Cephalopoda: Nautiloidea
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Proc. R. Soc. B (2007) 274, 1143–1152 doi:10.1098/rspb.2006.0353 Published online 20 February 2007 Enigmatic dual symbiosis in the excretory organ of Nautilus macromphalus (Cephalopoda: Nautiloidea) Mathieu Pernice1,*, Silke Wetzel2, Olivier Gros3, Renata Boucher-Rodoni1 and Nicole Dubilier2 1UMR 5178 Biologie des Organismes Marins et Ecosyste`mes, De´partement Peuplements et Milieux Aquatiques, Muse´um National d’Histoire Naturelle, 55 rue Buffon, 75005 Paris, France 2Max Planck Institute for Marine Microbiology, 28359 Bremen, Germany 3UMR 7138 Syste´matique, Adaptation, Evolution, De´partement de Biologie, universite´ des Antilles et de la Guyane B. P. 592, 97159 Pointe a` Pitre Cedex, Guadeloupe, France Symbiosis is an important driving force in metazoan evolution and the study of ancient lineages can provide an insight into the influence of symbiotic associations on morphological and physiological adaptations. In the ‘living fossil’ Nautilus, bacterial associations are found in the highly specialized pericardial appendage. This organ is responsible for most of the excretory processes (ultrafiltration, reabsorption and secretion) and secretes an acidic ammonia-rich excretory fluid. In this study, we show that Nautilus macromphalus pericardial appendages harbour a high density of a b-proteobacterium and a coccoid spirochaete using transmission electron microscopy, comparative 16S rRNA sequence analysis and fluorescence in situ hybridization (FISH). These two bacterial phylotypes are phylogenetically distant from any known bacteria, with ammonia-oxidizing bacteria as the closest relatives of the b-proteobacterium (above or equal to 87.5% sequence similarity) and marine Spirochaeta species as the closest relatives of the spirochaete (above or equal to 89.8% sequence similarity), and appear to be specific to Nautilus. FISH analyses showed that the symbionts occur in the baso-medial region of the pericardial villi where ultrafiltration and reabsorption processes take place, suggesting a symbiotic contribution to the excretory metabolism. Keywords: symbiosis; cephalopods; Nautilus; excretion; bacteria; 16S rRNA 1. INTRODUCTION physiology and metabolism is a means of understanding The acquisition and maintenance of one or more organisms the success and eventual demise of the ammonoids by another often leads to novel structures and metabolic (Boutilier et al. 1996; Staples et al. 2000). pathways. Hence, the study of symbiotic systems has In contrast to other extant cephalopods in which several resulted in exciting discoveries, such as the role of symbiotic organs are involved in excretion, in Nautilus, only the luminescent bacteria in the morphogenesis of the light organ pericardial appendages are responsible for most of the in squid (Euprymna scolopes/Vibrio fischeri association; three excretory processes (ultrafiltration, reabsorption and McFall-Ngai & Ruby 2000) or in the assimilation of secretion; Martin 1983). The main end product of inorganic molecules from hydrothermal vents by giant excretion is ammonia (Boucher-Rodoni 1989) and the tube worms (Riftia pachyptila with its sulphide-oxidizing contractile villi of the four pericardial appendages collect symbionts; Cavanaugh et al. 1981; Felbeck et al. 1981). the blood from capacious sinuses (figure 1b) and secrete The evolutionary history of both the partners is also a the acidic ammonia-rich fluid (up to 200 p.p.m., Schipp & major component of the symbiotic relationship, symbiosis Martin 1987) into the pericardial coelom from which it is being an important driving force of metazoan evolution finally excreted into the mantle cavity (figure 1a; Mangold (McFall-Ngai 2002). Therefore, the study of symbiotic et al. 1989). associations in ancient lineages might provide an insight Each pericardial appendage consists of numerous into the origin of major physiological adaptations. Thus, finger-like villi that are divided into an apical region the symbiotic associations in the excretory organs of covered with microvilli and a basal-medial region with Nautilus (Schipp et al. 1985) are of particular interest, as crypt-like invaginations. The basal-medial villus segment these invertebrates are considered as ‘living fossils’ from is filled with a branched lacunar system (Schipp et al. the Cambrian (542–500 Myr ago). They are also the only 1985). An ultrastructural study of Nautilus pericardial appendages revealed symbiotic bacteria forming a dense extant cephalopods with an external chambered shell fringe on the outer epithelium of the villi and its baso- (filled with over 90% nitrogen gas) resembling that of medial invaginations (Schipp et al. 1985). Only a single extinct ammonoids (figure 1a; Norman 2000). A better bacterial morphotype was observed with a thin rod shape comprehension of their life history, behaviour, ecology, (0.25–0.35!1.5–2.2 mm) and a typical gram-negative cell wall (Schipp et al. 1990). The presence of bacteria in * Author for correspondence ([email protected]). pericardial appendages, an ecological niche rich in Received 4 December 2006 1143 This journal is q 2007 The Royal Society Accepted 23 January 2007 1144 M. Pernice et al. Influence of symbiosis on adaptation (a)(b) siphuncle cameral jaws liquid blood in veins: + metabolic wastes VC G pericardial RS appendage EBV (with RA ABV symbionts) excretory organ PA heart with symbionts funnel blood in arteries DA H gills mantle cavity Figure 1. Morphology of Nautilus macromphalus.