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.

Sea-level changes and palaeo-ranges: reconstruction of ancient shorelines and river drainages and the phylogeography of the Australian land crayfish Engaeus sericatus Clark (Decapoda: Parastacidae )

Historical sea levels have been influential in shaping the phylogeography of freshwater-limited taxa via palaeodrainage and palaeoshoreline connections. In this study, we demonstrate an approach to phylogeographic analysis incorporating historical sea-level information in a nested clade phylogeographic analysis (NCPA) framework, using burrowing freshwater crayfish as the model organism. Our study area focuses on the Bass Strait region of southeastern Australia, which is marine region encompassing a shallow seabed that has emerged as a land bridge during glacial cycles connecting mainland Australia and Tasmania. Bathymetric data were analysed using Geographical Information Systems (GIS) to delineate a palaeodrainage model when the palaeocoastline was 150 m below present-day sea level. Such sea levels occurred at least twice in the past 500 000 years, perhaps more often or of larger magnitude within the last 10 million years, linking Victoria and Tasmania. Inter-locality distance measures confined to the palaeodrainage network were incorporated into an NCPA of crayfish (Engaeus sericatus Clark 1936) mitochondrial 16S rDNA haplotypes. The results were then compared to NCPAs using present-day river drainages and traditional great-circle distance measures. NCPA inferences were cross-examined using frequentist and Bayesian procedures in the context of geomorphological and historical sea-level data. We found distribution of present-day genetic variation in E. sericatus to be partly explained not only by connectivity through palaeodrainages but also via present-day drainages or overland (great circle) routes. We recommend that future studies consider all three of these distance measures, especially for studies of coastally distributed species.

Commensalism used by freshwater crayfish species to survive drying in seasonal habitats

Gramastacus insolitus is a very small non-burrowing Australian freshwater crayfish with a restricted distribution, occurring almost exclusively in seasonal habitats throughout its range. It is listed as a threatened species but its strategy for surviving dry periods was unknown. Eight seasonal surveys of crayfish distribution showed that members of G. insolitus were never found at sites that were outside the distribution of two larger burrowing freshwater crayfish species, Geocharax falcata and Cherax destructor. Excavation of 80 burrows of members of G. falcata and C. destructor in three different seasonal habitats in the Grampians National Park, Victoria, Australia, revealed that individuals of G. insolitus found refuge from drying by estivating in cracks and shallow depressions at the side of the main burrow tunnels constructed by larger species. Members of G. insolitus were not found estivating at the surface, such as under fallen wood, nor was it usually found in crayfish burrows unoccupied by the host crayfish. This study indicates that members of G. insolitus are commensal upon larger crayfish species, using their burrows to survive the seasonal drying of their habitat. Conservation strategies for populations of G. insolitus will need to consider co-existing species of burrowing crayfish.

Habitat use by five sympatric Australian freshwater crayfish species (Parastacidae)

1. The Grampians National Park in Victoria is a 'hot spot' for freshwater crayfish diversity, with seven species from six genera occurring in sympatry. Few studies have examined how multiple species of freshwater crayfish co-exist across landscapes consisting of a mosaic of perennial and seasonal habitats. Despite their endemicity and likely key role in freshwaters, the ecology and biology of these crayfish remains unknown.

2. This study determined the distribution and habitat use of five crayfish species (Euastacus bispinosus, Cherax destructor, Geocharax falcata, Gramastacus insolitus and Engaeus lyelli). Seasonal sampling surveys ascertained whether crayfish distribution was related to habitat type, environmental or physicochemical variables, catchment or season.

3. Distribution was directly related to habitat type and the environmental and physicochemical variables that characterised habitats. Engaeus lyelli, G. falcata and G. insolitus occurred predominantly in floodplain wetlands and flooded vegetation habitats, E. bispinosus occurred only in flowing soft-sediment channels and C. destructor was found in all catchments and habitat types studied. Gramastacus insolitus co-occurred with G. falcata at all sites except two, so no distinct habitat separations were apparent for these two species.

4. A high percentage cover of boulders was the best indicator of crayfish absence, and discriminated between habitat types and crayfish species: it was probably a surrogate for a larger range of environmental and physicochemical variables. Catchment and season did not affect crayfish distribution.

