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.

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).

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.

Constraints upon the response of fish and crayfish to environmental flow releases in a regulated headwater stream network

In dry climate zones, headwater streams are often regulated for water extraction causing intermittency in perennial streams and prolonged drying in intermittent streams. Regulation thereby reduces aquatic habitat downstream of weirs that also form barriers to migration by stream fauna. Environmental flow releases may restore streamflow in rivers, but are rarely applied to headwaters. We sampled fish and crayfish in four regulated headwater streams before and after the release of summer-autumn environmental flows, and in four nearby unregulated streams, to determine whether their abundances increased in response to flow releases. Historical data of fish and crayfish occurrence spanning a 30 year period was compared with contemporary data (electrofishing surveys, Victoria Range, Australia; summer 2008 to summer 2010) to assess the longer-term effects of regulation and drought. Although fish were recorded in regulated streams before 1996, they were not recorded in the present study upstream or downstream of weirs despite recent flow releases. Crayfish (Geocharax sp. nov. 1) remained in the regulated streams throughout the study, but did not become more abundant in response to flow releases. In contrast, native fish (Gadopsis marmoratus, Galaxias oliros, Galaxias maculatus) and crayfish remained present in unregulated streams, despite prolonged drought conditions during 2006-2010, and the assemblages of each of these streams remained essentially unchanged over the 30 year period. Flow release volumes may have been too small or have operated for an insufficient time to allow fish to recolonise regulated streams. Barriers to dispersal may also be preventing recolonisation. Indefinite continuation of annual flow releases, that prevent the unnatural cessation of flow caused by weirs, may eventually facilitate upstream movement of fish and crayfish in regulated channels; but other human-made dispersal barriers downstream need to be identified and ameliorated, to allow native fish to fulfil their life cycles in these headwater streams.

Effect of intermittent feeding on growth in early juveniles of the crayfish Cherax quadricarinatus

Three experiments were conducted with juveniles of the crayfish Cherax quadricarinatus to investigate the effect of intermittent feeding regimes on growth and the ability to tolerate the shortage of food. In experiment 1, stage III juveniles were assigned to one of seven intermittent feeding groups (from FS1: 1 day fed/1 day non-fed to FS7: 7 days fed/7 days non-fed) and two control groups, continuously fed (CF) and continuously starved (CS) animals; this experiment comprised a short-term intermittent feeding period until the first molt, followed by a continuous feeding period. In the experiment 2, stage III juveniles were assigned to one of three intermittent feeding groups (FS2 to FS4) and one control group (CF); it consisted of a prolonged intermittent feeding period, until the end of the experiment In the experiment 3, stage VI and VII juveniles were assigned to one of three intermittent feeding groups (FS2 to FS4) and one control (CF); it also consisted of a prolonged intermittent feeding period. The red claw crayfish juveniles were able to tolerate periods of intermittent feeding and underwent compensatory growth after continuous feed was re-established. The ability of crayfish to tolerate intermittent feeding was influenced by developmental stage and duration of the intermittent feeding period. Stage III juveniles survived, but decreased growth, when subjected to prolonged intermittent feeding. However, they showed full compensatory growth when the intermittent feeding period was short and followed by continuous feeding. on the other hand, stage VI-VII tolerated 60 days of prolonged intermittent feeding without any change in growth and survival. The hepatosomatic index (based on wet weight) values of the treatments and the control were similar, suggesting that intermittent feeding may not be considered a nutritional stress condition. The relative pleon weight (based on wet weight) values of the treatments and control were similar suggesting low use of nutrients from the muscle to increase the chance for survival. The juveniles of C quadricarinatus can tolerate relatively long periods of low food availability and this is an important adaptation for their survival in changing/unpredictable environments and an attribute favorable for the production of the species. (C) 2011 Elsevier B.V. All rights reserved.

EFFECT OF FOOD SHORTAGE ON GROWTH, ENERGETIC RESERVES MOBILIZATION, AND WATER QUALITY IN JUVENILES OF THE REDCLAW CRAYFISH, CHERAX QUADRICARINATUS, REARED IN GROUPS

The aim of this study is to analyze the impact of food shortage on growth performance, by means of energetic reserves (proteins, glycogen and lipids) mobilization and hepatopancreas cells analysis in C. quadricarinatus juveniles maintained in groups, as well as the effect on culture water quality. Two experiments were performed, each of them with two feeding regimes during 45 days. The Control feeding regime, in which crayfish were fed daily (once a day) throughout the experimental period (DF), and the Cyclic feeding regime, in which juveniles were fed for 2 or 4 days (once a day) followed by 2 or 4 days of food deprivation (2F/2D and 4F/4D, respectively) in repeated cycles. Cyclic feeding influenced growth, biochemical composition from hepatopancreas and muscle, and water quality. Juveniles cyclically fed were unable to maintain a normal growth trajectory during 45 days. Apparent feed conversion ratio, apparent protein efficiency ratio, hepatosomatic index and relative pleon mass were similar in cyclic and daily fed animals and no structural damage was found in the hepatopancreas of juveniles subjected to cyclic feeding. The novelty of this study was the significant accumulation of proteins in pleonal muscle in both cyclic feeding regimes (approx. 18%) suggesting that the storage of this constitutive material during food shortage may be an adaptation for a compensatory growth when food becomes abundant again. The cyclic feeding regimes had a positive effect on water quality decreasing inorganic nitrogen concentration. This was due to the reduction in the amount of animal excretes and feces in the group that received approx. 50% less feed. Additionally, water pH was higher in cyclic feeding tanks, as a result of lower organic matter decomposition and consequent release of CO2. Accordingly, total ammonia in the water was significantly lower for the cyclic feeding regimes compared to their respective controls. This study suggests that the protocol of cyclic feeding could be applied at least 45 days in 1 g juveniles maintained in group conditions, without affecting the energetic reserves and hepatopancreas structure, emphasizing the high tolerance of this species to food restriction.

The crayfish plasma clotting protein: A vitellogenin-related protein responsible for clot formation in crustacean blood

Coagulation in crayfish blood is based on the transglutaminase-mediated crosslinking of a specific plasma clotting protein. Here we report the cloning of the subunit of this clotting protein from a crayfish hepatopancreas cDNA library. The ORF encodes a protein of 1,721 amino acids, including a signal peptide of 15 amino acids. Sequence analysis reveals that the clotting protein is homologous to vitellogenins, which are proteins found in vitellogenic females of egg-laying animals. The clotting protein and vitellogenins are all lipoproteins and share a limited sequence similarity to certain other lipoproteins (e.g., mammalian apolipoprotein B and microsomal triglyceride transfer protein) and contain a stretch with similarity to the D domain of mammalian von Willebrand factor. The crayfish clotting protein is present in both sexes, unlike the female-specific vitellogenins. Electron microscopy was used to visualize individual clotting protein molecules and to study the transglutaminase-mediated clotting reaction. In the presence of an endogenous transglutaminase, the purified clotting protein molecules rapidly assemble into long, flexible chains that occasionally branch.