Summary

Recently it has become evident that invertebrates may mount a highly variable immune response that is dependent on which pathogen is involved. The molecular mechanisms behind this diversity are beginning to be unravelled and in several invertebrate taxa immune proteins exhibiting a broad range of diversity have been found. In some cases, evidence has been gathered suggesting that this molecular diversity translates into the ability of an affected invertebrate to mount a defence that is specifically aimed at a particular pathogen.

Introduction

Invertebrates need to defend themselves against a variety of pathogens. It is obvious that invertebrates are efficient at finding countermeasures against intruding microbes in spite of, in a number of cases, relying on immune systems that lack many of the components familiar from mammalian immunology. Understanding of invertebrate immunity has for some time been dominated by the idea that a relatively small number of germ-line-derived pattern-recognition proteins bind to a few molecules, in particular the major constituents of cell walls or other surface structures of potential pathogens and this initial recognition event in turn sets in motion a limited number of relatively fixed early responses such as phagocytosis, encapsulation, coagulation, melanisation and the production of oxygen radicals and other short-lived toxic compounds, followed by more long-term effects such as the onset of antimicrobial peptide (AMP) synthesis. This general response to compounds such as peptidoglycans, lipopolysaccharides, beta-1,3-glucans and double-stranded RNA (dsRNA), which are present in many microorganisms, certainly constitutes the backbone of invertebrate immunity, but from recent research a more complex picture is starting to emerge. Separate bacterial strains or species may, in the same host, trigger an immune response that differs considerably, both quantitatively and in terms of which immune effectors are used (Ayres and Schneider, 2008). Many invertebrates have the capacity to synthesise immune proteins with an enormous range of sequence variability. Together this seems to suggest that invertebrate immune reactions to pathogens may be as varied and complex as their vertebrate counterparts. The existence of such hypervariable proteins has led to speculation that they could constitute part of a system that would allow immune memory, or at least immune specificity, in invertebrates. Although there are some intriguing data suggesting the possibility of an immune memory or immune priming in invertebrates (e.g. Sadd and Schmid-Hempel, 2006), so far any mechanisms that point to the existence of immune memory in any invertebrate have not been presented. However, what is clear is that invertebrates can mount immune defences that vary in duration and specificity depending on which pathogen the animal is exposed to (e.g. Kurtz and Franz, 2003; Little et al., 2005).

We will discuss some aspects of these complexities concerning variable invertebrate immune proteins and some recent progress made in the field in this Commentary.

A varied immune response: variable lymphocyte receptors in jawless vertebrates

Although not invertebrates, the existence of an adaptive immune system in lampreys and hagfish based on variable molecules other than immunoglobulins indicates that a common function – that is, the production of diversified and clonally expressed receptors of antigens – may be achieved by means other than those in use in the immune system of jawed vertebrates. Lamprey lymphocytes possess variable lymphocyte receptors (VLRs) containing multiple leucine-rich repeats (LRRs). These can be combined by gene conversion events into molecules unique to separate clones of lymphocytes (Pancer et al., 2004). A single lymphocyte normally expresses a single type of VLR protein. Theoretically, this system can produce as much diversity as seen in mammalian B- and T-cell receptors. The similarities with the adaptive immune system of jawed vertebrates are underscored by the fact that a soluble VLR form capable of agglutinating antigen is produced in these animals as well and that these jawless vertebrates appear to have what have been likened to T-like and B-like lymphocytes (for a review, see Boehm et al., 2012). The existence of similar LRR-based adaptive immune systems in invertebrate deuterostomes is an exciting possibility but remains to be proven. Nonetheless, the lamprey example shows that an immune system capable of clonal expansion could obviously be based on proteins with antibody-like functions other than immunoglobulins. Equally intriguing is the fact that LRR-containing proteins form the core of some other immune/recognition systems such as the plant R-proteins (proteins that confer resistance towards specific pathogens with a corresponding avirulence protein), the intracellular NOD (nucleotide oligomerisation domain)-like receptors and the transmembrane toll-like receptors (TLRs), the last two of which are present in many animal groups. It seems that LRR domains are a major alternative to immunoglobulin domains for achieving ligand binding and specificity in the evolution of pattern recognition.