5. These crayfish species varied in their degree of habitat specialisation from strongly generalist (C. destructor) to occupying only a specific habitat type (E. bispinosus). Some species appeared specialised for seasonal wetlands (G. insolitus and G. falcata). Overlap in site occupancy also varied: G. insolitus and G. falcata distributions were strongly associated, whereas C. destructor appeared to occur opportunistically across habitats, both alone and co-occurring with all the other species.

6. Management strategies to conserve multiple species of crayfish co-existing within landscapes will need to incorporate a range of perennial and seasonal habitat types to ensure sufficient space is available for species to maintain different occupancy patterns. Given that water resources are under increasing pressure and are strongly regulated within the Grampians National Park, this may present a conservation challenge to water managers in this location.

A genetic investigation on translocation of Australian commercial freshwater crayfish, cherax destructor

The Australian freshwater crayfish, Cherax destructor is cultured commercially and has been translocated throughout much of Australia. Previous investigation on C. destructor using 16S rRNA sequences of samples collected from natural environments has revealed a significant phylogeographic structure in this species with three well supported geographically non-overlapping clades, namely ‘northern’ C. d. destructor, ‘southern’ C. d. destructor and C. d. albidus. Movement of individuals beyond their natural range of distribution may have adverse effects on genetic integrity of the species. In the present study, aspects of translocations of the species were genetically investigated. Sequences of the 16S rRNA gene region of themitochondrial DNA (mtDNA) were obtained fromsamples collected in nine quasi-natural waterbodies, supplemented with sequences of samples obtained from 31 natural waterbodies examined in a previous study. Results of phylogeographic analysis provide evidence that certain haplotypes from major clades of C. destructor have been translocated. The findings of this study have important implications for the conservation and management of genetic diversity within C. destructor.

Ontogeny of putative GABAergic neurons and their receptors in the nervous system of the crayfish Cherax destructor

Inhibitory neurons exert control the expression of many aspects of behaviour by regulating the effectiveness of excitatory neural function. By comparison with excitatory neural systems, relatively little is known about the development of inhibitory neurons and the influence which these neurons exert on the development of other neural systems. Two issues which relate to the development of inhibitory neurons are of particular interest. First, a paradox arises when inhibitory neurons are considered in terms of modern models of synaptic development which involve activity-dependent mechanisms of synaptic plasticity. Second, there is some evidence that inhibitory neurotransmitters may act in a special trophic manner during the early development of nervous systems. Investigations of these issues would be greatly facilitated in a neural system in which it was possible to experimentally control aspects of the development of individual pre- and postsynaptic cells. The aim of the results presented in this thesis was to characterise the normal development of one such system: the GABAergic inhibitory system of the Australian freshwater crayfish, Cherax destructor. The ontogeny of the inhibitory neurotransmitter GABA across the embryonic period of 30% to 100% development was investigated using immunohistochemical techniques. GABA-like immunoreactive cells and fibres were first detected in the embryonic brain region. The expression of GABA-like immunoreactivity progressed along a rostro-caudal gradient, with GABA-like immunoreactive cells being detected in the most anterior thoracic ganglia at 45% development and in all ganglia by 65% development. GABA-like immunoreactive fibres were evident in peripheral nerves as early as 55% development and ramified extensively throughout the neuropil of the nervous system by 65% development. By contrast, immunoreactivity to the primary excitatory neurotransmitter, glutamate, was not detected until 60-65% development. Glutamate-like immunoreactivity at 60-65% development was evident only in the form of punctate staining in the midline of the ventral nerve cord. Cell body staining was observed only at 90% development and was restricted to only a few cells on the periphery of the ventral nerve cord. Radio-labelled ligand binding methods and autoradiography were used to study the expression of putative GABA receptors in the Cherax embryos from 30% to 100% development. Specific binding was evident in the earliest embryos studies at 30% development. There was an initial increase in binding from 30% to 40% development, followed by a dramatic drop to almost zero binding at 50-55% development. This was followed by a gradual increase in binding levels with age, reaching a plateau at 85% development. Preliminary pharmacological evaluation of binding indicated that at least three GABA receptor types were expressed during embryonic development. Methods for culturing, dissociated neural tissues explanted form Cherax embryos at 85% development were established. The success of cultures was demonstrated by neurite extension, and neuronal networks in which neurons appeared to form connections with other neurons and with explanted muscle cells after two days in culture. Immunohistochemical studies demonstrated that some explanted neurons expressed GABA-like immunoreactivity within two days of explanting. These studies have provided a comprehensive description of the development of GABAergic neurons and their receptors in Cherax destructor embryos. The very early expression of GABA-like immunoreactivity, coupled with the early onset of specific GABA binding, strongly indicates that the GABAergic neurons are functional and able to exert an effect on other cells during much of the period of nervous system development in crayfish embryos. These results support the hypothesis that inhibitory neurons may play an important role as regulators of the overall process of assembly and maturation of the nervous system and provide a substantial basis for future experimental studies in which the specific action of inhibitory neurons on the development of discrete components of the crayfish nervous system may be investigated.

