved components such as immunityrelated signaling molecules. There is increasing evidence to support an extended role for insect AMPs beyond defense against pathogens, including the management of beneficial endosymbionts. The total number of AMPs varies among insects with completed genome sequences, providing intriguing examples of immunity gene expansion and loss. This plasticity is discussed in the context of recent developments in evolutionary ecology suggesting that the maintenance and deployment of immune responses reallocates resources from other fitness-related traits thus requiring fitness trade-offs. Based on our recent studies using both model and non-model insects, I propose that insect immunity genes can be lost when alternative defense strategies with a lower fitness penalty have evolved, such as the so-called social immunity in bees, the chemical sanitation of the microenvironment by some beetles, and the release of antimicrobial secondary metabolites in the hemolymph. Conversely, recent studies provide evidence for the expansion and functional diversification of insect AMPs and proteinase inhibitors to reflect coevolution with a changing pathosphere and/or adaptations to habitats or food associated with microbial contamination. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Insects are the most successful group of organisms on earth in terms of species diversity. This evolutionary success reflects their ability to adapt when faced with a constantly changing and diverse spectrum of pathogens and parasites, the so-called pathosphere (Evans and Schwarz, 2011). Protection from infection is mediated by a complex array of molecules and layered cellular mechanisms that establish the immune system and provide the capacity to recognize and combat virulent microbes, which often evolve rapidly due to their short generation times and small genomes. Insects are powerful tools for the discovery and analysis of new and interesting aspects of immunology such as immune priming and environmental effects on immunity, including the impact of the diet and microbiota (Chambers and Schneider, 2012). Indeed, the environment can influence the selection processes during host-parasite coevolution (Wolinska and King, 2009). The evolution of pathogens and parasites is driven by their need to avoid, overwhelm or suppress the host immune system (Lazzaro, 2008; Schmid-Hempel, 2008) but this imposes selection on the host to evolve corresponding genetic accommodations ⇑ Tel.: +49 641 9937600; fax: +49 641 9937609. E-mail address: [email protected]
(Schulte et al., 2010). The resulting reciprocal genetic adaptations evolve subject to the genes of both interactors (Little et al., 2010) that are sometimes described as an ‘arms race’ and this is a typical feature of the coevolution between pathogens/parasites and their hosts which explains why genes encoding particular components of the host immune system evolve faster than other parts of its genome (Lazzaro and Little, 2009). The ability of insects to respond to rapidly evolving pathogens and parasites requires a dynamic immune system providing at least comparable genetic plasticity (Sackton et al., 2007). Recent studies provide evidence that the functions of the insect immune system can be expanded beyond defense against pathogens and parasites to include the management of endosymbionts. The trade-off between the need to keep microbial symbionts alive while maintaining the ability to recognize and combat microbial pathogens has also contributed to the plasticity of insect immunity (Haine et al., 2008; Login et al., 2011). Many insect species have now been characterized by genome sequencing and transcriptomic analysis of the innate immune system, inviting comparative studies that address the expansion and reduction of immunity gene repertoires in different insect taxa. This review highlights the evolutionary plasticity of insect immunity in terms of gene family dynamics and functional diversification specifically relating to genes encoding antimicrobial peptides or small proteins (AMPs). Naturally-occurring AMPs are ancient
0022-1910/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jinsphys.2012.08.018
Please cite this article in press as: Vilcinskas, A. Evolutionary plasticity of insect immunity. Journal of Insect Physiology (2012), http://dx.doi.org/10.1016/ j.jinsphys.2012.08.018
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chemical defense molecules used by eukaryotic cells to ward off viral, bacterial, fungal and protozoon pathogens (Zasloff, 2002). The functions of AMPs in vertebrates can be expanded beyond killing of pathogens to include, for example, roles in lipopolysaccharide neutralization, immunomodulation, wound repair, induction of apoptosis or angiogenesis (Guani-Guerra et al., 2010; Riedl et al., 2011; Pulido et al., 2012; Yeung et al., 2011). Some AMP families such as the defensins are evolutionarily conserved in all animal taxa, from ancient multicellular organisms to humans (Altincicek and Vilcinskas, 2007b; Wiesner and Vilcinskas, 2010), whereas others appear more restricted in distribution, e.g. the gloverins are only found in the order Lepidoptera and the coleoptericins are only found in the order Coleoptera. The immunity-related functions of AMPs are not limited to the direct killing of microbes, and may also include immunomodulatory activities (Easton et al., 2009) and the control of endosymbionts (Login et al., 2011). Insect AMPs are generally cationic molecules with fewer than 100 amino acid residues, usually between 12 and 50 (Bulet and Stöcklin, 2005). They are placed into three classes according to their structure or the unusual abundance of particular amino acids: (1) linear alpha-helical peptides lacking cysteine residues; (2) peptides with a beta-sheet globular structure stabilized by intramolecular disulfide bridges; and (3) peptides with an unusually large proportion of proline or glycine residues.
2. Insights from comparative genomics The completion of the Drosophila melanogaster genome sequence allowed the total number of AMPs produced by an insect to be estimated for the first time. At least 34 AMPs belonging to eight families (including lysozymes) were identified. Five genes encode cecropins, which are small, linear and amphipathic peptides 35–38 amino acid residues in length, forming helical structures with antibacterial and antifungal activity. The other D. melanogaster AMP families have more specific activities. For example, the Drosophila defensin is most potent against Gram-positive bacteria, while attacin a and drosomycin are active against Gram-negative bacteria and fungi, respectively (Tzou et al., 2002), whereas metchnikowin is active against Gram-positive bacteria and fungi (Levashina et al., 1995; Rahnamaeian et al., 2009). The first comparative analysis of immunity gene repertoires in two insects became possible following the completion of the genome sequence of Anopheles gambiae, a dipteran malaria vector. The Toll signal transduction pathway, which is involved in both development and immunity, is conserved in A. gambiae and D. melanogaster, whereas immunity genes related to recognition have diversified in these species despite their occupation of the same taxonomic order. Interestingly, only 15 AMPs (including lysozymes) were identified in A. gambiae, less than half the number in D. melanogaster, possibly reflecting different selection pressures based on the different life styles of these species (Christophides et al., 2002). The genome sequence of the yellow fever and Dengue fever vector Aedes aegypti allowed the immunity gene repertoires of three dipteran species to be compared, leading to a pivotal study addressing the evolutionary plasticity of insect immunity. The combined application of multiple large-scale bioinformatics, manual curation and phylogenetic analysis, led to the discovery of slowly and rapidly evolving immunity-related gene families belonging to different functional classes (Waterhouse et al., 2007). There was lower relative sequence divergence in genes representing signal transduction proteins compared to genes involved in microbe recognition. The latter were characterized by species-dependent expansions and reductions in gene families and even novel species-restricted immunity genes. For example, the D. melanogaster genome contains 13 genes encoding peptidoglycan-recognition proteins and
nine encoding Toll homologs, whereas the A. gambiae genome encodes seven peptidoglycan-recognition proteins and 12 Toll homologues and the A. aegypti genome encodes eight peptidoglycanrecognition proteins and 11 Toll homologs. This study also established the varying evolutionary dynamics between different classes of effector molecules. AMPs showed more rapid diversification and greater species specificity than enzymes involved in defense reactions. Only three of the seven D. melanogaster AMP families were found in mosquitoes, namely the attacins, cecropins and defensins, whereas the gambicins were only found in mosquitoes, highlighting the dynamic evolution of AMPs at least within the Diptera (Waterhouse et al., 2007). The completion of 12 Drosophila spp. genome sequences allowed the evolutionary dynamics of immunity genes to be investigated across multiple species within a genus for the first time and highlighted the relatively recent evolutionary origin of several novel immune-related genes and gene families (Hahn et al., 2007). In agreement with earlier findings, this study showed that genes encoding effector molecules are more likely to vary in copy number across species than those encoding immunity-related signaling molecules. This study also provided evidence for positive selection imposed by pathogens on immunity-related recognition proteins in Drosophila spp. (Sackton et al., 2007). Theoretically, the expansion of immunity-related gene families should occur in species whose lifestyle promotes the spread of infections. For example, high population densities and social contacts accelerate the horizontal transmission of pathogens and parasites. Therefore, eusocial insects such as the honeybee Apis mellifera should be protected by an arsenal of effector molecules reflecting the high density of individuals in a beehive, their genetic homogeneity and their mutual feeding behavior, which should facilitate the rapid transmission of pathogens vectored by workers collecting nectar and pollen. However, the analysis of immunityrelated