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.

Detection, movement and persistence of invertebrate eDNA in groundwater.

Sampling groundwater biodiversity is difficult because of limited access and issues with species identification. Environmental DNA (eDNA) provides a viable alternative to traditional sampling approaches, however limited knowledge of the abundance and fate of DNA in groundwater hinders the interpretation of data from these environments. Groundwater environments are dark and have lower oxygen concentrations and microbial activity than surface waters. Consequently, assumptions about DNA fate in surface ecosystems may not apply to groundwaters. Here, we test the longevity and transport of eDNA in groundwater within a static microcosm and a flow-through mesocosm. A variety of invertebrates were placed within a mesocosm and microcosm to enable DNA shedding, and then removed. DNA persisted for up to 5 weeks after their removal in the static experiment and was detected between 9 and 33 days in the flow-through experiment. Sediments and water both proved important for eDNA detection. Crustacean DNA was detected sporadically and unpredictably, whereas non-crustacean DNA was detected more frequently despite their lower densities. We suggest that detecting crustaceans poses a challenge to utilising eDNA approaches for stygofauna monitoring. This is confounded by the scarcity of sequences for stygofauna in reference databases. Further research is needed before eDNA alone can be routinely employed for stygofauna detection.

Groundwater Ecosystems Vary with Land Use across a Mixed Agricultural Landscape.

Changes in surface land use may threaten groundwater quality and ecosystem integrity, particularly in shallow aquifers where links between groundwater and surface activities are most intimate. In this study we examine the response of groundwater ecosystem to agricultural land uses in the shallow alluvial aquifer of the Gwydir River valley, New South Wales, Australia. We compared groundwater quality and microbial and stygofauna assemblages among sites under irrigated cropping, non-irrigated cropping and grazing land uses. Stygofauna abundance and richness was greatest at irrigated sites, with the composition of the assemblage suggestive of disturbance. Microbial assemblages and water quality also varied with land use. Our study demonstrates significant differences in the composition of groundwater ecosystems in areas with different surface land use, and highlights the utility of groundwater biota for biomonitoring, particularly in agricultural landscapes.

Assessing the chronic toxicity of atrazine, permethrin, and chlorothalonil to the cladoceran Ceriodaphnia cf. dubia in laboratory and natural river water.

The majority of ecotoxicological data are generated from standard laboratory-based experiments with organisms exposed in nonflowing systems using highly purified water, which contains very low amounts of dissolved organic matter and suspended particulates. However, such experimental conditions are not ecologically relevant. Thus, there is a need to develop more realistic approaches to determining toxicity, including both lethal and sublethal effects. This research provides information on the effect of natural water constituents, such as suspended particulates and dissolved organic matter, in river water (RW) on the chronic toxicity (7-day reproductive impairment) of the pesticides atrazine, chlorothalonil, and permethrin to the freshwater cladoceran Ceriodaphnia cf. dubia. Standard bioassays were conducted under standard laboratory and more environmentally realistic conditions (using RW). The 7-day IC25 (reproduction impairment) values of atrazine, chlorothalonil, and permethrin to C. cf. dubia ranged from 862.4 to >1000, 51.3 to 66.4, and 0.19 to 0.23 µg/L, respectively. Using the Globally Harmonized System of Classification and Labelling of Chemicals, atrazine is classified as moderately to highly toxic, whereas permethrin and chlorothalonil were both highly toxic. The presence of dissolved organic matter and suspended particles in natural RW did not significantly (p > 0.05) change the toxicity of any of the pesticides to C. cf. dubia compared with that tested in laboratory water (LW). For the tested pesticides, toxicity testing in LW provided an adequate estimate of the hazard posed.

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.

Agricultural practices linked to shifts in groundwater microbial structure and denitrifying bacteria.

