IntroductionInvertebrate communities are central to many environmental monitoring programs. In freshwater ecosystems, aquatic macroinvertebrates are collected, identified and then used to infer ecosystem condition. Yet the key step of species identification is often not taken, as it requires a high level of taxonomic expertise, which is lacking in most organizations, or species cannot be identified as they are morphologically cryptic or represent little known groups. Identifying species using DNA sequences can overcome many of these issues; with the power of next generation sequencing (NGS), using DNA sequences for routine monitoring becomes feasible.ResultsIn this study, we test if NGS can be used to identify species from field-collected samples in an important bioindicator group, the Chironomidae. We show that Cytochrome oxidase I (COI) and Cytochrome B (CytB) sequences provide accurate DNA barcodes for chironomid species. We then develop a NGS analysis pipeline to identifying species using megablast searches of high quality sequences generated using 454 pyrosequencing against comprehensive reference libraries of Sanger-sequenced voucher specimens. We find that 454 generated COI sequences successfully identified up to 96% of species in samples, but this increased up to 99% when combined with CytB sequences. Accurate identification depends on having at least five sequences for a species; below this level species not expected in samples were detected. Incorrect incorporation of some multiplex identifiers (MID’s) used to tag samples was a likely cause, and most errors could be detected when using MID tags on forward and reverse primers. We also found a strong quantitative relationship between the number of 454 sequences and individuals showing that it may be possible to estimate the abundance of species from 454 pyrosequencing data.ConclusionsNext generation sequencing using two genes was successful for identifying chironomid species. However, when detecting species from 454 pyrosequencing data sets it was critical to include known individuals for quality control and to establish thresholds for detecting species. The NGS approach developed here can lead to routine species-level diagnostic monitoring of aquatic ecosystems.

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The integron/gene cassette system contributes to lateral gene transfer of genetic information in bacterial communities, with gene cassette-encoded proteins potentially playing an important role in adaptation to stress. Class 1 integrons are a particularly important class as they themselves seem to be broadly disseminated among the Proteobacteria and have an established role in the spread of antibiotic resistance genes. The abundance and structure of class 1 integrons in freshwater sediment bacterial communities was assessed through sampling of 30 spatially distinct sites encompassing different substrate and catchment types from the Greater Melbourne Area of Victoria, Australia. Real-time PCR was used to demonstrate that the abundance of intI1 was increased as a result of ecosystem perturbation, indicated by classification of sample locations based on the catchment type and a strong positive correlation with the first principal component factor score, comprised primarily of the heavy metals zinc, mercury, lead and copper. Additionally, the abundance of intI1 at sites located downstream from treated sewage outputs was associated with the percentage contribution of the discharge to the basal flow rate. Characterization of class 1 integrons in bacteria cultured from selected sediment samples identified an association with complete Tn402-like transposition modules, and the potential for coselection of heavy-metal and antibiotic resistance mechanisms in benthic environments.

1. We investigated the distribution of chironomid taxa in urban wetlands in the greater Melbourne area, Australia, to test if their distribution was influenced by sediment pollution and other environmental variables. 2. For identification of the Chironomidae, DNA markers generated via polymerase chain reaction-restriction fragment length polymorphism of cytochrome c oxidase sub unit I (COI) were validated against morphology and reference specimens for more than 5000 chironomids representing over 80 species. DNA-based identification generally concurred with morphological separation, but also indicated the existence of cryptic diversity in some genera. 3. Non-metric multidimensional scaling (NMS) and canonical correspondence analysis (CCA) showed chironomid assemblages were structured among wetlands and could be linked to several habitat characteristics. However, Chironomidae assemblages were only weakly linked to sediment pollution. 4. Logistic regressions identified potential bioindicators of sediment pollution. Riethia stictoptera, Tanytarsus inextentus, Coelopynia and Chironomus 'februarius' were negatively associated and Chironomus duplex was positively associated with sediment pollution. Thresholds for the pollution sensitivities of specific species were mostly similar to those established with previous microcosm tests. 5. Several other environmental factors influenced the distribution of specific chironomid taxa. Salinity, substratum type and submerged and riparian vegetation were particularly important. 6. We conclude that specific chironomid taxa rather than assemblages have potential as bioindicators of sediment pollution provided their ecological preferences are considered and their pollution sensitivities are characterized using multiple methods. The integration of DNA-based techniques should facilitate accurate and rapid identification of bioindicators species.

