Multiple bacterial partners in symbiosis with the nudibranch mollusk Rostanga alisae

The discovery of symbiotic associations extends our understanding of the biological diversity in the aquatic environment and their impact on the host’s ecology. Of particular interest are nudibranchs that unprotected by a shell and feed mainly on sponges. The symbiotic association of the nudibranch Rostanga alisae with bacteria was supported by ample evidence, including an analysis of cloned bacterial 16S rRNA genes and a fluorescent in situ hybridization analysis, and microscopic observations. A total of 74 clones belonging to the phyla α-, β-, γ-Proteobacteria, Actinobacteria, and Cyanobacteria were identified. FISH confirmed that bacteriocytes were packed with Bradyrhizobium, Maritalea, Labrenzia, Bulkholderia, Achromobacter, and Stenotrophomonas mainly in the foot and notum epidermis, and also an abundance of Synechococcus cyanobacteria in the intestinal epithelium. An ultrastructural analysis showed several bacterial morphotypes of bacteria in epidermal cells, intestine epithelium, and in mucus layer covering the mollusk body. The high proportion of typical bacterial fatty acids in R. alisae indicated that symbiotic bacteria make a substantial contribution to its nutrition. Thus, the nudibranch harbors a high diversity of specific endo- and extracellular bacteria, which previously unknown as symbionts of marine invertebrates that provide the mollusk with essential nutrients. They can provide chemical defense against predators.

www.nature.com/scientificreports/ evidence for an endo-and extracellular bacterial consortium, which is more complex than other marine symbioses described before. The results are discussed with a special emphasis on the bacterial diversity and on the potential role of the discovered symbionts. This study is the first multifaceted investigation of symbiotic association of bacteria with a nudibranch species.

Methods
Specimens. Specimens of the nudibranch R. alisae (Fig. 1A) and sponge O. pennata were collected in the Peter the Great Bay, Sea of Japan, by SCUBA diving, at the depth of 1-6 m year-round in 2017-2019. They were then kept in a tank or used immediately for analyses. The nudibranchs were dissected by razor and separate tissues were excised for the analyses under stereomicroscope Leica ES2 (Germany).
DNA amplification, cloning, and sequencing. Total genomic DNA was extracted from the foot, intestine, and notum tissues using the DNeasy Plant MiniKit protocol (Qiagen, Hilden, Germany). The procedures of DNA amplification, cloning, and sequencing were described previously 13,14 . A 1.5-kb fragment of the 16S rRNA bacterial genes was amplified with universal primers 22 : 5'-agagtttgatcatggctcag-3' (27F, forward) and 5'-ggttaccttgttacgactt-3' (1492R, reverse). The PCR reactions were carried out in final volumes of 25 µl using TaKaRa Ex Taq™ in accordance with the manufacturer's description (Takara Biotechnology Co., Ltd.). The PCR reaction mixtures were placed in a DNA thermal cycler (Eppendorf, Mastercycler Gradient), incubated for 5 min at 94 °C and subjected to 32 cycles of denaturation, annealing, and extension: at 94 °C for 30 s, at 52 °C for 30 s, and at 72 °C for 1.5 min, with a final 7-min extension period at 72 °C. The PCR products for the 16S rRNA gene were cloned (TOPO TA cloning kit, Invitrogen, Calif.) and sequenced by the dideoxy chain-termination technique using a Dye Terminator chemistry and separated on an ABI PRISM 377 automated DNA sequencer (Perkin Elmer). The sequences of both strands were determined for each clone, using overlapping internal primers spaced, on average, 500 nucleotides. At least two independent PCR amplifications were sequenced in both directions to correct for possible cloning or sequencing errors. The 16S rRNA sequences have been deposited in GenBank under accession numbers MZ410589-MZ410616. The sequences were assembled using the program SeqMan (Lasergene, DNASTAR, Inc.). Multiple alignment was carried out manually and using the program CLUSTAL W 23 . Putative chimeras were identified using the Bellerophon software 24 . Two programs available online, RDP Classifier 25 and Greengenes 26 , were used to uncover the bacterial affinities. The maximum-likelihood tree of the 16S rRNA sequences of Rostanga alisae symbionts was constructed with the program IQ-TREE 27 using the TIM3 + F + I + G4 model of nucleotide substitution.