Molecular systematic studies of freshwater crayfish of the the genus Cherax

This study investigated the molecular systematics and zoogeography of freshwater crayfish species (yabbies) of the genus Cherax throughout Australia. Using DNA sequencing of up to five gene regions and phylogenetic methods of analyses, this study provided new insights into the taxonomy and evolution of this important group of Australian freshwater invertebrates.

Ecology of the freshwater crayfish of the Grampians National Park

The ecology of five species of freshwater crayfish occurring in a variety of habitat types in the Grampians National Park was investigated. The study examined the numbers of each species living in the region, how they interact, when they reproduce, and what they eat. Management and conservation requirements were proposed.

Seasonal variations of total lipid and fatty acid contents in muscle, gonad and digestive glands of farmed jade tiger hybrid abalone in Australia

The lipid and fatty acid (FA) contents of muscle, gonad and digestive glands (DG) of Jade Tiger hybrid abalone were studied over the four seasons. Higher contents of total lipid and saturated fatty acids (SFA) were found in summer from muscle. For gonad the higher total lipid content was found in summer and spring whereas the SFA content peaked in summer only. For DG the higher contents of total lipid and SFA were recorded in all seasons except autumn. Winter samples showed significantly higher content of PUFA in all three types of tissue. High contents of eicosapentaenoic acid (EPA, 20:5 n−3), docosapentaenoic acid (DPA, 22:5 n−3) and docosahexaenoic acid (DHA, 22:6 n−3) were recorded in winter from muscle, although no marked variations were observed from gonad. For DG the high content of DHA was also observed in winter whilst EPA and DPA maintained high levels in all seasons except summer.

Ecological notes on the East Gippsland burrowing crayfish Engaeus orientalis, including burrow structure and associated fauna

Despite Australia having a high diversity of freshwater crayfish species, the ecology of many of these species remains poorly known, particularly burrowing crayfish of the genus Engaeus. Biological information on colour, behaviour, burrow structure, associated burrow fauna and habitat of the East Gippsland Burrowing Crayfish Engaeus orientalis obtained during incidental observations in 2007 and 2008 is provided. The burrow structure took the form of radiating runways under a rock slab, while the burrow location was in a semi-disturbed site away from water. Both are atypical for this species.

Development of twenty-four novel microsatellite markers for the freshwater crayfish, Geocharax gracilis, using next generation sequencing

The crayfish Geocharax gracilis is an important inhabitant of natural and agricultural drainage systems in south-eastern Australia. To investigate population structure, genetic diversity and patterns of connectivity in natural and human-altered ecosystems, we isolated and characterised 24 microsatellite loci using next generation sequencing. Loci were initially tested for levels of variation based on 12 individuals from across the species’ geographical range. A further 33 individuals from a single population were used to test for departures from Hardy–Weinberg equilibrium and linkage disequilibrium. We detected high to moderate levels of genetic variation across most loci with a mean allelic richness of 8.42 and observed heterozygosity of 0.629 (all samples combined). We found no evidence for linkage disequilibrium between any loci and only three loci (Geo01, Geo24 and Geo47) showed significant deviations from Hardy–Weinberg expectations. These same three loci, plus two additional loci (Geo06 and Geo28), also showed the presence of null alleles. These 24 variable markers will provide an important tool for future population genetic assessments in natural and human altered environments.