Animal rearing and dissection
Shipworms of the species Lyrodus pedicellatus from the Atlantic lineage  were used for our work. Samples were collected from the pier of Portsmouth Harbour (50° 47′ 47″ N, 1° 01′48″ W). Larvae from the original wood were used to infest logs of Scots pine, which were kept in tanks in the laboratories of the Institute of Marine Science, University of Portsmouth. To rear the animals, the water was taken directly from the Langstone Harbour (34 PSU salinity) and kept aerated and at a temperature of 15–18 °C using a flow-through system. The wood logs were opened by splitting them with a hammer and screwdriver, and the animals were then extracted with tweezers, placed in sea water containing EDTA-free protease inhibitors (1% v/v, Thermo Scientific) and kept on ice until dissection to anesthetise them. Species identification was performed using the pallets as described in . The dissections were performed using a stereomicroscope (Leica MZ6) after removing the mantle to expose the organs.
Scanning and transmission electron microscopy
Ten shipworms ranging in size from 4 to 8 cm were freshly extracted for all the microscopy experiments. For transmission electron microscopy, caecum, gills and food groove samples were fixed for 1–2 h at room temperature in primary fixative (4% formaldehyde (w/v), 2.5% (w/v) glutaraldehyde in 100 mM sodium phosphate buffer pH 7.2), and then washed (3 × 10 min) in 100 mM sodium phosphate buffer pH 7.2. Samples were then incubated in secondary fixative (1% osmium tetroxide in 100 mM sodium phosphate buffer pH 7.2) for 1 h on ice and dehydrated through a graded ethanol series (15 min each), followed by two washes (5 min each) in epoxy propane. Samples were infiltrated with a series of epoxy propane/Epon araldite (25%, 50%, 75% Epon Araldite with a minimum of 1 h at each stage, all at 30 °C) plus at least two changes of Epon araldite resin over 24 h at 30 °C, and polymerised at 60 °C for 48 h in flat embedding moulds. Pale gold (70–90 nm) ultra-thin sections were cut with a Diatome diamond knife using a Leica Ultracut UCT microtome and mounted on hexagonal 200-mesh nickel grids. Sections were post-stained with 2% (w/v) aqueous uranyl acetate (10 min) followed by lead citrate (5 min) in a carbon dioxide-free chamber and viewed using a FEI Tecnai 12 BioTWIN G2 TEM operating at 120 kV. Images were captured using AnalySIS software and a Megaview III CCD camera.
For scanning electron microscopy, the samples were fixed in 4% (v/v) glutaraldehyde in a cacodylate buffer (0.2 M sodium cacodylate, 0.3 M sodium chloride, 2 mM calcium chloride) for 2 h at room temperature and then rinsed once in buffer for 30 min. Samples were then taken through an ethanol dehydration series (50-70-100% ethanol and twice in 100% acetone, each stage for 30 min), critical point dried and then mounted on aluminium stubs using adhesive carbon tabs. Sputter coating was carried out under an argon atmosphere using a gold and palladium target, at a voltage of 1.4 kV using a current of approximately 18 mA for 3 min. Specimens were examined using a Zeiss MA10 Scanning Electron Microscope with an accelerating voltage of 20 kV and the Zeiss Smart software.
Embedding for immunolabeling proved difficult and several attempts were made to allow resin infiltration and at the same time preserve antigenicity. Freshly dissected shipworm tissues (food groove, gills and caecum) were fixed with 4% paraformaldehyde, 0.2% glutaraldehyde in sodium cacodylate buffer (0.2 M sodium cacodylate, 0.3 M sodium chloride, 2 mM calcium chloride, pH 7.4) on ice in a vacuum chamber for 2 h, then on a rotator without vacuum for a further 12 h at 4 °C. Samples were washed in 0.2 M sodium cacodylate buffer (three washes of 20 min each) and dehydrated through a graded ethanol series initially on ice (50%) and subsequently at − 20 °C on a rotator (70%, 90%, 100%) with 20 min at each stage and two changes of 100% ethanol. Ethanol was gradually replaced with LR Gold resin (1:2, 1:1, 2:1 resin:ethanol) with 1 h at each stage, followed by three changes of 100% LR Gold resin, 12 h each, all at − 20 °C on a rotator. Tissues were embedded in closed gelatine capsules and polymerised with UV light at − 20 °C for 24 h, followed by 24 h at − 10 °C. Pale gold (70–80 nm) ultra-thin sections were cut with a Diatome diamond knife, using a Leica Ultracut UCT microtome, and mounted on hexagonal 200-mesh nickel grids. All immunolabeling steps were achieved by floating grids on droplets of reagent. Sections were incubated in blocker (3% BSA in phosphate-buffered saline—PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4, pH 7.0) for 30 min at ambient temperature before incubation with primary antibodies against the bacterial LpsGH5_8 (see further down for details on antibodies production) diluted 1:100 in 1% BSA in PBS at 30 °C for 1 h, followed by washing with PBS at ambient temperature (three brief washes followed by three washes of 10 min each). Sections were incubated in secondary antibody (goat anti-rabbit IgG conjugated to 10 nm gold) diluted 1:100 in 1% BSA in PBS for 1 h at 30 °C, followed by washes with PBS as before, and subsequently with ultrapure water. All immunolabeling procedures included negative controls treated exactly as the samples: both pre-immune controls (diluted 1:100 in 1% BSA in PBS) and buffer only (1% BSA in PBS). Sections were post-stained with 2% (w/v) aqueous uranyl acetate (10 min), then lead citrate (5 min) in a carbon dioxide-free chamber and viewed using a FEI Tecnai 12 BioTWIN G2 operating at 120 kV. Images were captured using AnalySIS software and a Megaview III CCD camera.
