Genetic mechanisms of bone digestion and nutrient absorption in the bone-eating worm Osedax japonicus inferred from transcriptome and gene expression analyses
© The Author(s). 2017
Received: 29 April 2016
Accepted: 6 December 2016
Published: 13 January 2017
Bone-eating worms of the genus Osedax (Annelida, Siboglinidae) have adapted to whale fall environments by acquiring a novel characteristic called the root, which branches and penetrates into sunken bones. The worms lack a digestive tract and mouth opening, and it has been suggested that Osedax degrade vertebrate bones and uptake nutrients through acidification and secretion of enzymes from the root. Symbiotic bacteria in the root tissue may have a crucial role in the metabolism of Osedax. However, the molecular mechanisms and cells responsible for bone digestion and nutrient uptake are still unclear, and information on the metabolic interaction between Osedax and symbiotic bacteria is limited.
We compared transcriptomes from three different RNA samples from the following tissues: trunk + palps, root + ovisac, and larva + male. A Pfam domain enrichment analysis revealed that protease- and transporter-related genes were enriched in the root + ovisac specific genes compared with the total transcriptome. Through targeted gene annotation we found gene family expansions resulting in a remarkably large number of matrix metalloproteinase (mmp) genes in the Osedax compared with other invertebrates. Twelve of these Osedax mmp genes were expressed in the root epidermal cells. Genes encoding various types of transporters, including amino acid, oligopeptide, bicarbonate, and sulfate/carboxylate transporters, were also expressed in root epidermal cells. In addition, amino acid and other metabolite transporter genes were expressed in bacteriocytes. These protease and transporter genes were first expressed in root tissues at the juvenile stage, when the root starts to develop.
The expression of various proteinase and transporter genes in the root epidermis supports the theory that the root epidermal cells are responsible for bone digestion and subsequent nutrient uptake. Expression of transporter genes in the host bacteriocytes suggests the presence of metabolic interaction between Osedax and symbiotic bacteria.
The deep sea is one of the few remaining frontiers in the field of biology. Since the discovery of an invertebrate community in the Galapagos Rift in 1977 , several chemosynthetic ecosystems have been found in hydrothermal vents and hydrocarbon seeps worldwide [2, 3]. In these environments, many endemic species, such as vestimentiferan tubeworms, vesicomyid clams, and Rimicaris shrimps, have been reported . These organisms have evolved to consume new nutrient sources, with chemosynthetic energy obtained through symbiosis with chemosynthetic microbes (reviewed in [4, 5]). In addition to vents and seeps, another type of deep-sea community has also been discovered, referred to as the whale-fall ecosystem . When a carcass of a large vertebrate (e.g., a whale) sinks to the sea floor, the huge source of organic material harbors a variety of organisms. Initially, mobile scavengers such as sharks, hagfishes, and crustaceans aggregate and consume the soft tissue of the carcass . After the bones of the carcass are exposed, enigmatic marine worms of the genus Osedax colonize on the bones .
Here, we performed transcriptome analysis and examined the spatial and developmental expression patterns of genes related to digestion and nutrient uptake in the bone-eating worm Osedax japonicus, which can be reared for multiple generations under laboratory conditions . Comparing the transcriptomes from the three different tissues: root + ovisac (root), trunk + palp (trunk), and larva + male, we identified the genes that were specifically expressed in the root. We investigated the expression patterns of genes of interest that were considered related to bone digestion and nutrient uptake. The results revealed novel aspects of the molecular mechanisms of the metabolic process of Osedax worms.
Sequencing of O. japonicus transcriptome
We sequenced three types of RNA samples: trunk, root, and larva + male (Fig. 1a–d) using a HiSeq2000 sequencer (Illumina, San Diego, CA, USA). Males of O. japonicus are dwarf males and are approximately 500 μm in length (Fig. 1c). Owing to the small amounts of total RNA obtained from males and larvae, these two sample types were combined for sequencing. A total of 200,523,188 reads were obtained and the basic information regarding the raw data and assembly is shown in the supporting information (Additional file 1: Figure S1, Additional file 2: Table S1). The raw reads were assembled into 57,194 contigs and 38,913 subcomponents by Trinity software  with a contig N50 of 1383 bp. Although the read assembly was still incomplete, we considered these subcomponents to be distinct genetic loci. We set the threshold for gene expression intensity (fragments per kilobase per million reads, [FPKM] ≥1, see Methods) to eliminate fluctuations or background noise and 22,541 genes were found above the threshold in at least one sample. Among 22,541 genes, the numbers of trunk, root, and larva + male-specific genes were 825, 1214, and 5485, respectively. The 22,541 genes were compared against UniprotKB with BLASTX and 9448 genes were homologous to known functional genes. Among these 9448 genes, 185, 223, and 1258 genes were trunk, root, and larva + male specific, respectively. The list of root specific gene is shown in the supporting information (Additional file 3: Figure S2, Additional file 4: Table S2). Raw data are available in the DDBJ sequence Read Archive (DRA003880).
