Presence and activity of endo-ß-1,4-mannase, an important digestive carbohydrase within the digestive fluid of terrestrial crustaceans.

Within the midgut gland of the Christmas Island red crab, Gecarcoidea natalis, a single transcript for a GH5_10 endo-ß-1,4-mannase had the highest expression out of all of the carbohydrase enzymes (Gan et al. in Mar Biotechnol 20:654-665, 2018). The activity, and potential digestive importance of this hemicellulase, compared with other carbohydrases, has yet to be established. The digestive fluid of G. natalis contained substantial endo-ß-1,4-mannase activities (630 ± 55 (6) nmol reducing sugars. min-1. mg-1 protein). It was present as a single isozyme of 66.3 ± 0.7 kDa (n = 6). Endo-ß-1,4-mannase activities were higher than that for lichenase and endo-ß-1,4-glucanase but lower than that for ß-1,3-glucanase and amylase. The digestive fluid was able to hydrolyse, galactomannan, into its component monosaccharides. Hence, this confirms expression data that this enzyme is one of the most important digestive cellulases/ hemicellulases. Expression of GH5_10 endo-ß-1,4-mannase mRNA was consistent with that of a digestive enzyme, as it was expressed in the digestive midgut gland but not in muscle and gill. Endo-ß-1,4-mannase activities were also present within the digestive fluid of the terrestrial hermit crabs, Coenobita perlatus and Coenobita brevimanus. Endo-ß-1,4-mannase activities (1351 ± 136 (n=3) nmol reducing sugars. min-1 mg-1 protein for C. perlatus. 665 ± 32 n=(5) nmol reducing sugars. min-1 mg-1 protein for C. brevimanus) were higher than that for endo-ß-1,4-glucanase and amylase but were lower than ß-1,3-glucanase activities. Animals within the terrestrial hermit crab family, Coenobitidae consume legume and palm seeds which contain substantial amounts of mannan. Hence, high endo-ß-1,4-mannase activities suggest that digestion of mannan within these species may represent an important source of carbohydrate.

Review: The structure and function of cellulase (endo-ß-1,4-glucanase) and hemicellulase (ß-1,3-glucanase and endo-ß-1,4-mannase) enzymes in invertebrates that consume materials ranging from microbes, algae to leaf litter.

This review discusses the reaction catalysed, and the structure and function of the cellulase, endo-ß-1,4-glucanase and the hemicellulase enzymes, ß-1,3-glucanase and endo-ß-1,4-mannase that are present in numerous invertebrate groups with a diverse range of feeding specialisations. These range from microbial deposit and filter feeders, micro and macrophagous algal feeders, omnivores to herbivorous leaf litter and wood feeders. Endo-ß-1,4-glucanase from glycosyl hydrolase family 9 (GH9) digests cellulose like ß-1,4-glucans from a range of materials. As it hydrolyses crystalline cellulose very slowly, it is a poor cellulase. Where tested, the enzyme has dual endo-ß-1,4-glucanase and lichenase activity. Its presence does not necessarily indicate the ability of an animal to digest cellulose. It only indicates the ability to digest ß-1,4-glucans and its function, which is discussed in this review, should be considered with reference to the substrates present in the diet. ß-1,3-glucanase (laminarinase) belongs to glycosyl hydrolase family 16 (GH16) and hydrolyses ß-1.3-glucans. These polysaccharides are present in the cell walls of algae, protozoans and yeast, and they also occur as storage polysaccharides within protozoans and algae. Depending on their site of expression, these enzymes may function as a digestive enzyme or may be involved in innate immunity. Enzymes present in the digestive fluids or tissues, would be digestive. Haemolymph GH16 proteins may be involved in innate immunity through the activation of the phenol oxidase system. Insect GH16 proteins expressed within the haemolymph have lost their catalytic residues and function as ß-glucan binding proteins. In contrast, crustacean GH16 proteins expressed within the same tissue, have retained the catalytic residues and thus possibly their ß-1,3-glucanase activity. The potential function of which is discussed. Endo-ß-1,4-mannase from glycosyl hydrolase family 5, subfamily 10 (GH5_10) hydrolyses mannan, glucomannan and galactomannan. These hemicelluloses are present in the cell walls of plants and algae and also function as storage polysaccharides within legume and palm seeds. They are digestive enzymes whose high expression in some species suggests they are a major contributor to hemicellulose digestion. They may also provide the animal with substantial amounts of monosaccharides for energy.

