Carolyn Smith-Keune

Effects of chlorpyrifos on cholinesterase activity and stress markers in the tropical reef fish Acanthochromis polyacanthus

Tropical coastal ecosystems, including the Great Barrier Reef (GBR) of Australia are increasingly threatened by pollution; yet few studies have investigated the sensitivity of GBR species to these pollutants. Here we exposed juveniles of the tropical reef fish Acanthochromis polyacanthus (spiny damselfish) to three concentrations of the insecticide chlorpyrifos (CPF) and measured (i) muscle cholinesterase (ChE) activity; (ii) hepatic glutathione-S-transferase (GST) activity; and (iii) coenzyme Q (CoQ) redox balance, after 6h and 96h of exposure. After 96h, muscle ChE activity was significantly inhibited by 26%, 49% and 53% when fish were exposed to 1, 10 or 100μg/L CPF, respectively. Muscle ChE characterization revealed three types of ChEs, including two atypical forms. Hepatic CoQ antioxidant form significantly increased at 10μg/L after 6h of exposure, potentially demonstrating an early response to CPF-induced oxidative stress in liver. Hepatic GST was not affected by CPF exposure.

Molecular discrimination of Perna (Mollusca: Bivalvia) species using the polymerase chain reaction and species-specific mitochondrial primers.

This work was prompted by the need to be able to identify the invasive mussel species, Perna viridis, in tropical Australian seas using techniques that do not rely solely on morphology. DNA-based molecular methods utilizing a polymerase chain reaction (PCR) approach were developed to distinguish unambiguously between the three species in the genus Perna. Target regions were portions of two mitochondrial genes, cox1 and nad4, and the intergenic spacer between these that occurs in at least two Perna species. Based on interspecific sequence comparisons of the nad4 gene, a conserved primer has been designed that can act as a forward primer in PCRs for any Perna species. Four reverse primers have also been designed, based on nad4 and intergenic spacer sequences, which yield species-specific products of different lengths when paired with the conserved forward primer. A further pair of primers has been designed that will amplify part of the cox1 gene of any Perna species, and possibly other molluscs, as a positive control to demonstrate that the PCR is working.

Oceanographic Currents and Local Ecological Knowledge Indicate, and Genetics Does Not Refute, a Contemporary Pattern of Larval Dispersal for The Ornate Spiny Lobster, Panulirus ornatus in the South-East Asian Archipelago

Here we utilize a combination of genetic data, oceanographic data, and local ecological knowledge to assess connectivity patterns of the ornate spiny lobster Panulirus ornatus (Fabricius, 1798) in the South-East Asian archipelago from Vietnam to Australia. Partial mitochondrial DNA control region and 10 polymorphic microsatellites did not detect genetic structure of 216 wild P. ornatus samples from Australia, Indonesia and Vietnam. Analyses show no evidence for genetic differentiation among populations (mtDNA control region sequences ΦST = -0.008; microsatellite loci FST = 0.003). A lack of evidence for regional or localized mtDNA haplotype clusters, or geographic clusters of microsatellite genotypes, reveals a pattern of high gene flow in P. ornatus throughout the South-East Asian Archipelago. This lack of genetic structure may be due to the oceanography-driven connectivity of the pelagic lobster larvae between spawning grounds in Papua New Guinea, the Philippines and, possibly, Indonesia. The connectivity cycle necessitates three generations. The lack of genetic structure of P. ornatus population in the South-East Asian archipelago has important implications for the sustainable management of this lobster in that the species within the region needs to be managed as one genetic stock.

Isolation and characterization of 16 microsatellite loci in the humbug damselfish, Dascyllus aruanus (family Pomacentridae)

Here we report on 16 microsatellite loci designed for the damselfish Dascyllus aruanus. All loci were tested on 98 individuals and were polymorphic (seven to 35 alleles). Expected heterozygosity ranged from 0.705 to 0.942. Six loci showed Hardy‐Weinberg disequilibrium due to the occurrence of null alleles. Cross‐species amplifications conducted within the genus Dascyllus (D. carneus, D. strasburgi, D. trimaculatus) lead to polymorphic fragments in 32 out of 48 tests. These 16 loci will enable future research into the behavioural ecology and population ecology of Dascyllus aruanus throughout the Indo‐Pacific.

Temperature: a prolonged confounding factor on cholinesterase activity in the tropical reef fish Acanthochromis polyacanthus.

Cholinesterase activity usually decreases in fish exposed to anticholinesterase compounds such as organophosphate and carbamate pesticides. Here we show that tropical reef fish Acanthochromis polyacanthus (or spiny damsel) also exhibits a decrease in ChE activity when exposed to elevated temperature from 28°C to 32°C or 34°C after 4 days. We further demonstrate that the decline persists even after 7 days of recovery at control temperature. This is the first report of a drop in ChE activity in fish as temperature increases. Our results strongly suggest the need for long-term monitoring of water temperature in the field prior to sampling A. polyacanthus for toxicology studies, as temperature is a prolonged and confounding factor for ChE activity in this species.