Irrigation enhances the connectivity between the surface and groundwater by facilitating the transport of energy sources and oxygen. When combined with fertilisers, the impact on groundwater microbial communities and their interactions with nitrogen cycling in aquifers is poorly understood. This study examines the impact of different landuses (irrigated and non-irrigated) on groundwater microbial communities. A total of 38 wells accessing shallow aquifers in three sub-catchments of the Murray Darling Basin, Australia, were sampled for water chemistry and microbial community structure using environmental DNA (eDNA) techniques. All sub-catchments showed evidence of intense irrigation and groundwater contamination with total nitrogen, nitrates and phosphorus concentrations often well above background, with total nitrogen concentrations up to 70 mg/L and nitrate concentration up to 18 mg/L. Across sub-catchments there was high microbial diversity, with differences in community structure and function between catchments and landuses. Of the 1100 operational taxonomic units (OTUs) recorded, 47 OTUs were common across catchments with species from Woesearchaeota, Nitrospirales, Nitrosopumilales and Acidobacter taxonomic groups contributing greatly to groundwater microbial communities. Within non-irrigated sites, groundwaters contained similar proportions of nitrifying and denitrifying capable taxa, whereas irrigated sites had significantly higher abundances of microbes with nitrifying rather than denitrifying capabilities. Microbial diversity was lower in irrigated sites in the Macquarie catchment. These results indicate that irrigated landuses impact microbial community structure and diversity within groundwaters and suggest that the ratios of denitrifying to nitrifying capable microbes as well as specific orders (e.g., Nitrososphaerales) may be useful to indicate long-term nitrogen contamination of groundwaters. Such research is important for understanding the biogeochemical processes that are key predictors of redox state and contamination of groundwater by N species and other compounds. This will help to predict human impacts on groundwater microbial structure, diversity, and ecosystem functions, aiding the long-term management groundwater resources.

Dynamics of microbiotic patterns reveal surface water groundwater interactions in intermittent and perennial streams.

Exchange between groundwater (GW), hyporheic zone waters (HZ) and surface waters (SW) is critical for water quality, quantity, and the ecological health and functioning of all three ecosystems. Hydrological exchange is particularly important in intermittent creeks, such as in the Murray Darling Basin, Australia, where stream reaches shift from losing to gaining depending on the volume of surface flows. In this study we used hydrochemistry to identify SW-GW exchange and combined this with eDNA data to analyse the response of eukaryote and prokaryote communities to differing flow conditions within intermittent and perennial stream reaches. Our study suggested that SW and GW microbial communities were only around 30% similar. Differences in microbiota between SW, HZ and GW habitats were driven by changes in relative abundances of surface water dominant organisms (such as those capable of photosynthesis) as well as anaerobic taxa typical of GW environments (e.g., methanogens), with GW and HZ microbial communities becoming increasingly different to those in SW as flow ceased in intermittent creeks. Fine-scale hydrologic changes were identified through microbial communities in the perennial Maules Creek, indicating the importance of GW-SW exchange to biotic communities. This study highlights the importance of flow in shaping microbial communities and biogeochemical cycling within intermittent creeks and their connected alluvial aquifers. Our results suggest that microbiota may prove a useful indicator of SW-GW exchange, and in some circumstances, may be more sensitive in demonstrating fine-scale changes in SW-GW interactions than water chemistry. This knowledge furthers our understanding of GW-SW exchange and its impacts on ecological health.

What do qualitative rapid assessment collections of macroinvertebrates represent? A comparison with extensive quantitative sampling.

It is a fundamental tenet of Rapid Biological Assessments (RBA) that the samples collected reflect the community from which they are drawn. As with any biological sampling, RBA collections are subject to sampling error resulting in the omission of some taxa. The aim of this study is to compare the composition of RBA samples with an estimate of community structure based on extensive quantitative sampling. We used logistic regression to explore the relationships between the frequency of a taxon being collected in an RBA sample and its biological and ecological traits, namely its abundance, distribution, body size and habit. RBA samples and quantitative estimates of community structure were made in riffles in the Kangaroo and Nepean Rivers, New South Wales, Australia. Single RBA samples may collect up to 63% of the taxa that are collected by extensive quantitative sampling at a site. The frequency of a taxon being recorded in an RBA sample was significantly and positively related to all traits tested indicating a bias in the collection methods towards large, abundant and widely distributed taxa. Accordingly, taxa missed by RBA sampling were generally small, narrowly distributed or rare. These findings enhance our understanding of what RBA samples represent, and the bias and source of errors associated with RBA sampling. This study also quantifies the utility of RBA methods for biodiversity assessment.

