The surprisingly complex immune gene repertoire of a simple sponge, exemplified by the NLR genes: A capacity for specificity?

Developmental and Comparative Immunology 48 (2015) 269−274

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Developmental and Comparative Immunology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d c i

Review

The surprisingly complex immune gene repertoire of a simple sponge, exemplified by the NLR genes: A capacity for specificity? Sandie M. Degnan * School of Biological Sciences, The University of Queensland, Brisbane, Qld., Australia

A R T I C L E

I N F O

Article history: Available online 21 July 2014 Keywords: NACHT domain Leucine rich repeats Pattern recognition receptors Porifera Amphimedon Symbiosis

A B S T R A C T

Most bacteria are not pathogenic to animals, and may instead serve beneficial functions. The requisite need for animals to differentiate between microbial friend and foe is likely borne from a deep evolutionary imperative to recognise self from non-self, a service ably provided by the innate immune system. Recent findings from an ancient lineage of simple animals – marine sponges – have revealed an unexpectedly large and diverse suite of genes belonging to one family of pattern recognition receptors, namely the NLR genes. Because NLRs can recognise a broad spectrum of microbial ligands, they may play a critical role in mediating the animal–bacterial crosstalk needed for sophisticated discrimination between microbes of various relationships. The building blocks for an advanced NLR-based immune specificity encoded in the genome of the coral reef sponge Amphimedon queenslandica may provide a specialisation and diversity of responses that equals, or even exceeds, that of vertebrate NLRs. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction It is increasingly apparent that bacteria permeate every aspect of animal biology. Our growing appreciation that all animals not only tolerate, but indeed require, colonisation by beneficial bacteria spotlights the need for a re-evaluation of animal–bacterial crosstalk as fundamental drivers in evolution, ecology, physiology and development (Bosch and McFall-Ngai, 2011; Fraune and Bosch, 2010). One tractable window into animal–bacterial crosstalk is the innate immune system. We perhaps are most familiar with the innate immune system as a ‘first line of defence’ against potential pathogens, yet its origin is deeply rooted in the broader imperative to recognise self from non-self, as required even by single-celled eukaryotes (Hoffmann et al., 1999; Medzhitov and Janeway, 2002). The demands on this kind of recognition system increased enormously with the origin of multicellularity, as evidenced by the independent emergence of functionally analogous innate immune systems in different multicellular eukaryote lineages, including plants and animals. This deep evolutionary perspective implies that the immune system likely originated and has evolved as much in the context of positive encounters, such as symbionts and food sources, as in the context of negative ones, such as pathogens (Klimovich, 2002; McFall-Ngai, 2007). Certainly the critical role of symbiotic bacteria in shaping the immune system continues to be revealed by numerous elegant studies such as those documenting dramatic

* Dr Sandie M. Degnan, PhD, School of Biological Sciences, The University of Queensland, Brisbane, Qld., Australia. Tel.: +61 7 33469005; fax: +61 7 33651655. E-mail address: [email protected] (S.M. Degnan). http://dx.doi.org/10.1016/j.dci.2014.07.012 0145-305X/© 2014 Elsevier Ltd. All rights reserved.

effects on mammalian immune systems of disruptions to gut microbiota (Clemente et al., 2012; Round and Mazmanian, 2009). Equally fascinating, a growing number of examples from vertebrates and insects reveal that the animal hosts appear to play an active role in shaping and regulating their gut microbiota (Rawls et al., 2006; Ryu et al., 2008). The ubiquity and the diversity of these relationships between animals and bacteria strongly suggest that all metazoans, with or without an adaptive immune system, might require sophisticated powers to discriminate between bacterial friend and foe. Although the host factors that regulate the establishment and maintenance of homeostatic symbiont relationships, or that target a pathogen for destruction, remain largely a mystery, the innate immune system that is common to all animals is an obvious target for investigation. Here I summarise recent findings on the innate immune gene complement of an ancient lineage of simple animals – the marine sponges – that suggest a capacity for specific interactions with diverse bacteria via an unexpectedly large and diverse suite of genes belonging to one family of pattern recognition receptors (PRRs), namely the NLR genes. 2. A window into microbe–animal interactions: the advantage of being a simple sponge Listening in on individual conversations between animals and bacteria will perhaps be most tractable in animal models with simple morphologies and diverse, easy-to-visualise relationships with bacteria. The sponges (phylum Porifera) are among the morphologically simplest animals, generally recognised as having no true tissues or organs, no nerves or gut, and a smaller number of differentiated cell