Transmission electron microscopy (TEM).
For TEM, the tissue samples were fixed with a 2.5% glutaraldehyde solution in a 0.1 M cacodylate buffer (pH 7.2) for 4 h at room temperature. After being rinsed in the buffer, the samples were post-fixed for 1 h with a 1% osmium tetroxide solution and dehydrated in a graduated series of ethanol and acetone, and then embedded in a mixture of Epon and Araldit epoxy resin (Sigma, USA). Ultra-thin sections (ca. 50 nm) were prepared on a Leica EM UC6 ultramicrotome, post-stained with a 0.5% uranyl acetate and lead citrate solution, and viewed using a Libra 120 electron microscope (Carl Zeiss, Germany). For TEM, 15 nudibranch individuals were used.

Fluorescent in situ hybridization (FISH).
For the FISH analysis, the foot, intestine, and mantle tissues were initially fixed at 4 °C in a 4% paraformaldehdye solution (pH 7.0) in a phosphate-buffered saline (PBS) for 8 h. The fixed samples were rinsed 3 × 1 h, impregnated with a 15% sucrose solution on PBS, placed in NEG 50 ™ (Thermo Scientific, USA), and frozen at -50 °C. The frozen tissues were sectioned (12-14 µm thick) on a Histostar 500 HM (Thermo Scientific) and placed onto glass slides treated with polylysine. A bacterial universal probe EUB338 was used along with a group of specific probes designed as symbiotic bacteria dominant in tissues of R. alisae (Table S1). Hybridization was performed at 46 °C for 3.5 h in a solution containing 900 mM NaCl, 20 mM Tris/HCl, 35% formamide, 0.01% SDS, and 3 ng/ml of the EUB338 probe along with one of the specific probes. After hybridization, the slides were washed twice at 48 °C for 15 min in a buffer containing 100 mM NaCl, 20 mM Tris/HCl (pH 8.0), 5 mM EDTA, and 0.0001% SDS. The sections were stained with a dilute 4′6'-diamidino-2-phenylindole (DAPI) solution (5 µg ml -1 ) for 1 min and examined by epifluorescence microscope using a LSM 780 laser scanning confocal microscope. For FISH microscopy, 5 nudibranch individuals were used.
Lipid analysis. Lipid extracts were prepared from homogenized tissues according to 28  www.nature.com/scientificreports/ using STATISTICA 5.1 (StatSoft, Inc., USA). A statistical probability of p < 0.05 was considered significant. Values are represented as mean ± standard deviation, n = 7.

Results
Histological analysis. Morphological analysis using a differential interference contrast (DIC) and epifluorescence microscopy of tissues ( Fig. 1B-E) revealed bacterial clusters (10 − 15 µm overage size), and individual single bacteria in the epidermis covering the notum (Fig. 1D) and foot (Fig. 1E). Bacteria were also visualized in the mucus layer covering the body surface (Fig. 1C).
