rapid-testing-resistance-timber-to-biodegradation-marine-wood-boring

Rapid Testing of Resistance of Timber to Biodegradation by Marine Wood-Boring Crustaceans

Wood-boring invertebrates rapidly destroy marine timbers and wooden coastal infrastructure, causing billions of dollars of damage around the globe every ...

The digestion of lignocellulose is attracting attention both in terms of basic research into its metabolism by microorganisms and animals, and also as a means of converting plant biomass into biofuels. Limnoriid wood borers are unusual because, unlike other wood-feeding animals, they do not rely on symbiotic microbes to help digest lignocellulose. The absence of microbes in the digestive tract suggests that limnoriid wood borers produce all the enzymes necessary for lignocellulose digestion themselves. In this study we report that analysis of ESTs from the digestive system of Limnoria quadripunctata reveals a transcriptome dominated by glycosyl hydrolase genes. Indeed, > 20% of all ESTs represent genes encoding putative cellulases, including glycosyl hydrolase family 7 (GH7) cellobiohydrolases. These have not previously been reported in animal genomes, but are key digestive enzymes produced by wood-degrading fungi and symbiotic protists in termite guts. We propose that limnoriid GH7 genes are important for the efficient digestion of lignocellulose in the absence of gut microbes. Hemocyanin transcripts were highly abundant in the hepatopancreas transcriptome. Based on recent studies indicating that these proteins may function as phenoloxidases in isopods, we discuss a possible role for hemocyanins in lignin decomposition.

Molecular insight into lignocellulose digestion by a marine isopod in the absence of gut microbes.

Die Verdauung von Lignocellulose zieht Aufmerksamkeit in Bezug auf die Grundlagenforschung in den Stoffwechsel von Mikroorganismen und Tiere, und auch als Mittel der Umwandlung von pflanzlichen Biomasse in Biokraftstoffe. Limnoriid Holz Bohrer sind ungew&ouml;hnlich, da im Gegensatz zu anderen Tieren Holz-f&uuml;ttern sie nicht auf symbiotische Mikroben Digest Lignocellulose helfen angewiesen sind. Die Mikroben im Verdauungstrakt nahe, dass Limnoriid Holz Bohrer alle f&uuml;r Lignocellulose Verdauung notwendigen Enzyme selber herstellen. In dieser Studie berichten wir in dieser Analyse von Esten aus dem Magen-Darm-System der Limnoria Quadripunctata zeigt eine Transkriptom Glycosyl-Hydrolase-gen dominiert. Tats&auml;chlich &gt; 20 % von allen Esten stellen Gene Kodierung vermeintliche Cellulasen, einschlie&szlig;lich Glycosyl-Hydrolase-Familie 7 (GH7) Cellobiohydrolases. Diese wurden nicht zuvor in tierischen Genome gemeldet, aber wichtige Verdauungsenzyme produziert von Holz-erniedrigende Pilze und symbiotische Einzellern in Termite Mut. Wir schlagen vor, Limnoriid GH7 Gene wichtig f&uuml;r die effiziente Verdauung von Lignocellulose in Ermangelung der Darm-Mikroben sind. H&auml;mocyanin Abschriften waren sehr reichlich in der Hepatopankreas Transkriptom. Basierend auf aktuelle Studien, die darauf hinweist, dass diese Proteine als Phenoloxidases in Wasserasseln fungieren k&ouml;nnen, diskutieren wir eine m&ouml;gliche Rolle f&uuml;r Hemocyanins in Lignin Aufspaltung.

Molekulare Einblick in Lignocellulose Verdauung durch eine marine Kroatien in Ermangelung der Darm-Mikroben.