Antibody production and purification
Two milligrams of purified recombinant bacterial LpsGH5_8 (see further sections for cloning, expression and purification) were used to raise polyclonal antibodies in rabbits (ProteoGenix, France). To enrich the serum for antigen-specific antibodies, affinity columns were made using recombinant LpsGH5_8. Recombinant protein preparations were dialysed against coupling buffer (0.1 M NaHCO3, 0.5 M NaCl, pH 8.3) and bound to CNBr-activated Sepharose™ 4 Fast Flow resin (GE Healthcare Life Sciences) followed by affinity purification of an aliquot of the crude antibody serum according to the resin manufacturer’s instructions. The pre-immune serum was subject to the same purification procedure. Purified antibody and pre-immune serum fractions were characterised for their affinity by western blotting using both recombinant LpsGH5_8 and L. pedicellatus caecum fluids (Additional file 5: Fig. S5). Fractions showing the highest titre and no unspecific binding were selected for immunogold labelling.
One adult Lyrodus pedicellatus (Quatrefages, 1849) specimen, measuring 3.6 cm in length and 1.62 cm in width, was used for micro-CT scanning. The specimen was reared at the Institute of Marine Sciences, University of Portsmouth, UK, and extracted from wood in 2012. The sample was fixed in 4 % v/v glutaraldehyde in a cacodylate buffer (0.2 M sodium cacodylate, 0.3 M sodium chloride, 2 mM calcium chloride) for 1 h at room temperature, rinsed three times in buffer for 10 min each, post-fixed in 1 % w/v aqueous osmium tetroxide for 1 h and rinsed three times in seawater for 10 min each. Samples were then immediately ethanol dehydrated and dried with hexamethyldisilazane (HMDS).
The specimen was mounted onto the sample holder and secured using glue, and scanned at the Ghent University Centre for X-ray Tomography (UGCT), Woodlab-UGent, using a scanner developed at UGCT. The scanner consisted of two X-ray tubes and two X-ray detectors, specifically designed to obtain very high-resolution scans as well as scans of larger objects. Scans were carried out using a microfocus X-ray tube in combination with a Varian flat-panel detector with an exposure time of 1500 ms, a rotation angle of 0.25° resulting in an average scan time of 45–60 min and an approximate voxel pitch of 2.5 μm. Details of the scanner are outlined in Masschaele et al.  and Van den Bulcke et al. [64, 65]. Due to large specimen size, two stacked scans were performed. The dataset was reconstructed using the Octopus software package with beam hardening correction. The two reconstructed volumes were then loaded in VGStudio MAX and stitched into a single stack of cross-sections. All resulting image and video analysis was performed using visualisation software myVGL.
RNA extraction and sequencing
Gills, digestive glands and caecum from three healthy adult L. pedicellatus were dissected and prepared for the paper published by Sabbadin and colleagues , using Ribosomal RNA depletion with a RiboZero™ Magnetic Gold Kit (Epidemiology) (Epicentre) in order to isolate both eukaryotic and prokaryotic mRNA. RNA-Seq libraries were prepared from each mRNA sample according to the Ion Total RNA-Seq kit v2 (Thermo Fisher Scientific). Templates were synthesised from mRNA libraries using the Ion OneTouch 200 Template Kit v2 DL on a OneTouch system (Thermo Fisher Scientific) and sequenced on an Ion Torrent PGM™ using a 318 chip (IonPGM200Kit; Thermo Fisher Scientific). All raw sequence data are available in NCBI under BioProject PRJNA412369 (SRA files: SRR6106265, SRR6106266, SRR6106267, SRR6106268, SRR6106269, SRR6106270, SRR6106271, SRR6106272, SRR6106273).