Genetic affiliation of the bacterial 16S rRNA clones. The analysis of the cloned bacterial 16S rRNA genes allowed to obtain the most conservative and reliable scan of bacterial diversity, which made it possible to reveal bacteria that form mutualistic relationships with the host and to formulate the most parsimonious hypothesis concerning the functional importance of the symbiosis. On the basis of non-chimeric sequences (a total of 74 clones), 24 bacterial phylotypes were detected (with 97% identity cutoff) in tissues of R. alisae (Table S2). Phylogenetic analysis of each clone showed close matches with a multitude of bacteria ( Fig. 2) belonging to the classes α-Proteobacteria (15 clones), β-Proteobacteria (13 clones), γ-Proteobacteria (16 clones), Cyanobacteria (19 clones), and Actinobacteria (7 clones). Three rare phylotypes were associated with the classes δ-Proteobacteria (1 clone), Planctomycetes (1 clone), and Fusobacteria (2 clones). Three phylotypes (Bradyrhizobiaceae, Rhodobacteraceae, and Alcaligenaceae) were classified at the family level only. The rest of the phylotypes (except for those represented by single clones) belonged to 13 genera: Bradyrhizobium, Maritalea, Labrenzia, Burkholderia, Achromobacter, Stenotrophomonas, Synechococcus, Arthrobacter, Ilumatobacter, Aquicella, Lysobacter, Legionella, and Leptotrichia (Table S2). The phylogenetic analysis placed the bacterial phylotypes recovered from R. alisae within a clade of α-Proteobacteria that contains Labrenzia, Maritalea, and Bradyrhizobium. Phylotypes of symbiotic Labrenzia (transferred from Stappia) belong to a single cluster with the other species and strains of this genus. The closest relative of the nudibranch's symbiont Labrenzia is L. alba isolated from oyster and described as straight rods, motile by a subpolar flagellum, and chemoorganotrophic aerobe 29 . One of the three α-Proteobacterial symbionts was clustered with Maritalea porphyrae (transferred from Zhangella), isolated from the red alga Porphyra yezoensis 30 . Another member of α-Proteobacteria symbionts was clustered with B. pachyrhizi and B. elkanii, which were isolated from nodules of legume plants. Among the dominant β-Proteobacterial symbionts, one fell into the genus Burkholderia and another into Achromobacter. The dominant γ-Proteobacteria symbiont was phylogenetically located within the genus Stenotrophomonas, whose species are found ubiquitously worldwide, particularly in close association with plants. The cyanobacterial 16S rRNA sequences from the nudibranch were grouped with a clade of sequences of marine Synechococcus spp. isolated from the water column. A sequencing analysis and BLAST searches retrieved close relation between nudibranch symbionts and actinobacteria Arthrobacter, Iamia, Ilumatobacter, and Kocuria.
Mosaic sets of common and tissue-specific bacterial phylotypes were detected in the foot, notum, and intestine. The clones were unequally distributed between the tissues studied. The deviation from equal proportions was highly significant (χ 2 = 25.33, df = 1, P < 0.001). Synechococcus (Cyanobacteria) had the most abundant clones, especially in the intestine. Maritalea, Labrenzia, and a member of Rhodobacteriacea were located exclusively in the foot, which was especially rich in bacterial clusters. They, consequently, were specific symbionts of the foot tissue. Bradyrhizobium and Burkholderia were predominantly detected in the notum. Phylotypes of Stenotrophomonas were localized exclusively in the integumentary tissues, foot, and notum, and only Achromobacter clones were evenly distributed over the tissues. Phylotypes of Actinobacteria were found only in integumentary tissues; therefore, the species-specific tissue distribution was evident. Thus, Iamia, Ilumabacter, and Kocuria were found only in the foot; in contrast, another member of Actinobacteria, Arthrobacter, could be considered as a specific bacterium of the notum. Other bacterial clones had mosaic distribution with no clear preference for a tissue.
In addition to predominant bacterial phylotypes, members of γ-Proteobacteria such as Coxiella, Aquicella, Acinetobacter, and Aliivibrio, as well as Desulfovibrio, Planctomycetes, and Leptotrichia, were also present as single clones mainly in the intestine. These are known as intracellular parasites of protozoa and animal cells 31 , which suggest that these species are unlikely to be true symbiotic bacteria, and their presence most probably resulted from the contamination of this tissue as reported for Aquicella siphonis parasitizing an amoeba 31 . FISH observations. A bacterial universal probe EUB338 revealed that the bacterial clusters were more abundant in cytoplasm of the foot epithelium in the skin fold between the foot and the notum (Fig. 3A). The presence and localization of bacterial symbionts in tissues was confirmed by FISH analysis using the genus-specific oligonucleotide probes (Table S1) for Maritalea, Labrenzia, Bradyrhizobium, Burkholderia, Achromobacter, Stenotrophomonas, and Cyanobacteria ( Fig. 3B-I).
