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

BACKGROUND: 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. RESULTS: Through a combination of transcriptomics, proteomics, X-ray microtomography, electron microscopy studies and in vitro biochemical characterisation, we show that wood-digesting enzymes produced by symbiotic bacteria are localised not only in the gills, but also in the lumen of the food groove, a stream of mucus secreted by gill cells that carries food particles trapped by filter feeding to the mouth. Bacterial CAZymes are also present in the crystalline style and in the caecum of their shipworm host, suggesting a unique pathway by which enzymes involved in a symbiotic interaction are transported to their site of action. Finally, we characterise in vitro four new bacterial glycosyl hydrolases and a lytic polysaccharide monooxygenase identified in our transcriptomic and proteomic analyses as some of the major bacterial enzymes involved in this unusual biological system. CONCLUSION: Based on our data, we propose that bacteria and their enzymes are transported from the gills along the food groove to the shipworm's mouth and digestive tract, where they aid in wood digestion.

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

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.

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

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.

Diversity, environmental requirements, and biogeography of bivalve wood-borers (Teredinidae) in European coastal waters.

BACKGROUND: Bivalve teredinids inflict great destruction to wooden maritime structures. Yet no comprehensive study was ever carried out on these organisms in European coastal waters. Thus, the aims of this study were to: investigate the diversity of teredinids in European coastal waters; map their past and recent distributions to detect range expansion or contraction; determine salinity-temperature (S-T) requirements of species; flag, for future monitoring, the species that pose the greatest hazard for wooden structures. RESULTS: A total of nine teredinid species were found established in European coastal waters. Seven were considered cryptogenic, of unknown origin, and two were considered alien species. Teredo navalis and Nototeredo norvagica were the species with the widest distribution in European waters. Recently, T. navalis has been reported occurring further east in the Baltic Sea but it was not found at a number of sites on the Atlantic coast of southern Europe. The Atlantic lineage of Lyrodus pedicellatus was the dominant teredinid in the southern Atlantic coast of Europe. In the Mediterranean six teredinid species occurred in sympatry, whereas only three of these occurred in the Black Sea. The species that pose the greatest hazard to wooden maritime structures in European coastal areas are T. navalis and the two lineages of L. pedicellatus. CONCLUSIONS: Combined data from field surveys and from the literature made it possible to determine the diversity of established teredinid species and their past and recent distribution in Europe. The environmental requirements of species, determined using climatic envelopes, produced valuable information that assisted on the explanation of species distribution. In addition, the observed trends of species range extension or contraction in Teredo navalis and in the two lineages of Lyrodus pedicellatus seem to emphasise the importance of temperature and salinity as determinants of the distribution of teredinids, whereas their life history strategy seems to play an important role on competition.Teredo navalis and pedicellatus-like Lyrodus species should be monitored due to their destructive capability. The two alien species may expand further their distribution range in Europe, becoming invasive, and should also be monitored.

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

BACKGROUND: 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. RESULTS: Five major muscle systems were identified: a velum retractor, foot retractor, larval retractor, a distinct mantle musculature and an adductor system. For a short period of larval life, an additional ventral larval retractor is present. Early in development, a velum muscle ring and an oral velum musculature emerge. In late stages the lateral and dorsal mantle musculature, paired finger-shaped muscles, an accessory adductor and a pedal plexus are formed. Similar to other bivalve larvae, L. pedicellatus exhibits three velum retractor muscles, but in contrast to other species, one of them disappears in early stages of L. pedicellatus. The remaining two velum retractors are considerably remodelled during late larval development and are most likely incorporated into the elaborate mantle musculature of the adult. CONCLUSIONS: To our knowledge, this is the first account of any larval retractor system that might contribute to the adult bodyplan of a (conchiferan) mollusk. A comparative analysis shows that a pedal plexus, adductors, a larval velum ring, velum retractors and a ventral larval retractor are commonly found among bivalve larvae, and thus most likely belong to the ground pattern of the bivalve larval musculature.

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

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.

Accumulation and exposure classifications of plastics in the different coastal habitats in the western Philippine archipelago.

Studies consistently ranked the Philippines as one of the top contributors of plastic wastes leaking into the ocean. However, most of these were based on probabilities and estimates due to lack of comprehensive ground-truth data, resulting also in the limited understanding of the contributing factors and drivers of local pollution. This makes it challenging to develop science-driven and locally-contextualized policies and interventions to mitigate the problem. Here, 56 sites from different coastal habitats in the western Philippine archipelago were surveyed for macroplastics standing stock, representing geographic regions with varying demography and economic activities. Clustering of sites revealed three potential influencing factors to plastic accumulation: population density, wind and oceanic transport, and habitat type. Notably, the amount and types of dominant plastics per geographic region varied significantly. Single-use plastics (food packaging and sachets) were the most abundant in sites adjacent to densely populated and highly urbanized areas (Manila Bay and eastern Palawan), while fishing-related materials dominated in less populated and fishing-dominated communities (western Palawan and Bolinao), suggesting the local industries significantly contributing to the mismanaged plastics in the surveyed sites. Meanwhile, isolated areas such as islands were characterized by the abundance of buoyant materials (drinking bottles and hygiene product containers), emphasizing the role of oceanic transport and strong connectivity in the oceans. Exposure assessment also identified single-use and fishing-related plastics to be of "high exposure (Type 4)" due to their high abundance and high occurrence. These increase their chances of encountering and interacting with organisms and habitats, thus, resulting into more potential harm. This study is the first comprehensive work done in western Philippines, and results will help contextualize local pollution, facilitating more effective management and policymaking.