genes in the completed A. melifera genome sequence revealed an unexpected reduction of genes contributing to the recognition and deterrence of pathogens (The Honeybee Genome Sequencing Consortium, 2006). The genome encoded three lysozymes and six AMPs in four families, namely apidaecin, abaecin, hymenoptaecin and defensin (Evans et al., 2006). The low number of honeybee AMPs relative to the previously-analyzed dipteran genomes was explained by the existence of alternative mechanisms providing protection from infectious diseases, such as the so-called social immunity which includes grooming and other hygienic behaviors. Sanitation of the beehive by the production or collection of antimicrobial compounds deposited with propolis or transmitted with royal jelly could also contribute to the antimicrobial defense of the colony (Le Conte et al., 2011). Targeted screening for AMPs in the Asiatic honeybee A. cerana, which does not produce propolis, revealed a more diverse AMP profile compared to A. mellifera (Xu et al., 2009). The authors found 11 cDNAs encoding two different abaecins, 13 cDNAs encoding four different apidaecins, 29 cDNAs encoding seven different defensins, and 34 cDNAs encoding 13 different hymenoptaecins, and they suggested the longer domestication history of A. mellifera as an alternative explanation for its smaller immunity gene repertoire. However, recent insights into the genome sequences of the leaf-cutter ant Atta cephalotes (Suen et al., 2011) and the termite Zootermopsis suggest that the AMP repertoire is often strikingly reduced in even nondomesticated eusocial insects compared to solitary hymenopteran species such as the parasitic wasp Nasonia vitripennis (Tian et al., 2010). The latter study identified at least 42 different AMPs whose sequences exhibit signatures suggesting that the evolutionary diversification of AMPs results from classic mechanisms such as gene duplication, exon duplication and exon shuffling. An important and often overlooked challenge is the bias towards known gene families introduced by analyzing the evolution-
Please cite this article in press as: Vilcinskas, A. Evolutionary plasticity of insect immunity. Journal of Insect Physiology (2012), http://dx.doi.org/10.1016/ j.jinsphys.2012.08.018
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ary plasticity of insect immunity using model insect genomes and current bioinformatics methods. The latter have predominantly been used to screen for homologues of immune-related genes known from Drosophila, implicating that all components from this model insect are known. But our knowledge about insect immunity, even in insects with a completely sequenced genome, is far from complete. For example, recent studies provide for the first time evidence for the presence of negative regulators in insect immunity. MicroRNAs have been identified in the model insects Drosophila and, Tribolium which suppress the expression of immune genes at the posttranscriptional level (Freitak et al., 2012; Fullaondo and Lee, 2012). The discovery of novel or unexpected genes requires complementary approaches such as experimental screening for immunity-related genes using proteomic and transcriptomic approaches. The first antimicrobial peptides from insect have been purified by classic biochemical approaches while transcriptomic analysis using next generation sequencing methods are currently widely used (Vogel et al., 2011a). I favor suppression subtractive hybridization (SSH) for this purpose because it has been successfully applied in my group to discover many new or unexpected immunity-related genes in model and non-model insects (Altincicek and Vilcinskas, 2007a,b; Altincicek and Vilcinskas 2009). This PCR-based method amplifies genes that are differentially expressed following treatments such as the injection of bacteria or fungi, while simultaneously suppressing housekeeping genes. For example, conventional bioinformatics identified four defensins in the genome of the model beetle Tribolium castaneum (Tribolium Genome Sequencing Consortium, 2008), whereas our SSH approach revealed additional thaumatin-like genes that were overlooked by the bioinformatics approach (Altincicek et al., 2008a). Thaumatins are antifungal peptides originally identified in plants. A recombinant T. castaneum thaumatin showed potent antifungal activity suggesting it helps to defend beetles against fungal pathogens (Altincicek et al., 2008a). Our discovery led to the identification of homologous sequences in the genome of the pea aphid Acyrthosiphon pisum (International Aphid Genomics Consortium, 2010) using a bioinformatics approach (Gerardo et al., 2010). Interestingly, neither the bioinformatics-based analysis of genomic and transcriptomic data nor our SSH-based experimental screening approach for immunity-related genes revealed the presence of common AMPs such as defensins encoded by the aphid genome (Altincicek et al., 2008a). In addition, piercing aphids with a septic needle did not induce detectable activity against living bacteria. We proposed that aphids lost AMPs during their evolution, and this is further supported by the finding that supposedly essential immunity-related genes such as those encoding components of immunity signaling pathways are also missing from the aphid genome (Gerardo et al., 2010). This unexpected finding by the International Aphid Genomics Consortium (2010) invites a discussion of the selective forces driving the loss and/or expansion of immunity-related genes in insects.
3. The loss of immunity-related genes Pea aphids appear to lack many known immunity-related genes that are considered crucial for sensing bacteria (e.g. peptidoglycan recognition proteins), as well as genes for AMPs (Gerardo et al., 2010) and genes encoding essential immunity-related signaling components, e.g. the immune deficiency (IMD) pathway that was discovered in D. melanogaster and which regulates antibacterial and antifungal defense genes (Kaneko and Silverman, 2005). This may ultimately reflect the unique ecological niche that aphids occupy, i.e. they feed exclusively on phloem sap that is rich in sugars but does not provide a complete profile of amino acids. Aphids are
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therefore dependent on symbiotic bacteria such as Buchnera aphidicola, which account for up to 20% of their dry mass and utilize some of the phloem-derived sugars, but in return synthesize essential amino acids that cannot be sourced from the aphid diet. The lack of antibacterial defenses may therefore reflect the coevolution of aphids and their primary and secondary bacterial symbionts (International Aphid Genomics Consortium, 2010). We proposed three hypotheses to explain why aphids can afford to reduce their antibacterial defenses. First, the uptake of microbes in the diet is limited because phloem sap is normally sterile (Altincicek et al., 2008a). This contrasts with insects that feed on leaves, because leaf surfaces teem with bacteria and fungi (Freitak et al., 2007). Second, aphids may rely on extracellular symbionts to provide antibacterial defenses, e.g. secondary symbionts such as Regiella insecticola and Hamiltonella defensa have been shown to provide protection against fungi and parasitic wasps, respectively (Oliver et al., 2005; Scarborough et al., 2005). Third, piercing aphids with a septic needle induces the production of viviparous offspring, suggesting that aphids invest in terminal reproduction rather than immunity following a pathogen challenge (Altincicek et al., 2008a). The last of these hypotheses implies that maintaining and deploying an immune response takes resources from other fitness-determining traits, and this has inspired research into the ecological selection pressure that drives the evolution of innate immunity in insects (Schmid-Hempel, 2005). The insect immune system is an excellent model allowing the investigation of tradeoffs resulting from investments into different fitness components, a virtual currency that combines survival with reproduction (Schmid-Hempel, 2011). According to this hypothesis, there are limited resources that can be used to improve fitness so any investment in the production of immunity-related proteins requires the reallocation of resources, i.e. their withdrawal from other fitnessrelated traits such as fecundity or adaptability (McKean et al., 2008). Such trade-offs inevitably result in negative selection against genes that do not increase fitness in a given environment, therefore genes representing particular immunity functions can disappear from the genome if their loss costs less in terms of overall fitness than their maintenance. In the pea aphid, we propose that the fitness penalty resulting from the loss of immunity-related genes is negated by the fitness benefits provided by bacterial symbionts (Gerardo et al., 2010). The evolutionary plasticity of insect immunity may partly reflect the vertical transmission of symbionts that provide fitness benefits exceeding the costs of both maintaining the symbionts and losing innate immunity (Haine, 2008). I postulate that the loss of formerly essential immunity-related genes can occur in insects that have evolved alternative and less resource-hungry mechanisms that provide adequate protection against pathogens and parasites, particularly by expanding beyond the maintenance and deployment of an immune system to encompass diverse strategies such as the sanitation of the microenvironment (Gross et al., 2008) or hygienic behavior in ant or bees (Ulgevig and Cremer, 2007; Le Conte et al., 2011). Insects can afford to lose immunity-related genes if genes that mediate other defense mechanisms (e.g. chemical protection) can compensate for the loss of a dynamic host defense without a fitness penalty. For example, herbivorous insects such as the larvae of the brassy willow leaf beetle Phratora vitellinae constitutively release volatile glandular secretions containing antimicrobial compounds sequestered from the host plant, which fumigate ingested food (Gross et al., 2008). This strategy reduces fitness costs because the uptake of bacteria by other herbivorous insects can trigger immune responses associated with life history trade-offs (Freitak et al., 2007). This balance of fitness costs may explain the independent evolution of cellular components that allow plant-derived molecules to be used for the antimicrobial defense of leaf beetles (Kirsch et al., 2011).