The group-specific probes for Achromobacter and Stenotrophomonas showed discrete patches of hybridization in epithelial cells of the foot and notum (Fig. 3B,C), whereas oligonucletide probes for Labrenzia and Maritalea hybridized strongly with bacteria that settle in the foot epithelium (Fig. 3D,E). Bacterial signals of Bradyrhizobium and Burkholderia were found only in notum epithelial cells (Fig. 3F,G). Each of the symbionts was localized in clusters and occurred within a single cluster. FISH analysis also revealed the presence of cyanobacteria in the epithelial cells of all studied tissues, but they were more represented in the intestinal epithelium (Fig. 3H,I). Thus, the characteristic tissue specificity of certain bacteria was revealed, which is consistent with the results of the 16S rRNA analysis (Table S2). The most common bacteriocytes were formed by Labrenzia and Stenotrophomonas, while Maritalea and Achromobacter were detected less frequently. www.nature.com/scientificreports/ www.nature.com/scientificreports/ The number of bacterial clusters detected by Eub388 was higher than that of clusters labeled with each of the group-specific probes. The total number of bacteriocytes detected by the group-specific oligonucleotide probes was generally comparable to the number of bacteriocytes detected by EUB338, which is consistent with the assessment of the phylogenetic composition of the symbionts' population.
TEM observations. To examine the spatial organization of the bacterial symbionts, we performed TEM of the foot, notum, and intestine tissues. TEM revealed dense clusters of rod-shaped gram-negative bacteria enclosed in the epithelium covering the foot and notum of R. alisae (Fig. 4). Bacteria were separated by a membrane and located within specialized vacuoles, referred to as bacteriocytes, which could occupy most of cytoplasm of a host cell. The number of bacteria per bacteriocyte reached a few dozen (Fig. 4A-C). Two main bacterial morphotypes formed bacteriocytes; however, bacteria within each vacuole were monomorphic.
The most common bacteriocytes were those containing gram-negative curved-rod lophotrichous bacteria (0.3-0.4 μm) having up to six flagella (Fig. 4B,C). Less common were bacteriocytes with large gram-negative rod-shaped bacteria (0.7 × 1.5 μm), many of which appeared to be dividing (Fig. 4D-F). In some bacteriocytes, membranes of neighboring bacteria came into contact. As a result of incomplete division, the rod-shaped bacteria can form chains of individuals connected to each other, suggesting their clonal origin within a single specialized vacuole (Fig. 4E,F).
Epithelial cells also contained vacuoles with single gram-negative bacteria (Fig. 5A). Their morphotypes were diverse and similar to that described from bacteriocytes above. The similarity between the morphotypes of single bacteria and bacteria forming clusters, as well as the presence of dividing bacteria in the bacteriocytes, suggests that the observed single bacteria are the initial stage of formation of a dense bacterial cluster within bacteriocyte.
Apart from bacteriocytes packed with bacteria, large bacteria (diameter 0.5-0.7 μm, length up to 2 μm) with a well-developed polysaccharide envelope were found mainly in the intestinal epithelium and, more rarely, in the integumentary epithelium ( Fig. 5B-E). Their cytoplasm contained concentrically arranged membranes of the thylakoid type, vacuoles, and granules ( Fig. 5C-D). Bacteria were single or formed chains of several individuals (Fig. 5C,D). Some individuals in the colony had a thick, hypertrophied peptidoglycan fibrillar envelope (Fig. 5D). The presence of thylakoid membranes, the ability to form chains, and the hypertrophied peptidoglycan envelope in some individuals, which is typical of heterocysts, allows classifying these microbes as cyanobacteria.