Characterization of the root transcriptome
To elucidate the features of O. japonicus transcriptome, we attempted functional annotation of novel sequence data using gene ontology (GO) annotation and a single enrichment analysis against the total O. japonicas transcriptome. GO term enrichment analysis showed that 11, nine, and six terms associated with “molecular function,” “cellular component,” and “biological process,” respectively, were significantly over-represented in the root-specific genes against all gene set backgrounds (Additional file 5: Table S3). The over-represented GO terms included hydrolase activity, transferase activity, transport, and catalytic activity, which were all related to the nutrient uptake and metabolic processes. Only two terms of “molecular function” and a term of “biological process” were under-represented in the root transcriptome (Additional file 5: Table S3).
Pfam domains enriched in the root transcriptome
Putative peptidoglycan binding domain
Peptidase family M13
Amino acid permease
Major Facilitator Superfamily
Sodium:sulfate symporter transmembrane region
Amino acid permease
Clostridium neurotoxin, N-terminal receptor binding
Dual-action HEIGH metallo-peptidase
Coagulation Factor Xa inhibitory site
Peptidase family M1
Insulin growth factor-like family
Peptidase family M13
Carboxypeptidase activation peptide
Pregnancy-associated plasma protein-A
Chitin binding Peritrophin-A domain
Metallo-peptidase family M12B Reprolysin-like
ABC transporter transmembrane region
Sodium:neurotransmitter symporter family
Concanavalin A-like lectin/glucanases superfamily
Metallo-peptidase family M12
Collagen triple helix repeat (20 copies)
WW domain-binding protein 1
ERAP1-like C-terminal domain
Sugar (and other) transporter
Expression of protease genes in the root
To identify cells responsible for protease secretion, we examined expression pattern of genes encoding protease, which degraded the ECM. Because Pfam domains related to the MMP protein were enriched in the root-specific gene set, we first examined the expression of mmp genes. From the transcriptome of all samples, 24 mmp genes containing the catalytic domain with a conserved zinc-binding motif were found. Among them, 22 mmp genes were detected from the root transcriptome and 13 mmp genes were expressed only in the root (Additional file 6, Table S4). We examined the expression of 16 mmp genes and performed double staining with a probe against the 16S rRNA gene of Neptunomonas japonica (Nj 16S rRNA) infected with O. japonicus to clarify the localization of bacteriocytes harboring symbiotic bacteria.
Expression of transporter genes in the root
SLC family transporters play crucial roles in the uptake and efflux of compounds such as sugars, amino acids, nucleotides, inorganic ions, and fatty acids [32, 33]. In the O. japonicus transcriptome, 40 families in the slc gene superfamily, with at least 233 genes, were identified (Additional file 8: Table S5). Although the slc gene nomenclature system has been defined in mammals , due to low resolution of the phylogenetic analyses and the likely lineage-specific molecular evolution of the slc genes, the genes were named in an Osedax-specific manner.
The expression patterns of other metabolite transporters were also examined. The members of the SLC5 family are glucose transporters that play an important role in sugar uptake . In O. japonicus, Oja-slc5a-1 was expressed in the bacteriocytes (Fig. 4l). The SLC16 family members are monocarboxylate transporters that catalyze the proton-linked transport of monocarboxylates such as lactate, pyruvate and ketone bodies . A member of the slc16 family gene, Oja-slc16a-1, was expressed in the bacteriocytes (Fig. 4m). The SLC13 family members are Na+ and SO4 2--carboxylate co-transporters, which translocate anions, or di- and tri-carboxylate citric acid cycle intermediates in vertebrates . Two slc13 genes, Oja-slc13a-1 and Oja-slc13a-2, were expressed in the root epidermis (Fig. 4n, o). Expression of Oja-slc13a-1 was widespread in root epidermis (Fig. 4n), whereas Oja-slc13a-2 was expressed only in a small number of root epidermal cells (Fig. 4o, arrowheads). The SLC4 family of bicarbonate transporters transfers the bicarbonate ion or a related species across the plasma membrane . Oja-slc4a-1 was expressed in the mesodermal cells in the root (Fig. 4p). A nucleus counterstain with DAPI detected positive Oja-slc4a-1 signals in the cell layer just underneath the epidermis, which was identified as a muscle layer (Fig. 4q, r, ). Oja-slc4a-2 was expressed in the root epidermis and non-symbiotic mesenchymal cells around bacteriocytes (Fig. 4s, arrows).