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Grab water samples, sediment samples, and 2,2,4-trimethylpentane passive samplers (TRIMPS) were used to determine the exposure to 97 pesticides in 24 southeast Australian stream sites over 5 months. Macroinvertebrate communities and selected microorganisms (bacteria, flagellates, ciliates, amoebas, nematodes, and gastrotrichs) were sampled to detect relationships with pesticide toxicity. Sediment samples had the highest estimated toxicities in terms of toxic units (TU) for Daphnia magna (TUDM) and for Selenastrum capricornutum (TUSC). The pesticide-selective SPEARpesticides and the general SIGNAL index for macroinvertebrates exhibited negative linear relationships (r(2) = 0.67 and 0.36, respectively) with pesticide contamination in terms of log maximum TUDM (log mTUDM), suggesting macroinvertebrate community change due to pesticide exposure. Pesticide contamination was the only measured variable explaining variation in ecological quality. Variation in the densities of several microbial groups was best explained by environmental variables other than log TUs. The log mTUDM values derived from sediment concentrations were most important to establish a link with effects on macroinvertebrates, whereas log mTUDM of grab water samples had only minor contribution. Current-use insecticides and fungicides can affect macroinvertebrate communities and monitoring of sediment and continuous water sampling is needed to detect these effects.

Fungicides are regularly applied in horticultural production systems and may migrate off-site, potentially posing an ecological risk to surface waterways. However, few studies have investigated the fate of fungicides in horticultural catchments. This study investigated the presence of 24 fungicides at 18 sites during a 5-month period within a horticultural catchment in southeastern Australia. Seventeen of the 24 fungicides were detected in the waterways, with fungicides detected in 63% of spot water samples, 44% of surface sediment samples, and 44% of the passive sampler systems deployed. One third of the water samples contained residues of two or more fungicides. Myclobutanil, trifloxystrobin, pyrimethanil, difenoconazole, and metalaxyl were the fungicides most frequently detected, being present in 16-38% of the spot water samples. Iprodione, myclobutanil, pyrimethanil, cyproconazole, trifloxystrobin, and fenarimol were found at the highest concentrations in the water samples (> 0.2 μg/l). Relatively high concentrations of myclobutanil and pyrimethanil (≥ 120 μg/kg dry weight) were detected in the sediment samples. Generally the concentrations of the fungicides detected were several orders of magnitude lower than reported ecotoxicological effect values, suggesting that concentrations of individual fungicides in the catchment were unlikely to pose an ecological risk. However, there is little information on the effects of fungicides, especially fungi and microbes, on aquatic ecosystems. There is also little known about the combined effects of simultaneous low-level exposure of multiple fungicides to aquatic organisms. Further research is required to adequately assess the risk of fungicides in aquatic environments.

A method using field-based microcosms was developed to determine the effects of contaminated sediments on aquatic macroinvertebrates. Fine sediments from nonpolluted, moderately polluted, and severely polluted bodies of water were placed in microcosms positioned within the littoral zone of a nonpolluted wetland near Melbourne (Victoria, Australia). In three experiments, 47 taxa, including 18 Chironomidae, 6 taxa from other Diptera families, and 7 Hemiptera taxa, colonized the microcosms, mostly via eggs deposited by flying adults. The effects of sediment type on the presence and abundance of common taxa were considered statistically. Pollution levels in sediments (indexed either by a principal components analysis or by the concentration of zinc, the predominant metal) resulted in reduced occurrence and abundance of eight taxa but had no effect on another five taxa. These findings were validated with an extensive field database for the distribution of macroinvertebrates and associated concentrations of zinc in sediments from streams and wetlands in the Melbourne region. The occurrence of eight taxa and the abundance of two taxa varied at similar zinc concentrations in sediments from both the microcosms and the field. Patterns for another two species did not match the microcosm results, but these groups contained multiple species with potentially diverse responses. The present results suggest that contaminant levels in sediments probably have a direct effect on the occurrence and abundance of macroinvertebrates in bodies of water in urban areas. The microcosm method can be used to gather information regarding the effects of sediment quality on macroinvertebrates in lentic habitats, particularly for indigenous species that cannot be easily reared or tested in laboratory conditions. Because almost all macroinvertebrates in microcosms develop from eggs, the most sensitive life stages (i.e., first and second instars) are exposed to polluted sediments.