Two reads to rule them all: Nanopore long read-guided assembly of the iconic Christmas Island red crab, Gecarcoidea natalis (Pocock, 1888), mitochondrial genome and the challenges of AT-rich mitogenomes.

Despite recent advances in sequencing technology, a complete mitogenome assembly is still unavailable for the gecarcinid land crabs that include the iconic Christmas Island red crab (Gecarcoidea natalis) which is known for its high population density, annual mass breeding migration and ecological significance in maintaining rainforest structure. Using sequences generated from Nanopore and Illumina platforms, we assembled the complete mitogenome for G. natalis, the first for the genus and only second for the family Gecarcinidae. Nine Nanopore long reads representing 0.15% of the sequencing output from an overnight MinION Nanopore run were aligned to the mitogenome. Two of them were >10 kb and combined are sufficient to span the entire G. natalis mitogenome. The use of Illumina genome skimming data only resulted in a fragmented assembly that can be attributed to low to zero sequencing coverage in multiple high AT-regions including the mitochondrial protein-coding genes (NAD4 and NAD5), 16S ribosomal rRNA and non-coding control region. Supplementing the mitogenome assembly with previously acquired transcriptome dataset containing high abundance of mitochondrial transcripts improved mitogenome sequence coverage and assembly reliability. We then inferred the phylogeny of the Eubrachyura using Maximum Likelihood and Bayesian approaches, confirming the phylogenetic placement of G. natalis within the family Gecarcinidae based on whole mitogenome alignment. Given the substantial impact of AT-content on mitogenome assembly and the value of complete mitogenomes in phylogenetic and comparative studies, we recommend that future mitogenome sequencing projects consider generating a modest amount of Nanopore long reads to facilitate the closing of problematic and fragmented mitogenome assemblies.

Transcriptome-Guided Identification of Carbohydrate Active Enzymes (CAZy) from the Christmas Island Red Crab, Gecarcoidea natalis and a Vote for the Inclusion of Transcriptome-Derived Crustacean CAZys in Comparative Studies.

The Christmas Island red crab, Gecarcoidea natalis, is an herbivorous land crab that consumes mostly fallen leaf litter. In order to subsist, G. natalis would need to have developed specialised digestive enzymes capable of supplying significant amounts of metabolisable sugars from this diet. To gain insights into the carbohydrate metabolism of G. natalis, a transcriptome assembly was performed, with a specific focus on identifying transcripts coding for carbohydrate active enzyme (CAZy) using in silico approaches. Transcriptome sequencing of the midgut gland identified 70 CAZy-coding transcripts with varying expression values. At least three newly discovered putative GH9 endo-ß-1,4-glucanase ("classic cellulase") transcripts were highly expressed in the midgut gland in addition to the previously characterised GH9 and GH16 (ß-1,3-glucanase) transcripts, and underscoring the utility of whole transcriptome in uncovering new CAZy-coding transcripts. A highly expressed transcript coding for GH5_10 previously missed by conventional screening of cellulase activity was inferred to be a novel endo-ß-1,4-mannase in G. natalis with in silico support from homology modelling and amino acid alignment with other functionally validated GH5_10 proteins. Maximum likelihood tree reconstruction of the GH5_10 proteins demonstrates the phylogenetic affiliation of the G. natalis GH5_10 transcript to that of other decapods, supporting endogenous expression. Surprisingly, crustacean-derived GH5_10 transcripts were near absent in the current CAZy database and yet mining of the transcriptome shotgun assembly (TSA) recovered more than 100 crustacean GH5_10s in addition to several other biotechnological relevant CAZys, underscoring the unappreciated potential of the TSA database as a valuable resource for crustacean CAZys.

ORDER within the chaos: Insights into phylogenetic relationships within the Anomura (Crustacea: Decapoda) from mitochondrial sequences and gene order rearrangements.