An ultrastructural analysis showed that the nudibranch bore various extracellular bacteria in the mucus layer of the foot and notum that were identified by a 16S rRNA gene sequence analysis as close relatives of the genera Arthrobacter, Iamia, Ilumatobacter, and Kocuria (Fig. 6). The most abundant were gram-negative curved rodshaped bacteria (0.5 × 1.5-2 μm) (Fig. 6A) with a long (up to 1 μm) contact stalk-like extension, usually directed to the apical surface of epithelial cells and providing a primary contact of bacteria with the apical membrane of host's epithelial cells before penetrating into cytoplasm (Fig. 6B). This confirms the presence of a similar bacterial morphotype in bacteriocytes in epithelial cells. Bacteria inhabiting the mucus layer often come into contact with the apical membrane of epithelial cells, which is probably the initial stage of their inoculation (Fig. 6C). Large gram-negative rods (0.7 × 3 μm) (Fig. 6D) with numerous electron-translucent vacuoles were much less common. Bacteria forming long branching filaments resembling fungal hyphae were observed on the surface of ciliated cells of the foot epithelium (Fig. 6E). Unusually thin, from 10 to 20 nm, non-septate filaments branched and formed round bubbles of 50-100 nm in diameter at the apex. Similar branching bacteria were also found in the apical cytoplasm of epithelial cells that indicates the presence of air and substrate mycelium of branching bacteria.
The presence of these symbionts was typical for all adult animals studied, although the pattern of distribution of bacteriocytes varied between individuals. The general occurrence of individuals with symbionts indicates an obligate rather than a facultative association between symbionts and the nudibranch.

Fatty acid analysis.
Fatty acid analysis is a useful tool to ascertain original nutrient sources acquired by partners of the association 5,32 . The integumentary tissues of notum and foot that were rich in symbiotic bacteria and the intestine with an abundance of cyanobacteria were analyzed separately but their fatty acid compositions showed no significant differences (Table S3). Both tissues exhibited exceptionally high concentrations of oddand branched fatty acids (OBFA) typical for bacteria (13.7 and 12.5%, respectively), and also an abundance of saturated (19.6 and 20.8%) and monounsaturated fatty acids (20.8 and 21.4%), mainly due to 16:0 and 18:1n-7, along with high levels of n-6 polyunsaturated fatty acids (PUFA) (20.2-20.5%) (Fig. 7A). Marked differences were found between the nudibranch and its prey, the sponge O. pennata. The sponge contained the highest concentration of very long chain fatty acids (VLCFA) (28.1%). The nudibranch was much more enriched in PUFA n-6, in particular in linoleic acid (18:2n-6), 20:4n-6, and 22:4n-6, while PUFA n-3 such as 22:6n-3 and 22:5n-3, were more abundant in the sponge. The level of 18:2n-6, typical for cyanobacteria 33 , and essential for the mollusks, was almost by an order of magnitude higher in R. alisae than in the sponge. The difference in the OBFA content between nudibranch and sponge was also pronounced (Fig. 7B). Moreover, the composition of these bacterial markers differed between the nudibranch and its prey, the sponge. The most noticeable difference was in the content of 17:0 (2.1% in R. alisae vs. 0.3% in the sponge) and anteiso-16:0 (2.0% vs. 0.2%, respectively). The acids, iso-15:0, 17:1∆5, iso-18:0, anteso-18:0, and cyclo-19:0, were found only in R. alisae. The predominance of OBFA, 16:0, and 18:1n-7, which are typical for bacteria, as well as differences in the composition of bacterial markers between the nudibranch and the sponge O. pennata indicates the important bacterial contribution to the diet of the nudibranch.
It seems unlikely that the sponge O. pennata is the only source of fatty acids in R. alisae, since their levels in the predator nudibranch were significantly higher than in its prey sponge. R. alisae tissues also contained twice as much bacterial marker cis-vaccenic acid (18:1n-7) as compared to that in the sponge. Moreover, some bacterial indicators (mainly anteiso- 15 www.nature.com/scientificreports/

Discussion
Symbiont diversity and distribution. The present study provides the first evidence of symbiosis in R.
