Critically low levels of genetic diversity in fragmented populations of the endangered Glenelg spiny freshwater crayfish Euastacus bispinosus

The Glenelg spiny freshwater crayfish Euastacus bispinosus is a large endangered freshwater invertebrate of southeastern Australia that has suffered major population declines over the last century. Disjunct populations in the state of South Australia are in a particularly critical condition, restricted to a few isolated rising-spring habitats and in an ongoing state of decline. We assessed genetic diversity and gene flow within E. bispinosus across its current range using allele frequencies from 11 nuclear microsatellite loci and DNA sequence data from a single mitochon -drial locus (cytochrome oxidase subunit I). Populations were characterized by low levels of genetic diversity and found to be highly structured, with gene flow restricted both within and across catchments, highlighting the species' vulnerability to further habitat fragmentation and the importance of managing environmental threats on local scales across its current natural range. South Australian populations were characterized by critically low levels of genetic diversity generally, highlighting their potential vulnerability to localized extinction. Holistic conservation efforts are necessary to conserve populations, including local habitat management and, potentially, translocations to increase genetic diversity and evolutionary potential, and reduce possible inbreeding effects and the threat of extinction.

The development of 10 novel polymorphic microsatellite markers through next generation sequencing and a preliminary population genetic analysis for the endangered Glenelg spiny crayfish, Euastacus bispinosus

The Glenelg spiny crayfish, Euastacus bispinosus, is an iconic freshwater invertebrate of south eastern Australia and listed as 'endangered' under the Environment Protection and Biodiversity Conservation Act 1999, and 'vulnerable' under the International Union for Conservation of Nature's Red List. The species has suffered major population declines as a result of over-fishing, low environmental flows, the introduction of invasive fish species and habitat degradation. In order to develop an effective conservation strategy, patterns of gene flow, genetic structure and genetic diversity across the species distribution need to be clearly understood. In this study we develop a suite of polymorphic microsatellite markers by next generation sequencing. A total of 15 polymorphic loci were identified and 10 characterized using 22 individuals from the lower Glenelg River. We observed low to moderate genetic variation across most loci (mean number of alleles per locus = 2.80; mean expected heterozygosity = 0.36) with no evidence of individual loci deviating significantly from Hardy-Weinberg equilibrium. Marker independence was confirmed with tests for linkage disequilibrium, and analyses indicated no evidence of null alleles across loci. Individuals from two additional sites (Crawford River, Victoria; Ewens Ponds Conservation Park, South Australia) were genotyped at all 10 loci and a preliminary investigation of genetic diversity and population structure was undertaken. Analyses indicate high levels of genetic differentiation among sample locations (F ST = 0.49), while the Ewens Ponds population is genetically homogeneous, indicating a likely small founder group and ongoing inbreeding. Management actions will be needed to restore genetic diversity in this and possibly other at risk populations. These markers will provide a valuable resource for future population genetic assessments so that an effective framework can be developed for implementing conservation strategies for E. bispinosus.

Genetic analyses reveal limited dispersal and recovery potential in the large freshwater crayfish Euastacus armatus from the southern Murray-Darling Basin

Understanding dispersal traits and adaptive potential is critically important when assessing the vulnerability of freshwater species in highly modified ecosystems. The present study investigates the population genetic structure of the Murray crayfish Euastacus armatus in the southern Murray–Darling Basin. This species has suffered significant population declines in sections of the Murray River in recent years, prompting the need for information on natural recruitment processes to help guide conservation. We assessed allele frequencies from 10 polymorphic microsatellite loci across 20 sites encompassing the majority of the species’ range. Low levels of gene flow were observed throughout hydrologically connected waterways, but significant spatial autocorrelation and low migration rate estimates reflect local genetic structuring and dispersal limitations, with home ranges limited to distances <50-km. Significant genetic differentiation of headwater populations upstream of barriers imposed by impoundments were also observed; however, population simulations demonstrate that these patterns likely reflect historical limitations to gene flow rather than contemporary anthropogenic impacts. Dispersal limitations, coupled with its biological traits, suggest that local populations are vulnerable to environmental disturbance with limited potential for natural recolonisation following population decline. We discuss the implications of these findings in the context of managing the recovery of the species.

Genetic diversity and translocation in the marron, Cherax tenuimanus (Smith): implications for management and conservation

Approximately 440 base pairs (bp) of the mitochondrial 16S ribosomal ribose nucleic acid (rRNA) coding region were sequenced from 13 marron, Cherax tenuimanus (Smith), samples from six locations and samples of two additional Cherax species from Western Australia. The results indicated that, with the exception of the Margaret River, no variation was found within or between marron populations. In contrast, marron from the Margaret River were found to be polymorphic for two divergent haplotypes (2.76% divergence). These findings were consistent with allozyme data that highlight the general lack of genetic variability within and between populations of this species apart from the Margaret River stocks. The genetic polymorphisms in the Margaret River stocks contrasted with earlier studies and indicated the recent translocation and mixing of genetically differentiated stocks within this river system. The implications of these findings for the conservation and management of genetic diversity within marron are discussed.