Microbial communities are sensitive indicators for freshwater sediment copper contamination.

Anthropogenic activities, such as mining and agriculture, have resulted in many freshwater systems having elevated concentrations of copper. Despite the prevalence of this contamination, and the vital ecological function of prokaryotes, just three studies have investigated prokaryote community responses to copper concentration in freshwater sediments. To address this, the current study investigated these communities in outdoor mesocosms spiked with varying copper concentrations. We profiled the prokaryotic communities at the taxonomic level, using next-generation high-throughput sequencing techniques, as well as their function, using baiting with leaf analogues, and Biolog Ecoplates for community-level physiological profiling. Sediments containing just 46 mg kg-1 of copper, had distinctly different microbial communities compared with controls, as determined by both DNA and RNA 16S ribosomal RNA gene (rRNA) profiling. In addition to this, sediment communities displayed a greatly reduced utilisation of carbon substrates under elevated copper, while the communities recruited onto leaf analogues were also disparate from those of control ponds. Given the vital role of prokaryotes in ecosystem processes, including carbon cycling, these changes are potentially of great ecological relevance, and are seen to occur well below the 'low risk' sediment quality guideline values (SQGV) used by regulatory bodies internationally.

Comparison of the fate and toxicity of chlorpyrifos--laboratory versus a coastal mesocosm system.

The widespread use of chlorpyrifos for pest control in urban and rural environments poses a risk of contamination to aquatic environments via runoff, spray drift or spillage. The aim of this study was to assess the fate of chlorpyrifos and its toxicity to common freshwater invertebrates in the laboratory and in stream mesocosms. Chlorpyrifos was rapidly lost from the test systems but the rates of loss varied considerably, such that losses in the mesocosms could not be reliably predicted from the static laboratory studies. This was likely due to the mass transport of chlorpyrifos from the mesocosm via stream flow. Chlorpyrifos was acutely toxic to all invertebrates tested with the cladoceran species (laboratory 48h LC(50) values 0.07-0.10 microg L(-1)) being most sensitive. Despite the differences in the dynamics of chlorpyrifos in the laboratory and mesocosm systems, the sensitivities of the mayfly Atalophlebia australis and the cladoceran Simocephalus vetulus were similar in the 2 systems.

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.

Depauperate macroinvertebrates in a mine affected stream: clean water may be the key to recovery.

Acid mine drainage (AMD) is frequently linked with changes in macroinvertebrate assemblages, but the relative contribution of water and sediment to toxicity is equivocal. We have shown that the macroinvertebrate fauna of Neubecks Ck, a mine impacted stream in New South Wales, Australia, was much poorer than in two reference streams. Multivariate RELATE analyses indicated that the patterns in the biological data were more strongly correlated with the concentrations of common metals in the surface water than the pore water of these streams. From this we hypothesised that the water was more toxic to the biota than the sediment and we tested this hypothesis with a sediment transplant experiment. Sediment from Neubecks Ck that was placed in reference streams retained high concentrations of metals throughout the experiment, yet supported a macroinvertebrate assemblage similar to that in the reference streams. Sediment from the reference streams that was placed in Neubecks Ck supported few, if any, animals. This indicates that water in Neubecks Ck is toxic to biota, but that sediment is able to support aquatic biota in clean water. Therefore, remediation should focus on improving water quality rather than sediment quality.

Spider webs as environmental indicators.

Analysis of spider webs from limestone arches in New South Wales, Australia showed levels of lead and zinc to be several times higher at Jenolan Caves than reference sites at Abercrombie and Wombeyan Caves. The high concentrations at Jenolan were attributed to emissions from motor vehicles that travel through the arch. Analysis of water-soluble ions in webs from Jenolan and Abercrombie Caves showed large differences between the sites, which could be explained by differences in the geology and biology of the cave environments. Spider webs proved useful indicators of environmental chemistry and undoubtedly have applications beyond the cave environments studied here.