The infraorder Anomura consists of a morphologically and ecologically heterogeneous group of decapod crustaceans, and has attracted interest from taxonomists for decades attempting to find some order out of the seemingly chaotic diversity within the group. Species-level diversity within the Anomura runs the gamut from the "hairy" spindly-legged yeti crab found in deep-sea hydrothermal vent environments to the largest known terrestrial invertebrate, the robust coconut or robber crab. Owing to a well-developed capacity for parallel evolution, as evidenced by the occurrence of multiple independent carcinization events, Anomura has long tested the patience and skill of both taxonomists attempting to find order, and phylogeneticists trying to establish stable hypotheses of evolutionary inter-relationships. In this study, we performed genome skimming to recover the mitogenome sequences of 12 anomuran species including the world's largest extant invertebrate, the robber crab (Birgus latro), thereby over doubling these resources for this group, together with 8 new brachyuran mitogenomes. Maximum-likelihood (ML) and Bayesian-inferred (BI) phylogenetic reconstructions based on amino acid sequences from mitogenome protein-coding genes provided strong support for the monophyly of the Anomura and Brachyura and their sister relationship, consistent with previous studies. The majority of relationships within families were supported and were largely consistent with current taxonomic classifications, whereas many relationships at higher taxonomic levels were unresolved. Nevertheless, we have strong support for a polyphyletic Paguroidea and recovered a well-supported clade of a subset of paguroids (Diogenidae + Coenobitidae) basal to all other anomurans, though this requires further testing with greater taxonomic sampling. We also introduce a new feature to the MitoPhAST bioinformatics pipeline (https://github.com/mht85/MitoPhAST) that enables the extraction of mitochondrial gene order (MGO) information directly from GenBank files and clusters groups based on common MGOs. Using this tool, we compared MGOs across the Anomura and Brachyura, identifying Anomura as a taxonomic "hot spot" with high variability in MGOs among congeneric species from multiple families while noting the broad association of highly-rearranged MGOs with several anomuran lineages inhabiting extreme niches. We also demonstrate the value of MGOs as a source of novel synapomorphies for independently reinforcing tree-based relationships and for shedding light on relationships among challenging groups such as the Aegloidea and Lomisoidea that were unresolved in phylogenetic reconstructions. Overall, this study contributes a substantial amount of new genetic material for Anomura and attempts to further resolve anomuran evolutionary relationships where possible based on a combination of sequence and MGO information. The new feature in MitoPhAST adds to the relatively limited number of bioinformatics tools available for MGO analyses, which can be utilized widely across animal groups.

cDNA sequences of GHF9 endo-ß-1,4-glucanases in terrestrial Crustacea.

This study aimed to sequence and identify a glycosyl hydrolase family 9 (GHF9) endo-ß-1,4-glucanase expressed in the midgut gland of the herbivorous gecarcinid land crab, Gecarcoidea natalis. Hence this would explain the gene responsible for the production of previously purified and characterised endo-ß-1,4-glucanases. Three different transcripts, two complete and one partial were sequenced from cDNA and an open reading frame of 1383bp was produced. Translated, this would produce a putative protein of 460 amino acid residues, including a 16 amino acid residue signal peptide. The mature protein (without signal peptide) is predicted to have a molecular mass of 47.6-47.7kDa; this closely matches the molecular mass (47.4kDa) of one of the three endo-ß-1,4-glucanase/lichenase enzymes purified previously from G. natalis. It is therefore proposed that the gene described here encodes one of the previously characterised enzymes. The presence of multiple transcripts suggests gene duplication. To confirm that the gene is widely expressed within the Crustacea, cDNA encoding a GHF9 endo-ß-1,4-glucanase was also sequenced in diverse crustaceans, the deposit feeding soldier crab, Mictyris platycheles and the terrestrial hermit crabs, Coenobita purlatus and C. brevimanus. An open reading frame of 1356bp was sequence from M. platycheles, while an incomplete open reading frames of 1384 and 1523bp were respectively sequenced from Coenobita brevimanus and C. perlatus. The midgut gland of M. platycheles contained activity (0.704±0.218µmol reducing sugars produced. min-1·mg-1 tissue wet weight) of a 26.3±0.3(5) endo-ß-1,4-glucanase isozyme (determined from activity staining). These species, particularly M. platycheles does not consume and digest significant amounts of plant cellulose. This implies that the ancestral enzyme is not a cellulase, but rather it may be involved in hydrolysing cellulose like polysaccharides within other organisms such as algae.