Extensive diversity among deuterostome putative immune (receptor) proteins: amphioxus variable region-containing chitin-binding proteins and the sea urchin SP185/333 proteins

Amphioxus and Ciona harbour a family of proteins containing two highly polymorphic immunoglobulin domains and a C-terminal chitin-binding protein, coined the VCBP family for variable region-containing chitin-binding protein. These VCBPs exhibit two hypervariable domains that have, based on detailed structural studies, been suggested to fold into a receptor-binding site (Prada et al., 2006). The amphioxus VCBP molecule has a strong overall structural resemblance to vertebrate immunoglobulin and T-cell receptors in spite of considerable differences in sequence and position of the hypervariable portions. However, the diversity among the VCBPs produced (from about five loci) is considerably higher in amphioxus than in Ciona (Dishaw et al., 2011). In addition, the number of genes coding for putative recognition proteins containing LRRs, such as nucleotide-binding oligomerisation domain-like receptors, is high in amphioxus (Fig. 1). One may also note, although the immunological importance remains unexplored, that the amphioxus genome demonstrates a considerable expansion of certain gene families such as C-type lectins (ca. 1200 members) and putative scavenger receptors (ca. 270 members) (Dishaw et al., 2012), which requires experimental testing. Encouragingly, in arthropods experimental data on both class B scavenger receptor involvement in bacterial phagocytosis (e.g. Aung et al., 2012) and on regulation of the melanisation reaction by a C-type lectin (Wu et al., 2013) has been obtained recently and it may be possible to determine the corresponding immunological roles for these classes of proteins by comparison with equivalent proteins from other taxa. In Ciona the VCBP is produced by the granular-type amoebocytes and by a population of the epithelial cells of the stomach and intestine. Less is known about the biological roles of the VCBPs but present data suggest a function in gut innate immunity where they stimulate amoebocyte phagocytosis (Dishaw et al., 2011). Other highly variable putative immune receptors are the members of the 185/333 gene cluster, originally described (Nair et al., 2005) in the purple sea urchin Strongylocentrotus purpuratus (and therefore sometimes called Sp185/333), which is present in other sea urchin species. The S. purpuratus 185/333 gene family contains about 50 loci. These genes appear to recombine, thus producing diversity. In addition, gene duplications and conversions may further increase the sequence diversity among 185/333 genes. Their mRNAs and predicted protein sequences show extensive sequence diversity and the number of sequence variations is increased further by post-transcriptional modifications. Transcription of the gene cluster increases dramatically (up to 75-fold) in immunologically challenged coelomocytes and the composition of the different 185/333 members produced depends on the elicitors used, such as exposure to lipopolysaccharide (LPS) or peptidoglycans or dsRNA (Dheilly et al., 2009), resulting in different 185/333 populations, although the functional role of the 185/333 proteins remains unknown. In spite of the absence of a membrane-spanning domain or glycosylphosphatidylinositol (GPI)-anchor motif that mediates binding of the protein to the cell membrane within the 185/333 sequence, these proteins are nevertheless frequently seen associated with the cell membrane and are not free in the coelomic fluid. The 185/333 proteins are heterogeneous in size and have a strong tendency to oligomerise. The functional significance of this is unclear at present, although it has been suggested that they may form bridges between immune cells (Brockton et al., 2008). They do not appear to bind bacteria in the coelomic fluid but an immunostaining study has suggested that gut amoebocytes phagocytose 185/333-covered bacteria (Dheilly et al., 2011). Conclusive evidence for the exact role of these proteins in sea urchin immunity is eagerly awaited.

Fig. 1.