alisae, a species of nudibranchs. This is the most multiple symbiosis that have ever been recorded for marine invertebrates. While many organisms establish an exclusively one-on-one relationship with a single microbial species or microbes belonging to the same functional group 5,12 , there are also organisms that harbor multiple microbial species, in which symbiont-symbiont and host-symbiont interactions occur. Six phylotypes of chemoautotrophic bacteria were reported for mussel Idas sp. from a cold seep area 11 and five extracellular symbionts for the gutless oligochaete worm Olavius algarvensis 34 . However, in these cases, symbioses involving bacteria and marine invertebrates are either endosymbiotic microbes co-occurring inside the host bacteriocytes 5,11 or ectosymbiotic microbes associated with the external surfaces of the animals 3,4,9,15,34 , with the exception of scaly-foot snail from hydrothermal vents having partnerships simultaneously with epi-and endosymbiontic microbes 35 . Bacterial symbionts in R. alisae have appeared to be more diverse than was previously known for marine invertebrates. It is evident that the detected symbiont phylotypes differ greatly from all other known symbionts found in marine invertebrates. Labrenzia (Rodobacteriales) and Maritalea (Rhizobiales) have not been recorded as forming symbiotic associations with invertebrates or plants so far, although other members of the families Rodobacteriales and Rhizobiales are well known symbionts 14 . Strains of Bradyrhizobium, Burkholderia, Achromobacter, and Stenotrophomonas are reported as symbionts of plants, interacting with a vast majority of nodulating legume species and efficient in biological nitrogen fixation 36 . This may be important when considering the nature of these symbionts in the nudibranch. Symbioses between cyanobacteria and marine organisms are commonly found among marine plants, fungi, sponges, ascidians, corals, and protists 37,38 . Synechococcus, identified as dominant symbiont clones of R. alisae (Table S2), is a unicellular cyanobacterium common in the marine environment, providing a range of beneficial functions including photosynthesis, nitrogen fixation, UV protection, and production of defensive toxins 8,9,37 . Symbiotic interactions between actinobacteria and their host have been observed in insects, human, animals, and plants, where the bacteria provide the host with protection against pathogens and produce essential nutrients 39 . However, none of the members of the clade Actinobacteria recorded in R. alisae are known to live symbiotically.   www.nature.com/scientificreports/ Arrangement of symbiotic association. Despite the high diversity of bacteria, they are well organized in the host. Dense clusters of rod-shaped bacteria, Labrenzia, Maritalea, Bradyrhizobium, Burcholderia, Achromobacter, and Stenotrophomonas, were found within host-derived vacuoles, referred to as bacteriocytes, inside epithelial cells of R. alisae (Fig. 3). Although such arrangement differs from that typical of bacteriocytes, which are usually considered as specialized cells of the hosts for harboring bacteria, it resembles that reported for scaly-food snail from hydrothermal vents, which harbor symbionts in the esophageal gland 35 . Bacteriocytes in the gastropod Lurifax vitreus found near hydrothermal vents also constitute a portion of the mantle epithelium; they have large vacuoles containing many live and dividing bacteria 40 . Each bacteriocyte was densely packed with certain symbionts, and the bacteriocytes were randomly distributed within the epithelium cells. A distinctly regular distribution pattern was observed in the gill epithelium of the mussel Bathymodiolus sp.: the thiotrophic symbionts occupy the apical region, and the methanotrophic symbionts are more abundant in the basal region of bacteriocytes 4 . In the mussel Idas sp., however, there is no spatial pattern of the six distinct bacterial phylotypes, and the symbionts are mixed within bacteriocytes 11 . Synechococcus dominated the cytoplasm of intestinal epithelium and, more rarely, epidermis cells, mainly as specialized cell type referred to as nitrogen-fixing heterocysts. They are visually similar to cyanobacteria from corals and sponges 8,37 .
The phylogenetic diversity and the spatial organization of the symbiotic community in R. alisae were determined by the 16S rRNA analysis, which was consistent with the results of FISH and TEM. Unlike most symbioses of marine invertebrates when bacteria house specialized host cells 5,11 or cover epidermis 7,15 , symbiotic association of R. alisae exhibited spatial partitioning between symbionts, which were unevenly distributed between the tissues (Table S2). It has been established that different members of the microbial community can complement each other in acquisition of various restrictive nutrients, confirming the importance of the functional diversity of symbionts 41 . Thus, Stenotrophomonas rhizophila and Bradyrhizobium build a beneficial association in the rhizosphere and can act synergistically on promoting growth and nutrient uptake of soybean 36 . Cyanobacteria can interact synergistically with beneficial members from the endophytic microbiome of rice seedlings 42 . The location of bacterium in the organism of R. alisae may, in fact, depend on the specific metabolic and ecological roles that the symbionts play, and also on the interaction with bacterium belonging to different physiological groups.