A glycosyl hydrolase family 16 gene is responsible for the endogenous production of ß-1,3-glucanases within decapod crustaceans.

To identify the gene responsible for the production of a ß-1,3-glucanase (laminarinase) within crustacea, a glycosyl hydrolase family 16 (GHF16) gene was sequenced from the midgut glands of the gecarcinid land crab, Gecarcoidea natalis and the freshwater crayfish, Cherax destructor. An open reading frame of 1098 bp for G. natalis and 1095 bp for C. destructor was sequenced from cDNA. For G. natalis and C. destructor respectively, this encoded putative proteins of 365 and 364 amino acids with molecular masses of 41.4 and 41.5 kDa. mRNA for an identical GHF16 protein was also expressed in the haemolymph of C. destructor. These putative proteins contained binding and catalytic domains that are characteristic of a ß-1,3-glucanase from glycosyl hydrolase family 16. The amino acid sequences of two short 8-9 amino acid residue peptides from a previously purified ß-1,3-glucanase from G. natalis matched exactly that of the putative protein sequence. This plus the molecular masses of the putative proteins matching that of the purified proteins strongly suggests that the sequences obtained encode for a catalytically active ß-1,3-glucanase. A glycosyl hydrolase family 16 cDNA was also partially sequenced from the midgut glands of other amphibious (Mictyris platycheles and Paragrapsus laevis) and terrestrial decapod species (Coenobita rugosus, Coenobita perlatus, Coenobita brevimanus and Birgus latro) to confirm that the gene is widely expressed within this group. There are three possible hypothesised functions and thus evolutionary routes for the ß-1,3-glucanase: 1) a digestive enzyme which hydrolyses ß-1,3-glucans, 2) an enzyme which cleaves ß-1,3-glycosidic bonds within cell walls to release cell contents or 3) an immune protein which can hydrolyse the cell walls of potentially pathogenic micro-organisms.

Digestive enzymes of two brachyuran and two anomuran land crabs from Christmas Island, Indian Ocean.

The digestive ability of four sympatric land crabs species (the gecarcinids, Gecarcoidea natalis and Discoplax celeste and the anomurans, Birgus latro and Coenobita perlatus) was examined by determining the activity of their digestive enzymes. The gecarcinids are detritivores that consume mainly leaf litter; the robber crab, B. latro, is an omnivore that preferentially consumes items high in lipid, carbohydrate and/or protein; C. perlatus is also an omnivore/detritivore. All species possess protease, lipase and amylase activity for hydrolysing ubiquitous protein, lipid and storage polysaccharides (glycogen and starch). Similarly all species possess enzymes such as N-acetyl-ß-D-glucosaminidase, the cellulases, endo-ß-1,4-glucanase and ß-glucohydrolase and hemicellulases, lichenase and laminarinase for the respective hydrolysis of structural substrates chitin, cellulose and hemicelluloses, lichenan and laminarin. Except for the enzyme activities of C. perlatus, enzyme activity could not be correlated to dietary preference. Perhaps others factors such as olfactory and locomotor ability and metabolic status may determine the observed dietary preferences. The digestive fluid of C. perlatus possessed higher endo-ß-1,4-glucanase, lichenase and laminarinase activities compared to that of the other species. Thus, C. perlatus may be efficient at digestion of cellulose and hemicellulose within plant material. Zymography indicated that the majority of protease, lipase, phosphatase, amylase, endo-ß-1,4-glucanase, ß-glucohydrolase and N-acetyl-ß-D-glucosaminidase isozymes were common to all species, and hence were inherited from a common aquatic ancestor. Differences were observed for the phosphatase, lipase and endo-ß-1,4-glucanase isozymes. These differences are discussed in relation to phylogeny and possible evolution to cope with the adoption of a terrestrial diet.

Chemiluminescence evidence supporting the selective role of ligands in the permanganate oxidation of micropollutants.