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types than most other animal phyla (Ereskovsky, 2010). Furthermore, sponges were one of the earliest groups of animals to become established as a discrete phyletic lineage, making them arguably the most ancient of all the extant animal phyla (Li et al., 1998; Love et al., 2009). The phylogenetic status of poriferans as sister group to the Eumetazoa makes them ideal for elucidating the origin and evolution of animal innate immunity, because traits shared between sponges and other animals likely reflect shared inheritance from the last common animal ancestor (Srivastava et al., 2010). In marine environments in particular, sponges are extremely important ecosystem engineers (Bell, 2008). That sponges have been continually shaping the health and ecology of the Earth’s oceans for more than 600 million years is in no small part due to the phylogenetically diverse microbes with which they associate (Hentschel et al., 2012; Webster and Taylor, 2011). It is increasingly well understood how beneficial symbioses with bacteria have conferred on sponges the ability, for example, to synthesise novel bioactive compounds (Piel et al., 2004; Wilson et al., 2014) and to remove organic carbon from the water column (De Goei et al., 2008). Indeed, one of the critical functions of feeding adult sponges is filtering the microbial-laden water column by pumping as much as 2000– 4000 litres/kg sponge/day through their body (Hentschel et al., 2012). Simultaneously, they normally house diverse – and often very dense – communities of symbiotic bacteria and other microbes that thrive in the extracellular matrix that constitutes most of a sponge’s body; these often exist alongside the very same sponge cells that digest food bacteria (Taylor et al., 2007). It is significant, given the rate of water flow through the sponge body, that bacterial symbionts ingested by their sponge hosts are left unharmed, but food-source bacteria taken in at the same time are consumed so efficiently that water expelled from the exhalant siphon of an actively feeding sponge is effectively sterile (Vacelet and Donadey, 1977; Wilkinson et al., 1984). This suggests that sponges are able to differentiate between, and respond appropriately to, beneficial microbes – both food sources destined for ingestion, and core symbionts destined for retention – as well as harmful pathogens that all may be encountered in the same incoming water currents (Wilkinson et al., 1984). The sponge innate immune system is likely to be a critical component of the animal–microbe crosstalk that leads to the correct assignment of incoming bacteria to either food, pathogen or symbiont categories. In fact, sponges have a surprisingly well-developed innate immune system that relies upon an unexpectedly large number of the same innate immunity genes familiar to us from the human genome (Gauthier et al., 2010; Hentschel et al., 2012; Müller and Müller, 2003; Srivastava et al., 2010; Wiens et al., 2007). Not surprisingly, then, sponges can produce a broad range of antimicrobial compounds (Blunt et al., 2013). These traits add to their complexity as a habitat for prokaryote symbionts, but also raise the possibility that the innate immune system holds the key to the sophisticated interactions necessary for the host to appropriately respond to the huge diversity of microbes that it encounters through its life. The assembly of the near-complete genome of the coral reef demosponge Amphimedon queenslandica (Srivastava et al., 2010) now permits identification of a full complement of sponge innate immune genes. Because they lack the surveillance and response of the vertebrate adaptive immune system, sponges rely on pattern recognition receptors (PRRs) of the innate immune system to detect and discriminate microbes, followed by the differential activation of a handful of immune signaling pathways to regulate downstream effector and defense mechanisms (Kurtz and Armitage, 2006). PRRs are charged with the critical task of functioning as first point of molecular contact between sponge host and incoming microbes; their binding to characteristic microbial ligands (Microbial- or PathogenAssociated Molecular Patterns (MAMPs/PAMPs) or endogenous

Danger Associated Molecular Patterns (DAMPs) typically triggers a signal transduction cascade that leads to transcription of immune response effector genes encoding products such as antibacterial proteins (Janeway and Medzhitov, 2002). In the absence of an adaptive immune system, one way for invertebrate animals such as sponges to keep up with the huge diversity of bacteria they encounter is to have a large and varied repertoire of innate immune PRRs. Might sponges use PRRs of their innate immune system as the basis for their sophisticated discrimination? If yes, one might hypothesise an extensive genome complement of PRRs. In fact, the complement of PRRs in A. queenslandica is astonishingly large for such a morphologically and behaviourally simple animal, and suggests a genuine potential for specificity in animal–bacterial conversations where none was thought to exist. 3. The extensive immune potential suggested by Amphimedon queenslandica PRRs is exemplified by a large NLR gene family with diversity on multiple levels One gene family that is particularly expanded, as compared to many more morphologically complex animals, is the family of Nucleotide-binding domain and Leucine-rich repeat containing genes (NLRs, known also as NOD [Nucleotide Oligomerisation Domain]like receptors) (Yuen et al., 2014). NLRs play a pivotal role in sensing a wide diversity of bacteria because they are capable of detecting and binding to a wide range of MAMPs/PAMPs, including bacterial and viral RNA, bacterial flagellin and peptidoglycan components of both Gram-negative and Gram-positive bacteria (Kaparakis et al., 2007). Most NLRs are intracellular – the cytosolic counterparts of TLRs – where they respond to bacteria that evade secreted or membrane-bound PRRs and thus invade the cell; they likely also respond to bacterial products remaining after phagocytosis (Franchi et al., 2009). NLRs are also capable of detecting and responding to endogenous DAMPs that are produced by the host and indicate injury or cellular stress (Stuart et al., 2013). Intriguingly, it more recently has been revealed that NLRs can also be responsive to non-invasive (extracellular) bacteria, although how contact occurs between the intracellular receptors and the extracellular bacterial ligands is not yet fully understood (Ferrand and Ferrero, 2013). Not least because of this diverse repertoire of microbial ligands that may initiate an NLR response from both outside and inside, NLRs are candidates for having critical roles in mediating all kinds of animal–bacterial interactions, including the differentiation between microbial friend and foe (Robertson et al., 2012). The A. queenslandica genome encodes at least 135 bona fide NLR genes (AqNLRs) (Yuen et al., 2014) that contain the central NACHT and C-terminal Leucine-rich repeat (LRR) domains that define this gene family (Ting et al., 2008). This expanded AqNLR gene family (for comparison, humans have 22 NLRs) reflects similarly large numbers of NLRs in some other aquatic invertebrates including corals, sea urchins and cephalochordates; in all of these cases, the high gene numbers appear to be the result of lineage-specific expansions (Hamada et al., 2012; Lange et al., 2011; Messier-Solek et al., 2010; Yuen et al., 2014). Interestingly, no bona fide NLR genes at all are encoded by the genomes of the ctenophores Mnemiopsis leidyi (Yuen et al., 2014, analysing the genome reported by Ryan et al., 2013) or Pleurobrachia bachei (Moroz et al., 2014), although both contain multiple NACHT domains. Whether this reflects lineagespecific gene loss or a basal position of the ctenophore clade (Moroz et al., 2014; Ryan et al., 2013) remains to be determined. A typical animal NLR has a tripartite architecture where, in addition to the defining NACHT and LRR domains, there is an N-terminal domain that is usually a member of the death-fold superfamily that includes the Caspase recruitment domain (CARD), Pyrin domain (PYD) and DEATH domain (Proell et al., 2008). Of the 135 bona fide AqNLRs, 48 have this tripartite architecture with an

