Impact of reduced-representation sequencing protocols on detecting population structure in a threatened marsupial.

Reduced-representation sequencing methods have wide utility in conservation genetics of non-model species. Several methods are now available that reduce genome complexity to examine a wide range of markers in a large number of individuals. We produced two datasets collected using different laboratory techniques, comprising a common set of samples from the greater bilby (Macrotis lagotis). We examined the impact of differing data filtering thresholds on downstream population inferences. We found that choice of restriction enzyme and data filtering thresholds, especially the rate of allowable missing data, impacted our ability to detect population structure. Estimates of FST were robust to alterations in laboratory and bioinformatic protocols while principal coordinates and STRUCTURE analyses showed variation according to the number of loci and percent missing data. We advise researchers using reduced-representation sequencing in conservation projects to examine a range of data thresholds, and follow these through to downstream population inferences. Multiple measures of population differentiation should be used in order to fully understand how data filtering thresholds influence the final dataset, paying particular attention to the impact of allowable missing data. Our results indicate that failure to follow these checks could impact conclusions drawn, and conservation management decisions made.

The toxicity of arsenic(III), chromium(VI) and zinc to groundwater copepods.

Groundwater ecosystems globally are threatened by anthropogenic contamination, yet there are few ecotoxicological data using obligate groundwater biota on which to base risk assessments. Copepods are found inhabiting aquifers of different geologies around the world and so are a useful taxon for use in ecotoxicological studies of groundwater. The aim of this study was to test the sensitivity of obligate groundwater copepods to metal contaminants (arsenic(III), chromium(VI) and zinc) in groundwater in static 96 h, 14 days and 28 days exposure tests. The copepods were variably sensitive to As, Cr and Zn, with Cr being the most toxic across all taxa. No taxon was consistently most sensitive and there was no apparent relationship between the hardness, pH and organic carbon concentration of the diluent water and the sensitivity of biota. As expected, toxicity increased with exposure period and we encourage the use of longer exposure periods in future toxicity tests with groundwater organisms to reflect the greater exposure periods likely to be associated with groundwater contamination.

Parasitic nematode communities of the red kangaroo, Macropus rufus: richness and structuring in captive systems.

Captive management practices have the potential to drastically alter pre-existing host-parasite relationships. This can have profound implications for the health and productivity of threatened species in captivity, even in the absence of clinical symptoms of disease. Maximising the success of captive breeding programmes requires a detailed knowledge of anthropogenic influences on the structure of parasite assemblages in captive systems. In this study, we employed two high-throughput molecular techniques to characterise the parasitic nematode (suborder Strongylida) communities of the red kangaroo, Macropus rufus, across seven captive sites. The first was terminal restriction fragment length polymorphism (T-RFLP) analysis of a region of rDNA encompassing the internal transcribed spacers 1 (ITS1), the 5.8S rRNA gene and the internal transcribed spacer 2 (ITS2). The second was Illumina MiSeq next-generation sequencing of the ITS2 region. The prevalence, intensity of infection, taxonomic composition and comparative structure of strongylid nematode assemblages was assessed at each location. Prevalence (P = <0.001) and mean infection intensity (df = 6, F = 17.494, P = <0.001) differed significantly between the seven captive sites. Significant levels of parasite community structure were observed (ANOSIM, P = 0.01), with most of the variation being distributed within, rather than between, captive sites. The range of nematode taxa that occurred in captive red kangaroos appeared to differ from that of wild conspecifics, with representatives of the genus Cloacina, a dominant nematode parasite of the macropodid forestomach, being detected at only two of the seven study sites. This study also provides the first evidence for the presence of the genus Trichostrongylus in a macropodid marsupial. Our results demonstrate that contemporary species management practices may exert a profound influence on the structure of parasite communities in captive systems.

Genetics and infection dynamics of Paratrichosoma sp in farmed saltwater crocodiles (Crocodylus porosus).