Please cite this article in press as: Vilcinskas, A. Evolutionary plasticity of insect immunity. Journal of Insect Physiology (2012), http://dx.doi.org/10.1016/ j.jinsphys.2012.08.018
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Fig. 1. Evolutionary plasticity of insect immunity displayed by extreme differences in constitutive antibacterial activity in the hemolymph of three coccinelid species: the native and non-invasive ladybird Adalia bipunctata (left), the native and invasive (in northern America) seven-spotted ladybird Coccinella septempunctata (middle), and the introduced and invasive harlequin ladybird Harmonia axyridis (right). Three microliters of hemolymph collected upon reflex-bleeding of each beetle were put into a whole punched in Petri-dishes containing agar which was inoculated with Micrococcus luteus bacteria. 24 h upon cultivation at 30 °C zones of bacterial growth inhibition indicate the presence of a strong constitutive antibacterial activity in the hemolymph of H. axyridis which is absent from A. bipunctata and C. septempunctata. Beetles were immobilized by keeping them for 30 min. in the refrigerator, and then put on the wholes to illustrate the corresponding size of inhibition zones. The antibacterial activity in the hemolymph of H. axyridis has been attributed to harmonine, a secondary metabolite, which displayed broad-spectrum antibacterial and antimalaria activity. The production and release within the hemolymph of harmonine putatively saves fitness costs associated with the synthesis of AMPs and may, therefore, contribute to the invasive success of H. axyridis (Röhrich et al., 2012).
Chemical defenses that compensate for or support the protection provided by the immune system are widespread in insects. An impressive example is the burying beetle Nicrophorus vespilloides, which feeds and reproduces on small vertebrate cadavers. The latter are buried in the soil after localization through volatiles emitted from the carcass. Theory predicts that the exposure of the beetles to high loads of soil and/or carrion-associated microbes would require an effective defense system. We have recently analyzed the oral and anal secretions produced by N. vespilloides beetles, which are used to chemically preserve the cadavers as a food source for their offspring. We found more than 30 secondary metabolites in the secretions, most of which have known antimicrobial activities (Degenkolb et al., 2011). These compounds solely mediate the sanitation and chemical preservation of the buried cadavers and thus help to reduce the fitness costs associated with the maintenance and deployment of immune responses (Vogel et al., 2011b). Other beetles such as the invasive harlequin ladybird Harmonia axyridis synthesize and secrete secondary metabolites with broad-spectrum antimicrobial activity into the hemolymph (Fig. 1), explaining its profound resistance against pathogens and parasites which may in turn explain its success as an invasive species (Röhrich et al., 2012). The loss of immunity-related genes thus appears to occur in insects that rely on other strategies with a lower overall fitness penalty for defense against pathogens and parasites.
4. The expansion of immunity-related gene families As stated above, the comparative analysis of insect genomes and immunity-related transcriptomes has provided numerous examples of the expansion of immunity-related gene families. The expansion of AMP genes has been attributed to gene duplication and divergence to produce multiple diverse paralogs (Bulmer
and Crozier, 2004; Dessanayake et al., 2007). Gene or domain duplication followed by recombination between homologous sequences appear to be the key mechanisms underlying the expansion of effector molecule repertoires in invertebrates (Froy and Gurevitz, 2003). Several models for the creation, maintenance and evolution of gene copies have been proposed, but there is no clear consensus on how gene duplications are fixed and maintained (Innan ana Kondrashov, 2010). The remarkable dynamics of AMP gene evolution in insects can be explained in part by adaptations in response to a changing pathosphere. It has been postulated that environmental heterogeneity and pathogen diversity can maintain the diversity of host immunity-related genes by favoring different genotypes at different times and places, favoring the presence of paralogs that can adapt rapidly by recombination as well as mutation (Lazzaro and Little, 2009). The degree of plasticity displayed by insects exposed to the same environmental changes can evolve and be adaptive if fitness is increased by the changing phenotype. A faster and more potent immune response against pathogens following a second exposure (immune priming) was first observed in vertebrates. The latter can be more or less specific. Parental investment in offspring encompasses their preparation to defend themselves against pathogens or parasites initially encountered by the adults. This phenomenon designated as specific transgenerational immune priming has first been discovered in the crustacean Daphnia magna (Little et al., 2003), and then insects such as the bumble bee (Sadd et al., 2005), the beetles Tenebrio molitor (Moret, 2006) and Tribolium castaeum (Roth et al., 2009, and the lepidopteran Trichoplusia ni (Freitak et al., 2009). However, the underlying mechanisms behind the transgenerational immune priming are poorly understood (Chambers and Schneider, 2012). The evolutionary plasticity of insect immunity allows insects to change their phenotype in response to changes in the environment. Phenotypic variation with a genetic basis is an essential prerequisite for evolution. In order to study the impact of the environment on insect immunity, we used rat-tailed maggots of the drone fly Eristalis tenax as a model system because these are the only animals that survive in extremely polluted aquatic habitats such as liquid manure storage pits and cesspools (Altincicek and Vilcinskas, 2007a). From an evolutionary point of view, it is advantageous to be the only survivor in habitats with extreme microbial loads, because this saves fitness costs associated with dealing with competitors and predators. However, this saving must be balanced against the increased costs of a robust antimicrobial defense strategy. I therefore postulated that the colonization of such habitats would be accompanied by corresponding adaptations in the immune system, and found that the immune-inducible E. tenax transcriptome revealed the rapid diversification of AMPs and signatures of positive selection in individual members such as the defensin homolog eristalin (Altincicek et al., 2008b). These observations support the hypothesis that the changing pathosphere is a strong selective force favoring immune gene expansion. The impact of environmental adaptations on the evolution of immune genes has recently been demonstrated by an independent and intriguing approach in which the emergence and loss of protein domains was reconstructed in 20 arthropod species of the pancrustacean clade (Moore and Bornber-Bauer, 2012). Proteins contain structurally and functionally independent domains that are evolutionarily conserved across taxa (Moore et al., 2008). Protein evolution in eukaryotes is predominantly characterized by the rearrangement of domains rather than their emergence or loss. The pancrustacean clade is often used to estimate the frequency of protein domain gain and loss because it encompasses a large number of species with completed genome sequences whose homologous relationships are well defined, and which differ in their degree of environmental adaptation. Despite the lower rate
Please cite this article in press as: Vilcinskas, A. Evolutionary plasticity of insect immunity. Journal of Insect Physiology (2012), http://dx.doi.org/10.1016/ j.jinsphys.2012.08.018
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of domain turnover relative to gene family turnover, this comparative genomics approach revealed that domain emergence is associated predominantly with environmental adaptations. The authors introduced a novel indirect gene ontology (GO) analysis method that allowed the quantitation of domain emergence in proteins related by defined GO terms and found that proteins related to innate immune responses were the most frequently represented (Moore and Bornber-Bauer, 2012).