Microplastics leaving a trace in mangrove sediments ever since they were first manufactured: A study from Indonesia mangroves.

Mangrove environments have been well recognized as marine litter traps. However, it is unclear whether mangrove sediments sink microplastics more effectively than other marine sediments due to active sedimentation. Furthermore, microplastics archives in mangrove sediments may provide quantitative data on the impact of human activities on environmental pollution throughout history. Microplastic abundance varied markedly between high and low anthropogenic activities. Both mangrove and adjacent mudflats sediments act as microplastic sequesters, despite having similar microplastic abundances and depth profiles. The decreasing trend of microplastics was observed until the sediment layers dated to the first-time plastic was manufactured in Indonesia, in the early 1950s, but microplastics remained present beneath those layers, indicating the downward movements. This discovery highlighted the significance of mangrove sediments as microplastic sinks. More research is needed to understand the mechanisms of microplastic deposition in sediments, as well as their fate and potential impact on mangrove sediment dwellers.

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 year. As treatments of wood with broad spectrum biocides, such as creosote and chromated copper arsenate (CCA), are now restricted in marine use by legislation, naturally durable timber species and novel preservation methods of wood are required. These methods undergo testing in order to meet regulatory standards, such as the European standard for testing wood preservatives against marine borers, EN 275. Initial investigation of durable timbers species or wood preservative treatments can be achieved quickly and inexpensively through laboratory testing, which offers many advantages over marine field trials that are typically costly, long-term endeavours. Many species of Limnoria (gribble) are marine wood-boring crustaceans. Limnoria are ideal for use in laboratory testing of biodegradation of wood by marine wood-borers, due to the practicality of rearing them in aquaria and the ease of measuring their feeding rates on wood. Herein, we outline a standardizable laboratory test for assessing wood biodegradation using gribble.

Biomimetic generation of the strongest known biomaterial found in limpet tooth.

The biomaterial with the highest known tensile strength is a unique composite of chitin and goethite (α-FeO(OH)) present in teeth from the Common Limpet (Patella vulgata). A biomimetic based on limpet tooth, with corresponding high-performance mechanical properties is highly desirable. Here we report on the replication of limpet tooth developmental processes ex vivo, where isolated limpet tissue and cells in culture generate new biomimetic structures. Transcriptomic analysis of each developmental stage of the radula, the organ from which limpet teeth originate, identifies sequential changes in expression of genes related to chitin and iron processing. We quantify iron and chitin metabolic processes in the radula and grow isolated radula cells in vitro. Bioinspired material can be developed with electrospun chitin mineralised by conditioned media from cultured radula cells. Our results inform molecular processes behind the generation of limpet tooth and establish a platform for development of a novel biomimetic with comparable properties.

Seasonal heterogeneity and a link to precipitation in the release of microplastic during COVID-19 outbreak from the Greater Jakarta area to Jakarta Bay, Indonesia.

To reduce microplastic contamination in the environment, we need to better understand its sources and transit, especially from land to sea. This study examines microplastic contamination in Jakarta's nine river outlets. Microplastics were found in all sampling intervals and areas, ranging from 4.29 to 23.49 particles m-3. The trend of microplastic contamination tends to increase as the anthropogenic activity towards Jakarta Bay from the eastern side of the bay. Our study found a link between rainfall and the abundance of microplastic particles in all river outlets studied. This investigation found polyethylene, polystyrene, and polypropylene in large proportion due to their widespread use in normal daily life and industrial applications. Our research observed an increase in microplastic fibers made of polypropylene over time. We suspect a relationship between COVID-19 PPE waste and microplastic shift in our study area. More research is needed to establish how and where microplastics enter rivers.

Priorities to inform research on marine plastic pollution in Southeast Asia.

Southeast Asia is considered to have some of the highest levels of marine plastic pollution in the world. It is therefore vitally important to increase our understanding of the impacts and risks of plastic pollution to marine ecosystems and the essential services they provide to support the development of mitigation measures in the region. An interdisciplinary, international network of experts (Australia, Indonesia, Ireland, Malaysia, the Philippines, Singapore, Thailand, the United Kingdom, and Vietnam) set a research agenda for marine plastic pollution in the region, synthesizing current knowledge and highlighting areas for further research in Southeast Asia. Using an inductive method, 21 research questions emerged under five non-predefined key themes, grouping them according to which: (1) characterise marine plastic pollution in Southeast Asia; (2) explore its movement and fate across the region; (3) describe the biological and chemical modifications marine plastic pollution undergoes; (4) detail its environmental, social, and economic impacts; and, finally, (5) target regional policies and possible solutions. Questions relating to these research priority areas highlight the importance of better understanding the fate of marine plastic pollution, its degradation, and the impacts and risks it can generate across communities and different ecosystem services. Knowledge of these aspects will help support actions which currently suffer from transboundary problems, lack of responsibility, and inaction to tackle the issue from its point source in the region. Being profoundly affected by marine plastic pollution, Southeast Asian countries provide an opportunity to test the effectiveness of innovative and socially inclusive changes in marine plastic governance, as well as both high and low-tech solutions, which can offer insights and actionable models to the rest of the world.

Distinguishing ten sympatric species of fiddler crab (Decapoda: Ocypodidae) using a suite of phenotypic characteristics.

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).

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

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.

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

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.

Uncovering the molecular mechanisms of lignocellulose digestion in shipworms.

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 Lyrodus pedicellatus 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 ß-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.

Hemocyanin facilitates lignocellulose digestion by wood-boring marine crustaceans.

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.

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

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.

Lignocellulose degradation mechanisms across the Tree of Life.

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.