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.

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.

Endogenous production of endo-β-1,4-glucanase by decapod crustaceans

The potential ability to produce cellulase enzymes endogenously was examined in decapods crustaceans including the herbivorous gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes, the amphibious freshwater crab Austrothelphusa transversa, the terrestrial hermit crab, Coenobita variabilis the parastacid crayfish Euastacus, and the crayfish Cherax destructor. The midgut gland of both G. natalis and D. hirtipes contained substantial total cellulase activities and activities of the cellulase enzymes endo-β-1,4-glucanase and β-glucosidase. With the exception of total cellulase and β-glucosidase from D. hirtipes, the enzyme activities within the midgut gland were higher than those within the digestive juice. Hence, the enzyme activities appear to reside predominantly within midgut gland, providing indirect evidence for endogenous synthesis of cellulase enzymes by this tissue. A 900 bp cDNA fragment encoding a portion of the endo-β-1,4-glucanase amino acid sequence was amplified by RT-PCR using RNA isolated from the midgut gland of C. destructor, Euastacus, A. transversa and C. variabilis. This provided direct evidence for the endogenous production of endo-β-1,4-glucanase. The 900 bp fragment was also amplified from genomic DNA isolated from the skeletal muscle of G. natalis and D. hirtipes, clearly indicating that the gene encoding endo-β-1,4-glucanase is also present in these two species. As this group of evolutionary diverse crustacean species possesses and expresses the endo-β-1,4-glucanase gene it is likely that decapod crustaceans generally produce cellulases endogenously and are able to digest 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.

Control of ovarian development in the yabby (Cherax destructor)

A study under controlled conditions of ovarian development and rematuration in the yabby (Cherax destructot) was undertaken. The purpose of the study was to improve fundamental understanding of the reproductive biology of the species and provide a basis for application to hatchery management in culture. A review was made of the current status of yabby culture in Australia and the present understanding of reproductive biology of decapod Crustacea. The review emphasised factors controlling several aspects of ovarian development, in particular the processes of vitellogenesis. The subsequent study was designed within the context of current hatchery practice and was based on existing knowledge of decapod reproduction, The sexual differentiation of the yabby after hatching was investigated by serial histological sections, and experiments were carried out to investigate the possibility of sex reversal of males. Most of this Investigation was concerned with removing the influence of the androgenic gland in directing male development, with the intent of observing the development of the elementary gonadal tissue into ovary. It was found that in contrast to other crustacean species, the sex of the yabby becomes fixed before the development of external secondary sexual characteristics, and before the androgenic gland can be discerned. Ovarian tissue developed in females at less than 8 weeks after hatching. A preliminary examination was undertaken for feminising parasites in gonadal tissue of a hermaphrodite yabby. Investigation of the ovary after spawning demonstrated that whilst the female was held under constant conditions of temperature and photoperiod, little rematuration occurred. Except for generation of previtellogenic oocytes during the first two days, the gonaciosomatic index remained low for up to 5 months after spawning. If the temperature of the female was reduced to 10°C and maintained constant, the previtellogenic oocytes were partially resorbed over a three week period. Rematuration then commenced, albeit at a low rate because of the reduced temperature, A method for standardising gonadosomatic indices was developed which took into account differences in hepatopancreatic nutrient reserves of individuals and loss of one or more appendages. This part of the study also considered constraints to rematuration and developed a method of accounting for differences in the ability of females to remature after spawning. Experiments were carried out to investigate the effect of crowding and temperature manipulation on initiating ovarian rematuration and to determine the rate of rematuration at 22°C once initiated. The duration of low temperature had no effect on rematuration; an overnight cooling was sufficient to initiate the process, Rematuration to the end of stage 2 vltellogenesis was substantially complete within 10 days. Crowding of females suppressed rematuration, but less than ideal water quality was not found to have any effect. The presence of a male initiated rematuration at a similar rate, but also led to stage 3 vitetlogenesis and spawning. A study was made of the pheromonal influence of the male through water borne factors without success. Rematuration could not be induced in ovigerous females. The literature review indicated that ovarian rematuration was under the control of an ovary stimulating hormone produced by the thoracic nerve ganglia. Attempts were therefore made to stimulate ovarian rematuration by incorporating the thoracic nerve into the diet of females. Attempts were also made to induce the release of ovary stimulating hormone from the thoracic nerve with 5-hydroxytryptamine, and also with octopamine. No effects were found, but a significant difference between the neurophysiology of the yabby and northern hemisphere crayfish was observed, and the implications of this finding are discussed. The study did not produce any conclusive evidence of an ovary stimulating hormone for the yabby. A model of ovarian rematuration which collects the findings of the experimental investigations was developed, and was used to suggest a hatchery broodstock management protocol. This model differs from existing models in that rematuration triggers and nutritional status are considered.

