Sean W. Corley

Genetic variation and structuring in the threatened koala populations of Southeast Queensland

Habitat fragmentation can act to cause reproductive isolation between conspecifics and undermine species’ persistence, though most studies have reported the genetic condition of populations that have already declined to a very small size. We examined genetic diversity within the vulnerable, declining koala (Phascolarctos cinereus) population in Southeast Queensland, Australia to determine the genetic impact of ongoing threatening processes. Five hundred and twelve koalas from ten Southeast Queensland Local Government Areas on the mainland and one island were genotyped at six polymorphic microsatellite loci. Based on Bayesian cluster analysis incorporating spatial data, the regional koala population was subdivided into six clusters, with location of major roads and rivers appearing to be consistent with being barriers to gene flow. The distribution of mtDNA control region haplotypes identified distinct coastal and inland clades suggesting that historically there was gene flow between koalas along the coast (though little interchange between coastal and inland animals). In contrast, koalas from the Koala Coast (Brisbane City, Logan City and Redland Shire) were shown by microsatellite analysis to be genetically distinct from adjacent areas. It is likely, therefore, that more recent reductions in population size and restricted gene flow through urbanisation have contributed to the genetic differentiation of koalas in the Koala Coast region.

Identification of a mutation in the para-sodium channel gene of the cattle tick Rhipicephalus microplus associated with resistance to flumethrin but not to cypermethrin

A mutation in the domain II S4-5 linker region of the para-sodium channel gene has been associated previously with synthetic pyrethroid (SP) resistance in the cattle tick (Rhipicephalus microplus) in Australia. This is a C→A mutation at nucleotide position 190, which results in a leucine to isoleucine amino acid substitution (L64I). In a survey of 15 cattle tick populations with known SP resistance status, sourced from Queensland and New South Wales in Australia, there was a strong relationship (r=0.98) between the proportion of ticks carrying the L64I homozygous resistant genotype and the survival percentage after exposure to a discriminating concentration of cypermethrin in the bioassay, as expected. However, among populations resistant only to flumethrin, the L64I homozygous genotype was not found. The sequence obtained for a 167 bp region including domain II S4-5 linker in flumethrin-resistant ticks identified a G→T non-synonymous mutation at nucleotide position 214 that results in a glycine to valine substitution (G72V). The frequency of the G72V homozygous genotype in each population was found to be moderately related to the survival percentage at the discriminating concentration of flumethrin in the larval packet test (r=0.74). However, a much stronger relationship between genotype and resistance to flumethrin was observed when the heterozygotes of L64I and G72V were added to the G72V homozygotes (r=0.93). These results suggest that there is an interaction between the two mutations in the same gene, such that flumethrin resistance might be conferred by either two copies of the G72V mutation or by being a L64I and G72V heterozygote.

Mutation in the RmβAOR gene is associated with amitraz resistance in the cattle tick Rhipicephalus microplus

Significance Amitraz is a widely used acaricide for the control of the cattle tick Rhipicephalus microplus, an important parasite of cattle in the tropics and subtropics. Here we describe in detail the evolution of amitraz resistance in replicated populations of ticks in the field, using divergent selection pressures with amitraz. We also demonstrate a close association between resistance to amitraz and a specific allele of the β-adrenergic octopamine receptor gene, which we propose confers resistance to amitraz. We aimed to describe the evolution of resistance to amitraz in Rhipicephalus microplus in the field and to test the association between amitraz resistance and the frequency of a mutation in the β-adrenergic octopamine receptor gene (RmβAOR). We established six populations of Rhipicephalus microplus ticks in similar paddocks by the admixture of ticks from strains known to be susceptible and resistant to amitraz and synthetic pyrethroids. Each population was managed using one of three acaricide treatment regimes: always amitraz, always spinosad, or rotation between amitraz and spinosad. We used microsatellites to elucidate population structure over time, an SNP in the para-sodium channel gene previously demonstrated to confer resistance to synthetic pyrethroids to quantify changes in resistance to synthetic pyrethroids over time, and a nonsynonymous SNP in the RmβAOR, a gene that we proposed to confer resistance to amitraz, to determine whether selection with amitraz increased the frequency of this mutation. The study showed panmixia of the two strains and that selection of ticks with amitraz increased the frequency of the RmβAOR mutation while increasing the prevalence of amitraz-resistance. We conclude that polymorphisms in the RmβAOR gene are likely to confer resistance to amitraz.