Nature of symbiosis. Symbiotic associations between microbes and invertebrates are acquired mainly in a nutrient-depleted environment where symbionts usually provide their hosts with essential nutrients and highenergy compounds 1 . In contrast to known symbioses between microbes and gutless invertebrates, which obtain nutrients exclusively from the bacteria, R. alisae, like most nudibranch species, is a sponge-eating predator. However, due to the lack of adipose tissue, sponges are distinguished by a low lipid content (0.4 to 1.5% of wet weight) 43 and also by specific proteinaceous spongin fibers and chitin, a polysaccharide similar to cellulose that can be indigestible for some predators, which together indicate their low nutritional value. Furthermore, R. alisae feeds exclusively on the sponge O. pennata; therefore, in habitats with low prey availability, this nudibranch has to survive starvation while searching for sponge assemblages. We suppose that symbiotic bacteria of R. alisae contribute to the utilization of low-quality food, similarly to symbiotic bacteria from the genera Rhodobacter, Burkholderia, and Aeromonas associated with the detritivorous isopod Asellus aquaticus 44 .
A fatty acid analysis, as a useful approach to clarifying the nature of symbiosis 5,20,32 , has confirmed the trophic interaction between symbionts and the nudibranch host (Table S2). Among the fatty acids of symbiotic bacteria in R. alisae, OBFA are a major acyl constituent of membranes in Stenotrophomonas 45 and also in Actinobacteria, Arthrobacter, Iamia, Ilumatobacter, and Kocuria 46 . Cis-vaccenic acid is a major component of Maritalea 30 . Omega-cyclohexyl tridecanoic acid (cyclo19:0) is specific to Bradyrhizobium 47 , Burkholderia, and Achromobacter 48 . Linoleic acid is produced by cyanobacteria including marine species of Synecoccocus 33 ; in nudibranch, it obviously serves as a precursor in the synthesis of arachidonic acid (20:4n-6), thus, providing additional evidence for the transfer of fatty acids from symbionts to the host. Mollusks are capable of converting linoleic acid to arachidonic acid, since they have enzymes required for its synthesis 21 . The presence of these bacteria-specific markers and the abundance of arachidonic acid confirm the metabolic role of symbionts in the nudibranch host.
Among nutrients, biologically available nitrogen can be considered a restrictive nutrient for the sponge-eating R. alisae, which can be acquired with the help of nitrogen-fixing symbionts, also referred to as diazotrophs. R. alisae harbors Bradyrhizobium, Burkholderia, Achromobacter, and Stenotrophomonas that are efficient in biological nitrogen fixation previously found to be associated with nodulating legume species 36 . Symbiotic nitrogen fixers are known to be associated with a variety of marine invertebrates such as wood-boring bivalves, corals, sponges, sea urchins, tunicates, and polychaetes 7,8,37 . Moreover, the protection of the enzyme nitrogenase that catalyzes N 2 fixation against oxygen is an important physiological requirement in bacteria such as symbiotic Bradyrhizobium, Burkholderia, Achromobacter, and Stenotrophomonas that are located in bacteriocytes and provide this protection. Synechococcus is known as a nitrogen-fixer 37,49 . It performs N 2 fixation in heterocysts where nitrogenase is restricted under oxic conditions. Indeed, heterocysts of Synechococcus are abundant in the intestine cells of R. alisae (Fig. 5B-D).