Schematic views of four invertebrate immune proteins exhibiting large sequence variability. From the top: Down syndrome cell adhesion molecule (Dscam), Toll-like receptor (TLR), fibrinogen-related protein (FREP) and nucleotide-binding oligomerisation domain-like receptor (NLR). Dscam: representation of Dscam from the crayfish Pacifastacus leniusculus. Ig, immunoglobulin (superfamily) domain; FN III, fibronectin type III domain; TM, transmembrane domain. The 2nd, 3rd and 7th Ig domains, as counted from the N-terminus, plus the TM domain provide the sequence variation through alternative splicing. The variable Ig domains are coloured to indicate this diversity. Theoretically, the crayfish Dscam can produce more than 22,000 different isoforms. One experimentally proven activity by Dscam is to bind to microorganisms and promote their uptake by host cells. TLR: one example of a sea urchin (Strongylocentrotus purpuratus) TLR. LRR, leucine-rich repeat; LRR-NT and LRR-CT, N-terminal and C-terminal leucine-rich repeat domain, respectively; TM, transmembrane domain; TIR, toll/interleukin-1 receptor domain. The sequence diversity is found within the LRR repeats. This species has more than 200 TLR genes and the number of LRRs and their sequences vary significantly between them. The precise function(s) of sea urchin TLRs is not well known but insect TLRs have very well-established roles in mediating immune reactions. A FREP 3-form from Biomphalaria glabrata. EGF, epidermal growth factor domain; IgSF1 and IgSF2, immunoglobulin super family domain 1 and 2, respectively; SCR, small contacting region; ICR, interceding region; FBG, fibrinogen domain. In B. glabrata there are 14 different FREP subfamilies with a varying number of loci (and domain organisation) per subfamily. FREP 3 gene products are very diverse through gene conversions and point mutations in the IgSF1, IgSF2 and ICR domains. The FREP proteins are calcium-dependent lectins and FREP3 in particular is associated with increased resistance towards parasites in the snail. NLR from the amphioxus Branchiostoma floridae. CARD, caspase recruitment domain; DD, death domain; NACHT, a domain predicted to contain nucleoside phosphatase activity and to include seven distinct sequence motifs; LRR, leucine-rich repeats. There are about 100 NLR genes in the B. floridae genome. They are intracellular putative pattern-recognition (i.e. binding-conserved sequence motifs characteristic of potential pathogens) proteins. Figure courtesy of Apiruck Watthanasurorot.

Fig. 1.

Schematic views of four invertebrate immune proteins exhibiting large sequence variability. From the top: Down syndrome cell adhesion molecule (Dscam), Toll-like receptor (TLR), fibrinogen-related protein (FREP) and nucleotide-binding oligomerisation domain-like receptor (NLR). Dscam: representation of Dscam from the crayfish Pacifastacus leniusculus. Ig, immunoglobulin (superfamily) domain; FN III, fibronectin type III domain; TM, transmembrane domain. The 2nd, 3rd and 7th Ig domains, as counted from the N-terminus, plus the TM domain provide the sequence variation through alternative splicing. The variable Ig domains are coloured to indicate this diversity. Theoretically, the crayfish Dscam can produce more than 22,000 different isoforms. One experimentally proven activity by Dscam is to bind to microorganisms and promote their uptake by host cells. TLR: one example of a sea urchin (Strongylocentrotus purpuratus) TLR. LRR, leucine-rich repeat; LRR-NT and LRR-CT, N-terminal and C-terminal leucine-rich repeat domain, respectively; TM, transmembrane domain; TIR, toll/interleukin-1 receptor domain. The sequence diversity is found within the LRR repeats. This species has more than 200 TLR genes and the number of LRRs and their sequences vary significantly between them. The precise function(s) of sea urchin TLRs is not well known but insect TLRs have very well-established roles in mediating immune reactions. A FREP 3-form from Biomphalaria glabrata. EGF, epidermal growth factor domain; IgSF1 and IgSF2, immunoglobulin super family domain 1 and 2, respectively; SCR, small contacting region; ICR, interceding region; FBG, fibrinogen domain. In B. glabrata there are 14 different FREP subfamilies with a varying number of loci (and domain organisation) per subfamily. FREP 3 gene products are very diverse through gene conversions and point mutations in the IgSF1, IgSF2 and ICR domains. The FREP proteins are calcium-dependent lectins and FREP3 in particular is associated with increased resistance towards parasites in the snail. NLR from the amphioxus Branchiostoma floridae. CARD, caspase recruitment domain; DD, death domain; NACHT, a domain predicted to contain nucleoside phosphatase activity and to include seven distinct sequence motifs; LRR, leucine-rich repeats. There are about 100 NLR genes in the B. floridae genome. They are intracellular putative pattern-recognition (i.e. binding-conserved sequence motifs characteristic of potential pathogens) proteins. Figure courtesy of Apiruck Watthanasurorot.