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Fig. 1. Structures of NLR and adaptor and effector proteins encoded by the A. queenslandica genome, showing some of the possible homotypic interactions between them. The characteristic tripartite NLR structure consists of an N-terminal signaling domain (either a DEATH domain in AqNLRDs or a CARD domain in AqNLRDs), a central nucleotidebinding and oligomerisation domain (always a NACHT domain), and a C-terminal agonist sensing/ligand-binding domain (always a Leucine Rich Repeat, LRR). The AqNLRX genes do not encode an N-terminal domain. The two different colours of LRRs represent the two very divergent forms of LRR that are distributed among the AqNLRs. The genome also encodes many Death Effector Domains (DED) domains – also in the death domain superfamily – that could be involved. Some of these are associated with DEATH domains and thus could act as adaptor proteins; some are associated with peptidase_C14 domains and thus could act as signalling/effector proteins via the DEDcontaining adaptor proteins. In concert, the divergent forms of LRR, two different signaling domains, and the large and diverse suite of potential adaptor and effector proteins hints at a potentially enormous repertoire of receptor-ligand, signalling and effector responses.

N-terminal domain that is always a CARD or DEATH domain (Yuen et al., 2014) (Fig. 1). The presence or nature of the N-terminal domain, however, does not predict the evolutionary relationship of AqNLR genes, strongly suggesting that N-terminal domain shuffling has played a very important role in AqNLR evolution. Phylogenetic analysis instead reveals discrete clades characterised by the nature of their C-terminal LRR domain (Yuen et al., 2014). This is significant because, based on their function in other protein contexts, combined with limited empirical evidence from some vertebrate NLRs (Monie, 2013), it is thought that LRRs are the part of the protein that interacts directly with bacterial ligands. Two clades of AqNLRs together contain thirteen (nine of which are tripartite) NLRs that all are characterised by “standard” LRRs easily recognisable by the sequenced-based Pfam LRR clan HMMs (CL0022) (Yuen et al., 2014). A third clade contains 122 NLR genes, all of which have quite divergent LRRs that are recognised only by HMM profiles in the Superfamily (SSF52047) and Gene3D (G3DSA:3.80.10.10) protein structure libraries that bear more similarity to the LRR domain of the ribonuclease inhibitor-like (RNIlike) superfamily (Yuen et al., 2014). This clade with the abundant and very divergent LRRs appears to be the result of recent, rapid expansion (by gene duplication) and diversification that have resulted in substantial within-clade diversity in the LRR domains relative to the NACHT domain (Yuen et al., 2014). The duplication and subsequent functional divergence of existing genes is an important mechanism for generation of new function, but the fitness costs of carrying extra gene copies may be substantial (Adler et al., 2014). Because of this, the maintenance of this large

number of recently duplicated genes, in addition to the rest of the AqNLR gene complement, suggests that these costs are being offset by strong positive selection for novel beneficial functions. One hypothesis is that their evolution has been/is being driven by a dynamic suite of ligand-binding conditions, as suggested for the evolution of the large family of innate immunity Toll-like receptor (TLR) genes that display highly variable LRRs in echinoderms (Buckley and Rast, 2012). In this context, it is helpful to consider multiple ways in which NLR-related functional diversity could be generated by standing genetic variation as a source of adaptation to novel microbial challenges as they arise. There are at least four mechanisms by which the AqNLRs could generate diversity sufficient to engage a level of specificity that could meet the needs of the sponge, as described below. (i) Allelic and between-locus diversity throughout the different domains of the protein, perhaps most especially in the microbial ligandbinding LRR regions As discussed above, the AqNLRs between them encode at least two very different forms of LRR, which could logically bind to very different microbial ligands. But, the diversity doesn’t end there. The AqNLR gene models have proven difficult to predict (Yuen et al., 2014) and AqNLR RNASeq data are equally difficult to assemble (personal observation), both of which suggest extensive intraspecific polymorphism in these genes. Although this is yet to be confirmed for the AqNLRs, it would be consistent with high intraspecific polymorphism in different PRR gene families, namely Toll-like

