Nondestructive DNA extraction from blackflies (Diptera: Simuliidae): retaining voucher specimens for DNA barcoding projects.

A nondestructive, chemical-free method is presented for the extraction of DNA from small insects. Blackflies were submerged in sterile, distilled water and sonicated for varying lengths of time to provide DNA which was assessed in terms of quantity, purity and amplification efficiency. A verified DNA barcode was produced from DNA extracted from blackfly larvae, pupae and adult specimens. A 60-second sonication period was found to release the highest quality and quantity of DNA although the amplification efficiency was found to be similar regardless of sonication time. Overall, a 66% amplification efficiency was observed. Examination of post-sonicated material confirmed retention of morphological characters. Sonication was found to be a reliable DNA extraction approach for barcoding, providing sufficient quality template for polymerase chain reaction amplification as well as retaining the voucher specimen for post-barcoding morphological evaluation.

Knowledge transfer initiative between molecular biologists and environmental researchers and regulators.

A Natural Environment Research Council (NERC) funded Knowledge Transfer (KT) workshop was held in the United Kingdom (UK) to identify the needs and opportunities in the application of molecular biology and 'omics' techniques to environmental monitoring and risk assessment. Attendees highlighted a lack of effective communication between end-users and researchers as well as difficulties with data interpretation as reasons behind the slow uptake of molecular biology and omics techniques. A number of promising areas in which new techniques could be implemented at a practical level in the very near future were identified, thereby raising the profile of these recent technologies and providing vital proof of concept work. Molecular taxonomy, bacterial source tracking and pre-screening of chemicals for potential toxicities were all viewed as areas in which omics and molecular techniques could have immediate value, with the aim of reducing cost, increasing efficiency and providing more comprehensive data of improved quality.

Using ecological diversity measures with bacterial communities.

Abstract There are many ecological diversity measures, but their suitability for use with highly diverse bacterial communities is unclear and seldom considered. We assessed a range of species richness and evenness/dominance indices, and the use of species abundance models using samples of bacteria from zinc-contaminated and control soils. Bacteria were assigned to operational taxonomic units (OTUs) using amplified ribosomal DNA restriction analysis of 236 clones from each soil. The reduced diversity apparent in the contaminated soil was reflected by the diversity indices to varying degrees. The number of clones analysed and the weighting given to rare vs. abundant OTUs are the most important considerations when selecting measures. Our preferences, arrived at using theory and practical experience, include: the log series index alpha; the Q statistic (but only if coverage is 50% or more); the Berger-Parker and Simpson's indices, although their ecological relevance may be limited; and, unexpectedly, the Shannon-Wiener and Shannon evenness indices, even though their meanings may not be clear and their values inaccurate when coverage is low. For extrapolation, the equation for the log series distribution seems the best for extrapolating from OTU accumulation curves while non-parametric methods, such as Chao 1, show promise for estimating total OTU richness. Due to a preponderance of single-occurrence OTUs, none of the five species abundance models fit the OTU abundance distribution of the control soil, but both the log and log normal models fit the less diverse contaminated soil. Species abundance models are useful, irrespective of coverage, because they address the whole distribution of a sample, aiding comparison by revealing overall trends as well as specific changes in particular abundance classes.

Molecular characterisation of bacteria in a wetland used to remove ammoniacal-N from landfill leachate.

Bacteria associated with leachate from a constructed, surface-flow wetland effective at removing ammonium-N, were characterised using molecular methods which bypass the need for cultivation. DNA was extracted from the sample and bacterial 16S rDNA sequences amplified and cloned. Ninety-six individual clones were re-amplified and analysed by restriction digestion and targeted sequencing. A collector's curve was used to estimate the total number of bacterial groups (operational taxonomic units, OTUs) in the sample at 47. Thirty-six clones representing 28 OTUs were sequenced and characterised by aligning them against published sequences. Of these, 10 were confidently identified, whilst for the remaining 18, the closest match was obtained. The sequences in 64% of the OTUs were similar to those of the Proteobacteria but no classical nitrifying bacteria were identified. However, alkaliphilic Methylomicrobium sp. that oxidise ammonium and bacteria from the genus Alcaligenes, at least one species of which is capable of heterotrophic nitrification, were detected.