Preliminary study on the use of synthetic substrate for juvenile stage production of the yabby, Cherax destructor (Clark) (Decapoda: Parastacidae)

Juvenile Cherax destructor (commonly called the yabby) (mean weight 48.3 mg) were cultured intensively (stocking density 360/m²) under controlled conditions for 48 days. The animals were provided with a combination of food (high protein pellets and/or natural feed organisms attached to a conditioned synthetic substrate) and refuge. Fastest growth and highest yield was recorded when both pellets and the conditioned synthetic material were provided. Although the yabbies sheltered in the synthetic substrate, it did not increase survival. Juvenile yabbies (< 200 mg) were able to graze on small organisms attached to the synthetic material but this ability appeared to decline as the yabbies grew to a larger size. The use of artificial substrates in the intensive nursery phase production of juvenile freshwater crayfish is discussed.

Utility of mitochondrial DNA sequences from four gene regions for systematic studies of Australian freshwater crayfish of the Genus Cherax (Decapoda: Parastacidae)

One of the most important requirements for systematic and phylogenetic studies is the identification of gene regions with the appropriate level of variation for the question of interest. Molecular phylogenetic and systematic studies of freshwater crayfish have made use of DNA sequences mainly from ribosomal genes, especially the 16S rRNA gene region. Thus, little information is available on other potentially useful mitochondrial gene regions for systematic studies in these animals. In this study, we look at nucleotide variation and phylogenetic relations within and between four species of freshwater crayfish of the genus Cherax from the southwest of Western Australia using four fragments amplified from the 16S rRNA, 12S rRNA, Cytochrome Oxidase I (COI), and Cytochrome b (Cyt b) gene regions. Samples of Engaeus strictifrons, Euastacus bispinosus, and Geocharax falcata were also sequenced for comparative purposes. The size of the fragments varied from 358 bp to 600 bp. Across all samples, the four fragments showed significant phylogenetic signal and showed similar proportions of variable sites (28.81–37.33%). Average divergence within species for the mitochondrial gene regions varied from 1.18% to 4.91%, with the 16S rRNA being the least variable and Cyt b the most variable. Average divergence between species ranged 7.63–15.53%, with 16S rRNA being the least variable and COI the most variable. At the generic level, average divergence ranged 17.21–23.82%. Phylogenetic analyses of the 16S rRNA, 12S rRNA, and COI regions generated four clades consistent with the presence of four species previously identified on the basis of allozyme and morphological studies. The relationships among samples were largely congruent across the data set, although some relationships remained unresolved. Not all samples could be amplified using the Cyt b primers, and some of those that were showed quite anomalous relationships, suggesting that one or more Cyt b pseudogenes were being amplified.

The taxonomy and phylogeny of the Cherax destructor complex (Decapoda : Parastacidae) examined using mitochondrial 16S sequences

This study uses nucleotide sequences from the 16S rRNA mitochondrial gene to investigate the taxonomy and phylogeny of freshwater crayfish belonging to the 'Cherax destructor' complex. The sequencing of an approximately 440-bp fragment of this gene region from freshwater crayfish sampled from 14 locations identified significant haplotype diversity. Phylogenetic analysis found three distinct clades that correspond to the species C. rotundus, C. setosus and C. destructor. C. rotundus is largely confined to Victoria, and C. setosus is restricted to coastal areas north of Newcastle in New South Wales. C. destructor is widely distributed in eastern Australia and shows significant phylogeographic structure, with three well supported clades. None of these clades, however, correspond to species previously recognised as C. esculus, C. davisi or C. albidus. The failure to genetically distinguish these morphologically defined species is consistent with reproductive information and morphological plasticity relating to habitat similar to that documented for other Cherax species.

Complete mitochondrial DNA sequence of the Australian freshwater crayfish, Cherax destructor (Crustacea: Decapoda: Parastacidae): a novel gene order revealed

The complete mitochondrial DNA sequence was determined for the Australian freshwater crayfish Cherax destructor (Crustacea: Decapoda: Parastacidae). The 15,895-bp genome is circular with the same gene composition as that found in other metazoans. However, we report a novel gene arrangement with respect to the putative arthropod ancestral gene order and all other arthropod mitochondrial genomes sequenced to date. It is apparent that 11 genes have been translocated (ND1, ND4, ND4L, Cyt b, srRNA, and tRNAs Ser(UGA), Leu(CUN), Ile, Cys, Pro, and Val), two of which have also undergone inversions (tRNAs Pro and Val). The ‘duplication/random loss’ mechanism is a plausible model for the observed translocations, while ‘intramitochondrial recombination’ may account for the gene inversions. In addition, the arrangement of rRNA genes is incompatible with current mitochondrial transcription models, and suggests that a different transcription mechanism may operate in C. destructor.

Molecular phylogeny and zoogeography of the freshwater crayfish genus Cherax Erichson (Decapoda: Parastacidae) in Australia

The evolutionary history and biogeography of freshwater-dependent taxa in Australia is of intrinsic interest given the present-day aridity of this continent. Cherax is the most widespread and one of the most species-rich of Australia's nine freshwater crayfish genera. The phylogenetic relationships amongst 19 of the 23 Australian Cherax were established from mitochondrial DNA sequences representing the 12S rRNA and 16S rRNA gene regions. The relationships among species support an initial east–west separation, followed by a north–south divergence in eastern Australia. Molecular clock estimations suggest that these divergences date back to the Miocene. The phylogenetic relationships support endemic speciation within geographical regions and indicate that long-distance dispersal has not led to recent speciation as previously hypothesized. This new evolutionary scenario is consistent with the climatic history of Australia and the evolutionary history of other similarly distributed freshwater-dependent organisms in Australia.