Crystalline style sacs were freshly dissected from 38 animals (which were then pooled together), flash frozen in liquid nitrogen and stored at − 80 °C. Total RNA was extracted using TRIzol® Reagent (Thermo Fisher Scientific), DNase treatment was carried out with Turbo DNA-free (Ambion), RNA was cleaned with RNA Clean & Concentrator™-5 (Zymo Research) and then quantified with a Qubit 3.0 Fluorometer and Agilent TapeStation. RNA depletion for both eukaryotic and prokaryotic ribosomal RNA was performed with the Ribo-ZeroTM Magnetic Gold Kit Epidemiology (Epicentre) and mRNA was then concentrated using RNA Clean & Concentrator™-5 (Zymo Research). The sequencing of the crystalline style sacs was performed at the Next Generation Sequencing Facility at the University of Leeds with HiSeq3000 using Illumina Technology to generate the required 150 bp paired end data. After rRNA depletion, library construction was completed using Illumina’s TruSeq stranded mRNA library protocol, starting at the RNA fragmentation step as suggested by Illumina.
Transcriptome assembly and analysis
Two meta-transcriptomes were assembled, using raw EST sequencing reads from digestive gland, caecum and gills (meta-transcriptome 1)  and from crystalline style sacs (meta-transcriptome 2), respectively. The raw EST sequencing reads were trimmed using Trimmmatic (part of the Galaxy tool [66, 67]) and were assembled into contigs using the Trinity software v. 2.8.3 .. Raw reads were mapped back onto the contigs and gene expression levels were calculated as TPM values (transcripts per kilobase million) using Salmon as part of the online tool Galaxy [66, 69] using standard parameters. Annotation of the contigs was performed by BlastX searches against the non-redundant database of the NCBI. The online software dbCAN (DataBase for automated Carbohydrate-active enzyme ANnotation)  was used to search for carbohydrate-active domains. Results with an e-value < 1e−10 and those with a CBM (Carbohydrate Binging Module) but no annotation were excluded, as well as CAZymes (Carbohydrate-Active enZYmes) belonging to the class of glycosyl transferases.
The following protocol describes the preparation and analysis of caecum and crystalline style samples. The caeca of five animals grown on Scots pine were dissected in 50 mM sodium phosphate buffer pH 7 and the content (food particles and enzymes) was isolated and pooled together. Similarly, 21 crystalline styles were dissected and washed three times in PBS buffer. Caecum and crystalline styles samples were then boiled for 10 min in denaturing buffer (1% SDS, 2.5% beta-mercapto ethanol, 175 mM DTT), centrifuged, and the supernatant was run into a 10% polyacrylamide gel. The protein bands were excised and digested with trypsin, and the resulting peptides were analysed by label-free LC-MS/MS. Tandem mass spectra were searched against the combined meta-transcriptomes (digestive gland, caecum, gills and crystalline style sac) of L. pedicellatus (which includes both eukaryotic and prokaryotic sequences) using the Mascot search programme. emPAI values were converted into molar percentages, the identified proteins were ranked based on relative abundance and annotated using BlastX versus non-redundant NCBI databases. CAZy annotation was carried out using the online tool dbCAN .
CAZy annotation was carried out using the online software dbCAN and results with an e-value < 1e−10, with a CBM but no CAZy module, and CAZymes belonging to the class of glycosyl transferases were excluded. Signal peptides were identified using the server SignalP 4.1 for either eukaryotes or gram-negative bacteria (www.cbs.dtu.dk/services/SignalP/).
Bacterial CAZymes cloning, expression, purification and biochemical characterisation
RNA was extracted from the gill tissue using the TRIzol® method (Thermo Fisher Scientific), cleaned with RNA Clean & Concentrator™-5 (Zymo Research) and a polyA tail was added using the poly(A) polymerase and protocol from Takara . cDNA was produced using the SuperScript® II reverse transcriptase (Thermo Fisher Scientific) with a oligo-dT primer and was purified with the Clontech NucleoSpin PCR Clean-up and gel extraction Kit (Clontech). The DNA sequences encoding the bacterial proteins LpsGH5_8, LpsGH11, LpsGH134a and LpsGH134b were amplified from cDNA without their signal peptide using the primers and PCR setting listed in Table S6 (Additional file 14) and they were cloned with the StrataClone Blunt PCR Cloning Kit (Stratagene); the sequences were verified by Sanger sequencing. LpsAA10A was not successfully cloned from the cDNA and therefore a synthetic version of the gene was codon optimised for E. coli expression by GeneArt.
