Genetic and molecular basis of the immune system in the brachiopod Lingula anatina

https://doi.org/10.1016/j.dci.2017.12.021Get rights and content

Highlights

  • Brachiopods have been poorly studied from an immunological point of view.

  • Genes related to the immune system have been identified in Lingula anatina.

  • Brachiopods display a complex immune system.

  • Intracellular immune signaling pathways are conserved in lophotrochozoans.

  • Lingula developed peculiar molecular mechanisms for microbe detection and killing.

Abstract

The extension of comparative immunology to non-model systems, such as mollusks and annelids, has revealed an unexpected diversity in the complement of immune receptors and effectors among evolutionary lineages. However, several lophotrochozoan phyla remain unexplored mainly due to the lack of genomic resources. The increasing accessibility of high-throughput sequencing technologies offers unique opportunities for extending genome-wide studies to non-model systems. As a result, the genome-based study of the immune system in brachiopods allows a better understanding of the alternative survival strategies developed by these immunologically neglected phyla. Here we present a detailed overview of the molecular components of the immune system identified in the genome of the brachiopod Lingula anatina. Our findings reveal conserved intracellular signaling pathways as well as unique strategies for pathogen detection and killing in brachiopods.

Introduction

The Lophotrochozoa superphylum (including mollusks, annelids, brachiopods, and other invertebrates) is one of the most diversified animal groups, only second to Ecdysozoa in numbers of living species. These organisms comprise a heterogeneous mix of monophyletic invertebrate phyla (Helmkampf et al., 2008, Philippe et al., 2005) that have virtually colonized all terrestrial and aquatic environments, displaying a remarkable biological plasticity and capability of adaptation. This diversity is exemplified by mollusks, which comprise over 20% of all marine species. Despite the ecological and evolutionary importance of lophotrochozoans, their immunological study has been limited compared to the other major groups of protostomes, such as Ecdysozoa. For what concerns Mollusca, early cellular studies (Canesi et al., 2002, Matricon-Gondran and Letocart, 1999, Takahashi and Muroga, 2008) have been followed by extensive molecular surveys in gastropods (Adema et al., 2017, Coustau et al., 2015, Pila et al., 2017), cephalopods (Castillo et al., 2015, Gestal and Castellanos-Martínez, 2015) and bivalves (Gerdol and Venier, 2015, Song et al., 2015, Zhang et al., 2015). A minor interest has been directed to Annelida, with studies targeting immune cells (Boidin-Wichlacz et al., 2011, Vetvicka and Sima, 2009) and immune genes (Altincicek and Vilcinskas, 2007, Nyholm et al., 2012, Tasiemski and Salzet, 2017) of polychaetes, oligochaetes and hirudinean worms.

Detailed data concerning the immune system of other lophotrochozoan phyla is lacking, except from occasional reports focused on specific gene families (Jeong et al., 2015). Brachiopods, comprising no more than 325 extant species, can be considered as part of these immunologically unexplored phyla. The phylogenetic position of brachiopods, in particular in relation with other lophotrochozoan phyla (i.e. Nemertea) has been a contentious matter for a very long time. The recent availability of fully sequenced genomes for brachiopods, nemerteans and phoronids finally allowed to clarify the phylogenetic position of Nemertea as a sister to the closely related phyla Brachiopoda and Phoronida (Fig. 1, panel E) (Luo et al., 2018).

Despite their current sporadic distribution, brachiopods were extremely abundant in ancient seas, with over 30,000 species known by fossil records. While brachiopods underwent a massive reduction in their distribution, being out-competed by bivalves as a consequence of the Permian-Triassic extinction, some species survived by developing a series of morphological and physiological adaptations (Posenato et al., 2014). All extant brachiopods are marine organisms, which mostly live in continental shallow waters, usually attached to the substrate with their pedicles (Fig. 1, panel A and B). Although brachiopods morphologically resemble bivalve mollusks, their two valves are dorso-ventrally oriented, in contrast to a lateral position in bivalves. Furthermore, like phoronids and bryozoans, they possess a horseshoe-shaped lophophore, a characteristic feeding structure which has long been used for the classification of these phyla within a taxonomic group named Lophophorata (Emig, 1997).

