applyparastyle “fig//caption/p[1]” parastyle “FigCapt” Zoological Journal of the Linnean Society, 2020, XX, 1–24. With 8 figures. Sleeping with the enemy: unravelling the symbiotic relationships between the scale worm Neopolynoe Downloaded from https://academic.oup.com/zoolinnean/advance-article/doi/10.1093/zoolinnean/zlaa146/6055457 by guest on 04 January 2021 chondrocladiae (Annelida: Polynoidae) and its carnivorous sponge hosts SERGI TABOADA1,2,3,*,†, , ANA SERRA SILVA2,4,5,†, , CRISTINA DÍEZ-VIVES2, LENKA NEAL2, JAVIER CRISTOBO1,6, PILAR RÍOS1,7, JON THOMASSEN HESTETUN8, BRETT CLARK2, MARIA ELEONORA ROSSI5, JUAN JUNOY1, JOAN NAVARRO9, and ANA RIESGO2 1Departamento de Ciencias de la Vida, Apdo. 20, Campus Universitario, Universidad de Alcalá, 28805 Alcalá de Henares, Spain 2Life Sciences Department, The Natural History Museum, Cromwell Road, London SW7 5BD, UK 3Departamento de Biología (Zoología), Universidad Autónoma de Madrid, Facultad de Ciencias, Cantoblanco 28049, Madrid, Spain 4Division of Biosciences, University College London, Gower Street, London WC1E 6BT, UK 5School of Earth Sciences, University of Bristol, Queens Road, Bristol BS8 1RJ, UK 6Instituto Español de Oceanografía, Centro Oceanográfico de Gijón, C/ Príncipe de Asturias 70 bis, 33212 Gijón, Asturias, Spain 7Instituto Español de Oceanografía, Centro Oceanográfico de Santander, Promontorio San Martín s/n, Apdo. 240, 39080 Santander, Spain 8NORCE Environment, NORCE Norwegian Research Centre, Nygårdsgaten 112 NO-5838 Bergen, Norway 9Instituto de Ciencias del Mar CSIC, Passeig Marítim de la Barceloneta 37–49, 08003 Barcelona, Spain Received 13 July 2020; revised 19 September 2020; accepted for publication 14 October 2020 The North Atlantic deep-water polynoid worm Neopolynoe chondrocladiae is involved in an exceptional symbiotic relationship with two hosts: the carnivorous sponges Chondrocladia robertballardi and Chondrocladia virgata. While this is an obligate symbiotic relationship, its real nature is unclear. We used a multidisciplinary approach to narrow down the type of symbiotic relationship between symbiont and hosts. Molecular connectivity analyses using COI and 16S suggest that N. chondrocladiae has high potential for dispersal, connecting sites hundreds of kilometres apart, likely aided by oceanographic currents. Microbial analyses on different anatomical parts of five Chondrocladia species suggest that the presence of the worm in C. robertballardi does not affect the microbiome of the sponge. MicroCT analysis on N. chondrocladiae show that it has dorsally oriented parapodia, which might prevent the worm from getting trapped in the sponge. A faecal pellet recovered from the worm suggests that the polynoid feeds on the crustacean prey captured by the sponge, something corroborated by our stable isotope analysis. Light and confocal microscopy images suggest that N. chondrocladiae elytra produce bioluminescence. We propose that the worm might use bioluminescence as a lure for prey (increasing the food available for both the sponge and the polynoid) and thus fuelling a mutualistic relationship. ADDITIONAL KEYWORDS: bioluminescence – confocal – microbiome – microCT – molecular connectivity – mutualism – stable isotopes – trophic relationships. INTRODUCTION *Corresponding author. E-mail: [email protected] Marine annelid polychaetes are known to engage in a †These authors contributed equally to this work. multitude of symbiotic relationships, with hundreds of © 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–24 1 2 S. TABOADA ET AL. examples in a plethora of families (Martín & Britayev, an adhesive surface due to the presence of velcro-like 1998, 2018). Despite the efforts of the scientific community anchorate isochaelae spicules, which trap the prey; over the past 20 years, which have doubled the number the prey is later engulfed by the sponge tissue and of described symbiotic associations in polychaetes, subsequently digested by symbiotic bacteria (Vacelet our understanding of the interactions between these & Boury-Esnault, 1995; Vacelet & Duport, 2004; Lee symbiotic worms and their respective hosts is still et al., 2012). Interestingly, two recent studies on the Downloaded from https://academic.oup.com/zoolinnean/advance-article/doi/10.1093/zoolinnean/zlaa146/6055457 by guest on 04 January 2021 scarce and inadequate (Martín & Britayev, 1998, 2018). microbial characterization of the sponge Chondrocladia In their latest review on symbiotic polychaetes, Martín grandis (Verrill, 1879) showed that specific bacterial & Britayev (2018) highlighted that after a period when taxa were enriched in different anatomical parts, most of the reported symbiotic relationships were thus suggesting different microbial functional roles