The selective increase in the oxidation rate of certain organic compounds with permanganate in the presence of environmental "ligands" and reduced species has been ascribed to the different reactivity of the target compounds toward Mn(III), which bears striking similarities to recent independent investigations into the use of permanganate as a chemiluminescence reagent. In spite of the importance of Mn(III) in the light-producing pathway, the dependence of the oxidation mechanism for any given compound on this intermediate could not be determined solely through the emission intensity. However, target compounds susceptible to single-electron oxidation by Mn(III) (such as bisphenol A and triclosan) can be easily distinguished by the dramatic increase in chemiluminescence intensity when a permanganate reagent containing high, stable concentrations of Mn(III) is used. The differences are accentuated under the low pH conditions that favor the chemiluminescence emission due to the greater reactivity of Mn(III) and the greater influence of complexing agents. This study supports the previously postulated selective role of ligands and reducing agents in permanganate oxidations and demonstrates a new approach to explore the chemistry of environmental manganese redox processes.

Isozymes from the herbivorous gecarcinid land crab, Gecarcoidea natalis that possess both lichenase and endo-ß-1,4-glucanase activity.

Three isozymes with both lichenase and endo-ß-1,4-glucanase activity were purified and characterised from the midgut gland of the herbivorous gecarcinid land crab, Gecarcoidea natalis. The three isozymes, termed 1a, 1b and 2, had respective molecular masses of 53 ± 0 (3), 43 ± 0 (3) and 47.4 ± 0(3) kDa. All isozymes possessed similar V(max) values and thus hydrolysed both carboxy methyl cellulose and lichenan equally. Furthermore the chromatography profiles for lichenase activities mirrored that for endo-ß-1,4-glucanase activities suggesting that the same enzyme possessed both activities. Given this, the endo-ß-1,4-glucanase enzymes described for other animals, may, like the isozymes described in this study, may be able to hydrolyse lichenan. However this ability needs to be confirmed. The main digestive function of these isozymes may be to hydrolyse hemicelluloses such as lichenan and mixed beta-D-glucan. All three isozymes randomly hydrolysed internal glycosidic bonds within carboxy methyl cellulose and lichenan to release short oligomers of 4-5 glucose units in length. They also hydrolysed cellotetraose to either two units of cellobiose or cellotriose and glucose. Cellotriose was hydrolysed to cellobiose and glucose. All three enzymes lacked ß-1,4-glucosidase activity as they could not hydrolyse cellobiose.

Accumulation and excretion of morphine by Calliphora stygia, an Australian blow fly species of forensic importance.

This study examined the ability of the forensically important blow fly, Calliphora stygia to actively excrete morphine, thereby maintaining a low morphine level within its body when fed on a diet containing morphine at low (7pmolg(-1)) and high (17.5pmolg(-1)) concentrations. Morphine was accumulated within the bodies of maggots (≈70% within the tissues) at concentrations which were lower than that of the meat (3-24%). The morphine content of the initial developing stages (second and third instar maggots) maintained on the high morphine diet was higher than those on the low morphine diet. Morphine was cleared from the body with negatively exponential kinetics (High morphine group: Morphine (pmolg(-1) wet weight)=8425e(-0.014t). Low morphine group: Morphine (pmolg(-1) wet weight)=2180e(-0.010t)). Clearance constants for morphine by animals in both groups were similar and thus both groups had a similar ability to excrete morphine. The Malpighian tubules of maggots were able to actively secrete morphine using a transport mechanism that transports small type II organic cations, such as morphine and quinine. The rate of morphine secretion by the Malpighian tubules could explain the clearance of the drug by the maggots. As the morphine was transported across the Malpighian tubules cells, a significant proportion was metabolised into a compound that is yet to be fully characterised.

The last piece in the cellulase puzzle: the characterisation of beta-glucosidase from the herbivorous gecarcinid land crab Gecarcoidea natalis.

A 160 kDa enzyme with beta-glucosidase activity was purified from the midgut gland of the land crab Gecarcoidea natalis. The enzyme was capable of releasing glucose progressively from cellobiose, cellotriose or cellotetraose. Although beta-glucosidases (EC 3.2.1.21) have some activity towards substrates longer than cellobiose, the enzyme was classified as a glucohydrolase (EC 3.2.1.74) as it had a preference for larger substrates (cellobiose

Functional morphology of the gastric mills of carnivorous, omnivorous, and herbivorous land crabs.