The In-Fusion HD cloning kit (Takara) was used for cloning LpsGH5_8, LpsGH134a and LpsGH134b into the vector pET52b+, which has N-terminal Strep-Tag II followed by the human rhinovirus (HRV) 3C protease cleavage site, and a C-terminal His-Tag. The vectors were then transformed into E. coli Rosetta-GamiTM2(DE3) competent cells by heat shock.
LpsGH11 was cloned into the vector pOPINS3C , which contains an N-terminal His-Tag, followed by a Halo-Tag for improved soluble expression and the HRV 3C protease cleavage site, and no signal peptide for secretion. The vector was transformed into Spodoptera frugiperda 9 (Sf9) insect cells by heat shock.
LpsAA10A was cloned, without its CBM, using the In-Fusion HD cloning kit (Takara) into a modified pET26 vector containing at the N-terminus the pelB leader sequence to direct protein production to the periplasm, and a C-terminal Strep-tag. The construct was transformed into RosettaTM2(DE3) competent cells by heat shock.
Expression and purification
The E. coli bacterial cells containing LpsGH5_8, LpsGH134a and LpsGH134b constructs were grown in LB broth supplemented with carbenicillin (50 μg/ml) and chloramphenicol (34 μg/ml) at 37 °C until OD600 = 0.7 and then induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) 1 mM and grown overnight at 20 °C and 200 rpm. After harvesting the cells were pelleted, suspended in phosphate-buffered saline (PBS) with 0.01 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) and were lysed by sonication. After addition of 5 mM MgCl2 and DNaseI (0.025 U/μl) and filtering through a 0.45-μm filter, the supernatant was run through a 5-ml StrepTrap column, washed with PBS and eluted with 2.5 mM desthiobiotin in PBS. The eluted fractions containing absorbance peaks were analysed by SDS/PAGE to confirm the presence of the recombinant protein, combined together and the strep-tag was removed with the HRV Turbo protease (ABnova) at a ratio of 1:100 overnight at 4 °C and gentle shaking. After removal of the HRV, gel filtration was performed with a HiLoad 16/600 Superdex 75 pg column (Ge Healthcare) and the relevant peaks were verified by SDS/PAGE.
Expression of LpsGH11 in the Sf9 insect cells was performed using the baculovirus expression system  with a virus dilution of 1:1000. Once harvested, the cells were pelleted, resuspended in the lysis buffer I-PER insect cell protein extraction reagent (Thermo Fisher Scientific) with 5 mM MgCl2 and DNaseI (0.025 U/μl) and incubated on ice for 10 min. They were then pelleted and the supernatant was affinity purified with a pre-equilibrated HisTrap 5-ml column. Ni affinity chromatography was run with an elution gradient of 30 to 500 mM imidazole. Fractions were collected and analysed by SDS PAGE. Fractions containing the protein were pooled and concentrated to 2.5 ml and were run on a HiLoad 16/600 Superdex 75 pg column (Ge Healthcare) and the relevant peaks were verified by SDS/PAGE. The fractions containing the protein were concentrated to 1 ml and the tags were removed with the HRV Turbo protease (ABnova) at a ratio of 1:100 overnight at 4 °C and gentle shaking. Purification was carried out manually using a 5-ml HisTrap column and a gradient of 20–500 mM imidazole. The fractions were run by SDS-PAGE to confirm tag cleavage.
E. coli bacterial cells expressing the LpsAA10A were grown in M9 Minimal Medium, containing 1% (w/v) glucose and the appropriate antibiotics at 37 °C until OD600 = 0.7. The culture was induced with IPTG (0.1 mM) and grown overnight at 20 °C. Cells were harvested by centrifugation, resuspended in 50 ml of 50 mM Tris-HCl/20% sucrose (pH 8.0) for each litre of original culture and kept on ice for 30 min. After centrifugation at 8000 rpm for 10 min the supernatant was discarded, the cells were resuspended in 50 ml of 5 mM MgSO4 for each litre of original culture and kept on ice for 30 min. After centrifugation the supernatant, containing the periplasmic fraction, was equilibrated with 0.2 M Na phosphate buffer pH 7.6 to a final concentration of 50 mM, applied to a 5-ml StrepTrap HP column, washed with binding buffer and eluted with 2.5 mM desthiobiotin. 5-fold excess copper was added as CuSO4, then unbound copper and desthiobiotin were removed by passing the protein in a HiLoad TM 16/600 Superdex 75 gel filtration column (Ge Healthcare) equilibrated with 10 mM sodium phosphate buffer pH 7.0. The protein was then concentrated using Microsep TM Advance Centrifugal Devices (Pall Corporation).