Improving ingestion of artficial diets by Cherax quadricarinatus Von Martens (Decapoda ; Parastacidae)

Several dietary binding agents were evaluated to produce an artificial moist diet which significantly improved water stability and ingestion efficiency, by redclaw crayfish, by up to 60%. Also behavioural feeding repertoire was described for the first time for this species. A bio-assay was then utilised to compare, and make recommendations on, various feed attractants.

Crustacean mitogenomics and arthropod systematics

Substantial new DNA data were obtained by sequencing the mitochondrial genomes of four crustacean species, resulting in the discovery of a novel gene order in freshwater crayfish. Investigation of evolutionary relationships using mitochondrial genomes challenged established theories of crustacean evolution and diversification in relation to the other major Arthropod groups.

Dynamics of endogenous food production in Cherax destructor Clark culture ponds

This study examined endogenous food production in crayfish culture ponds and the pond dynamics resulting from the interactions of pond bottom and water column parameters under a crop flooding strategy.

Genetic studies on Cherax destructor Clark in relation to aquaculture

Investigated three strains of the common freshwater crayfish (yabby), to determine the potential for genetic improvement of this species for aquaculture. Crossbreeding generated viable progeny and differences were found between strains in reproductive performance, sex ratios, morphology and overall genetic divergence measured by DNA sequencing.

Population structure and life history characteristics of euastacus bispinosus and cherax destructor (Parastacidae) in the Grampians National Park, Australia

Despite being the most widely distributed and cultured freshwater crayfish species in Australia, relatively little is known about the ecology of Cherax destructor outside of captivity. Similarly, few ecological studies have been conducted on the large and threatened stream dwelling Australian freshwater crayfish Euastacus bispinosus. A series of seasonal sampling surveys over two years investigated the population structure, life history and reproductive timing of C. destructor in fire dam habitats, and of E. bispinosus in channel (stream) habitats, in the Grampians National Park in south-west Victoria, Australia. Cherax destructor individuals in the largest size class (50 – 59.95 mm OCL) were not abundant during the study, while those belonging to the 0 – 9.95 mm OCL size class were more frequent in summer than at other times of year, suggesting synchronous recruitment in fire dam habitats. Individuals in the reproductively active mid-size classes were also frequent in summer. For E. bispinosus, sex ratios in spring always favoured females, although there were no clear trends for other times of year. Gravid E. bispinosus females were found in winter and spring throughout the study, and were sexually mature at a smaller size than has previously been reported.

Individual and sex-specific differences in intrinsic growth rate covary with consistent individual differences in behaviour

1.The evolutionary causes of consistent individual differences in behavior are currently a source of debate. A recent hypothesis suggests that consistent individual differences in life-history productivity (growth and/or fecundity) may covary with behavioral traits that contribute to growth-mortality trade-offs, such as risk-proneness (boldness) and foraging activity (voraciousness). It remains unclear, however, to what extent individual behavioral and life-history profiles are set early in life, or are a more flexible result of specific environmental or developmental contexts that allow bold and active individuals to acquire more resources. 2.Longitudinal studies of individually housed animals under controlled conditions can shed light on this question. Since growth and behaviour can both vary within individuals (they are labile), studying between-individual correlations in behaviour and growth rate requires repeated scoring for both variables over an extended period of time. However, such a study has not yet been done. 3.Here, we repeatedly measured individual mass 7-times each, boldness 40-times each, and voracity 8-times each during the first four months of life on 90 individually-housed crayfish (Cherax destructor). Animals were fed ad-libitum, generating a context where individuals can express their intrinsic growth rate (i.e. growth capacity), but in which bold and voracious behaviour is not necessary for high resource acquisition (crayfish can and do hoard food back to their burrow). 4.We show that individuals that were consistently bold over time during the day were also bolder at night, were more voracious, and maintained higher growth rates over time than shy individuals. Independent of individual differences, we also observed that males were faster growing, bolder, and more voracious than females. 5.Our findings imply that associations between bold behaviour and fast growth can occur in unlimited food contexts where there is no necessary link between bold behaviour and resource acquisition - offering support for the 'personality- productivity' hypothesis. We suggest future research should study links between consistent individual differences in behaviour and life-history under a wider range of contexts, in order to shed light on the role of biotic and abiotic conditions in the strength, direction and stability of their covariance. This article is protected by copyright. All rights reserved.