Terrestrial decapods consume a wide variety of plant and animal material. The potential adaptations of carnivorous, omnivorous, and herbivorous terrestrial crustaceans were studied by examining the functional morphology of the gastric mill. Two closely related species from each feeding preference group were examined to identify which features of the mill were due to phylogeny and which were due to adaptation. The morphology of the gastric mill matched the diet well; the gastric mills of the carnivorous species (Geograpsus grayi and Geograpsus crinipes) possessed a blunt, rounded medial tooth and flattened lateral teeth with a longitudinal grinding groove. These features make them well suited to a carnivorous diet of soft animal tissue as well as hard material, such as arthropod exoskeleton. In contrast, the mill of the herbivorous gecarcinids (Gecarcoidea natalis and Discoplax hirtipes) consisted of a medial tooth with sharp transverse ridges and lateral teeth with sharp interlocking cusps and ridges and no grinding surface. These features would efficiently shred fibrous plant material. The morphology of the mill of the omnivorous coenobitids (Coenobita perlatus and Birgus latro) was more generalized toward a mixed diet. However, the mill of B. latro was more adapted to deal with highly nutritious food items, such as nuts and heavily calcified decapods. Its mill possessed lateral teeth with extended ridges, which sat close to the calcified cardiopyloric valve to form a flattened floor. Hard items trapped in the mill would be crushed against this surface by the medial tooth.

Potential endocrine disruption of ovary synthesis in the Christmas Island red crab Gecarcoidea natalis by the insecticide pyriproxyfen.

The effect of the insecticide, pyriproxyfen on early ovary synthesis was examined in the Gecarcinid land crab, Gecarcoidea natalis. Crabs were fed a mixture of either leaf litter and bait containing 0.5% (wt/wt) pyriproxyfen (experimental groups), or a mixture of leaf litter and a control bait containing no pyriproxyfen (control groups), at simulated baiting doses of 2 kg ha(-1) and 4 kg ha(-1), during the period in which G. natalis synthesises its ovaries. A third group of crabs were fed ad libitum either the bait containing 0.5% Pypriproxyfen or the control bait. Pyriproxyfen affected early ovary development in G. natalis. The ovaries from crabs in the experimental groups at all baiting levels had a higher total nitrogen content and dry mass than the ovaries from crabs in the control groups. Pyriproxyfen affected the histology of the ovaries. Ovaries from animals in the experimental groups were more mature, containing more previtellogenic and early vitellogenic oocytes, of a larger diameter, than the ovaries from crabs in the control groups. Significant amounts of pyriproxyfen accumulated within the midgut gland and ovary, the hypothesised target tissues, while minor amounts of pyriproxyfen was accumulated in the muscle, a hypothesised non target tissue. Pyriproxyfen may have stimulated early ovary development and induced synthesis of yolk protein by mimicking methyl farnesoate and thus causing endocrine disruption. Given this, pyriproxyfen should not be used to control invasive insects in environments where gecarcinid and other land crab species are present.

Food utilisation and digestive ability of aquatic and semi-terrestrial crayfishes, Cherax destructor and Engaeus sericatus (Astacidae, Parastacidae).

Both Engaeus sericatus and Cherax destructor are omnivorous crayfishes consuming a variety of food items. Materials identified in the faeces of both E. sericatus and C. destructor consisted of mainly plant material with minor amounts of arthropod animals, algae and fungi. The morphology of the gastric mill of C. destructor suggests that it is mainly involved in crushing of food material while the gastric mill of E. sericatus appears to be better suited to cutting of food material. Given this, the gastric mill of E. sericatus may be better able to cut the cellulose and hemicellulose fibres associated with fibrous plant material. In contrast, the gastric mill of C. destructor appears to be more efficient in grinding soft materials such as animal protein and algae. Both species accumulated high amounts of lipids in their midgut glands (about 60% of the dry mass) which were dominated by triacylglycerols (81-82% of total lipids). The dominating fatty acids were 16:0, 16:1(n-7), 18:1(n-9), 18:2(n-6), and 18:3(n-3). The two latter fatty acids can only be synthesised by plants, and are thus indicative of the consumption of terrestrial plants by the crayfishes. The similarity analysis of the fatty acid patterns showed three distinct clusters of plants and each of the crayfish species. The complement of digestive enzymes, proteinases, total cellulase, endo-beta-1,4-glucanase, beta-glucosidase, laminarinase and xylanase within midgut gland suggests that both C. destructor and E. sericatus are capable of hydrolysing a variety of substrates associated with an omnivorous diet. Higher activities of total cellulase, endo-beta-1,4-glucanase and beta-glucosidase indicate that E. sericatus is better able to hydrolyse cellulose within plant material than C. destructor. In contrast to E. sericatus, higher total protease and N-acetyl-beta-D-glucosaminidase activity in the midgut gland of C. destructor suggests that this species is better able to digest animal materials in the form of arthropods. Differences in total cellulase and gastric mill morphology suggest that E. sericatus is more efficient at digesting plant material than C. destructor. However, the contents of faecal pellets and the fatty acid compositions seem to indicate that both species opportunistically feed on the most abundant and easily accessible food items.