Expression of protease and transporter genes in the larval and juvenile stages
Bone digestion by proteases
Vertebrate bones are made of both organic and inorganic components. One of the main inorganic components is calcium phosphate. A previous study reported that Osedax root epidermis cells are immunoreactive against anti-V-H+ ATPase and anti-CA antibodies . The V-H+ ATPase and CA are also expressed in the osteoclasts in vertebrates and are involved in the resorption of bones through acidification . Vertebrate bones and cartilage also contain many different organic components, with the majority being ECM proteins, such as various types of collagens. MMPs are a large family of zinc dependent endoproteinases and are able to degrade ECM components . Vertebrates have seven types of fibrillar collagens, encoded by eleven genes . Types I, V, and XXIV are components of mineralized bone, whereas types II, XI, and XXVII are components of cartilage . In this study, we found at least 24 MMPs in O. japonicus. Of these genes, 12 were expressed in the root epidermis. The number of mmp genes is small in invertebrates (two in the fly Drosophila melanogaster and seven in the ascidian Ciona intestinalis, ), except for in the sea urchin (; 26 in the purple sea urchin Strongylocentrotus purpuratus). In vertebrates, at least 25 different mmp genes have been identified with 24 of them present in humans . In spiralians, little is known about mmp genes. We surveyed the genomes of three spiralians: the pacific oyster Crassostrea gigas , the owl limpet Lottia gigantea, and a marine polychaete Capitella teleta , and found six, eight and five mmp genes, respectively.
This remarkable increase of MMPs followed by amino acid substitutions and expression in the root epidermis in O. japonicus most likely enables them to digest various types of ECMs in vertebrate bones. In addition to ECM degradation in bone and cartilage, MMPs play essential roles in morphogenesis, immune system functioning, and reproduction through the remodeling of ECMs in vertebrates and invertebrates [50, 51]. We detected expression of some Osedax mmp genes in root epidermis, bacteriocytes, and ovarian tissues. The peculiar shape of the root tissues implies that their morphogenesis is regulated by the repeated growth and remodeling of ECMs. Some MMPs may also have a function during the formation of the root tissue.
Nutrient uptake and metabolic interaction between host and symbiotic bacteria
The SLC superfamily is the largest group of membrane transporter proteins, and includes 52 families and 395 transporter genes in the human genome . The SLC transporters play essential roles in nutrient uptake in the intestine as well as in the translocation of various kinds of molecules in other tissues and cell types. The functions of each member of the SLC transporter are well characterized in humans ; however, little is known about the function of SLC transporters in invertebrates. Almost all families of the SLC transporters are conserved among bilaterians . In this study, we confirmed the expression of some slc genes encoding oligopeptide and amino acid transporters in the root epidermis of O. japonicus. In mammals, members of the proton-coupled oligopeptide transporter family (SLC15) are responsible for the absorption of dietary protein digestion products in the intestine . In O. japonicas, we found that two genes encoding SLC15 family transporters were expressed in the root epidermis. Root epidermal cells started to express one of two slc15 genes at the early juvenile stage, when the animals began a sessile lifestyle and started consuming vertebrate bones. We also detected expression of a gene encoding the amino acid transporter family (SLC6) in the root epidermis. These transporters are the candidate molecules playing a crucial role in nutrient uptake. Further functional analysis will unveil the molecular mechanisms for digestion and absorption by the root of Osedax worms.
In this study, we found the expression of genes encoding amino acid, glucose, and monocarboxylate transporters in bacteriocytes. The genome sequence of Osedax symbionts showed that they encode a number of transporter genes for various substrates such as amino acids, peptides, and carbohydrates . The exact function of symbiotic bacteria in Osedax worms is still unclear; however, expression and possession of transporter genes in the host bacteriocytes and symbiont genome suggest the presence of metabolic interaction between Osedax and symbiotic bacteria (Fig. 6).
Because there is no comparable counterpart close relative of Osedax, the root is considered an evolutionary novelty. Here, we investigated tissue-specific transcriptome and the expression pattern of genes, which appear to be related to the function of the root. Expression of various proteinase including mmp and transporter genes in the root epidermis support the theory that the root epidermal cells are responsible for bone digestion and subsequent nutrient uptake. Expression of amino acid, sugar and other metabolite transporter genes in the host bacteriocytes suggests the presence of metabolic interaction between Osedax and symbiotic bacteria. Further studies using the O. japonicus and N. japonica system, which allows us to examine the biology of both species and the interactions between them, should provide essential information regarding the evolutionary novelty of Osedax.