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receptors (TLRs) and Scavenger Receptor Cysteine-Rich genes (SRCRs), in the sea urchin Strongylocentrotus purpuratus (Messier-Solek et al., 2010; Pancer, 2000). A similar situation seems to exist in the functionally analogous NLRs of plants, in which the LRR domain interacts directly with cognate microbial ligands in a highly specific manner that reflects allelic diversity with the LRR domains (Maekawa et al., 2011). Interestingly, however, plants have even a greater repertoire for perception of non-self than can be explained by simple receptorligand coevolution alone. This suggests that there may also be other mechanisms driving adaptive changes in plant NLR repertoires for detection of non-self (Maekawa et al., 2011). In vertebrate NLRs, there is little evidence of microbe-driven functional diversification of alleles, even in the LRR domain, but the emergence of the adaptive immune system in vertebrates may have replaced any such need. It is noteworthy here also that the presence of LRRs does not necessarily denote a role in MAMP-binding; some vertebrate NLRs have evolved roles beyond pattern recognition, including acting as signaling platforms that activate other facets of the immune system (Bonardi et al., 2012; Kufer and Sansonetti, 2011). (ii) N-terminal effector and signaling domain diversity and shuffling In their inactivated resting state, NLRs probably self-repress, by having the LRR folded back onto the NACHT domain thus preventing oligomerisation. Exposure to microbial ligands or DAMPS is thought to trigger conformational changes that expose the NACHT domain and thus enable oligomerisation adaptor proteins and recruitment of effector kinases and caspases, although there is very little empirical structural data available to confirm this. In vertebrates at least, these interactions lead usually to inflammatory or apoptotic responses (Schroder and Tschopp, 2010; Shaw et al., 2010). Most commonly in all animal genomes surveyed to date, N-terminal domains are members of the death-fold superfamily (Messier-Solek et al., 2010; Proell et al., 2008; Yuen et al., 2014), which includes the Caspase recruitment domain (CARD), Pyrin domain (PYD) and DEATH domain, suggesting functional convergence on these effector domains across the animal kingdom. The N-terminal effector domains of the AqNLRs confirm to this pattern – all are either DEATH or CARD domains – but the A. queenslandica genome also contains a diverse suite of other deathfold domain combinations (in ~460 non-NLR genes) that could interact with the AqNLRs as downstream adaptor and effector proteins (Yuen et al., 2014) (Fig. 1). An equivalent co-expansion of both NLR genes (118) and death-fold domain-containing genes (541) in the Branchiostoma genome has been interpreted as enhanced signaling potential (Huang et al., 2008; Messier-Solek et al., 2010), on the basis that complex synergies can occur at receptor, signaling and effector levels of the NLR immune response (Schulenburg et al., 2007). The number of possible combinations between 135 AqNLRs and ~460 potential adaptor and effector proteins encoded by the A. queenslandica genome hints at a potentially enormous repertoire of signalling and effector responses, in addition to the expansive receptor–ligand binding capabilities discussed in section (i) above. Interestingly, a small number of AqNLRs are also weakly predicted by bioinformatics to contain N-terminal transmembrane domains (Yuen et al., 2014); the same is true for anthozoan cnidarians (Hamada et al., 2012). This does not conform to the usual convergence on death-fold domains observed across the animal kingdom, and the significance of these is currently unclear. (iii) Heteromeric receptor complexes The N-terminal effector domain usually interacts with another homotypic protein to initiate the downstream innate immune responses (Kufer, 2008; Shaw et al., 2010). Indeed, the recruitment