Purification and characterisation of endo-beta-1,4-glucanase and laminarinase enzymes from the gecarcinid land crab Gecarcoidea natalis and the aquatic crayfish Cherax destructor.

Laminarinase and endo-beta-1,4-glucanase were purified and characterised from the midgut gland of the herbivorous land crab Gecarcoidea natalis and the crayfish Cherax destructor. The laminarinase isolated from G. natalis was estimated to have a molecular mass of 41 kDa by SDS-PAGE and 71 kDa by gel filtration chromatography. A similar discrepancy was noted for C. destructor. Possible reasons for this are discussed. Laminarinase (EC 3.2.1.6) from G. natalis had a V(max) of 42.0 micromol reducing sugars produced min(-1) mg protein(-1), a K(m) of 0.126% (w/v) and an optimum pH range of 5.5-7, and hydrolysed mainly beta-1,3-glycosidic bonds. In addition to the hydrolysis of beta-1,3-glycosidic bonds, laminarinase (EC 3.2.1.39) from C. destructor was capable of significant hydrolysis of beta-1,4-glycosidic bonds. It had a V(max) of 19.6 mumol reducing sugars produced min(-1) mg protein(-1), a K(m) of 0.059% (w/v) and an optimum pH of 5.5. Laminarinase from both species produced glucose and other short oligomers from the hydrolysis of laminarin. Endo-beta-1,4-glucanase (EC 3.2.1.4) from G. natalis had a molecular mass of 52 kDa and an optimum pH of 4-7. It mainly hydrolysed beta-1,4-glycosidic bonds, but was also capable of significant hydrolysis of beta-1,3-glycosidic bonds. Two endo-beta-1,4-glucanases, termed 1 and 2, with respective molecular masses of 53+/-3 and 52 kDa, were purified from C. destructor. Endo-beta-1,4-glucanase 1 was only capable of hydrolysing beta-1,4-glycosidic bonds and had an optimum pH of 5.5. Endo-beta-1,4-glucanases from both species produced some glucose, cellobiose and other short oligomers from the hydrolysis of carboxymethyl cellulose.

A review of feeding and nutrition of herbivorous land crabs: adaptations to low quality plant diets.

This paper reviews the nutritional ecology, the digestive physiology, and biochemistry of herbivorous land crabs and the adaptations that they possess towards a diet of plant material. Land crab species that breathe air and forage out of water can be divided into three feeding specialisations: primarily carnivorous, deposit feeders feeding on micro-organisms and organic matter in the sediment, and herbivores consuming mainly plant material and its detritus. The last forms the focus of this review. The diets of the herbivores are low in nitrogen and high in carbon, are difficult to digest since they contain cellulose and hemicellulose, and may disrupt digestion due to the presence of tannins. Herbivorous crustaceans are able to efficiently utilise plant material as their primary nutrient source and are indeed able to meet their nitrogen requirements from it. Herbivorous land crabs display a range of adaptations towards a low nitrogen intake and these are discussed in this review. They also appear to endogenously produce cellulase and hemicellulase enzymes for the digestion of cellulose and hemicellulose. Generalised and specific adaptations allow them to inhibit the potentially negative digestive effects of tannins. To digest plant material, they possess a plastic digestive strategy of high food intake, short retention time, high assimilation of cell contents, and substantial digestion of cellulose and hemicellulose.

Endogenous production of endo-beta-1,4-glucanase by decapod crustaceans.