Fibrinogen-related proteins in molluscs and arthropods

Fibrinogen-related proteins (FREPs), originally discovered and characterised from echinostome parasite-carrying Biomphalaria snails, combine immunoglobulin-like and fibrinogen domains into one protein (Zhang et al., 2004). They have some similarity to vertebrate ficolins, which are lectins that activate the complement system and act as opsonins. In contrast to ficolin, Biomphalaria FREPs do not contain a collagen-like domain in their N-terminus. They are soluble carbohydrate-binding proteins with the capacity to bind parasites, such as the trematode parasites schistosomes, and to promote their elimination from the host. Different FREPs have different binding capacities and some large FREPs are mainly directed towards bacteria and yeasts. In B. glabrata there are 14 sub-families and further diversification is achieved by alternative splicing combined with high mutation rates and possible gene conversions among some of the FREP genes. In particular, FREP2 and FREP3 (Fig. 1) are highly variable (Hanington et al., 2010; Moné et al., 2010). This will create differences between individuals and populations of snails but also give rise to haemocytes that release different FREP isoforms within a single individual. In particular, FREP3 has been implicated as a major contributor to trematode resistance in Biomphalaria (Hanington et al., 2010; Hanington et al., 2012). FREPs have been found to bind to mucins (gel-forming glycoproteins) on the Schistosoma surface (Moné et al., 2010). As these mucins are polymorphic it is possible that there is an on-going arms race between snail and parasite where some mucin and FREP genes may be forced to evolve rapidly in order to keep the parasite capable of infection and to maintain the host's ability to recognise the trematode (Moné et al., 2010). Other molluscs may contain polymorphic FREPs that lack an immunoglobulin-like domain, such as the mussel Mytilus galloprovincialis (Romero et al., 2011). The mussel FREPs display extensive sequence variation and interestingly purified FREPs display opsonic activity in in vitro assays with mussel haemocytes (Romero et al., 2011).

A second, large group of FREPs, which lack immunoglobulin domains, is also found in invertebrates. In the mosquito Anopheles gambiae, whose genome possesses at least 59 FREP genes, the influence of these proteins on the immune capacity of the animal has been extensively investigated by RNA interference (Dong and Dimopoulos, 2009). A reduction in FREP production causes an increase in the vulnerability of mosquitoes to microbial infection. At least some mosquito FREPs bind to bacterial surfaces while forming dimers. Whether they act as opsonins remains to be established, but their association with haemocyte membranes could indicate that this is the case. A similar expansion of the number of FREP genes has occurred in several Drosophila species (Middha and Wang, 2008) as well as in some mosquito species, possibly involving tandem duplications of the fibrinogen domain (Dong and Dimopoulos, 2009). Proteins with fibrinogen domains participate in coagulation and immune reactions in many taxa. Ficolin in mammals binds microbes and activates the complement system. Tachylectin 5A in horseshoe crabs exhibits significant sequence similarities with ficolins and is present at a high concentration in the haemolymph, where it probably constitutes the first line of defence against infection through its strong multivalent binding capacity for cell surface N-acetylglucose amine moieties, whereby microorganisms may become agglutinated, i.e. forming aggregates (Gokudan et al., 1999; Kairies et al., 2001). In a crustacean, two ficolin-like proteins were recently demonstrated to bind to Gram-negative bacteria and this event was followed by the clearance of the bacteria (Wu et al., 2011).

