Differential effects of arginine, glutamate and phosphoarginine on Ca2+-activation properties of muscle fibres from crayfish and rat

The effects of two amino acids, arginine which has a positively charged side-chain and glutamate which has a negatively charged side-chain on the Ca²⁺-activation properties of the contractile apparatus were examined in four structurally and functionally different types of skeletal muscle; long- and short-sarcomere fibres from the claw muscle of the yabby (a freshwater decapod crustacean), and fast- and slow-twitch fibres from limb muscles of the rat. Single skinned fibres were activated in carefully balanced solutions of different pCa (-log₁₀[Ca²⁺]) that either contained the test solute (“test”) or not (“control”). The effect of phosphoarginine, a phosphagen that bears a nett negative charge, was also compared to the effects of arginine. Results show that (i) arginine (33-36 mmol l^-1) significantly shifted the force–pCa curve by 0.08–0.13 pCa units in the direction of increased sensitivity to Ca²⁺-activated contraction in all fibre types; (ii) phosphoarginine (9–10 mmol l^-1) induced a significant shift of the force–pCa curve by 0.18–0.24 pCa units in the direction of increased sensitivity to Ca²⁺ in mammalian fast- and slow-twitch fibres, but had no significant effects on the force–pCa relation in either long- or short-sarcomere crustacean fibres; (iii) glutamate (36–40 mmol l^-1), like arginine affected the force–pCa relation of all fibre types investigated, but in the opposite direction, causing a significant decrease in the sensitivity to Ca²⁺-activated contraction by 0.08–0.19 pCa units; (iv) arginine, phosphoarginine and glutamate had little or no effect on the maximum Ca²⁺-activated force of crustacean and mammalian fibres. The results suggest that the opposing effects of glutamate and arginine are not related to simply their charge structure, but must involve complex interactions between these molecules, Ca²⁺ and the regulatory and other myofibrillar proteins.

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.

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.

Arsenic speciation in the freshwater crayfish, Cherax destructor Clark

Arsenic is a proven carcinogen that is found in the soil in gold mining regions at concentrations that can be thousands of times greater than gold. During mining arsenic is released into the environment, easily entering surrounding water bodies.
The yabby (Cherax destructor) is a common freshwater crustacean native to Australia's central and eastern regions. Increasing aquaculture and export of these animals has led us to question the effects of mine contamination on the yabbies themselves and to assess any potential risks to consumers. This study determined the species of arsenic present in a number of organs from the yabby. Several arsenic contaminated dam sites in the goldfields of western Victoria were sampled for yabby populations. Yabbies from these sites were collected and analysed for arsenic speciation using high performance liquid chromatography–inductively coupled plasma-mass spectrometry (HPLC–ICP-MS). Results showed that type of exposure influenced which arsenic species was present in each organ, and that as arsenic exposure increased the prevalence of inorganic arsenic species, mostly As(V), within the tissues increased. The bioaccessibility of the arsenic present in the abdominal muscle (the edible portion for humans) of the yabbies was assessed. It was found that the majority of the bioaccessible arsenic was present as inorganic As (III) and As(V).