Animal collection and laboratory culture
We obtained O. japonicus specimens from whale bones collected at a depth of 226 m off Cape Noma Misaki via a remotely operated vehicle (ROV), Hyper-Dolphin, on the research vessel (R/V) Natsushima on March 28 and April 13, 2012. Osedax japonicus specimens were kept in 100-L tanks at 11 °C in the laboratory together with whale bones. Embryos and males were collected from the mucus of females. Larval settlement was induced by adding small pieces of vertebrate bone to dishes where the larvae were kept . To induce the infection of N. japonica, we incubated the bones with N. japonicus overnight at 20 °C. After a brief wash with filtered artificial sea water, the bones were cultured with larvae.
RNA extraction and sequencing
We extracted total RNA from three samples: the female trunk + palps (trunk), the root + ovisac (root), and larvae + adult males, using QIAzol reagent followed by column purification using RNeasy Mini Kit (Qiagen, Limburg, Netherlands) according to the manufacturer’s instructions. The quality and concentration of total RNA was analyzed by gel electrophoresis and a NanoDrop Spectrophotometer (Agilent, Santa Clara, CA, USA), respectively. Only samples that met the manufacturer’s criteria (BGI: http://www.bgi.com/us/services/genomics/whole-transcriptome-sequencing/) were then processed for the RNA-seq analysis. Short read sequences were obtained using a Hiseq2000 sequencer (Illumina, San Diego, CA, USA) according to the manufacturer’s procedures. High quality bases (quality score ≥20) were used in following analyses. The raw sequence data are available in the DDBJ sequence Read Archive (DRA003880).
Three fastq files obtained from the samples were combined into a dataset and de novo assembly of the O. japonicus transcriptome without a reference genome was performed using Trinity (version r2013-02-25) . To obtain normalized gene expression intensities (in FPKM), reads from each of the three samples were mapped onto the Trinity assembly with the bowtie , and analyzed with the RNA-seq by Expectation-Maximization (RSEM: version 1.2.3; ) and edgeR . In the assembly procedure, putative alternative splicing variants were estimated as different contigs per sub-component; however we merged variants from one sub-component based on the “%comp_fpkm” values of the RSEM output when we estimated the tissue specificity of each sub-component. The Maser analytical pipelines on the National Institute of Genetics Cell Innovation program (http://cell-innovation.nig.ac.jp/) were used for the following functional estimations of the assembled Trinity contigs.
GO enrichment analysis was employed to identify GO terms associated with a subset of genes and to test whether this association (enrichment) is significantly different from what would be expected by chance given a background gene set (in this case, the entire gene set). GO terms were assigned to nucleotide contigs of the Osedax assembly against a set of the protein database of UniprotKB that showed BLASTX hits with a threshold E-value of 10-10. FatiGo was employed for the statistical enrichment test of root specific genes against the pool of all transcriptome based on the annotated subcomponents after a false discovery rate < 0.01 correction .
For the protein annotation analyses of Osedax sequences, the longest open reading frames and corresponding amino acid sequences per subcomponents were predicted using a Perl script, with sequences longer than 30 amino acids used for downstream analysis. Transmembrane domains and signal sequence regions were predicted with TMHMM 2.0  and SignalP 4.1 . Pfam domains were assigned based on the results of a HMMER (3.1b2; ) search against the Pfam-A database , with a threshold E-value of 10-5. The statistical analyses of the Pfam enrichment analysis were performed using R (3.1.2), with the Q-value package (1.43.0; ). The p-values of the enriched domains in the root-specific gene set against the pool of three transcriptomes were calculated using a hypergeometric distribution, with a false discovery rate < 0.05 correction. The Pfam enrichment analyses were performed using domains that were assigned to at least five subcomponents.
Phylogenetic analyses of genes
For the analysis counterpart of Osedax, we searched for mmp genes from three spiralians genomes already published: the pacific oyster Crassostrea gigas , the owl limpet Lottia gigantea, and a marine polychaete Capitella teleta , using BLAST on the genome browsers of the animals. We first performed TBLASTN against each genome using Osedax MMP sequences as queries (an E-value of < 1e−10). Gene orthologies were confirmed by the phylogenetic analyses described later.
Fragments of genes were amplified by PCR (the primer sequences and accession numbers are in Additional file 9: Table S6). Gene orthologies were inferred by maximum likelihood analyses and multiple alignments. Amino-acid alignments were made with MAFFT ver. 7 . Sequences were trimmed by trimAl . Maximum likelihood analyses were performed with RAxML ver. 8 .
