Christopher M. Austin

The promise and peril of chemical probes

Chemical probes are powerful reagents with increasing impacts on biomedical research. However, probes of poor quality or that are used incorrectly generate misleading results. To help address these shortcomings, we will create a community-driven wiki resource to improve quality and convey current best practice.

Phylogenetic relationships of the globally distributed freshwater prawn genus Macrobrachium (Crustacea: Decapoda: Palaemonidae): biogeography, taxonomy and the convergent evolution of abbreviated larval development

There has hitherto been little research into evolutionary and taxonomic relationships amongst species of the freshwater prawn genus Macrobrachium Bate across its global distribution. Previous work by the authors demonstrated that the endemic Australian species did not evolve from a single ancestral lineage. To examine whether other regional Macrobrachium faunas also reflect this pattern of multiple origins, the phylogeny of 30 Macrobrachium species from Asia, Central/South America and Australia was inferred from mitochondrial 16S rRNA sequences. Phylogenetic relationships demonstrate that, despite some evidence for regional diversification, Australia, Asia and South America clearly contain Macrobrachium species that do not share a common ancestry, suggesting that large‐scale dispersal has been a major feature of the evolutionary history of the genus. The evolution of abbreviated larval development (ALD), associated with the transition from an estuarine into a purely freshwater lifecycle, was also mapped onto the phylogeny and was shown to be a relatively homoplasious trait and not taxonomically informative. Other taxonomic issues, as well as the evolutionary origins of Macrobrachium, are also discussed.

Phylogenetic relationships among the Australian and New Zealand genera of freshwater crayfishes (Decapoda : Parastacidae)

We sequenced approximately 500 base pairs of DNA from the 16S region of the mitochondrial genome to estimate relationships among the freshwater crayfish genera of Australia and New Zealand. In total, 35 sequences were obtained, representing 32 species and all 10 genera native to Australia and New Zealand. From these sequences, maximum likelihood, minimum evolution and parsimony estimates of phylogenetic relationships among the genera were obtained and compared with previous hypotheses concerning the relationships among the crayfish genera. Our results support the monophyly of each genus (except perhaps Euastacus) and the organisation of these genera into three major clades: the first clade contains the genera Engaeus, Tenuibranchiurus, Geocharax, Gramastacus, and Cherax; the second clade contains the genera Paranephrops, Parastacoides, Euastacus, and Astacopsis; and the third clade contains the genus Engaewa. We reject the ecological hypothesis of Riek for two major clades of crayfish species. Finally, we provide a checklist of the Australian and New Zealand species as they are currently recognised.

Perspectives on validation of high-throughput assays supporting 21st century toxicity testing

In vitro high-throughput screening (HTS) assays are seeing increasing use in toxicity testing. HTS assays can simultaneously test many chemicals but have seen limited use in the regulatory arena, in part because of the need to undergo rigorous, time-consuming formal validation. Here we discuss streamlining the validation process, specifically for prioritization applications. By prioritization, we mean a process in which less complex, less expensive, and faster assays are used to prioritize which chemicals are subjected first to more complex, expensive, and slower guideline assays. Data from the HTS prioritization assays is intended to provide a priori evidence that certain chemicals have the potential to lead to the types of adverse effects that the guideline tests are assessing. The need for such prioritization approaches is driven by the fact that there are tens of thousands of chemicals to which people are exposed, but the yearly throughput of most guideline assays is small in comparison. The streamlined validation process would continue to ensure the reliability and relevance of assays for this application. We discuss the following practical guidelines: (1) follow current validation practice to the extent possible and practical; (2) make increased use of reference compounds to better demonstrate assay reliability and relevance; (3) de-emphasize the need for cross-laboratory testing; and (4) implement a web-based, transparent, and expedited peer review process.