to death-fold domains of other homotypic proteins appears to be central to cell death and inflammatory signalling pathways, in particular (Kersse et al., 2011; Schroder and Tschopp, 2010). However, in plants, some NLRs act together to confer pathogen resistance, suggestive of a mechanism whereby formation of a heteromeric receptor complex further extends the repertoire of NLR-mediated recognition (Eitas and Dangl, 2010). In vertebrates, limited evidence also exists for NLRs forming heteromers (Damiano et al., 2004), hinting that the same strategy may be available to animal NLRs. No evidence currently exists for heteromeric NLR receptors in A. queenslandica specifically, but the large number and diversity of the AqNLRs means that the potential for such a mechanism to increase the recognition spectrum of this gene family is enormous. (iv) Indirect stimulus Limited empirical evidence currently exists for the direct interaction of microbial ligands with animal NLRS (Monie, 2013), although LRR domains have been shown to directly bind microbial structures when they exist in other protein contexts (e.g., TLRs). The extensive evidence that at least some animal NLRs can respond to surprisingly diverse microbial structures suggests that their mode of stimulus recognition may in fact be indirect (Schroder and Tschopp, 2010). Indirect detection of non-self has been empirically demonstrated in several plant NLRs that appear to sense modified host proteins (‘modified self’), which themselves act as effector targets (Maekawa et al., 2011). Detecting modified self means that NLRs need respond only to the action of effectors, rather than to their structure. This could provide a selective advantage over direct perception by maximising the ability of a limited number of innate immunoreceptors to cope with highly variable, and ever-changing, microbial effector repertoires (Maekawa et al., 2011; Schroder and Tschopp, 2010). Not least because of the similarity between plants and sponges – sessile, attached multicellular organisms with no adaptive immune system and probably no specialised immune cells – this model of indirect interaction should be a focus of future research. 4. Vertical transmission of stable primary symbionts may imply both specificity and memory of the sponge innate immune system Like many sponges, A. queenslandica sexual reproduction is characterised by internal fertilisation and subsequent brooding of developing embryos inside chambers scattered through the body (Degnan et al., 2008). One consequence of this is that embryos are in close contact with maternal tissue for several weeks before mature larvae are released to swim freely in the external water column. This extended brooding period provides ample opportunity for the vertical inheritance of bacterial symbionts, as has been documented in a great many species of marine sponge (Schmitt et al., 2011; Thacker and Freeman, 2012); vertical inheritance of a core symbiont community is also known for A. queenslandica (personal observation; Fieth et al., ms in prep). Intriguingly, however, in all sponge species so far examined, only a small selection of obligate symbiont bacterial species are vertically inherited, despite the adult sponges hosting a large and diverse bacterial taxonomic community that also includes many horizontally-acquired taxa (Schmitt et al., 2011). Most often, these select vertically-inherited species are isolated from the broader microbiome into specialised cells, or parts of cells, that ensure the bacteria are deposited directly into oocytes or developing embryos (Thacker and Freeman, 2012). This implies specific recognition of these stable symbionts, likely via the innate immune system, that leads to a downstream effect of protection and transmission. That vertically-inherited symbionts in particular tend

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to be very stable across generations also raises the possibility of some degree of immune memory that would facilitate inter-generational recognition of symbionts. Soon after metamorphosing from free-swimming larvae into the sessile, benthic adult body plan, sponges begin to feed. Now the new individual suddenly finds itself awash with the myriad of microbes in the water column that it is actively drawing into its own body. Might the early encounters between embryo and verticallytransmitted symbionts prime the sponge immune system to specifically recognise and respond appropriately to these “good” bacteria, in a manner different from the food bacteria, and the potential pathogens, that are encountered in these later stages of the life cycle? The capacity for an innate immune response to show memory characteristics after an initial encounter with a pathogen is an accepted fact in plant immunology; excitingly, it also is now strongly suggested by a limited but growing number of studies in invertebrates (Netea, 2014; Netea et al., 2011; Quintin et al., 2014). We currently have no understanding as to whether or not the sponge AqNLRs show any evidence of specific memory, but functional immunology experiments conducted alongside transcriptional assays should help to shed some light. Such investigations will benefit from the natural experimental systems provided by the vertical inheritance of obligate symbionts during early development, and the full onslaught of aquatic microbes once the sponge begins to actively feed. 5. Summary and perspectives The tremendous abundance and diversity of the AqNLRs, superficially similar at a structural level to the many fewer vertebrate NLRs, are consistent with an involvement in immunity. Coupled with the abundance and diversity of potential adaptor and effector proteins, it is apparent that this morphologically simple marine sponge has an NLR complement with the capacity to recognise a vast array of microbial ligands and engage a diversity of downstream responses. The building blocks for a sophisticated NLR-based immune specificity are encoded in the A. queenslandica genome, with the power to generate an innate immune response of greater specialisation and diversity than vertebrate NLRs. Perhaps this simple sponge reflects to its full capacity the NLR-mediated detection of polymorphic non-self, the need for which was functionally replaced in vertebrates by the emergence of the adaptive immune system. As we become more and more appreciative that most bacteria are not pathogenic and may serve beneficial, or even necessary, functions for the host, the question of how animal hosts differentiate between microbial friend and foe begs louder. In invertebrate animals generally, the distinction between innate and adaptive immunity is becoming less and less clear, as the former increasingly reveals an unexpectedly remarkable plasticity of responses (Dong et al., 2012; Sun et al., 2014; Ziauddin and Schneider, 2012). Because of their capacity to recognise a diversity of ligands, both within and without the cell, NLRs may well be critical players in mediating the animal–bacterial crosstalk required for sophisticated discrimination between microbes of various relationships (Robertson et al., 2012). As the likely sister group to all other animals, sponges provide invaluable models for deciphering these conversations, with potential to reveal something about the diversity of vocabulary available to the animal kingdom since its origin in a sea of microbes more than 600 million years ago. Acknowledgements I thank members of the joint Degnan Labs for their enthusiasm and curiosity on all matters relating to sponge immunity and interactions with microbes, and especially Jo Bayes, Benedict Yuen and

