Simon M. Cragg

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.

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.

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.

Swimming behaviour of the larvae of Pecten maximus (L.) (Bivalvia)

Laboratory observations indicate that larvae of Pecten maximus (L.) pass through three phases of swimming behaviour before metamorphosis. Trochophores and early veligers generally swim upwards and, in shallow containers, tend to accumulate at the surface. Their swimming cilia beat without interruption so swimming is continuous. After 3 days of development, pauses in ciliary beating occur intermittently and the veliger becomes capable of withdrawing into its shell. It alternately swims up a vertically orientated spiral and sinks. This type of swimming behaviour is also displayed by the pediveliger, but this larva also periodically swims close to, or crawls on, the substratum. The tendency to accumulate at the surface becomes less pronounced as larvae grow older. By the pediveliger stage it is replaced by an increasing tendency to accumulate close to the substratum.

Developments in the understanding of the biology of marine wood boring crustaceans and in methods of controlling them.

The most economically important wood boring Crustacea belong to the isopod families Sphaeromatidae and Limnoriidae, both of which have been recently revised and a number of new species have been recognised. Other wood boring crustaceans have now been recognised from tropical mangrove sites. Limnoriids are found from temperate to tropical waters, but appear to be restricted to waters with salinities close to that of seawater. Wood-boring species of Sphaeroma on the other hand can tolerate extremely low salinities, but are restricted to sub-tropical and tropical waters. Approaches to borer control that have proved effective against teredinids (use of naturally durable timber, copper-chrome-arsenic or creosote treatment, surface coatings) have been found under certain circumstances to be ineffective against Limnoria and Sphaeroma. A number of additives to conventional preservatives have been tested, with some insecticides showing evidence of enhancing Limnoria control. The question of crustacean borer nutrition may hold the key to problems of their control. Sphaeromatid borers are capable of filter-feeding and thus may never ingest the treatments applied to wood. Limnoriids do ingest wood, but the role of wood degrading tunneling bacteria, and soft-rotting ascomycete and deuteromycete fungi occurring in the wood they digest remains to be fully elucidated. The source or sources of wood-degrading enzymes that permit digestion of wood particles requires further investigation. The microecology of borer burrows has an important bearing on the availability of nitrogen for borers. Further insights into the problems posed by these borers may be obtained with a better understanding of their ecology. A better testing protocol for preservatives has been developed as a result of knowledge of the natural vertical distribution of Sphaeroma. Behavioural studies indicate that settlement on wood by Limnoria is enhanced by factors derived from conspecifics and from wood-inhabiting microorganisms.

The ultrastructure of the statocysts in the pediveliger larvae of Pecten maximus (L.)(Bivalvia)

The pediveliger of Pecten maximus (L.) has a pair of statocysts situated at the base of the foot on either side of a bilobed pedal ganglion. The statocysts consist of a spherical sac connected to the mantle cavity by a cylindrical ciliated canal. Within the sac there are statoconia which are variable both in shape and structure. The cells of the sac are joined by septate desmosomes. There is a non-ciliated cell in each sac containing a variety of granules some of which resemble certain of the statoconia. The remainder of the sac is composed of hair cells, which bear a circular array of radiating cilia. The basal bodies and horizontal striated roots of these cilia are directed radially. The hair cells give rise to thin processes which probably join together to form the static nerve. This nerve runs from the static canal to the pleural ganglion.

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

BackgroundBivalve 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.ResultsA 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.ConclusionsCombined 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.

Laboratory screening of tropical hardwoods for natural resistance to the marine borer Limnoria quadripunctata: The role of leachable and non-leachable factors

Abstract The marine borer Limnoria ingests essential wood components including the extractives the wood contains. Some extractives may confer borer resistance on certain timbers. Feeding by Limnoria correlates with the rate of production of faecal pellets. The faecal pellet production rate and mortality on over 40 test timbers and non-resistant Pinus sylvestris sapwood was measured over 15 days. By placing animals in leachate from wood and with wood in flowing seawater, the effects of leaching-resistant and water-soluble compounds were measured. Some previously untested timbers affected Limnoria as strongly as timbers reputed for durability in marine construction. Wood of Minquartia guianensis, Nectandra rubra and Bruguiera gymnorhiza caused high mortality, and pellet production on them was less than 10% of production on P. sylvestris. Suppressed feeding rates, but with no heavy mortality, were observed on known durable species such as Chlorocardium rodiei, Dicorynia guianensis, Lophira alata and Nauclea trillesii, but also on Cynometra ananta, Distemonanthus benthamianus, Enterolobium schomburgkii, Goupia glabra, Hymenaea courabil, Mammea africana, Shorea sp. and Sacoglottis guianensis. Leachate from B. gymnorhiza, G. glabra, H. coubaril, N. rubra and Shorea sp. caused high mortality. These short-term bioassays thus detected clear differences between wood species in their resistance to Limnoria that matched findings from long-term marine trials, while indicating new species worthy of detailed testing.

Chapter 2 Development, physiology, behaviour and ecology of scallop larvae

Publisher Summary This chapter discusses the development, physiology, behavior, and ecology of larvae of the Superfamily Pectinoidea. Studies of scallop development have focused on species with commercial potential from shallow temperate to tropical waters, which, with one exception, show typical planktotrophic development. The egg and shell dimensions are used to provide evidence of mode of development. The timing of stages of larval development varies considerably from species to species, though information on early stages is sparse. The putative constraints of food supply, depth, and patchyness of adult environment, temperature also affects scallop life history characteristics. Scallop larvae develop a range of putative sense organs during larval life. The fluorescence microscopy techniques have provided a new understanding of the larval nervous system. Experimental investigation of sensory function yields insight into settlement behavior. Advances in larval biochemistry relate to lipid metabolism and to the role of neurotransmitters and neuromodulators, especially in metamorphosis control. Larval rearing techniques have refined with much attention being paid to bacteria as sources of disease and as protection against disease.

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

BackgroundThe 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.ResultsFive 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.ConclusionsTo 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.