Systematics of the freshwater crayfish genus Cherax Erichson (Decapoda: Parastacidae) in south-western Australia: electrophoretic, morphological and habitat variation

A detailed study of electrophoretic, morphological and habitat variation amongst species of Cherax in south-western Australia supported the recognition of only five of the eight species currently recognised and revealed that morphological and habitat variation within these crayfish is more extensive and complicated than was previously realised. Within several species morphological and habitat variation was found to be as great as that between species. Furthermore, a major component of the morphological variability, both within and between species, was found to be associated with habitat variation. Three of the five species of Cherax recognised in this study correspond to the consistently recognised and widespread species, C. preissii Erichson, C. quinquecarinatus (Gray) and C. tenuimanus Smith. The two other species are C. crassimantus Riek and C. glaber Riek which have restricted distributions in the extreme south-west of Western Australia. The species C. glabrimanus Riek and C. neocarinatus Riek could not be distinguished from C. quinquecarinatus, nor could C. plebejus (Hess) be distinguished from C. preissii. On a general level, the results of this study question the value of morphological information in systematic studies of freshwater crayfish. Morphologically based taxonomic studies of freshwater crayfish need to be interpreted with caution because, firstly, taxonomic characters may be far more variable than realised; secondly, morphological and habitat differences cannot necessarily be equated with specific distinctions; and thirdly, genetically distinct species that occupy similar habitats need not be morphologically distinct.

Integrated shotgun sequencing and bioinformatics pipeline allows ultra-fast mitogenome recovery and confirms substantial gene rearrangements in Australian freshwater crayfishes

BackgroundAlthough it is possible to recover the complete mitogenome directly from shotgun sequencing data, currently reported methods and pipelines are still relatively time consuming and costly. Using a sample of the Australian freshwater crayfish Engaeus lengana, we demonstrate that it is possible to achieve three-day turnaround time (four hours hands-on time) from tissue sample to NCBI-ready submission file through the integration of MiSeq sequencing platform, Nextera sample preparation protocol, MITObim assembly algorithm and MITOS annotation pipeline.ResultsThe complete mitochondrial genome of the parastacid freshwater crayfish, Engaeus lengana, was recovered by modest shotgun sequencing (1.2 giga bases) using the Illumina MiSeq benchtop sequencing platform. Genome assembly using the MITObim mitogenome assembler recovered the mitochondrial genome as a single contig with a 97-fold mean coverage (min. = 17; max. = 138). The mitogenome consists of 15,934 base pairs and contains the typical 37 mitochondrial genes and a non-coding AT-rich region. The genome arrangement is similar to the only other published parastacid mitogenome from the Australian genus Cherax.ConclusionsWe infer that the gene order arrangement found in Cherax destructor is common to Australian crayfish and may be a derived feature of the southern hemisphere family Parastacidae. Further, we report to our knowledge, the simplest and fastest protocol for the recovery and assembly of complete mitochondrial genomes using the MiSeq benchtop sequencer.

Identification of Rays through DNA Barcoding: An Application for Ecologists

DNA barcoding potentially offers scientists who are not expert taxonomists a powerful tool to support the accuracy of field studies involving taxa that are diverse and difficult to identify. The taxonomy of rays has received reasonable attention in Australia, although the fauna in remote locations such as Ningaloo Reef, Western Australia is poorly studied and the identification of some species in the field is problematic. Here, we report an application of DNA-barcoding to the identification of 16 species (from 10 genera) of tropical rays as part of an ecological study. Analysis of the dataset combined across all samples grouped sequences into clearly defined operational taxonomic units, with two conspicuous exceptions: the Neotrygon kuhlii species complex and the Aetobatus species complex. In the field, the group that presented the most difficulties for identification was the spotted whiptail rays, referred to as the ‘uarnak’ complex. Two sets of problems limited the successful application of DNA barcoding: (1) the presence of cryptic species, species complexes with unresolved taxonomic status and intra-specific geographical variation, and (2) insufficient numbers of entries in online databases that have been verified taxonomically, and the presence of lodged sequences in databases with inconsistent names. Nevertheless, we demonstrate the potential of the DNA barcoding approach to confirm field identifications and to highlight species complexes where taxonomic uncertainty might confound ecological data.