Down syndrome cell adhesion molecule in arthropods

Down syndrome cell adhesion molecule (Dscam), belonging to the immunoglobulin super family, was originally found to specify neuron–neuron contacts in humans and other animals. When it was discovered that Dscam proteins are also produced in insect haemocytes and that they can bind to bacteria (Watson et al., 2005; Dong et al., 2006), the discovery aroused significant interest amongst immunologists as it is possible to form tens of thousands of different Dscam isoforms (Fig. 1) that could potentially bind a large array of antigens by alternatively splicing exons. In the mosquito A. gambiae, Dscam affects the insect's resistance to both bacteria and Plasmodium parasites. In the mosquito, Dscam expression and transcript diversity increase about 24 h after exposure to Plasmodium, coinciding with the parasite crossing the gut epithelium of the insect (Smith et al., 2011). In freshwater crayfish, different Dscam proteins are produced after injection of Escherichia coli and Staphylococcus aureus, respectively (Watthanasurorot et al., 2011). Each bacterial species triggers the production of a group of Dscam proteins with similar but not identical sequences; sequence analysis shows that E. coli and S. aureus induce Dscam transcripts clustered into two discernible clades in a phylogram. Using recombinant forms of these proteins it was shown that the Dscam isoforms bound their corresponding bacteria with a higher avidity; that is, a bacterium may selectively induce the production of Dscam isoforms with a higher capacity to promote phagocytosis and clearance of a particular bacterial species (Watthanasurorot et al., 2011). However, a well-defined role in immunity for Dscam awaits further data. Sequence analyses of arthropod Dscams reveal extensive polymorphisms in putative pathogen binding sites but have failed to provide clear evidence for an arms race with pathogens (Brites et al., 2011), although this does not exclude an immune function for Dscam.

TLRs in invertebrates

TLRs are important pattern-recognition transmembrane proteins in mammals. The nine mammalian TLRs bind specific pathogen-associated molecular patterns (PAMPs) on the cell surface or in the endosomes. In fruit-flies – where their importance in immunity was originally discovered (Lemaitre et al., 1996) – the TLR ligand is not a PAMP but a processed cytokine, Spätzle. A proteolytic cascade is initiated by pattern-recognition proteins that bind to bacterial peptidoglycans or β-glucans of fungal origin and initiate processing of the pro-form of the Spätzle protein into its active Toll-binding form. This in turn initiates synthesis of AMPs, which are active primarily against some gram-positive bacteria and fungi, in the insect. Similar proteolytic cascades mediate AMP synthesis, prophenoloxidase (melanisation) activation and the activation of other immune effectors in many insect species and crustaceans (for reviews, see Cerenius et al., 2008; Cerenius et al., 2010) and possibly in other invertebrates as well. Some of these cascades, such as the Toll cascade and the proPO-cascade, are interconnected in order to coordinate the different arms of the innate defence, although there will be considerable differences in the way in which this is achieved in different insect species (Kan et al., 2008; Buchon et al., 2009; Roh et al., 2009).

Thus, TLR function differs between mammals, where the effect in many cases is mediated by direct pattern recognition, and arthropods, where indirect pattern recognition and a proteolyic cascade trigger activation of the receptor. In at least some arthropod species (e.g. Drosophila), TLRs have been assigned a role in early development by serving as receptors for proteins governing cell differentiation during embryogenesis. In Drosophila, Spätzle is a ligand for Toll during both embryogenesis and AMP induction but the up-stream events that finally lead to cleavage of pro-Spätzle into Spätzle differ (Morisato and Anderson, 1995; Lemaitre et al., 1996). However, in one recent study the direct binding of an arbovirus to the Drosophila Toll-7 receptor was demonstrated (Nakamoto et al., 2012). Caution must therefore be exercised when assigning a function for newly discovered TLRs in different phyla. The discovery of large expansions of the TLR family in the genomes of some animals, notably amphioxus and sea urchins (Fig. 1), questioned whether they act as receptors for foreign material as in mammals. If this is the case they would theoretically be capable of discriminating between hundreds of different PAMPs. Two different ascidian (Ciona) TLRs were recently transfected into a mammalian cell culture and found to mediate activation of the NF-κ B pathway (a highly conserved intracellular pathway that is involved in activating expression of a number of immunity-related genes in both invertebrates and vertebrates) in cell culture in the presence of putative ligands such as flagellin, heat-killed bacteria or dsRNA (Sasaki et al., 2009). Although these results need to be corroborated using sea urchins, they seem to indicate that sea urchin TLRs and their vertebrate counterparts function in the same way and not, as in insects, via proteolytic processing by extracellular cascades. Furthermore, these data could indicate that there is a difference between deuterostomes and protostomes in how TLRs are recruited for use in their respective immune systems.