Inheritance of molecular markers and sex in the Australian freshwater crayfish, Cherax destructor Clark

Inheritance of three kinds of molecular genetic markers (mtDNA, random-amplified polymorphic DNAs (RAPDs) and allozymes) and sex were investigated in crossbreeding experiments between three populations of the Australian freshwater crayfish Cherax destructor. Crossbreeding did not disrupt the ively maternally inherited, and allozyme and RAPD markers were transmitted following expected Mendelian principles for co-dominant and dominant traits respectively. Unlike these three markers, sex ratios were found to be distorted by crossbreeding in some families. Two crossbred families produced only females. The implications of these findings for freshwater crayfish population genetics, taxonomy and aquaculture are discussed.

The systematics of freshwater crayfish of the genus Cherax Erichson (Decapoda : Parastacidae) in eastern Australia re-examined using nucleotide sequences from 12S rRNA and 16S rRNA genes

Nucleotide sequence data were used to re-examine systematic relationships and species boundaries within the genus Cherax from eastern Australia. Partial sequences were amplified from the 12S (~365 bp) and 16S (~545 bp) rRNA mitochondrial gene regions. Levels of intra- and inter-specific divergence for Cherax species were very similar between the two gene regions and similar to that reported for other freshwater crayfish for 16S rRNA. Phylogenetic analyses using the combined data provided strong support for a monophyletic group containing 11 eastern Australian species and comprising three well-defined species-groups: the 'C. destructor' group containing three species, the 'C. cairnsensis' group containing four species and the 'C. cuspidatus' group containing two species. Cherax dispar and C. robustus are distinct from all other species and each other. In addition, two northern Australian and a New Guinean species were placed in the 'Astaconephrops' group, which is the sister-group to the eastern Australian Cherax lineage. Several relationships were clarified, including: the status of northern and southern C. cuspidatus as separate species; a close relationship between C. cairnsensis and C. depressus; the validity of C. rotundus and C. setosus as separate species and their close affinities with C. destructor; and the distinctiveness of the northern forms of Cherax. The analysis of the 12S rRNA and 16S rRNA data is highly concordant with the results of previous allozyme studies.

Phylogeny of the Australian freshwater crayfish Cherax destructor-complex (Decapoda:Parastacidae) inferred from four mitochondrial gene regions

The phylogenetic relationships among 32 individuals of Australian freshwater crayfish belonging to the Cherax destructor-complex were investigated using a dataset comprising sequences from four mitochondrial gene regions: the large subunit rRNA (16S rRNA), cytochrome oxidase I (COI), adenosine triphosphatase 6 (ATPase 6), and cytochrome oxidase III (COIII). A total of 1602 bp was obtained, and a combined analysis of the data produced a tree with strong support (bootstrap values 94–100%) for three divergent lineages, verifying the phylogenetic hypotheses of relationships within the C. destructor species-complex suggested in previous studies. Overall, sequences from the 16S rRNA gene showed the least variation compared to those generated from protein coding genes, which presented considerably greater levels of divergence. The level of divergence within C. destructor was found to be greater than that observed in other species of freshwater crayfish, but interspecific variation among species examined in the present study was similar to that reported previously.

Population genetic studies on Australian freshwater crayfish, Cherax destructor (Crustacea: Parastacidae) using allozyme and RAPD markers

Allozyme and Random Amplified Polymorphic DNA (RAPD) variation was surveyed in the freshwater crayfish Cherax destructor Clark, an ecologically and commercially important species that is widespread throughout the freshwater systems of central Australia. At the intra-population level, allozymes revealed a similar level of variation to that found in other freshwater crayfish; RAPDs showed less diversity than allozymes, which was unexpected. At the inter-population level, both techniques revealed significant population structure, both within and between drainages. RAPD results were consistent with phylogeographic patterns previously identified using mtDNA. Although allozyme data showed little geographic pattern in relation to genetic variation based on multidimensional-scaling (MDS) plots on matrices of genetic distance, results of AMOVA and Mantel tests indicated significant population structuring. Each of the mtDNA lineages proposed in a previous study also showed significant genetic structure at similar levels as revealed by RAPDs but different levels by allozymes. These results reject hypotheses previously put forward on genetic homogenisation within the species due to wide-scale translocation. The implications of the findings for conservation and aquaculture of C. destructor are also discussed.

Total arsenic accumulation in yabbies (Cherax Destructor Clark) exposed to elevated arsenic levels in Victorian gold mining areas, Australia

Arsenic is a proven carcinogen often found at high concentrations in association with gold and other heavy metals. The freshwater yabby, Cherax destructor Clark (Decapoda, Parastacidae), is a ubiquitous species native to Australia's central and eastern regions, with a growing international commercial market. However, in this region of Australia, yabby farmers often harvest organisms from old mine tailings dams with elevated environmental arsenic levels. Yabbies exposed to elevated environmental arsenic were found to accumulate and store as much as 100 μg/g arsenic in their tissues. The accumulation is proportional to the concentration of arsenic in the sediment and is high enough to be of concern for people who eat the yabbies. A comparison of arsenic levels in wild and lab-fed animals also was performed. Although there was no significant difference in the level of arsenic in the various organs of the wild animals, the animals purchased from a yabby farm showed a significantly higher arsenic concentration in their hepatopancreas (3.7 ± 0.9 μg/g) compared to other organs (0.6–1.8 μg/g). Furthermore, after a 40-d exposure to food containing 200 to 300 μg/g inorganic arsenic, arsenate (As[V])-exposed animals showed a significant increase in tissue-specific arsenic accumulation, whereas arsenite (As[III])-exposed animals showed a lower, nonsignificant increase in As uptake, primarily in the hepatopancreas. These results have important implications for yabby growers and consumers alike.

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.

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-β-1,4-glucanase, β-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-β-1,4-glucanase and β-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-β-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.

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.