fmars-07-00674 August 19, 2020 Time: 20:21 # 1 ORIGINAL RESEARCH published: 21 August 2020 doi: 10.3389/fmars.2020.00674 Chloroflexi Dominate the Deep-Sea Golf Ball Sponges Craniella zetlandica and Craniella infrequens Throughout Different Life Stages Kathrin Busch1, Erik Wurz2, Hans Tore Rapp3†, Kristina Bayer1, Andre Franke4 and Ute Hentschel1,5* 1 GEOMAR Helmholtz Centre for Ocean Research Kiel, Kiel, Germany, 2 Marine Animal Ecology Group, Wageningen University and Research, Wageningen, Netherlands, 3 Department of Biological Sciences, K.G. Jebsen Centre for Deep Sea Research, University of Bergen, Bergen, Norway, 4 Institute of Clinical Molecular Biology (IKMB), Kiel, Germany, 5 Faculty of Mathematics and Natural Sciences, Christian-Albrechts-Universität zu Kiel, Kiel, Germany Deep-sea sponge grounds are underexplored ecosystems that provide numerous goods and services to the functioning of the deep-sea. This study assessed the prokaryotic diversity in embryos, recruits, and adults of Craniella zetlandica and Craniella infrequens, common and abundant representatives of deep-sea sponge grounds in the Edited by: North Atlantic. Our results reveal that symbiont transmission in the two Craniella sponge Joana R. Xavier, species likely occurs vertically, as highly similar microbial consortia have been identified University of Porto, Portugal in adults, embryos, and recruits. Moreover, transmission electron microscopy revealed Reviewed by: Cristiane Cassiolato Pires high abundances of sponge-associated microorganisms, among which Chloroflexi Hardoim, (SAR202) were identified as common representatives by amplicon sequencing and São Paulo State University, Brazil Lucia Bongiorni, fluorescence in situ hybridization (FISH). Equal diversity metrices, a similar overall Institute of Marine Science (CNR), Italy prokaryotic community composition and a distinct dominance of the phylum Chloroflexi *Correspondence: within all life stages are the key findings of our analyses. Information such as presented Ute Hentschel here provide understanding on the recruitment of deep-sea sponge holobionts which is [email protected] needed to develop integrated management tools of such vulnerable marine ecosystems. †Deceased March 07, 2020 Keywords: vulnerable marine ecosystems, sponges, Craniella, Chloroflexi, symbiosis, early life stages, amplicon Specialty section: sequencing, fluorescence in situ hybridization This article was submitted to Deep-Sea Environments and Ecology, a section of the journal INTRODUCTION Frontiers in Marine Science Received: 23 March 2020 A fundamental process influencing the fitness of many organisms is the establishment of host- Accepted: 23 July 2020 microbial colonization during development (Mortzfeld et al., 2016). Microbial consortia enable Published: 21 August 2020 the respective animal host to expand its range of nutrition (Nicholson et al., 2012), defense Citation: (Flórez et al., 2015), immunity (Eberl, 2010), and development (Koropatnick et al., 2004). Host- Busch K, Wurz E, Rapp HT, microbe interactions may be regulated from early developmental stages and transmitted through Bayer K, Franke A and Hentschel U generations (reviewed in Rosenberg and Zilber-Rosenberg, 2011; McFall-Ngai et al., 2013). In (2020) Chloroflexi Dominate the general, beneficial microbial symbionts are expected to be transmitted vertically (from parents Deep-Sea Golf Ball Sponges Craniella zetlandica and Craniella infrequens to offspring) in order to assure an establishment of a compatible partnership (Björk et al., Throughout Different Life Stages. 2019 and references therein). Further, probability of vertical transmission is expected to increase Front. Mar. Sci. 7:674. with increasing dependence of the host on its microbial partners (Ewald, 1987; Bull et al., doi: 10.3389/fmars.2020.00674 1991; Yamamura, 1993; Douglas, 1994; Thompson, 1994; Herre et al., 1999; Wilkinson, 2001; Frontiers in Marine Science| www.frontiersin.org 1 August 2020| Volume 7| Article 674 fmars-07-00674 August 19, 2020 Time: 20:21 # 2 Busch et al. Chloroflexi Dominate Craniella Life Stages Sachs et al., 2011). In the marine environment symbioses are dominant microbial phyla differ between different life stages common and host-microbe interactions have been assessed at (adults, embryos, and recruits) of the two sponge species. different developmental stages for a variety of model organisms, Knowledge about deep-sea sponge holobiont recruitment and including cnidarians (Apprill et al., 2009; Mortzfeld et al., 2016), plasticity across life stages is crucial to enable, in the long term, echinoderms (Galac et al., 2016; Carrier et al., 2019), cephalopods an adequate conservation of sponge populations from vulnerable (Montgomery and McFall-Ngai, 1994), molluscs (Endow and