Nitrate assimilation is one of the major processes of nitrogen acquisition by many heterotrophic bacteria and cyanobacteria 50,51 . Symbionts of R. alisae can play an important role in the process of nitrate utilization through denitrification, dissimilatory nitrate reduction, and assimilatory nitrate reduction as a nitrogen source and synthesize it into organic nitrogen. The nitrate reducers, Labrenzia 52 , Stenotrophomonas 53 , Maritalea 30 , and Rhodobacteraceae 29 are widely represented in R. alisae. Synechococcus also utilizes nitrate, nitrite, or ammonium for growth 50 . Thus, symbiotic bacteria may play a significant role in the N-budget of the nudibranch mollusk.
The www.nature.com/scientificreports/ carbon monoxide (CO) oxidizers 54,55 . Despite the toxicity of CO for multicellular organisms, numerous aerobic and anaerobic microorganisms can use CO as a source of energy and/or carbon for cell growth 56 . The marine worm Olavius algarvensis establishes symbiosis with chemosynthetic bacteria using CO, a substrate previously not known to play a role in symbiotic associations with marine invertebrates, as an energy source 57 . We do not rule out that the R. alisae symbionts also might exploit CO as carbon and energy source. Despite this, assumption may seem impossible taking in account the CO toxicity, but, since many invertebrates (mollusks, tube worm, etc.) use toxic sulfate, thiosulfate, and methane as an energy source 1,15 , this hypothesis is worth to be addressed. An important component of skeleton in marine sponges of the family Microcionidae, including O. pennata, is the structural polysaccharide chitin 58 . Some bacteria are capable of hydrolyzing chitin via the activity of chitinolytic enzymes and can utilize chitin as a source of carbon, nitrogen, and/or energy 59 . Chitinase activity was documented for strains of Labrenzia 60 , Burkholderia 61 , Arthrobacter 62 , Achromobacter 63 , Stenotrophomonas 64 , Alcaligenes 65 , and actinobacteria 59 associated with R. alisae. Thus, these bacteria can work synergistically to digest chitin and spongin, contributing to feeding success of the host nudibranch which depends solely on low-quality, nitrogen-and carbon-deficient food available.
Furthermore, direct evidence has confirmed that many bioactive compounds from invertebrates are produced by symbiotic microorganisms 66,67 . Many biologically active compounds including toxic and deterrent secretions have been identified in nudibranchs of the family Discodorididae 68 . Symbiotic bacteria may exhibit toxic activity to provide the host nudibranch with chemical defense against predators and environment. Bacteria, especially actinobacteria, living in a symbiotic relationship with R. alisae may help the host in defense, since nudibranch lack a shell, and secondary metabolites of bacteria can provide them with chemical defense against predators and environment, as has been reported for some marine invertebrates 2,9,10 .
In complex associations, the integration and coexistence of symbionts depend on supplementary partnerships and mutual contribution to the host's metabolism 41 . The most intensively studied cases are highly specialized associations, where both partners can only exist in close relationship with one another. The relatively high diversity of microbes in R. alisae complicates understanding the complex pattern of molecular and cellular interactions between the host and its symbionts.

Conclusion
The current study demonstrates that the nudibranch R. alisae is a unique species among the marine animals, as it harbors a wide range of phylogenetically distinct but physiologically similar bacteria. These bacteria, known as aerobic chemoheterotrophs or facultative anaerobes, are capable of nitrogen fixation and/or denitrification under anaerobic conditions, oxidizing of carbon monoxide, hydrolyzing of chitin, and can use chitin as a source of carbon, nitrogen, and/or energy. They may also exhibit toxic activity and may provide chemical defense for the host nudibranch. A combination of such characteristic features as the dense population of diverse endo-and extracellular bacteria, the specific diet that is extremely poor in organic nutrients and energy, and the abundance of diverse fatty acids specific for bacteria and cyanobacteria in its composition suggest the existence of nutritional symbiosis in this nudibranch species. Overall, the data obtained expand our understanding of the biological role of the symbiosis of Metazoa with bacteria.

Data availability
The datasets generated or analyzed during the current study are included in this published article and its Supplementary Information files. All sequences are available in GenBank under accession numbers MZ410589-MZ410616. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.