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.