Can non-destructive DNA extraction of bulk invertebrate samples be used for metabarcoding?

BACKGROUND: High throughput DNA sequencing of bulk invertebrate samples or metabarcoding is becoming increasingly used to provide profiles of biological communities for environmental monitoring. As metabarcoding becomes more widely applied, new reference DNA barcodes linked to individual specimens identified by taxonomists are needed. This can be achieved through using DNA extraction methods that are not only suitable for metabarcoding but also for building reference DNA barcode libraries. METHODS: In this study, we test the suitability of a rapid non-destructive DNA extraction method for metabarcoding of freshwater invertebrate samples. RESULTS: This method resulted in detection of taxa from many taxonomic groups, comparable to results obtained with two other tissue-based extraction methods. Most taxa could also be successfully used for subsequent individual-based DNA barcoding and taxonomic identification. The method was successfully applied to field-collected invertebrate samples stored for taxonomic studies in 70% ethanol at room temperature, a commonly used storage method for freshwater samples. DISCUSSION: With further refinement and testing, non-destructive extraction has the potential to rapidly characterise species biodiversity in invertebrate samples, while preserving specimens for taxonomic investigation.

Detecting invertebrate species in archived collections using next-generation sequencing.

Invertebrate biodiversity measured at mostly family level is widely used in biological monitoring programmes to assess anthropogenic impacts on ecosystems. However, next-generation sequencing (NGS) could allow development of new more sensitive biomonitoring tools by allowing rapid species identification. This could be accelerated if archived invertebrate collections and environmental information from past programmes are used to understand species distributions and their environmental responses. In this study, we take archived macroinvertebrate samples from two sites collected on multiple occasions and test whether NGS can successfully detect species. Samples had been stored in 70% ethanol at room temperature for up to 12 years. Three amplicons ranging from 197 to 274 bps within the DNA barcode region were amplified from samples and compared to DNA barcoding libraries to identify species. We were able to amplify partial DNA barcodes from most samples, and species were often detected with multiple amplicons. However, some singletons and taxa poorly covered by DNA barcoding were missed. This suggests additional DNA barcodes will be required to fill 'gaps' in current DNA barcode libraries for aquatic macroinvertebrates and/or that it may not be possible to detect all taxa in a sample. Furthermore, older samples often detected fewer taxa and were less reliable for amplification, suggesting NGS is best used on samples within 8 years of collection. Nevertheless, many common taxa with existing DNA barcodes were reliably identified with NGS and were often present at sites across multiple years, showing the potential of NGS for detecting common and abundant species in archived material.

Toxicant mixtures in sediment alter gene expression in the cysteine metabolism of Chironomus tepperi.

Sediment contamination can pose risks to the environment, and sediment toxicity tests have been developed to isolate the impact of sediment from other factors. Mixtures of contaminants often occur in sediments, and traditional endpoints used in toxicity testing, such as growth, reproduction, and survival, cannot discern the cause of toxicity from chemical mixtures because of complex interactions. In urban waterways, the synthetic pyrethroid bifenthrin and the metal copper are commonly found in mixtures, so the present study was designed to investigate how these contaminants cause toxicity in mixtures. To investigate this, Chironomus tepperi was exposed to environmentally relevant concentrations of copper and bifenthrin-spiked sediments in a 2-way factorial mixture for 5 d. Growth and expression profiles of cysteine metabolism genes were measured after exposure. Growth increased at low copper concentrations, decreased at high copper concentrations, and was unaffected by bifenthrin exposures. Copper exposures induced possible cellular repair by upregulating S-adenosylmethionine synthetase expression and downregulating expression of S-adenosylhomocysteine hydrolase and cystathionine-ß-synthase. Metallothionein upregulation was also observed. Bifenthrin exposure altered cysteine metabolism to a lesser extent, downregulating cystathionine-ß-synthase and γ-glutamylcysteine synthase. Synergistic, antagonistic, and dose-dependent interactions were observed, and there was evidence of conflicting modes of action and limited substrate production. These findings demonstrate how contextual gene expression changes can be sensitive and specific identifiers of toxicant exposure in mixtures. Environ Toxicol Chem 2017;36:691-698. © 2016 SETAC.

Genes involved in cysteine metabolism of Chironomus tepperi are regulated differently by copper and by cadmium.

Freshwater invertebrates are often exposed to metal contamination, and changes in gene expression patterns can help understand mechanisms underlying toxicity and act as pollutant-specific biomarkers. In this study the expressions of genes involved in cysteine metabolism are characterized in the midge Chironomus tepperi during exposures to sublethal concentrations of cadmium and copper. These metals altered gene expression of the cysteine metabolism differently. Both metals decreased S-adenosylhomocysteine hydrolase expression and did not change the expression of S-adenosylmethionine synthetase. Cadmium exposure likely increased cystathionine production by up-regulating cystathionine-ß-synthase (CßS) expression, while maintaining control level cysteine production via cystathionine-γ-lyase (CγL) expression. Conversely, copper down-regulated CßS expression and up-regulated CγL expression, which in turn could diminish cystathionine to favor cysteine production. Both metals up-regulated glutathione related expression (γ-glutamylcysteine synthase and glutathione synthetase). Only cadmium up-regulated metallothionein expression and glutathione S-transferase d1 expression was up-regulated only by copper exposure. These different transcription responses of genes involved in cysteine metabolism in C. tepperi point to metal-specific detoxification pathways and suggest that the transsulfuration pathway could provide biomarkers for identifying specific metals.

Environmental monitoring using next generation sequencing: rapid identification of macroinvertebrate bioindicator species.

INTRODUCTION: Invertebrate 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. RESULTS: In 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. CONCLUSIONS: Next 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.