Real-time PCR as a surveillance tool for the detection of Trichinella infection in muscle samples from wildlife

Trichinella nematodes are the causative agent of trichinellosis, a meat-borne zoonosis acquired by consuming undercooked, infected meat. Although most human infections are sourced from the domestic environment, the majority of Trichinella parasites circulate in the natural environment in carnivorous and scavenging wildlife. Surveillance using reliable and accurate diagnostic tools to detect Trichinella parasites in wildlife hosts is necessary to evaluate the prevalence and risk of transmission from wildlife to humans. Real-time PCR assays have previously been developed for the detection of European Trichinella species in commercial pork and wild fox muscle samples. We have expanded on the use of real-time PCR in Trichinella detection by developing an improved extraction method and SYBR green assay that detects all known Trichinella species in muscle samples from a greater variety of wildlife. We simulated low-level Trichinella infections in wild pig, fox, saltwater crocodile, wild cat and a native Australian marsupial using Trichinella pseudospiralis or Trichinella papuae ethanol-fixed larvae. Trichinella-specific primers targeted a conserved region of the small subunit of the ribosomal RNA and were tested for specificity against host and other parasite genomic DNAs. The analytical sensitivity of the assay was at least 100 fg using pure genomic T. pseudospiralis DNA serially diluted in water. The diagnostic sensitivity of the assay was evaluated by spiking 10 g of each host muscle with T. pseudospiralis or T. papuae larvae at representative infections of 1.0, 0.5 and 0.1 larvae per gram, and shown to detect larvae at the lowest infection rate. A field sample evaluation on naturally infected muscle samples of wild pigs and Tasmanian devils showed complete agreement with the EU reference artificial digestion method (k-value=1.00). Positive amplification of mouse tissue experimentally infected with T. spiralis indicated the assay could also be used on encapsulated species in situ. This real-time PCR assay offers an alternative highly specific and sensitive diagnostic method for use in Trichinella wildlife surveillance and could be adapted to wildlife hosts of any region.

Generation of Full-Length cDNAs for Eight Putative GPCnR from the Cattle Tick, R. microplus Using a Targeted Degenerate PCR and Sequencing Strategy

We describe here a rapid and efficient method for the targeted isolation of specific members of gene families without the need for cloning. Using this strategy we isolated full length cDNAs for eight putative G-protein coupled neurotransmitter receptors (GPCnR) from the cattle tick Rhipicephalus (Boophilus) microplus. Gene specific degenerate primers were designed using aligned amino acid sequences of similar receptor types from several insect and arachnid species. These primers were used to amplify and sequence a section of the target gene. Rapid amplification of cDNA ends (RACE) PCR was used to generate full length cDNA sequences. Phylogenetic analysis placed 7 of these sequences into Class A G-protein coupled receptors (GPCR) (Rm_α2AOR, Rm_β2AOR, Rm_Dop1R, Rm_Dop2R, Rm_INDR, Rm_5-HT7R and Rm_mAchR), and one into Class C GPCR (Rm_GABABR). Of the 7 Class A sequences, only Rm_mAchR is not a member of the biogenic amine receptor family. The isolation of these putative receptor sequences provides an opportunity to gain an understanding of acaricide resistance mechanisms such as amitraz resistance and might suggest possibilities for the development of new acaricides.

Molecular biology of amitraz resistance in cattle ticks of the genus i Rhipicephalus i

Amitraz is an important product for the control of cattle ticks around the world. In comparison with other products for the control of ticks, it is quite affordable and it has a rapid knock-down effect. It binds with and activates adrenergic neuro-receptors of animals and it inhibits the action of monoamine oxidases (MAO). Resistance to amitraz has been documented in Rhipicephalus microplus, R. decoloratus and R. appendiculatus. Four mechanisms of resistance have been proposed, each of which is supported by evidence but none of which has been definitively confirmed as the cause of resistance in the field. The proposed mechanisms include genetic target site insensitivity in two G protein-coupled receptors, the beta-adrenergic octopamine receptor (BAOR) and the octopamine/tyramine receptor (OCT/Tyr), increased expression or activity of monoamine oxidases and increased expression or activity of the ATP binding cassette transporter.