5. Functional shifts among immunity-related effector genes The evolutionary plasticity of insect immunity can be expanded beyond the diversification or loss of immunity-related effector molecules to include functional shifts, i.e. the adaptation of proteins to take on novel functions. The AMPs present in a single insect species can often be grouped into different gene families originating from a single ancestral gene. The above mentioned duplication and divergent evolution of AMP genes is associated with functional shifts of the resulting paralogs that can ultimately lead to the formation of new AMP families (Tian et al., 2010; Yang et al., 2011). Predicted functional shifts among effector molecules are not easy to confirm because individual AMPs are difficult to purify as test for their antimicrobial spectrum, but this can be addressed by the expression of AMPs as recombinant proteins. This approach has recently been used to examine the functional divergence of AMP families in the silkworm Bombyx mori (Yang et al., 2011). The authors found that the gloverins evolved slowly and their antimicrobial properties were largely conserved, whereas the cecropin and moricin families showed diverse activities. This type of shifting has previously been observed among the defensins, most of which show activity against Gram-positive and/or Gramnegative bacteria, but some of which can also inhibit fungi, including defensins that act exclusively against mycelia-forming fungi (Langen et al., 2006). The heterologous expression of insect AMPs has become a screening tool for the discovery of peptides that inhibit human pathogens (Ratcliffe et al., 2011) particularly multidrug-resistant pathogens and emerging diseases (Vilcinskas, 2011). Insect AMPs could also be used to engineer disease-resistant plants (Vilcinskas and Gross, 2005). In addition to AMPs, other effector molecules in insects can allow the experimental assessment of immunity-related evolutionary plasticity, such as inhibitors of pathogen or parasite proteinases. Virtually all pathogens use proteolytic enzymes to digest host proteins for nutrition and to degrade host defense molecules such as AMPs (Armstrong, 2006; Potempa and Robert, 2009). Such enzymes are known as virulence factors when they are required to accomplish successful infections. For example, parasitic fungi produce and secrete an array of proteolytic enzymes enabling them to infect insects directly via the protein-containing exoskeleton (Vilcinskas, 2010). The known substrate specificity and the controlled expression profiles of invasive proteinases indicate that the diversification and functional shifting among these fungal enzymes is driven by adaptation to different host ranges. However, only proteinases that cannot be completely inactivated by corresponding host proteinase inhibitors in the cuticle, tissues or hemolymph can operate as virulence factors (Vilcinskas, 2010). This complementarity between virulence and defense factors results in the negative selection of pathogen-associated proteinases for which a surplus of corresponding inhibitors is present in the host, because the secretion of such enzymes would waste resources. In turn, emerging proteinases with no corresponding inhibitors in the host would be subject to positive selection because they would function successfully even when produced in low quantities (Vilcinskas, 2010). This hypothesis explains the evolution of particular M4 family microbial metalloproteinases such as thermolysin
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because most animals have not evolved corresponding inhibitors during host-parasite coevolution. Therefore, thermolysin-like metalloproteinases such as aureolysin, pseudolysin and vibriolysin are virulence factors in many human pathogens (Adekoya and Sylte, 2009). Only insects, particularly the greater wax moth Galleria mellonella, have evolved a peptide that can specifically inhibit thermolysin-like metalloproteinases. This insect metalloproteinase inhibitor (IMPI) is synthesized and secreted into the hemolymph along with other AMPs where it protects them from degradation by microbial metalloproteinases (Vilcinskas and Wedde, 2002). The IMPI sequence does not match any other proteins in public databases although screening the Pfam database of protein families for conserved domains revealed significant similarity to a trypsin inhibitor-like (TIL) cysteine-rich domain (Clermont et al., 2004). IMPI has thus been assigned to protease inhibitor family I8, a cysteine-rich trypsin inhibitor-like family in the MEROPS database of peptidases, although it is not a serine proteinase inhibitor (Rawlings et al., 2004). Our recent research provides evidence for the presence of a family of IMPI paralogs in G. mellonella resulting from gene and/or domain duplications and for functional shifts within this family (Wedde et al., 2007). Furthermore, we recently analyzed the crystal structure of IMPI in a complex with thermolysin, revealing an unprecedented inhibition mechanism that was previously thought to be restricted to serine proteinase inhibitors. The recruitment of one class of protease inhibitors to regulate another class is a case of divergent evolution based on the hyper-variability of enzyme-contacting regions, strongly suggesting a recent ‘arms race’ and providing another intriguing example for the plasticity of insect immunity. Our results suggest that IMPI originates from reciprocal selection in an antagonistic environment, in which the insect host developed a novel defense against the weapons of microbes (Arolas et al., 2011). The specificity of proteinases and their inhibitors is easily determined, making them powerful tools to study the dynamics of host-parasite coevolution directly at the frontier where the antagonists interact. 6. Concluding remarks The comparative analysis of insect genomes and immunityrelated transcriptomes has revealed the remarkable plasticity of the insect innate immune system, including remarkable examples of gene loss, gene functional adaptation, and gene family expansion under the unique selection pressures of different ecological niches. Gene loss may result from a trade-off between immunity and other traits whose maintenance has a lower fitness penalty in response to particular environmental changes. Deciphering the functional shifts of immunity-related effector molecules such as AMPs and protease inhibitors that act against pathogen virulence factors has expanded our knowledge about evolutionary plasticity in insects even further by revealing unanticipated dynamic interactions. In order to explore the mechanisms underlying complex immunity-related phenomena in insects such as the rapid reciprocal adaptations during host-parasite-coevolution, trans-generational immune priming and gender specific-responses, we have started to examine the role of epigenetic mechanisms in insect immunity. Acknowledgements The author acknowledges project funding provided by the German Research Foundation (Deutsche Forschungsgemeinschaft) within the DFG priority program 1399 ‘‘Host-parasite-coevolution - rapid reciprocal adaptation and its genetic base’’ (VI 219/3-1) and by the Hessian Ministry of Science and Art via the LOEWE excellence initiative which encompasses a generous grant for the research focus ‘‘Insect Biotechnology’’.
