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Additional file 12: Fig. S7. Characterisation of the recombinant bacterial CAZymes encoded by the endosymbionts. DNS reducing sugars assays showing activities on a number of substrates for LpsGH5_8, LpsGH11, LpsGH134a and LpsGH134b. CMC = carboxymethyl cellulose, LBG = locust bean gum. The nanomoles of sugars released by the different proteins cannot be compared quantitatively as different amounts were used for the assay. File format .DOCX.

Additional file 8: Table S2. CAZy families. List of the CAZy families mentioned in the results, with the description of the activities that have been recorded for enzymes listed in each family. The information has been gathered from the CAZy database ( http://www.cazy.org ). File format .DOCX.

Additional file 2: Fig. S2. Searching for the opening of the duct of Deshayes in the shipworm digestive system. Left, transverse sections through the shell valve, from the anterior to posterior adductor muscle (A-E). Right, 3D rendered model of the whole shipworm, with the green highlighted region showing the position of the transverse cross section displayed on the left. File format .DOCX.

Additional file 6: Fig. S6. Immunogold labelling of LpsGH5_8 in L. pedicellatus using pre-immune serum (negative control). A) SEM image illustrating the anatomical position of the gills, food groove, caecum and mouth in the shipworm body. B-D) Immunogold labelling of the lumen of the food groove (B), lumen of the caecum (C), and the gills (D) performed with pre-immune serum. No gold particles are observed in any of the samples. B = bacteria. File format .DOCX.

Additional file 7: Table S1. Details of the proteins identified through proteomic analysis of the crystalline style. The annotation was performed against the NCBI non-redundant database, the signal peptides were identified with the Signal P4.1 server and the CAZy domains were searched using dbCAN. Many of the contigs were out of frame or not full length (particularly the bacterial ones) and therefore the identification of the signal peptide was not possible. File format .DOCX.

Additional file 4: Fig. S4. Bacterial symbionts in L. pedicellatus. A) TEM image of a gill bacteriocyte. Arrows indicate the numerous rod-shaped bacteria in the cell. B) Close-up view of gill bacteria (TEM). C) Detailed TEM showing a cross section of a gill bacterium and some of its features (periplasmic space and membranes). File format .DOCX.

Additional file 5: Fig. S5. SDS-PAGE and nitrocellulose western blot obtained by loading 0.6 ��g of the recombinant purified bacterial LpsGH5_8 (without the appended CBM). A) Coomassie Brilliant Blue stained SDS-PAGE gel of the purified protein. B) Western blot detection performed with the purified pre-immune serum. C) Western blot detection performed with the purified anti-protein serum. File format .DOCX.

Additional file 3: Fig. S3. Scanning electron microscopy of L. pedicellatus. A dissected specimen with the mantle removed, showing the food groove connecting the gills to the mouth. File format .DOCX.

Additional file 9: Table S3. CAZy families of the caecum. Table showing the relative abundance (calculated from the emPAI score) of eukaryotic and prokaryotic CAZy families identified in the proteomic analysis of the caecum content of L. pedicellatus. File format .DOCX.

Woody (lignocellulosic) plant biomass is an abundant renewable feedstock, rich in polysaccharides that are bound into an insoluble fiber composite with lignin. Marine crustacean woodborers of the genus Limnoria are among the few animals that can survive on a diet of this recalcitrant material without relying on gut resident microbiota. Analysis of fecal pellets revealed that Limnoria targets hexose-containing polysaccharides (mainly cellulose, and also glucomannans), corresponding with the abundance of cellulases in their digestive system, but xylans and lignin are largely unconsumed. We show that the limnoriid respiratory protein, hemocyanin, is abundant in the hindgut where wood is digested, that incubation of wood with hemocyanin markedly enhances its digestibility by cellulases, and that it modifies lignin. We propose that this activity of hemocyanins is instrumental to the ability of Limnoria to feed on wood in the absence of gut symbionts. These findings may hold potential for innovations in lignocellulose biorefining., Marine woodborers can digest woody biomass without the help of gut microbiota but the mechanism has remained unclear. Here, the authors provide evidence that the woodborer’s respiratory protein hemocyanin plays a central role in wood digestion and may offer a route toward biorefining of woody plant biomass.

Thermobia domestica belongs to an ancient group of insects and has a remarkable ability to digest crystalline cellulose without microbial assistance. By investigating the digestive proteome of Thermobia, we have identified over 20 members of an uncharacterized family of lytic polysaccharide monooxygenases (LPMOs). We show that this LPMO family spans across several clades of the Tree of Life, is of ancient origin, and was recruited by early arthropods with possible roles in remodeling endogenous chitin scaffolds during development and metamorphosis. Based on our in-depth characterization of Thermobia’s LPMOs, we propose that diversification of these enzymes toward cellulose digestion might have endowed ancestral insects with an effective biochemical apparatus for biomass degradation, allowing the early colonization of land during the Paleozoic Era. The vital role of LPMOs in modern agricultural pests and disease vectors offers new opportunities to help tackle global challenges in food security and the control of infectious diseases., LPMOs catalyze the oxidative breakdown of polysaccharides, thereby facilitating biomass degradation. By analyzing the digestive proteome of firebrats, the authors here identify a yet uncharacterized LPMO family and provide phylogenetic, structural and biochemical insights into its origin and functions.

Additional file 1. Additional tables, figures, and additional text.