The potential ability to produce cellulase enzymes endogenously was examined in decapods crustaceans including the herbivorous gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes, the amphibious freshwater crab Austrothelphusa transversa, the terrestrial hermit crab, Coenobita variabilis the parastacid crayfish Euastacus, and the crayfish Cherax destructor. The midgut gland of both G. natalis and D. hirtipes contained substantial total cellulase activities and activities of the cellulase enzymes endo-beta-1,4-glucanase and beta-glucosidase. With the exception of total cellulase and beta-glucosidase from D. hirtipes, the enzyme activities within the midgut gland were higher than those within the digestive juice. Hence, the enzyme activities appear to reside predominantly within midgut gland, providing indirect evidence for endogenous synthesis of cellulase enzymes by this tissue. A 900 bp cDNA fragment encoding a portion of the endo-beta-1,4-glucanase amino acid sequence was amplified by RT-PCR using RNA isolated from the midgut gland of C. destructor, Euastacus, A. transversa and C. variabilis. This provided direct evidence for the endogenous production of endo-beta-1,4-glucanase. The 900 bp fragment was also amplified from genomic DNA isolated from the skeletal muscle of G. natalis and D. hirtipes, clearly indicating that the gene encoding endo-beta-1,4-glucanase is also present in these two species. As this group of evolutionary diverse crustacean species possesses and expresses the endo-beta-1,4-glucanase gene it is likely that decapod crustaceans generally produce cellulases endogenously and are able to digest cellulose.

Presence and properties of cellulase and hemicellulase enzymes of the gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes.

Digestive juice from the herbivorous gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes exhibited total cellulase activity and activities of two cellulase enzymes; endo-beta-1,4-glucanase and beta-1,4-glucosidase. These enzymes hydrolysed native cellulose to glucose. The digestive juice of both species also contained laminarinase, licheninase and xylanase, which hydrolysed laminarin, lichenin and xylan, respectively, to component sugars. The pH optima of beta-1,4-glucosidase, endo-beta-1,4-glucanase and total cellulase from G. natalis were 4-5.5, 5.5 and 5.5-7, respectively. In the digestive juice from D. hirtipes, the corresponding values were 4-7, 5.5-7 and 4-9, respectively. The pH of the digestive juice was 6.69+/-0.03 for G. natalis and 6.03+/-0.04 for D. hirtipes and it is likely that the cellulases operate near maximally in vivo. In G. natalis, total cellulase activity and endo-beta-1,4-glucanase activity were higher than in D. hirtipes, and the former species can thus hydrolyse cellulose more rapidly. beta-1,4-glucosidase from G. natalis was inhibited less by glucono-d-lactone (K(i)=11.12 mmol l(-1)) than was the beta-1,4-glucosidase from D. hirtipes (K(i)=4.53 mmol l(-1)). The greater resistance to inhibition by the beta-1,4-glucosidase from G. natalis may contribute to the efficiency of the cellulase system in vivo by counteracting the effects of product inhibition and possibly dietary tannins. The activity of beta-1,4-glucosidase in the digestive juice of D. hirtipes was higher than that of G. natalis.

Dietary assimilation and the digestive strategy of the omnivorous anomuran land crab Birgus latro (Coenobitidae).

On Christmas Island, Indian Ocean, the diet of robber crabs, Birgus latro (Linnaeus) was generally high in fat, storage polysaccharides or protein and largely comprised fruits, seeds, nuts and animal material. The plant items also contained significant amounts of hemicellulose and cellulose. In laboratory feeding trials, crabs had similar intakes of dry matter when fed artificial diets high in either fat or storage polysaccharide, but intake was lower on a high protein diet. Assimilation coefficients of dry matter (69-74%), carbon (72-81%), nitrogen (76-100%), lipid (71-96%) and storage polysaccharide (89-99%) were high on all three diets. B. latro also assimilated significant amounts of the chitin ingested in the high protein diet ( 93%) and hemicellulose (49.6-65%) and cellulose (16-53%) from the high carbohydrate and high fat diets. This is consistent with the presence of chitinase, hemicellulase and cellulase enzymes in the digestive tract of B. latro. The mean retention time (27.2 h) for a dietary particle marker ((57)Co-labelled microspheres) was longer than measured in leaf-eating land crabs. The feeding strategy of B. latro involves the selection of highly digestible and nutrient-rich plant and animal material and retention of the digesta for a period long enough to allow extensive exploitation of storage carbohydrates, lipids, protein and significant amounts of structural carbohydrates (hemicellulose, cellulose and chitin).