Please cite this article in press as: Vilcinskas, A. Evolutionary plasticity of insect immunity. Journal of Insect Physiology (2012), http://dx.doi.org/10.1016/ j.jinsphys.2012.08.018
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Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Evolutionary plasticity of insect immunity Andreas Vilcinskas ⇑ Institute of Phytopathology and Applied Zoology, Justus-Liebig-University of Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany
a r t i c l e
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Article history: Available online xxxx Keywords: Insects Immunity Evolutionary ecology Antimicrobial peptides Fitness costs Genomics
a b s t r a c t Many insect genomes have been sequenced and the innate immune responses of several species have been studied by transcriptomics, inviting the comparative analysis of immunity-related genes. Such studies have demonstrated significant evolutionary plasticity, with the emergence of novel proteins and protein domains correlated with insects adapting to both abiotic and biotic environmental stresses. This review article focuses on effector molecules such as antimicrobial peptides (AMPs) and proteinase inhibitors, which display greater evolutionary dynamism than conserved components such as immunityrelated signaling molecules. There is increasing evidence to support an extended role for insect AMPs beyond defense against pathogens, including the management of beneficial endosymbionts. The total number of AMPs varies among insects with completed genome sequences, providing intriguing examples of immunity gene expansion and loss. This plasticity is discussed in the context of recent developments in evolutionary ecology suggesting that the maintenance and deployment of immune responses reallocates resources from other fitness-related traits thus requiring fitness trade-offs. Based on our recent studies using both model and non-model insects, I propose that insect immunity genes can be lost when alternative defense strategies with a lower fitness penalty have evolved, such as the so-called social immunity in bees, the chemical sanitation of the microenvironment by some beetles, and the release of antimicrobial secondary metabolites in the hemolymph. Conversely, recent studies provide evidence for the expansion and functional diversification of insect AMPs and proteinase inhibitors to reflect coevolution with a changing pathosphere and/or adaptations to habitats or food associated with microbial contamination. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Insects are the most successful group of organisms on earth in terms of species diversity. This evolutionary success reflects their ability to adapt when faced with a constantly changing and diverse spectrum of pathogens and parasites, the so-called pathosphere (Evans and Schwarz, 2011). Protection from infection is mediated by a complex array of molecules and layered cellular mechanisms that establish the immune system and provide the capacity to recognize and combat virulent microbes, which often evolve rapidly due to their short generation times and small genomes. Insects are powerful tools for the discovery and analysis of new and interesting aspects of immunology such as immune priming and environmental effects on immunity, including the impact of the diet and microbiota (Chambers and Schneider, 2012). Indeed, the environment can influence the selection processes during host-parasite coevolution (Wolinska and King, 2009). The evolution of pathogens and parasites is driven by their need to avoid, overwhelm or suppress the host immune system (Lazzaro, 2008; Schmid-Hempel, 2008) but this imposes selection on the host to evolve corresponding genetic accommodations ⇑ Tel.: +49 641 9937600; fax: +49 641 9937609. E-mail address: [email protected]
(Schulte et al., 2010). The resulting reciprocal genetic adaptations evolve subject to the genes of both interactors (Little et al., 2010) that are sometimes described as an ‘arms race’ and this is a typical feature of the coevolution between pathogens/parasites and their hosts which explains why genes encoding particular components of the host immune system evolve faster than other parts of its genome (Lazzaro and Little, 2009). The ability of insects to respond to rapidly evolving pathogens and parasites requires a dynamic immune system providing at least comparable genetic plasticity (Sackton et al., 2007). Recent studies provide evidence that the functions of the insect immune system can be expanded beyond defense against pathogens and parasites to include the management of endosymbionts. The trade-off between the need to keep microbial symbionts alive while maintaining the ability to recognize and combat microbial pathogens has also contributed to the plasticity of insect immunity (Haine et al., 2008; Login et al., 2011). Many insect species have now been characterized by genome sequencing and transcriptomic analysis of the innate immune system, inviting comparative studies that address the expansion and reduction of immunity gene repertoires in different insect taxa. This review highlights the evolutionary plasticity of insect immunity in terms of gene family dynamics and functional diversification specifically relating to genes encoding antimicrobial peptides or small proteins (AMPs). Naturally-occurring AMPs are ancient
0022-1910/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jinsphys.2012.08.018
Please cite this article in press as: Vilcinskas, A. Evolutionary plasticity of insect immunity. Journal of Insect Physiology (2012), http://dx.doi.org/10.1016/ j.jinsphys.2012.08.018
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chemical defense molecules used by eukaryotic cells to ward off viral, bacterial, fungal and protozoon pathogens (Zasloff, 2002). The functions of AMPs in vertebrates can be expanded beyond killing of pathogens to include, for example, roles in lipopolysaccharide neutralization, immunomodulation, wound repair, induction of apoptosis or angiogenesis (Guani-Guerra et al., 2010; Riedl et al., 2011; Pulido et al., 2012; Yeung et al., 2011). Some AMP families such as the defensins are evolutionarily conserved in all animal taxa, from ancient multicellular organisms to humans (Altincicek and Vilcinskas, 2007b; Wiesner and Vilcinskas, 2010), whereas others appear more restricted in distribution, e.g. the gloverins are only found in the order Lepidoptera and the coleoptericins are only found in the order Coleoptera. The immunity-related functions of AMPs are not limited to the direct killing of microbes, and may also include immunomodulatory activities (Easton et al., 2009) and the control of endosymbionts (Login et al., 2011). Insect AMPs are generally cationic molecules with fewer than 100 amino acid residues, usually between 12 and 50 (Bulet and Stöcklin, 2005). They are placed into three classes according to their structure or the unusual abundance of particular amino acids: (1) linear alpha-helical peptides lacking cysteine residues; (2) peptides with a beta-sheet globular structure stabilized by intramolecular disulfide bridges; and (3) peptides with an unusually large proportion of proline or glycine residues.
2. Insights from comparative genomics The completion of the Drosophila melanogaster genome sequence allowed the total number of AMPs produced by an insect to be estimated for the first time. At least 34 AMPs belonging to eight families (including lysozymes) were identified. Five genes encode cecropins, which are small, linear and amphipathic peptides 35–38 amino acid residues in length, forming helical structures with antibacterial and antifungal activity. The other D. melanogaster AMP families have more specific activities. For example, the Drosophila defensin is most potent against Gram-positive bacteria, while attacin a and drosomycin are active against Gram-negative bacteria and fungi, respectively (Tzou et al., 2002), whereas metchnikowin is active against Gram-positive bacteria and fungi (Levashina et al., 1995; Rahnamaeian et al., 2009). The first comparative analysis of immunity gene repertoires in two insects became possible following the completion of the genome sequence of Anopheles gambiae, a dipteran malaria vector. The Toll signal transduction pathway, which is involved in both development and immunity, is conserved in A. gambiae and D. melanogaster, whereas immunity genes related to recognition have diversified in these species despite their occupation of the same taxonomic order. Interestingly, only 15 AMPs (including lysozymes) were identified in A. gambiae, less than half the number in D. melanogaster, possibly reflecting different selection pressures based on the different life styles of these species (Christophides et al., 2002). The genome sequence of the yellow fever and Dengue fever vector Aedes aegypti allowed the immunity gene repertoires of three dipteran species to be compared, leading to a pivotal study addressing the evolutionary plasticity of insect immunity. The combined application of multiple large-scale bioinformatics, manual curation and phylogenetic analysis, led to the discovery of slowly and rapidly evolving immunity-related gene families belonging to different functional classes (Waterhouse et al., 2007). There was lower relative sequence divergence in genes representing signal transduction proteins compared to genes involved in microbe recognition. The latter were characterized by species-dependent expansions and reductions in gene families and even novel species-restricted immunity genes. For example, the D. melanogaster genome contains 13 genes encoding peptidoglycan-recognition proteins and