Characterization of polymorphic microsatellites in the giant bulldog ant, Myrmecia brevinoda and the jumper ant, M. pilosula.

The ant genus Myrmecia Fabricius (Hymenoptera: Formicidae) is endemic to Australia and New Caledonia, and has retained many biological traits that are considered to be basal in the family Formicidae. Here, a set of 16 dinucleotide microsatellite loci were studied that are polymorphic in at least one of the two species of the genus: the giant bulldog ant, M. brevinoda Forel, and the jumper ant, M. pilosula Smith; 13 are novel loci and 3 are loci previously published for the genus Nothomyrmecia Clark (Hymenoptera: Formicidae). In M. brevinoda, the total of 12 polymorphic microsatellites yielded a total of 125 alleles, ranging from 3 to 18 with an average of 10.42 per locus; the observed and expected heterozygosities ranged from 0.4000 to 0.9000 and from 0.5413 to 0.9200, respectively. In M. pilosula, the 9 polymorphic loci yielded a total of 67 alleles, ranging from 3 to 12 with an average of 7.44 per locus; the observed and expected heterozygosities ranged from 0.5625 to 0.9375 and from 0.4863 to 0.8711, respectively. Five loci were polymorphic in both target species. In addition, 15 out of the 16 loci were successfully amplified in M. pyriformis. These informative microsatellite loci provide a powerful tool for investigating the population and colony genetic structure of M. brevinoda and M. pilosula, and may also be applicable to a range of congeners considering the relatively distant phylogenetic relatedness between M. pilosula and the other two species within the genus Myrmecia.

Phylogenetic signals and ecotoxicological responses: potential implications for aquatic biomonitoring.

Macroinvertebrates can be successfully used as biomonitors of pollutants and environmental health because some groups are sensitive whereas, others are relatively tolerant to pollutants. An issue of ongoing debate is what constitutes an appropriate group for biomonitoring; should the group represent species, genera or higher taxonomic levels? A phylogenetic framework can provide new insights into this issue. By developing phylogenies for chironomids and mayflies, this investigation shows that there is strong phylogenetic signal for pollution responses, and that phylogenetic nodes are common to tolerant and sensitive groups of species. A phylogenetic analysis of biotic indices developed for mayflies based on their response to organic pollution shows that mayfly families varied in pollution tolerance. In contrast, based on sediment zinc concentrations as an indicator of pollution tolerance, Australian chironomids tend to vary in tolerance at lower taxonomic levels. Published data on North American chironomids shows much of the signal for pollution responses is contained within genera rather than sub-families. Tools are now available to distinguish whether this signal reflects historical evolutionary constraints or environmental effects leading to common evolved responses. This suggests that ideally higher taxonomic levels should be used for biomonitoring when there are strong phylogenetic constraints at higher levels. Evolutionary considerations can therefore help to guide the development of macroinvertebrate biomonitors and provide insights into processes that produce sensitive and tolerant taxa.

Combining rapid bioassessment and field-based microcosms for identifying impacts in an urban river.

Rapid bioassessment indices based on macroinvertebrates are the most commonly used tools for assessing stream condition. However, once stream degradation has been detected, it is often difficult to identify which environmental stressors are most important because of changes in multiple correlated factors. In this study, we examined eight sites in an urban river watershed using a field-based microcosm experiment and the rapid bioassessment-based biotic index, SIGNAL. The experiment assessed the effects of polluted river sediment by examining the macroinvertebrate taxa that colonized sediments at an unpolluted wetland. Results were compared with an assessment of field-collected macroinvertebrates using SIGNAL, a biotic index that assigns pollution sensitivity scores to macroinvertebrate families, and environmental data, to determine whether sediment pollution or other factors such as habitat deterioration were likely to be influencing riverine macroinvertebrate communities. The microcosm results indicated that common species (Tanytarsus fuscithorax, Procladius paludicola, and Ablabesmyia notabilis) and the overall macroinvertebrate assemblage did not significantly change among sediments from different sites, with the exception of local effects on a few uncommon taxa (Chironomus pseudoppositus, Kiefferulus martini, Cladotanytarsus australomancus, Chaoboridae, Polypedilum "S1," and Tanytarsus belairensis). In contrast, SIGNAL showed a gradual trend of deterioration from upstream to downstream, decreasing from a score of 6.5 in upstream areas (unimpacted) to a score of 4.4 in the downstream sites (moderately impacted). This result combined with a significant correlation of SIGNAL scores to habitat data suggested that habitat deterioration rather than polluted sediment was likely to be responsible for the declining stream condition detected with the rapid bioassessment approach. The addition of the microcosms to other monitoring approaches could be useful for determining whether sediment pollution is influencing degraded urban streams.

High molecular weight petroleum hydrocarbons differentially affect freshwater benthic macroinvertebrate assemblages.

High-molecular weight petroleum hydrocarbons (HMWPHs) are a common pollutant in urban freshwater sediments. A previous study found that HMWPHs derived from synthetic oils are detrimental to aquatic benthic macroinvertebrates at concentrations greater than 840 mg/kg, but it is unclear what effect hydrocarbons derived from other sources have on these organisms. A field-based microcosm experiment was conducted to determine whether natural and other types of HMWPHs produce similar effects on indigenous benthic macroinvertebrates as was induced by synthetic motor oils. Ordinations and comparisons of sensitive species indicated that HMWPHs from different anthropogenic sources negatively affected macroinvertebrates, whereas naturally occurring hydrocarbons above the threshold of 840 mg/kg had no detrimental effect. This result suggests that total petroleum hydrocarbon content, which is often used to identify hydrocarbon pollutants, is a good conservative indicator of HMWPH concentrations affecting the biota.