Proteolytic cascades and regulation of immune responses

Our picture of invertebrate immune systems, even in the most extensively explored species, is still very fragmented. We have barely started to piece together the factors and reactions that together constitute the varied responses an animal can mount against pathogens. The presence of foreign material or ‘danger signals’ produced by an animal in response to damage inflicted upon it may trigger different sets of chains of reactions. This ultimately results in the activation of different effector mechanisms such as phagocytosis, coagulation, melanisation, release of short-lived toxic substances, synthesis of antimicrobial peptides and other mechanisms. From experience with vertebrates we know that proteolytic cascades are one mechanism for controlling these processes, as demonstrated by the complement and coagulation cascades. In invertebrates, one immune cascade that has been well characterised biochemically is the horseshoe crab coagulation system (Kawabata, 2010), which is exploited commercially in the limulus test for endotoxins. This cascade shows no close resemblance to mammalian cascades and may have developed independently. The evolutionary origin of the complement system has long been debated. It seems that the major constituents of the complement system were present early in deuterostome evolution; a ‘protocomplement’ containing complement component 3 (C3), complement factor B (Bf), mannose-binding lectin-associated serine protease (MASP) and several lectins is present in several urochordate species (Nonaka and Satake, 2010). In amphioxus, ficolin-mediated activation of C3 has been shown (Huang et al., 2011). Among protostomes, C3-like proteins – named thioester-containing proteins (TEPs) – have been implicated in immunity and in some species there is a relatively high number of TEP homologues; for example, 19 TEP homologues have been identified in in A. gambiae (Levashina et al., 2001). The protostome C3-like proteins lack several structural elements present in deuterostome C3 but both possess an internal thioester bond, which is important for their function. In addition to C3/C3-like proteins there is the thioester-containing broad specificity proteinase inhibitor α-2-macroglobulin (A2M), which is probably phylogenetically related to TEPs and C3. The presence in a single animal of a variety of TEP and A2M forms may allow some division of function. In crayfish, for example, a TEP gene is specifically expressed in tissues beneath the cuticle, such as the intestine and the gills, whereas two different A2Ms are produced by the haemocytes (Wu et al., 2013). Crayfish TEP was demonstrated to be important in combatting gastrointestinal infections whereas one of two A2Ms is crosslinked by transglutaminase to the clotting protein at wound sites, possibly to block proteinases secreted by microorganisms trying to invade via the wound site (Wu et al., 2013; Chaikeeratisak et al., 2012; Hall and Söderhäll, 1994). The data mentioned above and in numerous other publications undoubtedly will stimulate further searches for complement-like activities in protostomes.

The proPO-cascade bears some resemblance to the complement system in that it is triggered by lectins, is composed of serine proteinases and produces opsonic, toxic and lytic activities. As mentioned above, in the last few years we have witnessed several new discoveries that have revealed intriguing details of how the system is regulated both positively and negatively (reviewed in Cerenius et al., 2008; Cerenius et al., 2010); the precise manipulation of the system by several highly adapted pathogens (e.g. Crawford et al., 2012); and the possible link between the proPO-activating cascade and the system wound response by a proPO-activating proteinase as suggested by data obtained from Drosophila (Nam et al., 2012).

Different microorganisms trigger different immune responses

Despite possessing very similar PAMPS on their surfaces, different microbial strains are able to activate a variety of immune responses in invertebrates. However, it is easy to overlook this fact when experiments are performed by injecting large doses of an organism, such as E. coli, that are greater than would normally be encountered by an animal. Research using Drosophila and experimental infections with different types of bacteria demonstrates an equally diverse set of immune responses and outcomes of the infection (Ayres and Schneider, 2008). Experimental interference with separate immune pathways such as the imd, Toll or melanisation response affects different bacteria in various ways. It is also evident when testing in vitro that, for example, different AMPs or the products of the proPO-cascade affect different microorganisms in diverse ways.

One way of deciphering the reasons for the different immune responses elicited by even closely related microorganisms is to test the survival of series of mutants from a single pathogenic species in a host organism. By mutating structural components of the cell surface of the bacterium Aeromonas hydrophila, Noonin and co-workers were able to monitor the effect of specific structural motifs on bacterial survival in crustacean and insect hosts (Noonin et al., 2010). In this way crucial parts of the LPS structure that affect bacterial virulence in the host were identified. It was also possible to map the effects of the mutated bacteria on different components of the host proPO and thus on the melanisation reaction (Noonin et al., 2010). In a recent study using different mutants of Pseudomonas aeruginosa in an oral Drosophila infection model (not by injecting with a needle as is often the case in experimental set-ups), the quorum-sensing transcription factor RhlR was identified as a crucial component for establishing an infection (Limmer et al., 2011). The exact mechanism is still unknown, but RhlR is obviously affecting phagocytic clearance and consequently the establishment of an early infection.