A high through-put protocol for quantifying nucleic acids in individual microcrustaceans using new generation RNA and DNA specific dyes

Abstract Quantifying growth rate in microcrustacea is important to plankton biologists when determining primary productivity. RNA:DNA ratios have been widely used as an indicator of instantaneous growth rate in microcrustaceans in nutritional and environmental studies. It is important when measuring nucleic acids in these small organisms that it is done efficiently and with high sensitivity, so that small differences in nucleic acid content can be accurately detected. A technique is presented for the accurate, high-throughput determination of RNA and DNA content in individual microcrustacea, based on two new generation fluorescent dyes that bind preferentially to either RNA (Quant-iT™ RNA) or DNA (Quant-iT™ DNA). Contaminant interference (either from detergents or enzymes used in the chemical cell lysis process, or cell based contaminants such as proteins) can significantly affect fluorescence readings of these dyes. Through the development of our protocol, we show that proteinase K was an ineffective cell lysis method, whereas sarcosyl efficiently lysed cells and could be used when its final assay concentration was diluted to avoid interference with dye fluorescence. Organic solvents were also utilized to remove cell based contaminants that caused an interference with the Quant-iT™ DNA dye fluorescence. Through the use of specific RNA and DNA dyes and by achieving low levels of technical variation, this study developed a rapid and reliable method for nucleic acid extraction and quantification in Artemia.

A robust flow-cytometric protocol for assessing growth rate of hatchery-reared barramundi Lates calcarifer larvae

In this study, a flow-cytometric cell cycle analysis method to assess instantaneous growth rate of whole larvae of the Australian barramundi Lates calcarifer was developed and validated. High-resolution DNA measurements of either fresh, frozen or RNAlater-preserved larvae (gap0-gap1, G(0) -G(1), coefficient of variation (c.v.) < 3, 4 and 5%, respectively) enabled the deconvolution of the DNA histogram and assignment of the proportion of nuclei into cell cycle compartments G(0) -G(1), S (DNA synthesis) and G(2) -M (Gap2-Mitosis). This technique can be also used for individual fish tissues such as brain, liver, fin and muscle. For the first time, the combined proportion of replicating nuclei (into S and G(2) -M phases) of whole fish larvae and absolute growth rate in length (mm day(-1)) has been correlated in commercial aquaculture conditions. Fast growing L. calcarifer larvae had an overall hyperplasia advantage as indicated by a greater proportion of cells in the S+G(2) -M phase compared with slow growing larvae, which might explain the increasing differences in size during culture. In a fasting trial, larvae ceased growth while maintaining the constant initial rates of cell division throughout a 6 day period. For a highly fed fast growing control group, cell division rates significantly increased after day 4. Flow-cytometric cell cycle analysis of whole fish larvae may provide fish biologists and aquaculturists with a better understanding of how cell division rates influence early growth in natural and artificial environments.

Development, characterisation and cross-species amplification of 16 novel microsatellite markers for the endangered Black-throated Finch (Poephila cincta) in Australia

The Black-throated Finch (Southern) (Poephila cincta cincta) is threatened by the substantial landscape changes in northern Australia. We developed 16 polymorphic microsatellite markers using 454-shotgun whole-genome sequencing technology. We identified an average of 4.7 alleles per locus based on 63 wild caught individuals from Townsville, Queensland. Thirteen and 9 markers were also successfully cross-amplified in two confamilial species, the Double-barred Finch (Taeniopygia bichenovii) and the chestnut-breasted Mannikin (Lonchura castaneothorax) with 11 and 5 were polymorphic, respectively. These markers will help understand the population genetic structure of the endangered Black-throated Finch and determine genetic consequences of landscape changes for the species.

Genetic structure and diversity of the black-throated finch (Poephila cincta) across its current range

Abstract. Understanding the patterns of population connectivity and level of genetic diversity can facilitate the identification of both ecologically relevant populations and the spatial scales at which conservation management may need to focus. We quantified genetic variation within and among populations of black-throated finches across their current distribution. To quantify genetic structure and diversity, we genotyped 242 individuals from four populations using 14 polymorphic microsatellite markers and sequenced 25 individuals based on a 302-base-pair segment of mitochondrial control region. We found modest levels of genetic diversity (average allelic richness r = 4.37 ± 0.41 (standard error) and average heterozygosity HO = 0.42 ± 0.040 (standard error)) with no bottleneck signature among sampled populations. We identified two genetic groups that represent populations of two subspecies based on Bayesian clustering analysis and low levels of genetic differentiation based on pairwise genetic differentiation statistics (all FST, RST and Nei’s unbiased D values < 0.1). Our data suggest that genetic exchange occurs among sampled populations despite recent population declines. Conservation efforts that focus on maintaining habitat connectivity and increasing habitat quality to ensure a high level of gene flow on a larger scale will improve the species’ ability to persist in changing landscapes. Conservation management should also support continuous monitoring of the bird to identify any rapid population declines as land-use intensification occurs throughout the species’ range.