River-aquifer interactions and their relationship to stygofauna assemblages: a case study of the Gwydir River alluvial aquifer (New South Wales, Australia).

In contrast to surface water ecosystems, groundwater ecosystems are usually considered to have relatively stable conditions and physically inert environments. However, many groundwater ecosystems undergo substantial changes through space and time, related to fluxes in groundwater flow, exchange and nutrient imports. In this study we used hydrochemical data to: 1) determine the different hydrogeological conditions in an alluvial system, the shallow Gwydir River alluvial aquifer (located in Northern New South Wales, Australia); and 2) analyze the relationship between hydrochemical conditions and the composition of stygofauna assemblages in the aquifer. Using hydrochemical modeling and multivariate analyses, four main hydrogeological situations were defined as occurring in the aquifer. Bores were classified as having either a high, low or no influence from or exchange with the river. The latter group was further subdivided into those of low and high salinity. Further analysis combining the biological and hydrochemical data identified two main groups of samples. The first group was composed mainly of samples related to the aquifer groundwater which had higher richness and abundance of fauna compared to samples in the second group which was comprised of samples affected by stream water leakage and samples related to the highest salinities. These results suggest that more stable conditions (mainly related to steadier groundwater head levels) and lower nitrate concentrations promoted a more diverse and abundant stygofauna community.

A comparison of mixture toxicity assessment: examining the chronic toxicity of atrazine, permethrin and chlorothalonil in mixtures to Ceriodaphnia cf. dubia.

Pesticides predominantly occur in aquatic ecosystems as mixtures of varying complexity, yet relatively few studies have examined the toxicity of pesticide mixtures. Atrazine, chlorothalonil and permethrin are widely used pesticides that have different modes of action. This study examined the chronic toxicities (7-d reproductive impairment) of these pesticides in binary and ternary mixtures to the freshwater cladoceran Ceriodaphnia cf. dubia. The toxicity of the mixtures was compared to that predicted by the independent action (IA) model for mixtures, as this is the most appropriate model for chemicals with different modes of action. Following this they were compared to the toxicity predicted by the concentration addition (CA) model for mixtures. According to the IA model, the toxicity of the chlorothalonil plus atrazine mixture conformed to antagonism, while that of chlorothalonil and permethrin conformed to synergism. The toxicity of the atrazine and permethrin mixture as well as the ternary mixture conformed to IA implying there was either no interaction between the components of these mixtures and/or in the case of the ternary mixture the interactions cancelled each other out to result in IA. The synergistic and antagonistic mixtures deviated from IA by factors greater than 3 and less than 2.5, respectively. When the toxicity of the mixtures was compared to the predictions of the CA model, the binary mixture of chlorothalonil plus atrazine, permethrin plus atrazine and the ternary mixture all conformed to antagonism, while the binary mixture of chlorothalonil plus permethrin conformed to CA. Using the CA model provided estimates of mixture toxicity that did not markedly underestimate the measured toxicity, unlike the IA model, and therefore the CA model is the most suitable to use in ecological risk assessments of these pesticides.

Confirming the species-sensitivity distribution concept for endosulfan using laboratory, mesocosm, and field data.

In Australia, water-quality trigger values for toxicants are derived using protective concentration values based on species-sensitivity distribution (SSD) curves. SSD curves are generally derived from laboratory data with an emphasis on using local or site-specific data. In this study, Australian and non-Australian laboratory-species based SSD curves were compared and the concept of species protection confirmed by comparison of laboratory-based SSD curves with local mesocosm experiments and field monitoring data. Acute LC50 data for the organochlorine pesticide endosulfan were used for these comparisons; SSD curves were fitted using the Burr type III distribution. SSD curves indicated that the sensitivities of Australian fish and arthropods were not significantly different from those of corresponding non-Australian taxa. Arthropod taxa in the mesocosm were less sensitive than taxa in laboratory tests, which suggests that laboratory-generated single-species data may be used to predict concentrations protective of semifield (mesocosm) systems. SSDs based on laboratory data were also protective of field populations.