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Rebecca Fieth for contributing data that have helped to inform our thoughts on animal–microbe crosstalk in Amphimedon. Funding was gratefully received from the Australian Research Council Discovery scheme (DP110104601 and DP0985995). References Adler, M., Anjum, M., Berg, O.G., Andersson, D.I., Sandegren, L., 2014. High fitness costs and instability of gene duplications reduce rates of evolution of new genes by duplication-divergence mechanisms. Mol. Biol. Evol. doi:10.1093/molbev/ msu111. Bell, J.J., 2008. The functional roles of marine sponges. Est. Coast. Shelf Sci. 79, 341–353. doi:10.1016/j.ecss.2008.05.002. Blunt, J.W., Copp, B.R., Keyzers, R.A., Munro, M.H.G., Prinsep, M.R., 2013. Marine natural products. Nat. Prod. Rep. 30, 237–323. doi:10.1039/c2np20112g. Bonardi, V., Cherkis, K., Nishimura, M.T., Dangl, J.L., 2012. A new eye on NLR proteins: focused on clarity or diffused by complexity? Curr. Opin. Immunol. 24, 41–50. doi:10.1016/j.coi.2011.12.006. Bosch, T.C.G., McFall-Ngai, M.J., 2011. Metaorganisms as the new frontier. Zoology (Jena) 114, 185–190. doi:10.1016/j.zool.2011.04.001. Buckley, K.M., Rast, J.P., 2012. Dynamic evolution of toll-like receptor multigene families in echinoderms. Front. Immunol. 3, 1–16. doi:10.3389/ fimmu.2012.00136/abstract. Clemente, J.C., Ursell, L.K., Parfrey, L.W., Knight, R., 2012. The impact of the gut microbiota on human health: an integrative view. Cell 148, 1258–1270. doi:10.1016/j.cell.2012.01.035. Damiano, J.S., Oliveira, V., Welsh, K., Reed, J.C., 2004. Heterotypic interactions among NACHT domains: implications for regulation of innate immune responses. Biochem. J. 381, 213–219. doi:10.1042/BJ20031506. De Goei, J.M., Moodley, L., Houtekamer, M., Carballeira, N.M., van Duyl, F.C., 2008. Tracing 13C-enriched dissolved and particulate organic carbon in the bacteria containing coral reef sponge Halisarca caerulea: evidence for DOM feeding. Limnol. Oceanogr. 53, 1376–1386. Degnan, B.M., Adamska, M., Craigie, A., Degnan, S.M., Fahey, B., Gauthier, M., et al., 2008. The demosponge amphimedon queenslandica: reconstructing the ancestral metazoan genome and deciphering the origin of animal multicellularity. CSH Protoc. 2008 pdb.emo108. Dong, Y., Cirimotich, C.M., Pike, A., Chandra, R., Dimopoulos, G., 2012. Anopheles NF-κB-regulated splicing factors direct pathogen-specific repertoires of the hypervariable pattern recognition receptor AgDscam. Cell Host Microbe 12, 521–530. doi:10.1016/j.chom.2012.09.004. Eitas, T.K., Dangl, J.L., 2010. NB-LRR proteins: pairs, pieces, perception, partners, and pathways. Curr. Opin. Plant Biol. 13, 472–477. doi:10.1016/j.pbi.2010.04.007. Ereskovsky, A.V., 2010. The Comparative Embryology of Sponges, Springer Netherlands, Dordrecht. doi:10.1007/978-90-481-8575-7. Ferrand, J., Ferrero, R.L., 2013. Recognition of extracellular bacteria by NLRs and its role in the development of adaptive immunity. Front Immunol. 4, 344. doi:10.3389/fimmu.2013.00344. Franchi, L., Eigenbrod, T., Muñoz-Planillo, R., Nunez, G., 2009. The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis. Nat. Immunol. 10, 241–247. doi:10.1038/ni.1703. Fraune, S., Bosch, T.C.G., 2010. Why bacteria matter in animal development and evolution. Bioessays 32, 571–580. doi:10.1002/bies.200900192. Gauthier, M.E.A., Du Pasquier, L., Degnan, B.M., 2010. The genome of the sponge Amphimedon queenslandica provides new perspectives into the origin of Toll-like and interleukin 1 receptor pathways. Evol. Dev. 12, 519–533. doi:10.1111/j.1525142X.2010.00436.x. Hamada, M., Shoguchi, E., Shinzato, C., Kawashima, T., Miller, D.J., Satoh, N., 2012. The complex NOD-like receptor repertoire of the coral Acropora digitifera includes novel domain combinations. Mol. Biol. Evol. 30, 167–176. doi:10.1093/molbev/ mss213. Hentschel, U., Piel, J., Degnan, S.M., Taylor, M.W., 2012. Genomic insights into the marine sponge microbiome. Nat. Rev. Microbiol. 10, 641–654. doi:10.1038/ nrmicro2839. Hoffmann, J.A., Kafatos, F.C., Janeway, C.A., Ezekowitz, R., 1999. Phylogenetic perspectives in innate immunity. Science 284, 1313–1318. doi:10.1126/ science.284.5418.1313. Huang, S., Yuan, S., Guo, L., Yu, Y., Li, J., Wu, T., et al., 2008. Genomic analysis of the immune gene repertoire of amphioxus reveals extraordinary innate complexity and diversity. Genome Res. 18, 1112–1126. doi:10.1101/gr.069674.107. Janeway, C.A., Medzhitov, R., 2002. Innate immune recognition. Annu. Rev. Immunol. 20, 197–216. doi:10.1146/annurev.immunol.20.083001.084359. Kaparakis, M., Philpott, D.J., Ferrero, R.L., 2007. Mammalian NLR proteins; discriminating foe from friend. Immunol. Cell Biol. 85, 495–502. doi:10.1038/ sj.icb.7100105. Kersse, K., Verspurten, J., Vanden Berghe, T., Vandenabeele, P., 2011. The death-fold superfamily of homotypic interaction motifs. Trends Biochem. Sci. 36, 541–552. doi:10.1016/j.tibs.2011.06.006. Klimovich, V.B., 2002. Actual problems of evolutionary immunology. J. Evol. Biochem. Physiol. 38, 562–574. doi:10.1023/A:1022060916099. Kufer, T.A., 2008. Signal transduction pathways used by NLR-type innate immune receptors. Mol. Biosyst. 4, 380–386. doi:10.1039/b718948f. Kufer, T.A., Sansonetti, P.J., 2011. NLR functions beyond pathogen recognition. Nat. Immunol. 12, 121–128. doi:10.1038/ni.1985.