Substrates used for the DNS assay, or PACE: barley β-glucan (β-D-1,3-1,4-glucan), mannan (borohydride reduced), konjac glucomannan (β-D-1,4), larch arabinogalactan, wheat arabinoxylan, tamarind seed xyloglucan, potato galactan and galactan (Gal:Ara:Rha:Xyl:GalUA = 91:2:1.7:0.3:5), are all purchased from Megazyme; locust bean gum (LBG), carboxymethyl-cellulose (CMC), microcrystalline cellulose (Avicel) and beech wood xylan are purchased from Sigma-Aldrich. Phosphoric acid swollen cellulose (PASC) was prepared as in . Grass xylan (miscanthus stem alcohol-insoluble residues) was prepared as described in .
DNS-reducing sugar assays
The activity of LpsGH5_8, LpsGH11, LpsGH134a and LpsGH134b was determined by dinitrosalicylic acid (DNS)-reducing sugar assay on a range of polysaccharides (see paragraph “Substrates”). The 50-μl reactions were carried out in triplicates in 50 mM sodium phosphate buffer pH 6.0, 0.1% substrate and 3 μg of protein (0.5 μg for LpsGH11). They were incubated at 30 °C for 2 h with shaking at 320 rpm and then 9 μl of the reaction was added to 31 μl of DNS reagent and heated at 100 °C for 5 min. After cooling at room temperature and addition of 160 μl water, the 540 nm absorbance was measured in a micro-plate reader and the results were compared to a glucose standard curve. The A540 of the substrates was subtracted from that of the samples. The DNS reagent was prepared by mixing 0.75 g of dinitrosalycilic acid, 1.4 g NaOH, 21.6 g sodium potassium tartrate tetrahydrate, 0.53 mL phenol and 0.59 g sodium metabisulfite in 100 ml of distilled water, and it was filtered and kept in the dark before used.
Product analysis by mass spectrometry (MS)
Reactions with the purified LpsAA10A were carried out by mixing 4 mg mL − 1 substrate with purified copper-loaded enzyme (2 μM) and 4 mM electron donor (gallic acid), in 50 mM ammonium acetate buffer pH 6 in 2-mL plastic reaction tubes (reaction volume: 100 μL). The tubes were incubated for 24 h at 28 °C shaking at 1000 rpm, centrifuged at 14,000 rpm and the supernatant was collected for analysis through mass spectrometry. Briefly, 1 μl of supernatant was mixed with an equal volume of matrix solution (20 mg mL− 1 2,5-dihydroxybenzoic acid (DHB) in 50% acetonitrile plus 0.1% TFA), spotted on a SCOUT-MTP 384 target plate (Bruker) and analysed by positive-mode MALDI-TOF MS using an Ultraflex III matrix-assisted laser desorption ionisation-time of flight/time of flight (MALDI/TOF-TOF) instrument (Bruker).
Polysaccharide analysis by carbohydrate gel electrophoresis (PACE)
Purified enzyme at 20 μg/ml was mixed with 0.5% galactan, glucomannan, galactomannan, mannan or locust bean gum (LBG) or with 40 mg/ml of milled Scots pine wood (pre-treated in 0.5 N NaOH for 30 min at 90 °C and rinsed 5 times in 50 mM NaPO4 buffer) in 50 mM NaPO4 buffer pH 6.5 and incubated overnight at 30 °C with shaking. The samples were then centrifuged, supernatant was transferred to a new tube and undigested polysaccharides were removed by precipitation with 80% ethanol. Following centrifugation, supernatants were transferred to a new tube and dried.
Miscanthus stem AIR (alcohol-insoluble residues) was pre-treated in 4 M NaOH for 1 h at RT and neutralised with HCl. Resultant substrate at 1 mg/ml (of initial untreated AIR) was digested overnight at RT with various amounts of xylanase (3–40 μg/ml). All samples were purified on Nanosep 10 K and dried. Dried digestion products and manno-oligosaccharide and xylo-oligosaccharide standards and appropriate controls were labelled with 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS; Invitrogen, www.invitrogen.com) and separated by polyacrylamide gels, as described previously . PACE gels were visualised using a G-box (Syngene, www.syngene.com/). Experiments were carried out in triplicate, and the representative gels are shown.