In situ hybridization
Animals were fixed with 4% paraformaldehyde (PFA) in Mops buffer (0.1 M Mops, 0.5 M NaCl) at 4 °C, overnight. After several washes with PBST (i.e., PBS containing 0.1% Tween 20), samples were dehydrated through an ethanol series and stored in 80% ethanol at –20 °C. We used two in situ hybridization methods for visualizing spatiotemporal gene expression patterns. For the whole-mount in situ hybridization of larvae and juvenile females, samples were rehydrated and washed three times with PBST. The samples were digested with 1 μg/ml proteinase K/PBST for 20 min at 37 °C. After a brief wash with PBST, the samples were postfixed in 4% PFA/PBST for 10 min at room temperature (20–25 °C, RT), and washed three times with PBST. The samples were prehybridized in hybridization solution (50% formamide, 5× SSC, 5× Denhardt’s solution, 100 μg/ml yeast RNA, and 0.1% Tween 20) at 55 °C for 2 h and hybridized with a hybridization solution containing a digoxigenin (DIG)-labeled RNA probe at 55 °C for at least 16 h. For the negative control, an RNA probe of a gene, which was not expressed in the stages examined, was used (data not shown). The samples were washed with a solution of 50% formamide, 4× SSC, and 0.1% Tween 20 for 30 min twice; 50% formamide, 2× SSC, and 0.1% Tween 20 for 30 min twice; 2× SSC and 0.1% Tween 20 for 30 min twice; and 0.2× SSC and 0.1% Tween 20 for 30 min twice at 55 °C. The samples were washed with MABT (i.e., maleic acid buffer containing 0.1% Tween 20) three times, blocked in 2% blocking reagent (Roche, Indianapolis, IN, USA) in MABT for 60 min at RT, and incubated overnight at 4 °C with a 1:1500 dilution of anti-DIG-AP antibody (Roche) in the blocking buffer. Samples were then washed six times with MABT for 60 min and transferred into AP buffer (100 mM Tris pH 9.5, 100 mM NaCl, 50 mM MgCl2, and 10% dimethylformamide) without MgCl2 and dimethylformamide. After being washed twice with AP buffer, a chromogenic reaction was performed using nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP; Roche) in AP buffer until signals were visible. The reaction was stopped in PBST, postfixed in 4% PFA/PBST, rewashed with PBST, and mounted with 40% glycerol before being observed under a light microscope (IX71, Olympus).
For frozen sections, bones containing adult O. japonicas females were fixed overnight with 4% PFA/MOPS at 4 °C. The fixed samples were washed three times with PBS. We digested bones overnight with Morse’s solution at RT . After we had picked worms from the bones, they were washed three times with PBS and mounted in Tissue-Tek® O.C.T compound to use as frozen sections. The in situ hybridization protocol of the frozen sections of adult females was based on a previous report . For double staining, we used a fluorescein-labeled probe for hybridization. After the chromogenic reaction with NBT/BCIP described above, slides were incubated in 0.1 M glycine-HCl for 30 min to inactivate the alkaline phosphatase. Slides were washed three times with MAB, blocked in 2% blocking reagent in MAB for 60 min at RT, and incubated for 1 h at RT with a 1:2000 dilution of anti-fluorescein-AP antibody (Roche) in blocking buffer. They were washed six times with MAB for 60 min and transferred into AP buffer without MgCl2. After being washed twice with AP buffer, a chromogenic reaction was performed with 2-(4-iodophynyl)-5-(4-nitrophenyl)-3-pheniltetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate (INT/BCIP; Roche) in AP buffer until signals were visible. The reaction was stopped in PBST, postfixed in 4% PFA/PBS, rewashed with PBS, and mounted with 80% glycerol. The slides were then observed under a light microscope.
We are grateful to the captain and crew of the R/V Natsushima and the operation team of the ROV Hyper-Dolphin for animal collection. We thank to Hiroshi Wada for his help in informatics analysis, and Michiko Yano for her help in the molecular biological experiments. The authors utilized the NIG Cell Innovation program (http://cell-innovation.nig.ac.jp/index_en.html) for the informatics analysis. This work is supported by Grants-in-Aid for Research Activity Start-up 23870044 to NM.
Availability of data and materials
Raw short read sequences are available in the DDBJ Sequence Read Archive under the accession number DRA003880.
NM conceived the project, conducted the experiments, and drafted the manuscript. MAY conceived the project, performed the bioinformatic analysis, and drafted the manuscript. HK performed the bioinformatic analysis and drafted the manuscript. YF supervised the project. All authors have read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
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