274

S.M. Degnan/Developmental and Comparative Immunology 48 (2015) 269−274

Kurtz, J., Armitage, S., 2006. Alternative adaptive immunity in invertebrates. Trends Immunol. 27, 493–496. doi:10.1016/j.it.2006.09.001. Lange, C., Hemmrich, G., Klostermeier, U.C., Lopez-Quintero, J.A., Miller, D.J., Rahn, T., et al., 2011. Defining the origins of the NOD-like receptor system at the base of animal evolution. Mol. Biol. Evol. 28, 1687–1702. doi:10.1093/molbev/msq349. Li, C., Chen, J., Hua, T., 1998. Precambrian sponges with cellular structures. Science 279, 879–882. Love, G.D., Grosjean, E., Stalvies, C., Fike, D.A., Grotzinger, J.P., Bradley, A.S., et al., 2009. Fossil steroids record the appearance of Demospongiae during the Cryogenian period. Nature 457, 718–721. doi:10.1038/nature07673. Maekawa, T., Kufer, T.A., Schulze-Lefert, P., 2011. NLR functions in plant and animal immune systems: so far and yet so close. Nat. Immunol. 12, 817–826. doi:10.1038/ni.2083. McFall-Ngai, M., 2007. Adaptive immunity: care for the community. Nature 445, 153. doi:10.1038/445153a. Medzhitov, R., Janeway, C.A., 2002. Decoding the patterns of self and nonself by the innate immune system. Science 296, 298–300. doi:10.1126/science.1068883. Messier-Solek, C., Buckley, K.M., Rast, J.P., 2010. Highly diversified innate receptor systems and new forms of animal immunity. Semin. Immunol. 22, 39–47. doi:10.1016/j.smim.2009.11.007. Monie, T.P., 2013. NLR activation takes a direct route. Trends Biochem. Sci. 38, 131–139. doi:10.1016/j.tibs.2013.01.001. Moroz, L.L., Kocot, K.M., Citarella, M.R., Dosung, S., Norekian, T.P., Povolotskaya, I.S., et al., 2014. The ctenophore genome and the evolutionary origins of neural systems. Nature 510, 109–114. doi:10.1038/nature13400. Müller, W.E.G., Müller, I.M., 2003. Origin of the metazoan immune system: identification of the molecules and their functions in sponges. Integr. Comp. Biol. 43, 281–292. doi:10.1093/icb/43.2.281. Netea, M.G., 2014. Immunological memory in innate immunity. J. Innate Immun. 6, 117–118. Netea, M.G., Quintin, J., van der Meer, J.W.M., 2011. Trained immunity: a memory for innate host defense. Cell Host Microbe 9, 355–361. doi:10.1016/ j.chom.2011.04.006. Pancer, Z., 2000. Dynamic expression of multiple scavenger receptor cysteinerich genes in coelomocytes of the purple sea urchin. Proc. Natl Acad. Sci. U.S.A. 97, 13156–13161. doi:10.2307/123671?ref=search-gateway: 437489f13218c17b2cdb152423051a9f. Piel, J., Hui, D., Wen, G., Butzke, D., Platzer, M., Fusetani, N., et al., 2004. Antitumor polyketide biosynthesis by an uncultivated bacterial symbiont of the marine sponge Theonella swinhoei. Proc. Natl Acad. Sci. U.S.A. 101, 16222–16227. Proell, M., Riedl, S.J., Fritz, J.H., Rojas, A.M., Schwarzenbacher, R., 2008. The Nod-like receptor (NLR) family: a tale of similarities and differences. PLoS ONE 3, e2119. doi:10.1371/journal.pone.0002119. Quintin, J., Cheng, S.-C., van der Meer, J.W.M., Netea, M.G., 2014. Innate immune memory: towards a better understanding of host defense mechanisms. Curr. Opin. Immunol. 29, 1–7. Rawls, J.F., Mahowald, M.A., Ley, R.E., Gordon, J.I., 2006. Reciprocal gut microbiota transplants from zebrafish and mice to germ-free recipients reveal host habitat selection. Cell 127, 423–433. doi:10.1016/j.cell.2006.08.043. Robertson, S.J., Rubino, S.J., Geddes, K., Philpott, D.J., 2012. Examining host-microbial interactions through the lens of NOD: from plants to mammals. Semin. Immunol. 24, 9–16. doi:10.1016/j.smim.2012.01.001. Round, J.L., Mazmanian, S.K., 2009. The gut microbiota shapes intestinal immune responses during health and disease. Nat. Rev. Immunol. 9, 313–323. doi:10.1038/ nri2515.