nine encoding Toll homologs, whereas the A. gambiae genome encodes seven peptidoglycan-recognition proteins and 12 Toll homologues and the A. aegypti genome encodes eight peptidoglycanrecognition proteins and 11 Toll homologs. This study also established the varying evolutionary dynamics between different classes of effector molecules. AMPs showed more rapid diversification and greater species specificity than enzymes involved in defense reactions. Only three of the seven D. melanogaster AMP families were found in mosquitoes, namely the attacins, cecropins and defensins, whereas the gambicins were only found in mosquitoes, highlighting the dynamic evolution of AMPs at least within the Diptera (Waterhouse et al., 2007). The completion of 12 Drosophila spp. genome sequences allowed the evolutionary dynamics of immunity genes to be investigated across multiple species within a genus for the first time and highlighted the relatively recent evolutionary origin of several novel immune-related genes and gene families (Hahn et al., 2007). In agreement with earlier findings, this study showed that genes encoding effector molecules are more likely to vary in copy number across species than those encoding immunity-related signaling molecules. This study also provided evidence for positive selection imposed by pathogens on immunity-related recognition proteins in Drosophila spp. (Sackton et al., 2007). Theoretically, the expansion of immunity-related gene families should occur in species whose lifestyle promotes the spread of infections. For example, high population densities and social contacts accelerate the horizontal transmission of pathogens and parasites. Therefore, eusocial insects such as the honeybee Apis mellifera should be protected by an arsenal of effector molecules reflecting the high density of individuals in a beehive, their genetic homogeneity and their mutual feeding behavior, which should facilitate the rapid transmission of pathogens vectored by workers collecting nectar and pollen. However, the analysis of immunityrelated genes in the completed A. melifera genome sequence revealed an unexpected reduction of genes contributing to the recognition and deterrence of pathogens (The Honeybee Genome Sequencing Consortium, 2006). The genome encoded three lysozymes and six AMPs in four families, namely apidaecin, abaecin, hymenoptaecin and defensin (Evans et al., 2006). The low number of honeybee AMPs relative to the previously-analyzed dipteran genomes was explained by the existence of alternative mechanisms providing protection from infectious diseases, such as the so-called social immunity which includes grooming and other hygienic behaviors. Sanitation of the beehive by the production or collection of antimicrobial compounds deposited with propolis or transmitted with royal jelly could also contribute to the antimicrobial defense of the colony (Le Conte et al., 2011). Targeted screening for AMPs in the Asiatic honeybee A. cerana, which does not produce propolis, revealed a more diverse AMP profile compared to A. mellifera (Xu et al., 2009). The authors found 11 cDNAs encoding two different abaecins, 13 cDNAs encoding four different apidaecins, 29 cDNAs encoding seven different defensins, and 34 cDNAs encoding 13 different hymenoptaecins, and they suggested the longer domestication history of A. mellifera as an alternative explanation for its smaller immunity gene repertoire. However, recent insights into the genome sequences of the leaf-cutter ant Atta cephalotes (Suen et al., 2011) and the termite Zootermopsis suggest that the AMP repertoire is often strikingly reduced in even nondomesticated eusocial insects compared to solitary hymenopteran species such as the parasitic wasp Nasonia vitripennis (Tian et al., 2010). The latter study identified at least 42 different AMPs whose sequences exhibit signatures suggesting that the evolutionary diversification of AMPs results from classic mechanisms such as gene duplication, exon duplication and exon shuffling. An important and often overlooked challenge is the bias towards known gene families introduced by analyzing the evolution-
Please cite this article in press as: Vilcinskas, A. Evolutionary plasticity of insect immunity. Journal of Insect Physiology (2012), http://dx.doi.org/10.1016/ j.jinsphys.2012.08.018
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ary plasticity of insect immunity using model insect genomes and current bioinformatics methods. The latter have predominantly been used to screen for homologues of immune-related genes known from Drosophila, implicating that all components from this model insect are known. But our knowledge about insect immunity, even in insects with a completely sequenced genome, is far from complete. For example, recent studies provide for the first time evidence for the presence of negative regulators in insect immunity. MicroRNAs have been identified in the model insects Drosophila and, Tribolium which suppress the expression of immune genes at the posttranscriptional level (Freitak et al., 2012; Fullaondo and Lee, 2012). The discovery of novel or unexpected genes requires complementary approaches such as experimental screening for immunity-related genes using proteomic and transcriptomic approaches. The first antimicrobial peptides from insect have been purified by classic biochemical approaches while transcriptomic analysis using next generation sequencing methods are currently widely used (Vogel et al., 2011a). I favor suppression subtractive hybridization (SSH) for this purpose because it has been successfully applied in my group to discover many new or unexpected immunity-related genes in model and non-model insects (Altincicek and Vilcinskas, 2007a,b; Altincicek and Vilcinskas 2009). This PCR-based method amplifies genes that are differentially expressed following treatments such as the injection of bacteria or fungi, while simultaneously suppressing housekeeping genes. For example, conventional bioinformatics identified four defensins in the genome of the model beetle Tribolium castaneum (Tribolium Genome Sequencing Consortium, 2008), whereas our SSH approach revealed additional thaumatin-like genes that were overlooked by the bioinformatics approach (Altincicek et al., 2008a). Thaumatins are antifungal peptides originally identified in plants. A recombinant T. castaneum thaumatin showed potent antifungal activity suggesting it helps to defend beetles against fungal pathogens (Altincicek et al., 2008a). Our discovery led to the identification of homologous sequences in the genome of the pea aphid Acyrthosiphon pisum (International Aphid Genomics Consortium, 2010) using a bioinformatics approach (Gerardo et al., 2010). Interestingly, neither the bioinformatics-based analysis of genomic and transcriptomic data nor our SSH-based experimental screening approach for immunity-related genes revealed the presence of common AMPs such as defensins encoded by the aphid genome (Altincicek et al., 2008a). In addition, piercing aphids with a septic needle did not induce detectable activity against living bacteria. We proposed that aphids lost AMPs during their evolution, and this is further supported by the finding that supposedly essential immunity-related genes such as those encoding components of immunity signaling pathways are also missing from the aphid genome (Gerardo et al., 2010). This unexpected finding by the International Aphid Genomics Consortium (2010) invites a discussion of the selective forces driving the loss and/or expansion of immunity-related genes in insects.