Anatomically, brachiopods have an open circulatory system, consisting of a dorsal vessel and several interconnected blood sinuses, which deliver nutrients and oxygen to various parts of the body (Fig. 1, panel D). The blood and the colorless fluid contained in the coelomic cavity can mix, to some extent, as the two compartments communicate. In physiological conditions, three main types of circulating cells can be recognized in L. anatina: (i) erythrocytes, containing the respiratory pigment hemerythrin and abundant in the blood vessels of the mantle; (ii) spindle body cells, elongated and rich in fibres, the most abundant cell type in aquaria-maintained animals; (iii) amoebocytes, with a phagocytic activity, common in the coelomic cavity of the pedicle and characterized by the presence of electron-dense granules (Rowley and Hayward, 1985) (Fig. 1, panel C). The presence of amoeboid coelomocytes in the pedicle coelom of brachiopods has been confirmed in different species, whereas this cell type appears to be extremely rare in the mantle cavity (Heller, 1931, Morse, 1902, Prenant, 1928, Yatsu, 1902). Although some studies have further classified amoebocytes among the hyaline, eosinophilic and basophilic subtypes (Ohuye, 1938), other researchers have later pointed out that such morphological variations might be related to the degree of cell granulation (Rowley and Hayward, 1985). While brachiopod amoebocytes cells might be involved in immune functions (James et al., 1991), this has not been demonstrated. An immune-related function for these phagocytic cells would indeed be reminiscent to that of annelid coelomocytes, which are involved in the recognition and clearance of pathogens and waste material (Vetvicka and Sima, 2009). Brachiopod amoebocytes are also involved in the resorption of necrotic tissues (Chuang, 1983) and the shell regeneration process (Pan and Watabe, 1989).

The information available concerning pathogens and diseases affecting brachiopod species are also scarce and mostly derive from paleobiological observations. Brachiopods have been subject to intensive predation by drilling gastropods and/or polychaetes during their evolution (Baliński and Yuanlin, 2010, Baumiller and Bitner, 2004, Kiel, 2008, Leighton, 2003, Teichert, 1945) and predation by marine invertebrates appears to be rather relevant even in extant brachiopods under certain conditions (Tyler et al., 2013). Signs of recovery from shell drilling have been widely documented by fossil records (Alexander, 1986, Hiller, 2014, Peel, 2015), indicating that, despite the tissue damage sustained, brachiopods can survive and recover sub-lethal damages. Shell damage could lead to the exposure of injured tissues to the external environment, thereby enabling the direct contact with pathogens that are often responsible for disease outbreaks and even mass mortality events in other sessile marine invertebrates (Garnier et al., 2007, Paillard, 2004). Fossil records also show that Devonian brachiopods could be infested by non-boring vermiform metazoan parasites of uncertain taxonomic placement, as evidenced by the presence of tubular structures on the inner surface of valves, interpreted as the result of shell deposition on parasites infesting the mantle cavity and damaging the mantle tissue (Bassett et al., 2004, Vinn et al., 2014). Despite the little amount of reports available for parasitism in contemporary brachiopods, the observation of shells from dead animals suggests that many rhynchonelliform species living in Northern America suffer from significant parasitism by spionid polychaetes (Rodrigues, 2007). Furthermore, some brachiopod species have been also reported as occasional intermediate hosts of digenean trematodes (Cremonte et al., 2008).

At the same time, very little is known about mutualistic and commensalistic symbiosis in this phylum. Apart from epizoic foraminifers and other encrusting organisms which exploit feeding currents (Zumwalt and Delaca, 1980), a curious case of commensalism has been reported between Laqueus rubellus and the crab Pinnotheres laquei, which lives between the brachiopod valves without affecting shell deposition and mantle/lophopohore shape (Feldmann et al., 1996). The microbiota associated with brachiopod tissues represent a completely uncharted territory.

Overall, while the general knowledge of brachiopod biology is still very limited, this lophotrochozan phylum represents an interesting, yet unexplored target for immunological studies, providing an alternative model to mollusks and roundworms, which might help to improve our understanding of the evolution of immune strategies in metazoans.

Section snippets

Sequence data and identification of immune-related genes

The L. anatina genome and transcriptome assemblies were retrieved from a previous study (Luo et al., 2015). Briefly, the genome assembly was obtained by the use of a hybrid sequencing approach combining the outputs of Illumina, 454 Life Sciences reads and PacBio sequencing technologies. The initial assembly, performed with 454 and paired-end Illumina reads only, was carried out with Newbler. The preparation of Illumina mate-pair libraries, together with additional 8.5 Gb PacBio data (consisting

General remarks

The metazoan innate immune system is based on a pathway consisting of receptors, signaling and effector molecules whose concerted action activates a coordinated response towards invading microorganisms and parasites. The initial involvement of specific immune cells and humoral factors in the recognition of microbe- and pathogen-associated molecular patterns (MAMPs and PAMPs) further reinforces the response at a systemic level, leading to the recruitment of other specialized cells at the site of

Conclusions

The release of the genome of the brachiopod L. anatina permitted to identify a highly complex immune system, which consists of several hundred molecular players among PRRs, signaling adaptors, effector molecules and modulators of the immune response. We showed that the brachiopod immunome shares a remarkable similarity with that of invertebrate deuterostomes lacking adaptive immunity, and a less significant overlap with to that of arthropods. Therefore, the L. anatina immunome confirmed the

Acknowledgements

We thank Dr. Samuele Greco for graphical design assistance in Fig. 1. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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