Ryan, J.F., Pang, K., Schnitzler, C.E., Nguyen, A.-D., Moreland, R.T., Simmons, D.K., et al., 2013. The genome of the ctenophore Mnemiopsis leidyi and its implications for cell type evolution. Science 342, 1242592. doi:10.1126/science.1242592. Ryu, J.H., Kim, S.H., Lee, H.Y., Bai, J.Y., Nam, Y.D., Bae, J.W., et al., 2008. Innate immune homeostasis by the homeobox gene caudal and commensal-gut mutualism in Drosophila. Science 319, 777–782. doi:10.1126/science.1149357. Schmitt, S., Tsai, P., Bell, J., Fromont, J., Ilan, M., Lindquist, N., et al., 2011. Assessing the complex sponge microbiota: core, variable and species-specific bacterial communities in marine sponges. ISME J. 6, 564–576. doi:10.1038/ismej.2011.116. Schroder, K., Tschopp, J., 2010. The inflammasomes. Cell 140, 821–832. doi:10.1016/ j.cell.2010.01.040. Schulenburg, H., Boehnisch, C., Michiels, N.K., 2007. How do invertebrates generate a highly specific innate immune response? Mol. Immunol. 44, 3338–3344. doi:10.1016/j.molimm.2007.02.019. Shaw, P.J., Lamkanfi, M., Kanneganti, T.-D., 2010. NOD-like receptor (NLR) signaling beyond the inflammasome. Eur. J. Immunol. 40, 624–627. doi:10.1002/ eji.200940211. Srivastava, M., Simakov, O., Chapman, J., Fahey, B., Gauthier, M.E.A., Mitros, T., et al., 2010. The Amphimedon queenslandica genome and the evolution of animal complexity. Nature 466, 720–726. doi:10.1038/nature09201. Stuart, L.M., Paquette, N., Boyer, L., 2013. Effector-triggered versus pattern-triggered immunity: how animals sense pathogens. Nat. Rev. Immunol. 13, 199–206. doi:10.1038/nri3398. Sun, J.C., Ugolini, S., Vivier, E., 2014. Immunological memory within the innate immune system. EMBO J. doi:10.1002/embj.201387651. Taylor, M.W., Hill, R.T., Piel, J., Thacker, R.W., Hentschel, U., 2007. Soaking it up: the complex lives of marine sponges and their microbial associates. ISME J. 1, 187–190. doi:10.1038/ismej.2007.32. Thacker, R.W., Freeman, C.J., 2012. Sponge-microbe symbioses: recent advances and new directions. Adv. Mar. Biol. 62, 57–111. doi:10.1016/B978-0-12-3942838.00002-3. Ting, J.P.Y., Lovering, R.C., Alnemri, E.S., Bertin, J., Boss, J.M., Davis, B.K., et al., 2008. The NLR gene family: a standard nomenclature. Immunity 28, 285–287. doi:10.1016/j.immuni.2008.02.005. Vacelet, J., Donadey, C., 1977. Electron microscope study of the association between some sponges and bacteria. J. Exp. Mar. Bio. Ecol. 30, 301–314. doi:10.1016/00220981(77)90038-7. Webster, N.S., Taylor, M.W., 2011. Marine sponges and their microbial symbionts: love and other relationships. Environ. Microbiol. 14, 335–346. Wiens, M., Korzhev, M., Perovic-Ottstadt, S., Luthringer, B., Brandt, D., Klein, S., et al., 2007. Toll-like receptors are part of the innate immune defense system of sponges (demospongiae: Porifera). Mol. Biol. Evol. 24, 792–804. doi:10.1093/molbev/ msl208. Wilkinson, C.R., Garrone, R., Vacelet, J., 1984. Marine sponges discriminate between food bacteria and bacterial symbionts: electron microscope radioautography and in situ evidence. Proc. R. Soc. Lond. B Biol. Sci. 220, 519–528. doi:10.1098/ rspb.1984.0018. Wilson, M.C., Mori, T., Rückert, C., Uria, A.R., Helf, M.J., Takada, K., et al., 2014. An environmental bacterial taxon with a large and distinct metabolic repertoire. Nature 506, 58–62. Yuen, B., Bayes, J.M., Degnan, S.M., 2014. The characterization of sponge NLRs provides insight into the origin and evolution of this innate immune gene family in animals. Mol. Biol. Evol. 31, 106–120. doi:10.1093/molbev/mst174. Ziauddin, J., Schneider, D.S., 2012. Where does innate immunity stop and adaptive immunity begin? Cell Host Microbe 12, 394–395. doi:10.1016/j.chom.2012.10.004.