3. The loss of immunity-related genes Pea aphids appear to lack many known immunity-related genes that are considered crucial for sensing bacteria (e.g. peptidoglycan recognition proteins), as well as genes for AMPs (Gerardo et al., 2010) and genes encoding essential immunity-related signaling components, e.g. the immune deficiency (IMD) pathway that was discovered in D. melanogaster and which regulates antibacterial and antifungal defense genes (Kaneko and Silverman, 2005). This may ultimately reflect the unique ecological niche that aphids occupy, i.e. they feed exclusively on phloem sap that is rich in sugars but does not provide a complete profile of amino acids. Aphids are
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therefore dependent on symbiotic bacteria such as Buchnera aphidicola, which account for up to 20% of their dry mass and utilize some of the phloem-derived sugars, but in return synthesize essential amino acids that cannot be sourced from the aphid diet. The lack of antibacterial defenses may therefore reflect the coevolution of aphids and their primary and secondary bacterial symbionts (International Aphid Genomics Consortium, 2010). We proposed three hypotheses to explain why aphids can afford to reduce their antibacterial defenses. First, the uptake of microbes in the diet is limited because phloem sap is normally sterile (Altincicek et al., 2008a). This contrasts with insects that feed on leaves, because leaf surfaces teem with bacteria and fungi (Freitak et al., 2007). Second, aphids may rely on extracellular symbionts to provide antibacterial defenses, e.g. secondary symbionts such as Regiella insecticola and Hamiltonella defensa have been shown to provide protection against fungi and parasitic wasps, respectively (Oliver et al., 2005; Scarborough et al., 2005). Third, piercing aphids with a septic needle induces the production of viviparous offspring, suggesting that aphids invest in terminal reproduction rather than immunity following a pathogen challenge (Altincicek et al., 2008a). The last of these hypotheses implies that maintaining and deploying an immune response takes resources from other fitness-determining traits, and this has inspired research into the ecological selection pressure that drives the evolution of innate immunity in insects (Schmid-Hempel, 2005). The insect immune system is an excellent model allowing the investigation of tradeoffs resulting from investments into different fitness components, a virtual currency that combines survival with reproduction (Schmid-Hempel, 2011). According to this hypothesis, there are limited resources that can be used to improve fitness so any investment in the production of immunity-related proteins requires the reallocation of resources, i.e. their withdrawal from other fitnessrelated traits such as fecundity or adaptability (McKean et al., 2008). Such trade-offs inevitably result in negative selection against genes that do not increase fitness in a given environment, therefore genes representing particular immunity functions can disappear from the genome if their loss costs less in terms of overall fitness than their maintenance. In the pea aphid, we propose that the fitness penalty resulting from the loss of immunity-related genes is negated by the fitness benefits provided by bacterial symbionts (Gerardo et al., 2010). The evolutionary plasticity of insect immunity may partly reflect the vertical transmission of symbionts that provide fitness benefits exceeding the costs of both maintaining the symbionts and losing innate immunity (Haine, 2008). I postulate that the loss of formerly essential immunity-related genes can occur in insects that have evolved alternative and less resource-hungry mechanisms that provide adequate protection against pathogens and parasites, particularly by expanding beyond the maintenance and deployment of an immune system to encompass diverse strategies such as the sanitation of the microenvironment (Gross et al., 2008) or hygienic behavior in ant or bees (Ulgevig and Cremer, 2007; Le Conte et al., 2011). Insects can afford to lose immunity-related genes if genes that mediate other defense mechanisms (e.g. chemical protection) can compensate for the loss of a dynamic host defense without a fitness penalty. For example, herbivorous insects such as the larvae of the brassy willow leaf beetle Phratora vitellinae constitutively release volatile glandular secretions containing antimicrobial compounds sequestered from the host plant, which fumigate ingested food (Gross et al., 2008). This strategy reduces fitness costs because the uptake of bacteria by other herbivorous insects can trigger immune responses associated with life history trade-offs (Freitak et al., 2007). This balance of fitness costs may explain the independent evolution of cellular components that allow plant-derived molecules to be used for the antimicrobial defense of leaf beetles (Kirsch et al., 2011).
Please cite this article in press as: Vilcinskas, A. Evolutionary plasticity of insect immunity. Journal of Insect Physiology (2012), http://dx.doi.org/10.1016/ j.jinsphys.2012.08.018
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Fig. 1. Evolutionary plasticity of insect immunity displayed by extreme differences in constitutive antibacterial activity in the hemolymph of three coccinelid species: the native and non-invasive ladybird Adalia bipunctata (left), the native and invasive (in northern America) seven-spotted ladybird Coccinella septempunctata (middle), and the introduced and invasive harlequin ladybird Harmonia axyridis (right). Three microliters of hemolymph collected upon reflex-bleeding of each beetle were put into a whole punched in Petri-dishes containing agar which was inoculated with Micrococcus luteus bacteria. 24 h upon cultivation at 30 °C zones of bacterial growth inhibition indicate the presence of a strong constitutive antibacterial activity in the hemolymph of H. axyridis which is absent from A. bipunctata and C. septempunctata. Beetles were immobilized by keeping them for 30 min. in the refrigerator, and then put on the wholes to illustrate the corresponding size of inhibition zones. The antibacterial activity in the hemolymph of H. axyridis has been attributed to harmonine, a secondary metabolite, which displayed broad-spectrum antibacterial and antimalaria activity. The production and release within the hemolymph of harmonine putatively saves fitness costs associated with the synthesis of AMPs and may, therefore, contribute to the invasive success of H. axyridis (Röhrich et al., 2012).
Chemical defenses that compensate for or support the protection provided by the immune system are widespread in insects. An impressive example is the burying beetle Nicrophorus vespilloides, which feeds and reproduces on small vertebrate cadavers. The latter are buried in the soil after localization through volatiles emitted from the carcass. Theory predicts that the exposure of the beetles to high loads of soil and/or carrion-associated microbes would require an effective defense system. We have recently analyzed the oral and anal secretions produced by N. vespilloides beetles, which are used to chemically preserve the cadavers as a food source for their offspring. We found more than 30 secondary metabolites in the secretions, most of which have known antimicrobial activities (Degenkolb et al., 2011). These compounds solely mediate the sanitation and chemical preservation of the buried cadavers and thus help to reduce the fitness costs associated with the maintenance and deployment of immune responses (Vogel et al., 2011b). Other beetles such as the invasive harlequin ladybird Harmonia axyridis synthesize and secrete secondary metabolites with broad-spectrum antimicrobial activity into the hemolymph (Fig. 1), explaining its profound resistance against pathogens and parasites which may in turn explain its success as an invasive species (Röhrich et al., 2012). The loss of immunity-related genes thus appears to occur in insects that rely on other strategies with a lower overall fitness penalty for defense against pathogens and parasites.
4. The expansion of immunity-related gene families As stated above, the comparative analysis of insect genomes and immunity-related transcriptomes has provided numerous examples of the expansion of immunity-related gene families. The expansion of AMP genes has been attributed to gene duplication and divergence to produce multiple diverse paralogs (Bulmer
and Crozier, 2004; Dessanayake et al., 2007). Gene or domain duplication followed by recombination between homologous sequences appear to be the key mechanisms underlying the expansion of effector molecule repertoires in invertebrates (Froy and Gurevitz, 2003). Several models for the creation, maintenance and evolution of gene copies have been proposed, but there is no clear consensus on how gene duplications are fixed and maintained (Innan ana Kondrashov, 2010). The remarkable dynamics of AMP gene evolution in insects can be explained in part by adaptations in response to a changing pathosphere. It has been postulated that environmental heterogeneity and pathogen diversity can maintain the diversity of host immunity-related genes by favoring different genotypes at different times and places, favoring the presence of paralogs that can adapt rapidly by recombination as well as mutation (Lazzaro and Little, 2009). The degree of plasticity displayed by insects exposed to the same environmental changes can evolve and be adaptive if fitness is increased by the changing phenotype. A faster and more potent immune response against pathogens following a second exposure (immune priming) was first observed in vertebrates. The latter can be more or less specific. Parental investment in offspring encompasses their preparation to defend themselves against pathogens or parasites initially encountered by the adults. This phenomenon designated as specific transgenerational immune priming has first been discovered in the crustacean Daphnia magna (Little et al., 2003), and then insects such as the bumble bee (Sadd et al., 2005), the beetles Tenebrio molitor (Moret, 2006) and Tribolium castaeum (Roth et al., 2009, and the lepidopteran Trichoplusia ni (Freitak et al., 2009). However, the underlying mechanisms behind the transgenerational immune priming are poorly understood (Chambers and Schneider, 2012). The evolutionary plasticity of insect immunity allows insects to change their phenotype in response to changes in the environment. Phenotypic variation with a genetic basis is an essential prerequisite for evolution. In order to study the impact of the environment on insect immunity, we used rat-tailed maggots of the drone fly Eristalis tenax as a model system because these are the only animals that survive in extremely polluted aquatic habitats such as liquid manure storage pits and cesspools (Altincicek and Vilcinskas, 2007a). From an evolutionary point of view, it is advantageous to be the only survivor in habitats with extreme microbial loads, because this saves fitness costs associated with dealing with competitors and predators. However, this saving must be balanced against the increased costs of a robust antimicrobial defense strategy. I therefore postulated that the colonization of such habitats would be accompanied by corresponding adaptations in the immune system, and found that the immune-inducible E. tenax transcriptome revealed the rapid diversification of AMPs and signatures of positive selection in individual members such as the defensin homolog eristalin (Altincicek et al., 2008b). These observations support the hypothesis that the changing pathosphere is a strong selective force favoring immune gene expansion. The impact of environmental adaptations on the evolution of immune genes has recently been demonstrated by an independent and intriguing approach in which the emergence and loss of protein domains was reconstructed in 20 arthropod species of the pancrustacean clade (Moore and Bornber-Bauer, 2012). Proteins contain structurally and functionally independent domains that are evolutionarily conserved across taxa (Moore et al., 2008). Protein evolution in eukaryotes is predominantly characterized by the rearrangement of domains rather than their emergence or loss. The pancrustacean clade is often used to estimate the frequency of protein domain gain and loss because it encompasses a large number of species with completed genome sequences whose homologous relationships are well defined, and which differ in their degree of environmental adaptation. Despite the lower rate
Please cite this article in press as: Vilcinskas, A. Evolutionary plasticity of insect immunity. Journal of Insect Physiology (2012), http://dx.doi.org/10.1016/ j.jinsphys.2012.08.018
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of domain turnover relative to gene family turnover, this comparative genomics approach revealed that domain emergence is associated predominantly with environmental adaptations. The authors introduced a novel indirect gene ontology (GO) analysis method that allowed the quantitation of domain emergence in proteins related by defined GO terms and found that proteins related to innate immune responses were the most frequently represented (Moore and Bornber-Bauer, 2012).