Tolerance and symbioses

Awareness of the costs of mounting an immune response, in terms of both energy expenditure and the potential self-damage caused by highly reactive immune components, has raised interest in deciphering the mechanisms behind host tolerance of pathogenic organisms (Ayres and Schneider, 2012). Although little is currently known about the mechanisms of pathogen tolerance, deep-sequencing analyses have revealed the presence of many microbes that appear not to induce overt immune responses in their hosts. In some cases it is evident that the microorganism has developed mechanisms to cope with and even manipulate the hosts's immune system (e.g. Kleino et al., 2008; Lhocine et al., 2008). This raises the possibility that one controlled infection may protect the host against additional infections by other pathogens (e.g. Haine, 2008). The most thoroughly investigated microbial–invertebrate symbiosis is undoubtedly Wolbachia, an intracellular bacterium that is present in many arthropods and nematodes and has profound effects on its hosts (e.g. Werren et al., 2008). One reason for the interest in this particular interaction is the demonstration of increased resistance against Plasmodium and viruses in various mosquito species when Wolbachia is present (e.g. Hedges et al., 2010; Hughes et al., 2011). Other well-studied examples are the squid–Vibrio fischeri symbiosis and the pea aphid–Buchera symbiosis (for a review, see Nyholm and Graf, 2012). Symbiotic microorganisms may provide further advantages for the infected animal. A recent study provides evidence for a role of mutualistic Burkholderia bacteria in mediating insecticide resistance in the bean bug Riptortus pedestris (Kikuchi et al., 2012) and it is likely that deep-sequencing techniques will continue to reveal many more or less ‘hidden’ microorganisms in other invertebrates. These and numerous other examples demonstrate that the immune response in the simultaneous presence of several pathogens may be very complex, such that certain microbes may either stimulate or hinder an adequate response to additional pathogens or other environmental factors.

Concluding remarks

The picture of invertebrate immunity is growing increasingly complex. Invertebrates are capable of mounting a wide range of variable immune responses by activating different components of their immune arsenal when dealing with different pathogen species and varying doses of pathogens, or when different host tissues or organs are involved. Studies of all these interactions will require extensive effort and a large technical repertoire. We anticipate that detailed investigation of a few thoroughly characterised host–pathogen interactions will continue to provide further insights that will permit us to identify further elements of invertebrate responses to infection.

Footnotes

Funding

This work was financed by the Swedish Science Research Council [grant nos 621-2012-2418, 319-2010-6250 to K.S.].

Glossary

     
  • Antigen agglutination

    This is the binding of several microbial particles or antigen molecules by a multivalent (with several binding sites) antibody. Agglutination may limit the spread of microbes or antigen by retaining it in aggregates.

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  • Coagulation

    Coagulation is the formation of a clot or gel in the blood/haemolymph to prevent excessive bleeding and to entrap microorganisms. In mammals this is brought about by the conversion of soluble fibrinogen into insoluble fibrin. Data from three invertebrates, horseshoe crabs, crustaceans and fruit flies, show that even within arthropods, highly different molecular mechanisms are used to achieve haemolymph coagulation.

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  • Encapsulation

    In invertebrates a number of haemocytes attach to foreign objects, e.g. parasites or fungal spores, too large to be phagocytosed. Within the capsule many different immune reactions such as formation of reactive oxygen species and melanin synthesis take place.

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  • Granular amoebocyte

    In sea squirts, granular amoebocytes are one type of haemocyte characterised by granules of variable sizes in the cytoplasm. These cells may carry out phagocytosis and synthesise complement proteins. Other invertebrates have a variety of different types of granular haemocytes that participate in many immune reactions.

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  • Immunoglobulins

    These are proteins also called antibodies. They are produced by vertebrate lymphocytes and bind with high specificity to foreign material (antigens). Proteins containing immunoglobulin domains (i.e. that belong to the immunoglobulin superfamily) are present in invertebrates and some of them such as Dscam and haemolin are associated with immune reactions.