(a) Schematic of a longitudinal section through Nautilus.(b) Details of the inset in (a): simplified diagram of the excretory organ (with symbionts) and associated circulatory structures of Nautilus. ABV, afferent branchial vein; DA, dorsal aorta; EBV, efferent branchial vein; G, gill; H, heart; PA, pericardial appendage; RA, renal appendage; RS, renal sac; VC, vena cava. Inset, figure 4. ammonia, has led to several hypotheses about their were divided into two; one part was immediately frozen in potential role in the nitrogen metabolism of Nautilus, liquid nitrogen and stored at K808C for DNA extraction and such as (i) detoxification by the symbionts via ammonia the other fixed in 2% paraformaldehyde for CARD–FISH oxidation (Schipp et al. 1990) or (ii) the use of nitrogenous (see §2h). The last two pericardial appendages were fixed for waste by the bacteria to produce the nitrogen gas filling the transmission electron microscopy (see §2b). Nautilus shell and responsible for its neutral buoyancy (Boucher-Rodoni & Mangold 1994). (b) Transmission electron microscopy Nautilus pericardial appendage bacteria were physio- Pericardial appendages from the four individuals were fixed at logically characterized as Pseudomonodales after their 48C for 12 h in a fixative solution containing 3.2% isolation in artificial culture media (Schipp et al. 1990). glutaraldehyde in sterile seawater (pH 7.4) and washed in However, it is now widely acknowledged that culture 0.05% of NaN3 in sterile seawater (pH 7.4). After a brief methods reveal only a small fraction of the total bacterial rinse in 0.1 M pH 7.2 cacodylate buffer, adjusted to diversity (Amann et al. 1990; Pace 1997). The aim of the 900 mOsm with NaCl and CaCl2 to improve membrane present study was therefore to characterize the symbiotic preservation, samples were fixed for 45 min in 1% osmium bacteria in Nautilus macromphalus (Cephalopoda: Nauti- tetroxide at room temperature, then rinsed in distilled water, loidea) excretory organs using culture-independent mol- and post-fixed with 2% aqueous uranyl acetate for 1 h before ecular approaches. The phylogenetic position of bacteria embedding in epon–araldite. Semi-thin sections (0.5 mm associated with the pericardial appendages was investi- thick) and ultra-thin sections (60 nm thick) were obtained gated by comparative sequence analysis of the 16S rRNA using an Ultracut E microtome (LEICA). Transmission gene. Their localization within the organ was determined electron microscopy analyses were performed on a Philips by catalysed reporter deposition (CARD) fluorescence 201 microscope operated at 75 kV accelerating voltage. in situ hybridization (FISH) and transmission electron microscopy (TEM). We also examined the potential (c) DNA extraction function of the bacteria by investigating genes charac- DNA was extracted from the pericardial appendages of four teristic for nitrogen metabolism, namely amoA, nirS, N. macromphalus specimens by the method described by Zhou nirK and nosZ, as respective indicators for aerobic et al. (1996) using proteinase K for cell digestion and a ammonia oxidation, nitrite reduction and nitrous oxide standard chloroform–isoamyl alcohol extraction procedure. reduction (Braker et al. 1998; Scala & Kerkhof 1998; DNA was precipitated in isopropanol, washed with 70% Kowalchuck & Stephen 2001). The specificity of this ethanol, resuspended in 1! TE buffer and stored in aliquots bacterial–invertebrate association and its possible impli- at K208C. cations in Nautilus metabolism are discussed. (d) 16S rRNA gene PCR amplification 2. MATERIAL AND METHODS Bacterial 16S rRNA gene PCR amplification was per- (a) Specimen collection formed using GM3 (50-AGAGTTTGATCMTGGC-30) Four N. macromphalus individuals were collected on the outer and GM4 (50-TACCTTGTTACGACTT-30) bacterial pri- shelf of New Caledonia in September 2005. All specimens mers (Escherichia coli positions 8–23 and 1492–1477, were dissected aseptically and for each specimen, the four respectively, Muyzer et al. 1995). The reaction mixture pericardial appendages were processed as follows: two organs contained 50 pmol