Nature uses a diversity of glycoside hydrolase (GH) enzymes to convert polysaccharides to sugars. As lignocellulosic biomass deconstruction for biofuel production remains costly, natural GH diversity offers a starting point for developing industrial enzymes, and fungal GH family 7 (GH7) cellobiohydrolases, in particular, provide significant hydrolytic potential in industrial mixtures. Recently, GH7 enzymes have been found in other kingdoms of life besides fungi, including in animals and protists. Here, we describe the in vivo spatial expression distribution, properties, and structure of a unique endogenous GH7 cellulase from an animal, the marine wood borer Limnoria quadripunctata (LqCel7B). RT-quantitative PCR and Western blot studies show that LqCel7B is expressed in the hepatopancreas and secreted into the gut for wood degradation. We produced recombinant LqCel7B, with which we demonstrate that LqCel7B is a cellobiohydrolase and obtained four high-resolution crystal structures. Based on a crystallographic and computational comparison of LqCel7B to the well-characterized Hypocrea jecorina GH7 cellobiohydrolase, LqCel7B exhibits an extended substrate-binding motif at the tunnel entrance, which may aid in substrate acquisition and processivity. Interestingly, LqCel7B exhibits striking surface charges relative to fungal GH7 enzymes, which likely results from evolution in marine environments. We demonstrate that LqCel7B stability and activity remain unchanged, or increase at high salt concentration, and that the L. quadripunctata GH mixture generally contains cellulolytic enzymes with highly acidic surface charge compared with enzymes derived from terrestrial microbes. Overall, this study suggests that marine cellulases offer significant potential for utilization in high-solids industrial biomass conversion processes.

Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance.

Abstract The complete mitochondrial genome of Limnoria quadripunctata, a marine wood-eating isopod crustacean, was determined from whole genome sequence data. The mitogenome is 16,503 bp in length and contains 39 genes: 13 protein-coding, 2 ribosomal RNA, 22 tRNA, two of which are repeated and a control region. The start codon most commonly used by the Limnoria protein-coding genes is ATN, as is the case in the two other available complete isopod mitogenomes. The gene arrangement differs among these complete isopod mitogenomes, as does the AT-content of H-strand protein-coding genes. The latter observations, coupled with the considerable nucleotide diversity observed between the isopod mitogenomes, support the idea that each isopod species belongs to a distinct lineage as implied by their current placement in separate suborders.

The complete mitochondrial genome of Limnoria quadripunctata Holthuis (Isopoda: Limnoriidae).

The shipworm Lyrodus pedicellatus is a wood-boring bivalve with an unusual vermiform body. Although its larvae are brooded, they retain the general appearance of a typical bivalve veliger-type larva. Here, we describe myogenesis of L. pedicellatus revealed by filamentous actin labelling and discuss the data in a comparative framework in order to test for homologous structures that might be part of the bivalve (larval) muscular ground pattern.

Developmental dynamics of myogenesis in the shipworm Lyrodus pedicellatus (Mollusca: Bivalvia).

Uca cryptica Naderloo, Türkay & Chen, 2010, was originally described from four male specimens found in museum collections without any information regarding the site of collection. We present the first recorded field observations of this species and new morphological features. Specimens were observed and collected in the Wakatobi National Park, on the island of Kaledupa, Sulawesi Tenggara, Indonesia. Colouration of both males and females is described and ecology and distribution are noted. Uca cryptica has been seen coexisting with nine other species; one of the highest recorded numbers of Uca species living in sympatry. 

Uca cryptica Naderloo, Türkay & Chen, 2010 (Crustacea: Brachyura:<br />Ocypodidae) is no longer cryptic.

Organisms use diverse mechanisms involving multiple complementary enzymes, particularly glycoside hydrolases (GHs), to deconstruct lignocellulose. Lytic polysaccharide monooxygenases (LPMOs) produced by bacteria and fungi facilitate deconstruction as does the Fenton chemistry of brown-rot fungi. Lignin depolymerisation is achieved by white-rot fungi and certain bacteria, using peroxidases and laccases. Meta-omics is now revealing the complexity of prokaryotic degradative activity in lignocellulose-rich environments. Protists from termite guts and some oomycetes produce multiple lignocellulolytic enzymes. Lignocellulose-consuming animals secrete some GHs, but most harbour a diverse enzyme-secreting gut microflora in a mutualism that is particularly complex in termites. Shipworms however, house GH-secreting and LPMO-secreting bacteria separate from the site of digestion and the isopod Limnoria relies on endogenous enzymes alone. The omics revolution is identifying many novel enzymes and paradigms for biomass deconstruction, but more emphasis on function is required, particularly for enzyme cocktails, in which LPMOs may play an important role. 