classified as commensal (Martín & Britayev, 1998; in sponge metabolism (Verhoeven & Dufour, 2017; Britayev et al., 2014), there has been an increase in Verhoeven et al., 2017). Especially in the spheres, a the number of relationships identified as mutualistic greater abundance of Proteobacteria of the genera (many of which were formerly described as commensal Colwellia Deming et al., 1988 and Pseudoalteromonas or parasitic), which proves the efficacy of applying new Gauthier et al., 1995 was found; these bacteria are techniques and resources to the study of these symbiotic known to be involved in the hydrolysis of chitin, one associations. However, there are still many aspects of of the main components of crustacean exoskeletons symbiotic relationships involving polychaetes that need (Verhoeven et al., 2017). to be addressed in detail. Among them, for instance, In a recent study, Taboada et al. (2020) described is the scarce information available about behavioural morphological adaptations in both N. chondrocladiae observations based on living organisms (e.g. Martín and its hosts, two species of the genus Chondrocladia. et al., 2015), the nature of the trophic links between host These adaptations suggested the obligate nature of and symbiont (see: Martín & Britayev, 1998, 2018), and the symbiotic relationship established between the the phylogeographic and colonization patterns of the worm and the sponge. Neopolynoe chondrocladiae has symbiont, limited to the best of our knowledge to just specialized hooked chaetae in some segments, which two recent contributions (Lattig et al., 2017; Meca et al., could facilitate the attachment to, and navigation along, 2019). Difficulties in obtaining all this information are the branching body of the sponge host (Taboada et al., directly linked to the inherent limitations of studying 2020). These hooked chaetae have also been reported marine benthic organisms, which is particularly true for for other symbiotic polynoids (Pettibone, 1969; Martín deep-water species, whose collection and investigation & Britayev, 1998; Molodtsova et al., 2016; Ravara & are particularly challenging. Thus, there is a clear Cunha, 2016). Also, Taboada et al. (2020) described need to increase the information on known symbiotic open galleries in the stalk of the two sponges where relationships in polychaetes in order to provide a better N. chondrocladiae occurs, Chondrocladia virgata understanding of their true nature. Thomson, 1873 and Chondrocladia robertballardi Neopolynoe chondrocladiae (Fauvel, 1943) is a North Cristobo et al., 2015. Neopolynoe chondrocladiae worms Atlantic deep-water annelid of the family Polynoidae were found within these open galleries, which appeared Kinberg, 1856 involved in an exceptional symbiotic not to be excavated by the polynoid, but seemed to relationship with carnivorous sponges of the genus be produced by a gradual overgrowth of the sponge Chondrocladia Thomson, 1873 (Taboada et al., 2020). to accommodate the worm (Taboada et al., 2020). The symbiotic association between these organisms is Similar induced galleries, commonly known as ‘worm- noteworthy due to the fact that the symbiotic worm runs’, have been reported in gorgonian and hexacoral is a potential prey living inside its potential predator antipatharians, which are built by the host in order to and host. Members of the genus Chondrocladia belong provide shelter for the worm (Molodtsova & Budaeva, to the sponge family Cladorhizidae Dendy, 1922, and 2007; Britayev et al., 2014). Regarding the symbiotic are sponge carnivores feeding primarily on small relationship between N. chondrocladiae and its two crustaceans and polychaetes (Vacelet & Duport, 2004; Chondrocladia hosts, Taboada et al. (2020) hypothesized Vacelet, 2007). Chondrocladia sponges consist of that, apart from getting shelter from its host, the worm several anatomical parts: roots (used to anchor the might also be getting food that sponges trap in their animal to the sediment), axis (which can be subdivided spheres, while the worm might be providing a benefit in multiple branches) and terminal inflatable spheres to the sponge by cleaning its surface and/or dissuading sometimes sustained by short stems (Hestetun et al., potential predators, as described in other examples in 2016b). The spheres are used both for prey capture and the literature (e.g. Martín et al., 1992; Mortensen, 2001). reproduction, and maintain their turgidity thanks to In any case, despite the different sources of evidence a remnant aquiferous system with canals inside the provided, Taboada et al. (2020) hypothesized that the main axis (Kübler & Barthel, 1999). The spheres have nature of the symbiotic relationship between the worm © 2020 The Linnean Society