Allozyme evidence for a new species of freshwater crayfish of the genus Cherax Erichson (Decapoda : Parastacidae) from the south-west of Western Australia

The marron, Cherax tenuimanus (Smith), is one of the most easily recognisable members of the freshwater crayfish genus Cherax. Since its description in 1912, the taxonomy of the species has not been in dispute, but recent genetic studies have demonstrated that the species is not homogenous and consists of two genetically distinct forms. One of these forms is widespread and exploited via aquaculture and the other is restricted to a single river system, the Margaret River. This paper presents allozyme data, collected over a 19-year period, which documents the introduction of the widespread form into the Margaret River and the subsequent reproductive interactions between the two forms. These data indicate minimal interbreeding between the two forms of marron and so justify their recognition as distinct species. As the original description of the marron was based on specimens collected from the Margaret River, the form native to this river retains the name C. tenuimanus and a new species, Cherax cainii Austin is described for the common, widespread form of marron. An additional outcome of this study is that C. tenuimanus has been rapidly displaced by the introduced C. cainii within the Margaret River. Consequently, urgent conservation measures are required to protect C. tenuimanus and prevent its possible extinction.

Combined effects of shelter and density on the growth and survival of juveniles of the Australian freshwater crayfish, Cherax destructor Clark, part 2

Abstract A 31 day growth trial of newly independent (stage 3) yabbies (Cherax destructor) was conducted using three density levels, 4, 8 and 16 per 2.5 l aquarium, equivalent to 150, 300 and 600 m−2, and three temperatures (22, 25 and 28°C). Average final weight of yabbies was found to decline with increasing density, and to increase with increasing temperature. A significant interaction was found between density and temperature with the effects of temperature being reduced at higher densities. Analysis using yield produced similar results with the optimal combination of temperature and density found to be 25°C and 600 m−2. Survival was not affected by density, but declined with increasing temperature. The implications of these findings for optimal conditions for the intensive culture of juvenile yabbies are discussed.

Changes in the fatty acid profiles of hybrid red tilapia, Oreochromis mossambicus X O. niloticus, subjected to short-term starvation, and a comparison with changes in seawater raised fish

Juvenile hybrid red tilapia of mean weight 52.9 ± 2.80 g were starved for 45 days, and the liver and muscle fatty acid profiles of fed and starved fish determined on Days 0, 24 and 45. A corresponding group of fish were seawater adapted and were sampled on Day 45. In fed fish the total fatty acids in the livers (expressed in μg mg−1 lipid) decreased with growth (45 days), from 816 ± 16 to 600 ± 7 and 821 ± 25 to 589 ± 23 in females and males, respectively. This decrease was significant by the 24th day. In muscle, however, the amount of fatty acids in total lipid increased with growth, in females from 365 ± 21 to 489 ± 6 and in males from 387 ± 17 to 480 ± 17 μg mg−1 lipid. Compared with fed fish, during starvation the proportion of fatty acids in total lipid increased in both types of tissues but was still lower than at the initial level, significantly so in the liver. Twenty individual fatty acids were quantified as percent of total fatty acids in liver and muscle tissues of fish from different treatments during this study. In starved fish, liver monoenes decreased significantly (P < 0.05), from 33.0 to 16.3% and 35.6 to 9.5%, and the percentage of polyunsaturated fatty acids (PUFA) increased significantly from 18.3 to 39.9% and 16.9 to 46.2% in females and males, respectively. Comparable trends were also observed in muscle, but in muscle the percentage of PUFA tended to be higher than in the liver. The fatty acids that occurred in the highest proportion were oleic acid (18:1 n − 9), followed by palmitic acid (16:0) and docosahexaenoic acid (DHA; 22:6n − 3), collectively accounting for more than 50% of the total. The relative amount of PUFA, in particular DHA, increased considerably and very significantly with starvation. Principal component analysis of the fatty acid data effectively summarized the major differences among the experimental treatments, which included substantial differences in the fatty acid profiles between sexes, fed and starved animals and between fish raised in fresh- and seawater.