Lignocellulose degradation mechanisms across the Tree of Life.

Lignocellulose forms the structural framework of woody plant biomass and represents the most abundant carbon source in the biosphere. Turnover of woody biomass is a critical component of the global carbon cycle, and the enzymes involved are of increasing industrial importance as industry moves away from fossil fuels to renewable carbon resources. Shipworms are marine bivalve molluscs that digest wood and play a key role in global carbon cycling by processing plant biomass in the oceans. Previous studies suggest that wood digestion in shipworms is dominated by enzymes produced by endosymbiotic bacteria found in the animal's gills, while little is known about the identity and function of endogenous enzymes produced by shipworms. Using a combination of meta-transcriptomic, proteomic, imaging and biochemical analyses, we reveal a complex digestive system dominated by uncharacterized enzymes that are secreted by a specialized digestive gland and that accumulate in the cecum, where wood digestion occurs. Using a combination of transcriptomics, proteomics, and microscopy, we show that the digestive proteome of the shipworm  is mostly composed of enzymes produced by the animal itself, with a small but significant contribution from symbiotic bacteria. The digestive proteome is dominated by a novel 300 kDa multi-domain glycoside hydrolase that functions in the hydrolysis of β-1,4-glucans, the most abundant polymers in wood. These studies allow an unprecedented level of insight into an unusual and ecologically important process for wood recycling in the marine environment, and open up new biotechnological opportunities in the mobilization of sugars from lignocellulosic biomass.

Uncovering the molecular mechanisms of lignocellulose digestion in shipworms.

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.

Hemocyanin facilitates lignocellulose digestion by wood-boring marine crustaceans.

More than two-thirds of global biomass consists of vascular plants. A portion of the detritus they generate is carried into the oceans from land and highly productive blue carbon ecosystems-salt marshes, mangrove forests, and seagrass meadows. This large detrital input receives scant attention in current models of the global carbon cycle, though for blue carbon ecosystems, increasingly well-constrained estimates of biomass, productivity, and carbon fluxes, reviewed in this article, are now available. We show that the fate of this detritus differs markedly from that of strictly marine origin, because the former contains lignocellulose-an energy-rich polymer complex of cellulose, hemicelluloses, and lignin that is resistant to enzymatic breakdown. This complex can be depolymerized for nutritional purposes by specialized marine prokaryotes, fungi, protists, and invertebrates using enzymes such as glycoside hydrolases and lytic polysaccharide monooxygenases to release sugar monomers. The lignin component, however, is less readily depolymerized, and detritus therefore becomes lignin enriched, particularly in anoxic sediments, and forms a major carbon sink in blue carbon ecosystems. Eventual lignin breakdown releases a wide variety of small molecules that may contribute significantly to the oceanic pool of recalcitrant dissolved organic carbon. Marine carbon fluxes and sinks dependent on lignocellulosic detritus are important ecosystem services that are vulnerable to human interventions. These services must be considered when protecting blue carbon ecosystems and planning initiatives aimed at mitigating anthropogenic carbon emissions.

Vascular Plants Are Globally Significant Contributors to Marine Carbon Fluxes and Sinks.

Mangroves are located at the land-sea interface and are therefore confronted with human settlement in the coastal areas and associated pressures and uses. This unique habitat provides important ecosystem services to coastal communities worldwide, but the global decline of their surface area and their degradation over the past decades has put coastal communities even more at risk from the effects of climate change. This paper aims to present the first ecosystem services valuation of the mangroves of the French overseas Territories. We provide the economic value of mangroves for coastal protection, carbon sequestration, water purification and fish biomass production. We coupled a geospatial analysis of mangrove's distribution with the characterisation of land artificialisation behind mangroves. Then we developed a vulnerability index based on multiple indicators of exposure to environmental and anthropogenic stressors, mangroves' sensitivity to pressures, and mangroves' adaptive capacity to adjust their production functions accordingly. We estimated the monetary value of regulation and support services provided by mangroves in French overseas territories to be on average EUR 1.6 billion annually, 60% of which is carbon sequestration, 28% coastal protection, 7% water purification and 6% fish biomass production. When considering mangroves services without the vulnerability adjustment, the total value for those services would reach EUR 2 billion per year. Although much of the spatio-temporal variability in mangrove functioning could not be considered given the spatial scale of our study, these results demonstrate the value and socio-economic importance of mangroves to face and adapt from the effects of coastal change, at local and national scales, but also highlight the loss of services due to their vulnerability. This paper emphasises on the value of ecosystem services provided by mangroves to face coastal change so that a service-based approach to conservation would plead for increased national investment into their protection.