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  • Lectins

    These are proteins that bind carbohydrates normally without enzymatically modifying them. Different lectins bind specific sugar groups and may therefore serve as recognition factors of microbial compounds. Mannose-binding lectin and ficolin are two lectins that activate the complement system.

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  • Leucine repeats

    These are repeats of sequence motifs containing leucine residues within proteins. There are several families of leucine-rich repeat (LRR)-containing proteins with repeats of different length and composition. LRR proteins are involved in many protein–protein interactions. Examples of immune proteins containing LRRs are plant resistance factors and toll-like receptors in several animal groups.

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  • Lymphocytes

    These are the white blood cells responsible for the reactions of the adaptive immune system. In jawed vertebrates B lymphocytes (B-cells) produce antibodies and T lymphocytes (T-cells) carry out cell-mediated immune reactions. Both cell types propagate as clones where each clone synthesises a single antibody or T-cell receptor. This is the basis of the immunologic memory, a hallmark of adaptive immunity.

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  • Melanisation

    Melanisation is the synthesis of the dark pigment melanin from tyrosine and phenolic substances by a combination of enzymatic and non-enzymatic reactions. In many invertebrates the melanisation reaction is an important immediate response producing toxic intermediates and physically encapsulating parasites and microorganisms.

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  • Opsonic activity

    Opsonins are immune components that after binding to a foreign particle, e.g. a bacterium, will enhance the phagocytic uptake of that particle.

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  • Pathogen associated molecular pattern (PAMP)

    PAMPs are molecules derived from pathogens that are recognised by the innate immune system and thereby trigger the activation of immune reactions. Bacterial lipopolysaccharides, bacterial peptidoglycans, fungal β-1,3-glucans and viral double-stranded RNA are examples of PAMPs. It has been suggested that these molecules should instead be named microbial associated molecular patterns as they are present in pathogenic as well as non-pathogenic microorganisms.

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  • Phagocytosis

    This is the process by which particulate material is taken up (endocytosed) by cells. Immune cells dedicated to the phagocytosis of intruders seem to occur in many if not most invertebrates.

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  • proPO cascade

    This is a proteolytic cascade that terminates in the conversion of inactive prophenoloxidase into catalytically active phenoloxidase. The latter will catalyse the production of reactive intermediates that will finally lead to melanin production. This cascade constitutes an important immune reaction in several invertebrate groups and is triggered by e.g. minute amounts of microbial carbohydrates.

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  • Toll-like receptors (TLRs)

    The TLRs are cell surface or endosomal receptors which, in mammals, bind different microbial products through their leucine-rich domain. The fruit fly Toll controls early embryo development and the synthesis of antimicrobial peptides at later life cycle stages.

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  • Variable lymphocyte receptors (VLRs)

    These are receptors on the lymphocytes of jawless vertebrates responsible for binding antigens. In contrast to the receptors and antibodies of jawed vertebrates, these receptors are not immunoglobulins but contain multiple modules of leucine-rich repeats. These receptors have a high capacity for diversification; lampreys and other agnathans may produce different lymphocyte lineages and consequently possess an immunological memory.

List of abbreviations

     
  • A2M

    α-2-macroglobulin

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  • AMP

    antimicrobial peptide

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  • Bf

    complement factor B

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  • C3

    complement component 3

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  • Dscam

    Down syndrome cell adhesion molecule

  •  
  • dsRNA

    double-stranded RNA

  •  
  • FREP

    fibrinogen-related protein

  •  
  • LPS

    lipopolysaccharide

  •  
  • LRR

    leucine-rich repeat

  •  
  • MASP

    mannose binding lectin-associated serine protease

  •  
  • NOD

    nucleotide oligomerisation domain

  •  
  • PAMP

    pathogen-associated molecular pattern

  •  
  • RhlR

    regulatory protein of the rhlAB genes encoding rhamnosyltransferase

  •  
  • TEP

    thioester-containing protein

  •  
  • TLR

    toll-like receptor

  •  
  • VCBP

    variable region-containing chitin-binding protein

  •  
  • VLR

    variable lymphocyte receptor

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Competing interests

No competing interests declared.