marine ecosystems. Ohta, 1990; Wentrup et al., 2014), and annelids (Nussbaumer et al., 2006; Schimak et al., 2015). Sponges (Porifera) are evolutionarily ancient animals which MATERIALS AND METHODS first appeared between 650 and 700 million years ago (Love et al., 2009; Zumberge et al., 2018). Today, more than 9,000 Sampling species (according to the World Porifera Database) are classified Sponges of different developmental stages (adults, embryos, into four classes (Demospongiae, Calcarea, Homoscleromorpha, recruits) were collected during two research cruises onboard and Hexactinellida) and populate virtually all marine benthic the Norwegian research vessel RV G.O.Sars (“GS2017110” habitats from shallow tropical reefs to the polar seas and the and “GS2018108”) to the North Atlantic Ocean (Schulz Bank deep ocean (van Soest et al., 2020). Sponges are prominent seamount at 73.8◦N, 7.5◦E; and Stjernsund at 70.3◦N, 22.5◦E) examples of marine holobionts, consisting of diverse prokaryotic in July 2017 and August 2018. Samples were collected by communities with more than 63 prokaryotic phyla detected remotely operated vehicle (ROV AEgir, University of Bergen) within sponge hosts (Thomas et al., 2016; Moitinho-Silva et al., or Agassiz trawl at an average depth of 600 m below surface. 2017a). The prokaryotic communities of sponges are host species- Tissue chunks of adult sponges and recruits were subsampled specific and distinct from seawater prokaryotic communities onboard ship with sterile scalpels from the mesohyl, rinsed, flash- (Hentschel et al., 2002; Thomas et al., 2016). The transmission frozen and stored at −80◦C for molecular analyses. Additional of microbial sponge symbionts to the next generation occurs pieces of tissue were subsampled with biopsy punches and vertically or horizontally (De Caralt et al., 2007; Maldonado, preserved onboard ship for microscopy. Embryos were carefully 2007; Schmitt et al., 2008; Webster et al., 2010; Gloeckner picked out of the parent sponges (Figure 2) with sterile spring et al., 2013). Microbial communities have been detected in steel forceps using a stereomicroscope. After rinsing, 10 whole reproductive stages, such as oocytes, embryos, larvae, and embryos were each pooled per adult sponge to account for juveniles of various shallow water species (Ereskovsky et al., 2005; the small biomass, fixed in RNAlaterTM Stabilizing Solution Schmitt et al., 2007; Sharp et al., 2007; Gloeckner et al., 2013; (Thermo Fisher Scientific, Waltham, MA, United States), stored Björk et al., 2019) indicating that vertical transmission is an at 4◦C overnight, transferred to −20◦C on the next day important mechanism for the establishment and maintenance of and subsequently to −80◦C. In addition, intact embryos were sponge-microbe associations over evolutionary time scales. preserved onboard ship for microscopy. In terms of sample In comparison to sponges from shallow and often warm- numbers, we sampled eight adult C. infrequens sponges, of which water habitats, much less is known about sponges and their four were brooding embryos, and nine C. infrequens recruits. microbiomes from deep and cold environments. Sponges are During the cruise we further sampled eight adult C. zetlandica ubiquitous and abundant components of deep-sea benthic sponges of which one was brooding embryos. In addition to communities and can form structurally complex and diverse our cruise activities, we sampled nine C. zetlandica recruits in ecosystems, termed “sponge grounds” (Klitgaard and Tendal, September 2018 from an aquarium system (Bergen, Norway). The 2004; Xavier et al., 2015; Maldonado et al., 2016). Sponge grounds respective parent sponges had been maintained since summer are known to strongly support biodiversity, either directly by 2017 in 45 L tanks with sea-water flow through (1 L min−1) provision of shelter or indirectly via food webs, and further pumped from the adjacent fjord at 105 m depth below surface. perform a variety of ecosystem functions, among others via Seawater was treated with a 20 mm drum filter prior to flowing participation in bentho-pelagic coupling and biogeochemical through the holding tanks. Under these conditions spawning cycles (reviewed in Maldonado et al., 2016; Thompson and Fuller, was documented frequently and the new recruits had settled in 2020). Deep-sea sponge grounds (syn. “sponge aggregations” the tanks. C. zetlandica recruits from the aquarium system were or “sponge gardens”) have been flagged as vulnerable marine smaller (and thus most likely younger) than the in situ sampled ecosystems (VMEs) that are currently under pressure through C. infrequens recruits and therefore flash-frozen as a whole. In fishing and other human activities (Pham et al., 2019; Murillo our case the new sponge recruits