Mangrove ecological services at the forefront of coastal change in the French overseas territories.

Shipworms are marine xylophagus bivalve molluscs, which can live on a diet solely of wood due to their ability to produce plant cell wall-degrading enzymes. Bacterial carbohydrate-active enzymes (CAZymes), synthesised by endosymbionts living in specialised shipworm cells called bacteriocytes and located in the animal's gills, play an important role in wood digestion in shipworms. However, the main site of lignocellulose digestion within these wood-boring molluscs, which contains both endogenous lignocellulolytic enzymes and prokaryotic enzymes, is the caecum, and the mechanism by which bacterial enzymes reach the distant caecum lumen has remained so far mysterious. Here, we provide a characterisation of the path through which bacterial CAZymes produced in the gills of the shipworm Lyrodus pedicellatus reach the distant caecum to contribute to the digestion of wood.

Characterisation of the enzyme transport path between shipworms and their bacterial symbionts.

Ten species of fiddler crab are reported inhabiting the intertidal zone of a shore on Kaledupa Island, Indonesia. This is one of the highest recorded numbers of fiddler crab species living in sympatry, equating to over two-thirds of those known from the Wallacea biogeographic region and more than half of all those recorded from Indonesia. The descriptions to identify and distinguish these ten species are provided using a suite of characters e.g., carapace, major cheliped, male gonopods, gastric mills, life colouration in males and females, and notes on their ecology and distribution. Specimens were observed and collected in the Wakatobi National Park, near the village of Ambeua on Kaledupa island, Sulawesi Tenggara, Indonesia. Gastric mills are described for the first time for Gelasimus jocelynae, Paraleptuca crassipes, Tubuca coarctata, T. demani and T. dussumieri. A tabulation of anatomical features and colouration for all species in this study is provided as a support for field studies. It identifies features that support the recently proposed taxonomic revision of fiddler crabs by Shih et al. (2016).

Article	Year
Molecular insight into lignocellulose digestion by a marine isopod in the absence of gut microbes. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 20212162	2010
Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 23733951	2013
The complete mitochondrial genome of Limnoria quadripunctata Holthuis (Isopoda: Limnoriidae). Mitochondrial DNA\| PubMed ID: 24409843	2014
Developmental dynamics of myogenesis in the shipworm Lyrodus pedicellatus (Mollusca: Bivalvia). Frontiers in zoology\| PubMed ID: 25558273	2014
Uca cryptica Naderloo, Türkay & Chen, 2010 (Crustacea: Brachyura:<br />Ocypodidae) is no longer cryptic. Zootaxa\| PubMed ID: 26249996	2015
Lignocellulose degradation mechanisms across the Tree of Life. Current opinion in chemical biology\| PubMed ID: 26583519	2015
Uncovering the molecular mechanisms of lignocellulose digestion in shipworms. Biotechnology for biofuels\| PubMed ID: 29527236	2018
Hemocyanin facilitates lignocellulose digestion by wood-boring marine crustaceans. Nature communications\| PubMed ID: 30510200	2018
Vascular Plants Are Globally Significant Contributors to Marine Carbon Fluxes and Sinks. Annual review of marine science\| PubMed ID: 31505131	2020
Mangrove ecological services at the forefront of coastal change in the French overseas territories. The Science of the total environment\| PubMed ID: 33158516	2021
Characterisation of the enzyme transport path between shipworms and their bacterial symbionts. BMC biology\| PubMed ID: 34724941	2021
Distinguishing ten sympatric species of fiddler crab (Decapoda: Ocypodidae) using a suite of phenotypic characteristics. Zootaxa\| PubMed ID: 34810920	2021

Simon M. Cragg

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