Paratrichosoma-associated helminthiasis has been identified in saltwater crocodiles under intensive farming conditions. The development of sustainable integrated management practices is dependent on a detailed understanding of Paratrichosoma population genetics and infection dynamics. This study investigated the genetic relationships of Paratrichosoma sp in a population of commercially farmed saltwater crocodiles, Crocodylus porosus, in northern Australia. 18S ribosomal RNA gene sequence data were obtained from Paratrichosoma sp eggs present in the epidermis of infected animals. A high level of genetic diversity was distributed within the Paratrichosoma sp population (241 variable positions in the 1094 bp alignment), indicating an accelerated rate of nucleotide base-pair substitutions in this genus of nematodes. Several possible environmental correlates of the incidence and intensity of helminthiasis, including season, rainfall, and mean monthly temperature, were investigated by visual inspection of crocodile skins. Stepwise logistic regression revealed a significant negative linear relationship (P = 0.011, R (2) = 32.69 %) between mean monthly rainfall and the incidence of monthly Paratrichosoma-associated helminthiasis. Variation in the severity of Paratrichosoma-associated helminthiasis could not be explained by any of the independent environmental variables included within an ordinal regression analysis. The large genetic diversity in these nematodes indicates a high probability of anthelmintic resistant alleles occurring in the population. We discuss how the spread of these alleles may be mitigated by adopting targeted treatment protocols.

Towards the molecular characterisation of parasitic nematode assemblages: an evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis.

Identifying factors which regulate temporal and regional structuring within parasite assemblages requires the development of non-invasive techniques which facilitate both the rapid discrimination of individual parasites and the capacity to monitor entire parasite communities across time and space. To this end, we have developed and evaluated a rapid fluorescence-based method, terminal restriction fragment length polymorphism (T-RFLP) analysis, for the characterisation of parasitic nematode assemblages in macropodid marsupials. The accuracy with which T-RFLP was capable of distinguishing between the constituent taxa of a parasite community was assessed by comparing sequence data from two loci (the ITS+ region of nuclear ribosomal DNA and the mitochondrial CO1) across ∼20 species of nematodes (suborder Strongylida). Our results demonstrate that with fluorescent labelling of the forward and reverse terminal restriction fragments (T-RFs) of the ITS+ region, the restriction enzyme Hinf1 was capable of generating species specific T-RFLP profiles. A notable exception was within the genus Cloacina, in which closely related species often shared identical T-RFs. This may be a consequence of the group's comparatively recent evolutionary radiation. While the CO1 displayed higher sequence diversity than the ITS+, the subsequent T-RFLP profiles were taxonomically inconsistent and could not be used to further differentiate species within Cloacina. Additionally, several of the ITS+ derived T-RFLP profiles exhibited unexpected secondary peaks, possibly as a consequence of the restriction enzymes inability to cleave partially single stranded amplicons. These data suggest that the question of T-RFLPs utility in monitoring parasite communities cannot be addressed without considering the ecology and unique evolutionary history of the constituent taxa.

Nematode community structure in the brush-tailed rock-wallaby, Petrogale penicillata: implications of captive breeding and the translocation of wildlife.

Despite an increasing appreciation of the disease risks associated with wild-life translocations, the effects which captive breeding programs exert on parasite communities remain understudied. This may be attributed, in part, to the current lack of rapid and cost-effective techniques for comparing parasite assemblages between host populations. Terminal restriction fragment length polymorphism (T-RFLP) analysis of the rDNA region encompassing the internal transcribed spacers (ITS-1 and ITS-2) and 5.8S rRNA gene was used to characterise bursate nematode communities (suborder Strongylida) across two captive and two non-captive colonies of the threatened brush-tailed rock-wallaby, Petrogale penicillata. A clone library was constructed and a restriction enzyme selected to differentiate the predominant operational taxonomic units (OTUs) by the unique peak profiles they generated. The prevalence, intensity of infection and comparative structure of strongylid assemblages was evaluated for each of the host colonies. Compared to wild conspecifics, captive wallabies exhibited a reduced prevalence of infection and significantly lower faecal egg counts. T-RFLP revealed that a high proportion of the OTUs co-occurred across three of the four study locations. Despite this, the composition of strongylid assemblages was significantly different between the colonies, even when host translocation events had occurred. These results suggest that captive breeding programs may exert a profound impact on parasitic helminth assemblages. Developing efficient techniques for characterising community dynamics in potentially pathogenic organisms is critical to the long term success of species recovery efforts worldwide.