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

DNA microarray profiling of genes differentially regulated by the histone deacetylase inhibitors vorinostat and LBH589 in colon cancer cell lines

<p>Abstract</p> <p>Background</p> <p>Despite the significant progress made in colon cancer chemotherapy, advanced disease remains largely incurable and novel efficacious chemotherapies are urgently needed. Histone deacetylase inhibitors (HDACi) represent a novel class of agents which have demonstrated promising preclinical activity and are undergoing clinical evaluation in colon cancer. The goal of this study was to identify genes in colon cancer cells that are differentially regulated by two clinically advanced hydroxamic acid HDACi, vorinostat and LBH589 to provide rationale for novel drug combination partners and identify a core set of HDACi-regulated genes.</p> <p>Methods</p> <p>HCT116 and HT29 colon cancer cells were treated with LBH589 or vorinostat and growth inhibition, acetylation status and apoptosis were analyzed in response to treatment using MTS, Western blotting and flow cytometric analyses. In addition, gene expression was analyzed using the Illumina Human-6 V2 BeadChip array and Ingenuity^®Pathway Analysis.</p> <p>Results</p> <p>Treatment with either vorinostat or LBH589 rapidly induced histone acetylation, cell cycle arrest and inhibited the growth of both HCT116 and HT29 cells. Bioinformatic analysis of the microarray profiling revealed significant similarity in the genes altered in expression following treatment with the two HDACi tested within each cell line. However, analysis of genes that were altered in expression in the HCT116 and HT29 cells revealed cell-line-specific responses to HDACi treatment. In addition a core cassette of 11 genes modulated by both vorinostat and LBH589 were identified in both colon cancer cell lines analyzed.</p> <p>Conclusion</p> <p>This study identified HDACi-induced alterations in critical genes involved in nucleotide metabolism, angiogenesis, mitosis and cell survival which may represent potential intervention points for novel therapeutic combinations in colon cancer. This information will assist in the identification of novel pathways and targets that are modulated by HDACi, providing much-needed information on HDACi mechanism of action and providing rationale for novel drug combination partners. We identified a core signature of 11 genes which were modulated by both vorinostat and LBH589 in a similar manner in both cell lines. These core genes will assist in the development and validation of a common gene set which may represent a molecular signature of HDAC inhibition in colon cancer.</p

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-β-synthase (CβS) expression, while maintaining control level cysteine production via cystathionine-γ-lyase (CγL) expression. Conversely, copper down-regulated CβS 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.6 page(s

cyst_metab_LC data

Concentrations of amine containing metabolites involved in the cysteine metabolism of Chironomus tepperi deployed in microcosms. Detected by LCM

Chironomus_dryweight_raw

Dry weight data for deployed Chironomus tepper

Gene expression data

Gene expression data of the cysteine metabolism of Chironomus tepperi deployed in microcosm