Characterisation and cross-amplification of 21 novel microsatellite loci for the dusky shark, Carcharhinus obscurus

The dusky shark Carcharhinus obscurus risks excessive fisheries exploitation worldwide due to its low productivity. Genetic monitoring is an effective way of resolving species stock structure, genetic diversity, and forensically identifying processed animals. Here we present the first C. obscurus species-specific microsatellite loci. Twenty-one di- to tetra-nucleotide loci with between 2 and 20 alleles per locus were developed. Observed heterozygosity ranged from 0.28 to 0.91 with only one locus slightly deviating from Hardy–Weinberg equilibrium. No significant evidence for null alleles or linkage disequilibrium was detected. These loci were cross-amplified in three related species. Seventeen, twelve, and eighteen loci exhibited polymorphism in sandbar shark Carcharhinus plumbeus, spinner shark Carcharhinus brevipinna, galapagos shark Carcharhinus galapagensis, respectively. Investigations into C. obscurus will benefit from these loci which possess attributes suitable for population-scale and individual-scale analyses. Additionally locus cross-amplification will facilitate research for these species with few existing microsatellites and similarly vulnerable life-histories.

Isolation and characterization of mimosine, 3, 4 DHP and 2, 3 DHP degrading bacteria from a commercial rumen inoculum

The presence of the toxic amino acid mimosine in Leucaena leucocephala restricts its use as a protein source for ruminants. Rumen bacteria degrade mimosine to 3,4‐ and 2,3‐dihydroxypyridine (DHP), which remain toxic. Synergistes jonesii is believed to be the main bacterium responsible for degradation of these toxic compounds but other bacteria may also be involved. In this study, a commercial inoculum provided by the Queenslands Department of Agriculture, Fisheries, and Forestry was screened for isolation and characterization of mimosine, 3,4‐ and 2,3‐DHP degrading bacterial strains. A new medium for screening of 2,3‐DHP degrading bacteria was developed. Molecular and biochemical approaches used in this study revealed four bacterial isolates – Streptococcus lutetiensis, Clostridium butyricum, Lactobacillus vitulinus, and Butyrivibrio fibrisolvens – to be able to completely degrade mimosine within 7 days of incubation. It was also observed that C. butyricum and L. vitulinus were able to partially degrade 2,3‐DHP within 12 days of incubation, while S. lutetiensis, was able to fully degrade both 3,4 and 2,3 DHP. Collectively, we concluded that S. jonesii is not the sole bacterium responsible for detoxification of Leucaena. Comprehensive screening of rumen fluid of cattle grazing on Leucaena pastures is needed to identify additional mimosine‐detoxifying bacteria and contribute to development of more effective inoculums to be used by farmers against Leucaena toxicity.

Characterisation and cross-amplification of 19 novel microsatellite loci for the sandbar shark, Carcharhinus plumbeus

Conservation of vulnerable shark species that are also heavily exploited, such as the sandbar shark Carcharhinus plumbeus is a pressing issue, and molecular genetics is an increasingly important population monitoring and species identification tool. Here we present 19 C. plumbeus microsatellite loci with a broad range of alleles per locus (3–22), and observed heterozygosities (0.17–0.95). Null alleles were not apparent and if present, were at frequencies <0.041. None of the loci showed significant deviation from Hardy–Weinberg equilibrium or indications of linkage disequilibrium. Cross-species amplification of these loci in three other carcharinids, dusky shark Carcharhinus obscurus, spinner shark Carcharhinus brevipinna, and galapagos shark Carcharhinus galapagensis showed polymorphism in 13, 13, and 14 loci respectively. These highly diverse loci will assist both individual-specific and population-wide genetic investigations for these species.