Endogenous production of endo-β-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-β-1,4-glucanase and β-glucosidase. With the exception of total cellulase and β-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-β-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-β-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-β-1,4-glucanase is also present in these two species. As this group of evolutionary diverse crustacean species possesses and expresses the endo-β-1,4-glucanase gene it is likely that decapod crustaceans generally produce cellulases endogenously and are able to digest cellulose.

Cadmium, copper, mercury, and zinc concentrations in tissues of the King Crab (Pseudocarcinus gigas) from southeast Australian waters

The concentrations of cadmium, copper, mercury, and zinc were determined in muscle (body, claw, and leg), hepatopancreas, and gill tissues of Pseudocarcinus gigas, an exceptionally large, long-lived, and deep-dwelling crab species. The accumulation patterns observed are discussed in terms of both intra- and interspecies variations, with particular attention to the possible consequences of the extreme size and depth range of P. gigas. Metal concentrations did not depend significantly on sex of the crab. Significant differences between tissues were detected for all metals, and the distribution of metal between the tissues was different for each metal. Significant correlations were found between metal concentrations in the various tissues and crab size, and these are discussed and rationalised. The concentrations of mercury and zinc in muscle tissue increased with crab size and were high compared to other crab species. The concentrations of cadmium and copper present in edible tissues were not especially high compared to other crab species, but the concentration of cadmium in the hepatopancreas is of dietary concern.

Habitat use by the hymenosomatid crab Amarinus lacustris (Chilton) in two south-eastern Australian rivers

The hymenosomatid crab Amarinus lacustris is abundant in some south-eastern Australian rivers; however, little is known of its ecology. Patterns of habitat use by crabs in rivers may be affected by seasonal changes in river discharge. This study investigates population characteristics, timing of reproduction and patterns of habitat use by A. lacustris in five riffle and pool habitats from each of the Hopkins and Merri Rivers in south-west Victoria, Australia, sampled over a twelve-month period. Distribution of Amarinus lacustris was similar between the two rivers, but log-linear modelling showed that there was a strong association between crab sex, habitat occupied and time of year because female A. lacustris showed a shift from riffle to pool habitats during March and April, coinciding with the non-gravid period of the year. Male crabs also showed a change in relative occurrence, occurring most often in riffles during winter–spring (July–November) but being equally common in both habitats in summer–autumn (January–May). These patterns are probably the result of the reproductive cycle of A. lacustris, which appears to show both ontogenetic and sex-related changes in habitat use during its life cycle, taking advantage of seasonal fluctuations in flow regime that may assist egg/larval development and dispersal.

Excretory and storage purines in the Anomuran land crab Birgus latro; guanine and uric acid

Birgus latro excretes nitrogenous waste as a mixture of urate and guanine and not predominantly urate as believed previously. The presence of guanine in faeces was confirmed by enzymatic derivatisation of guanine to xanthine with guanase. This is the first report of significant excretion of guanine outside the Chelicerata. The ratios of urate to guanine within the excreta of animals in field situations (natural diets) and in the laboratory (a range of artificial diets) were 3:2. Rates of excretion of both urate and guanine increased when experimental crabs were fed an artificial diet high in nitrogen. Significant amounts of guanine were also measured in tissues of B. latro, but only urate was present in equivalent tissues of the closely related species Coenobita brevimanus. Coenobita brevimanus did not excrete any significant amount of purines with the faeces.

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 (⁵⁷Co-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).

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

Laminarinase and endo-β-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 µmol 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 β-1,3-glycosidic bonds. In addition to the hydrolysis of β-1,3-glycosidic bonds, laminarinase (EC 3.2.1.39) from C. destructor was capable of significant hydrolysis of β-1,4-glycosidic bonds. It had a V_max of 19.6 µmol 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-β-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 β-1,4-glycosidic bonds, but was also capable of significant hydrolysis of β-1,3-glycosidic bonds. Two endo-β-1,4-glucanases, termed 1 and 2, with respective molecular masses of 53±3 and 52 kDa, were purified from C. destructor. Endo-β-1,4-glucanase 1 was only capable of hydrolysing β-1,4-glycosidic bonds and had an optimum pH of 5.5. Endo-β-1,4-glucanases from both species produced some glucose, cellobiose and other short oligomers from the hydrolysis of carboxymethyl cellulose.

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.

A life history model for the giant crab Pseudocarcinus gigas

This study examined P. gigas populations from Cape Naturaliste (Western Australia) to Flinders Island (Tasmania), investigated reproduction, developed a unique tag to study their movement and growth, and collected new environmental information that allowed the development of a model of the life history for this iconic endemic Australian species.

Spermatogenesis in the blue swimming crab, Portunus pelagicus, and evidence for histones in mature sperm nuclei

Spermatogenesis in the blue swimming crab, Portunus pelagicus, is described by light and electron microscopy. The testis is composed of anterior (AT) and posterior (PT) lobes, that are partitioned into lobules by connective tissue trabecula, and further divided into zones (germinal, transformation and evacuation), each with various stages of cellular differentiation. The vas deferens is classified into three distinct regions: anterior (AVD), median (MVD), and posterior (PVD), on the presence of spermatophores and two secretions, termed substance I and II. Based on the degree and patterns of heterochromatin, spermatogenesis is classified into 13 stages: two spermatogonia (SgA and SgB), six primary spermatocytes (leptotene, zygotene, pachytene, diplotene, diakinesis, and metaphase), a secondary spermatocyte (SSc), three spermatids (St 1–3), and a mature spermatozoon. Spermatid stages are differentiated by chromatin decondensation and the formation of an acrosomal complex, which is unique to brachyurans. Mature spermatozoa are aflagellated, and have a nuclear projection and a spherical acrosome. AUT-PAGE and Western blots show that, during chromatin decondensation, there is a reduction of most histones, with only small amounts of H2B and H3 remaining in mature spermatozoa.

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

A 160 kDa enzyme with β-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 β-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<cellotriose=cellotetraose). It was able to synthesise some cellotetraose by the transglycosylation of smaller substrates – another common feature of glucohydrolases. The interaction between the glucohydrolase described here and the endo-β-1,4-glucanases described previously for G. natalis provides a complete model for cellulose hydrolysis in crustaceans and possibly in other invertebrates. After mechanical fragmentation by the gastric mill, multiple endo-β-1,4-glucanases would initially cleave β-1,4-glycosidic bonds within native cellulose, releasing small oligomers, including cellobiose, cellotriose and cellotetraose. The glucohydrolase would then attach to these oligomers, progressively releasing glucose. The glucohydrolase might also attach directly to crystalline cellulose to release glucose from free chain ends. This two-enzyme system differs from the traditional model, which suggests that total cellulose hydrolysis requires the presence an endo-β-1,4-glucanse, a cellobiohydrolase and a β-glucosidase

Seasonal winds drive water temperature cycle and migration patterns of Southern Australian giant crab Pseudocarcinus gigas

The giant crab Pseudocarcinus gigas occurs along the continental shelf break of southern Australia. During the summer alongshore winds cause cooler water to upwell onto the shelf, and the crabs move from deeper water onto the shelf where there is more food. The combination of a preferred thermal niche and a depth-stratified food supply defines the favorable foraging environments that enhance the growth of P. gigas. Climate change is expected to cause a southerly shift of the austral subtropical high-pressure belt, and modelers have predicted more upwelling-favorable winds. The associated increase in the circulation of cooler water across the shelf is likely to provide P. gigas with an increased access to benthic food resources and their growth rate may increase in some regions.