5. Functional shifts among immunity-related effector genes The evolutionary plasticity of insect immunity can be expanded beyond the diversification or loss of immunity-related effector molecules to include functional shifts, i.e. the adaptation of proteins to take on novel functions. The AMPs present in a single insect species can often be grouped into different gene families originating from a single ancestral gene. The above mentioned duplication and divergent evolution of AMP genes is associated with functional shifts of the resulting paralogs that can ultimately lead to the formation of new AMP families (Tian et al., 2010; Yang et al., 2011). Predicted functional shifts among effector molecules are not easy to confirm because individual AMPs are difficult to purify as test for their antimicrobial spectrum, but this can be addressed by the expression of AMPs as recombinant proteins. This approach has recently been used to examine the functional divergence of AMP families in the silkworm Bombyx mori (Yang et al., 2011). The authors found that the gloverins evolved slowly and their antimicrobial properties were largely conserved, whereas the cecropin and moricin families showed diverse activities. This type of shifting has previously been observed among the defensins, most of which show activity against Gram-positive and/or Gramnegative bacteria, but some of which can also inhibit fungi, including defensins that act exclusively against mycelia-forming fungi (Langen et al., 2006). The heterologous expression of insect AMPs has become a screening tool for the discovery of peptides that inhibit human pathogens (Ratcliffe et al., 2011) particularly multidrug-resistant pathogens and emerging diseases (Vilcinskas, 2011). Insect AMPs could also be used to engineer disease-resistant plants (Vilcinskas and Gross, 2005). In addition to AMPs, other effector molecules in insects can allow the experimental assessment of immunity-related evolutionary plasticity, such as inhibitors of pathogen or parasite proteinases. Virtually all pathogens use proteolytic enzymes to digest host proteins for nutrition and to degrade host defense molecules such as AMPs (Armstrong, 2006; Potempa and Robert, 2009). Such enzymes are known as virulence factors when they are required to accomplish successful infections. For example, parasitic fungi produce and secrete an array of proteolytic enzymes enabling them to infect insects directly via the protein-containing exoskeleton (Vilcinskas, 2010). The known substrate specificity and the controlled expression profiles of invasive proteinases indicate that the diversification and functional shifting among these fungal enzymes is driven by adaptation to different host ranges. However, only proteinases that cannot be completely inactivated by corresponding host proteinase inhibitors in the cuticle, tissues or hemolymph can operate as virulence factors (Vilcinskas, 2010). This complementarity between virulence and defense factors results in the negative selection of pathogen-associated proteinases for which a surplus of corresponding inhibitors is present in the host, because the secretion of such enzymes would waste resources. In turn, emerging proteinases with no corresponding inhibitors in the host would be subject to positive selection because they would function successfully even when produced in low quantities (Vilcinskas, 2010). This hypothesis explains the evolution of particular M4 family microbial metalloproteinases such as thermolysin
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because most animals have not evolved corresponding inhibitors during host-parasite coevolution. Therefore, thermolysin-like metalloproteinases such as aureolysin, pseudolysin and vibriolysin are virulence factors in many human pathogens (Adekoya and Sylte, 2009). Only insects, particularly the greater wax moth Galleria mellonella, have evolved a peptide that can specifically inhibit thermolysin-like metalloproteinases. This insect metalloproteinase inhibitor (IMPI) is synthesized and secreted into the hemolymph along with other AMPs where it protects them from degradation by microbial metalloproteinases (Vilcinskas and Wedde, 2002). The IMPI sequence does not match any other proteins in public databases although screening the Pfam database of protein families for conserved domains revealed significant similarity to a trypsin inhibitor-like (TIL) cysteine-rich domain (Clermont et al., 2004). IMPI has thus been assigned to protease inhibitor family I8, a cysteine-rich trypsin inhibitor-like family in the MEROPS database of peptidases, although it is not a serine proteinase inhibitor (Rawlings et al., 2004). Our recent research provides evidence for the presence of a family of IMPI paralogs in G. mellonella resulting from gene and/or domain duplications and for functional shifts within this family (Wedde et al., 2007). Furthermore, we recently analyzed the crystal structure of IMPI in a complex with thermolysin, revealing an unprecedented inhibition mechanism that was previously thought to be restricted to serine proteinase inhibitors. The recruitment of one class of protease inhibitors to regulate another class is a case of divergent evolution based on the hyper-variability of enzyme-contacting regions, strongly suggesting a recent ‘arms race’ and providing another intriguing example for the plasticity of insect immunity. Our results suggest that IMPI originates from reciprocal selection in an antagonistic environment, in which the insect host developed a novel defense against the weapons of microbes (Arolas et al., 2011). The specificity of proteinases and their inhibitors is easily determined, making them powerful tools to study the dynamics of host-parasite coevolution directly at the frontier where the antagonists interact. 6. Concluding remarks The comparative analysis of insect genomes and immunityrelated transcriptomes has revealed the remarkable plasticity of the insect innate immune system, including remarkable examples of gene loss, gene functional adaptation, and gene family expansion under the unique selection pressures of different ecological niches. Gene loss may result from a trade-off between immunity and other traits whose maintenance has a lower fitness penalty in response to particular environmental changes. Deciphering the functional shifts of immunity-related effector molecules such as AMPs and protease inhibitors that act against pathogen virulence factors has expanded our knowledge about evolutionary plasticity in insects even further by revealing unanticipated dynamic interactions. In order to explore the mechanisms underlying complex immunity-related phenomena in insects such as the rapid reciprocal adaptations during host-parasite-coevolution, trans-generational immune priming and gender specific-responses, we have started to examine the role of epigenetic mechanisms in insect immunity. Acknowledgements The author acknowledges project funding provided by the German Research Foundation (Deutsche Forschungsgemeinschaft) within the DFG priority program 1399 ‘‘Host-parasite-coevolution - rapid reciprocal adaptation and its genetic base’’ (VI 219/3-1) and by the Hessian Ministry of Science and Art via the LOEWE excellence initiative which encompasses a generous grant for the research focus ‘‘Insect Biotechnology’’.
Please cite this article in press as: Vilcinskas, A. Evolutionary plasticity of insect immunity. Journal of Insect Physiology (2012), http://dx.doi.org/10.1016/ j.jinsphys.2012.08.018
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Please cite this article in press as: Vilcinskas, A. Evolutionary plasticity of insect immunity. Journal of Insect Physiology (2012), http://dx.doi.org/10.1016/ j.jinsphys.2012.08.018