Metabarcoding of Freshwater Invertebrates to Detect the Effects of a Pesticide Spill
Total Page：16
File Type：pdf, Size：1020Kb
Received: 14 June 2017 | Revised: 9 October 2017 | Accepted: 16 October 2017 DOI: 10.1111/mec.14410 ORIGINAL ARTICLE Metabarcoding of freshwater invertebrates to detect the effects of a pesticide spill Carmelo Andujar 1,2,3 | Paula Arribas1,2,3 | Clare Gray2 | Catherine Bruce4 | Guy Woodward2 | Douglas W. Yu5,6 | Alfried P. Vogler1,2 1Department of Life Sciences, Natural History Museum, London, UK Abstract 2Department of Life Sciences, Imperial Biomonitoring underpins the environmental assessment of freshwater ecosystems College London, Ascot, UK and guides management and conservation. Current methodology for surveys of 3Grupo de Ecología y Evolución en Islas, Instituto de Productos Naturales y (macro)invertebrates uses coarse taxonomic identification where species-level reso- Agrobiología (IPNA-CSIC), San Cristóbal de lution is difficult to obtain. Next-generation sequencing of entire assemblages la Laguna, Spain (metabarcoding) provides a new approach for species detection, but requires further 4NatureMetrics Ltd, CABI Site, Egham, Surrey, UK validation. We used metabarcoding of invertebrate assemblages with two fragments 5State Key Laboratory of Genetic of the cox1 “barcode” and partial nuclear ribosomal (SSU) genes, to assess the Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, effects of a pesticide spill in the River Kennet (southern England). Operational Kunming, Yunnan, China taxonomic unit (OTU) recovery was tested under 72 parameters (read denoising, 6 School of Biological Sciences, University of filtering, pair merging and clustering). Similar taxonomic profiles were obtained East Anglia, Norwich, Norfolk, UK under a broad range of parameters. The SSU marker recovered Platyhelminthes and Correspondence Nematoda, missed by cox1, while Rotifera were only amplified with cox1. A refer- Carmelo Andujar, Grupo de Ecologíay Evolución en Islas, Instituto de Productos ence set was created from all available barcode entries for Arthropoda in the BOLD ́ Naturales y Agrobiologia (IPNA-CSIC), San database and clustered into OTUs. The River Kennet metabarcoding produced Cristóbal de la Laguna, Spain. Email: [email protected] matches to 207 of these reference OTUs, five times the number of species recog- nized with morphological monitoring. The increase was due to the following: greater Funding information NERC, Grant/Award Number: NE/M021955 taxonomic resolution (e.g., splitting a single morphotaxon “Chironomidae” into 55 named OTUs); splitting of Linnaean binomials into multiple molecular OTUs; and the use of a filtration-flotation protocol for extraction of minute specimens (meiofauna). Community analyses revealed strong differences between “impacted” vs. “control” samples, detectable with each gene marker, for each major taxonomic group, and for meio- and macrofaunal samples separately. Thus, highly resolved taxonomic data can be extracted at a fraction of the time and cost of traditional nonmolecular methods, opening new avenues for freshwater invertebrate biodiversity monitoring and molecular ecology. KEYWORDS biomonitoring, community ecology, DNA barcoding, freshwater ecosystems, invertebrates 1 | INTRODUCTION species assemblages. Biomonitoring therefore is required to assess the ecological status of fresh waters and to enforce their protection The freshwater biota is affected by a host of natural environmental through legislation, such as the Water Framework Directive (WFD) drivers and, increasingly, anthropogenic disturbances that alter local of the European Union and the US Clean Water Act (European | Molecular Ecology. 2017;1–21. wileyonlinelibrary.com/journal/mec © 2017 John Wiley & Sons Ltd 1 2 | ANDUJAR ET AL. Commission 2000; United States 1972). However, this field of 2015; Lanzen, Lekang, Jonassen, Thompson, & Troedsson, 2016; applied ecology is still largely reliant on techniques that were devel- Leray & Knowlton, 2015) and freshwater sediments and the water oped over a century ago, albeit with some statistical advances, column (Bista et al., 2017; Elbrecht & Leese, 2015). Metabarcoding tweaks and adjustments in the intervening years, and has been can thus provide the elusive species resolution desired for roundly criticized for failing to adapt to a rapidly changing world (Fri- biomonitoring of entire ecosystems and also for capturing the large berg et al., 2011). The vast majority of biomonitoring schemes still proportion of organisms that are either too small to see or identify relies on identifying macro-invertebrates by eye, or at best via using traditional sorting and microscopy techniques (Bohan et al., microscopy, to a coarse level of taxonomic resolution, and the 2017; Creer et al., 2010; Hajibabaei, Baird, Fahner, Beiko, & Golding, molecular revolution that is overtaking mainstream ecology has yet 2016; Ji et al., 2013). to be embraced (Bohan et al., 2017; Pauls et al., 2014). Because of Here, we applied metabarcoding to study the consequences of the need for rapid and cost-effective approaches, it is routine prac- an insecticide spill on invertebrate freshwater communities in a large tice that many taxa are not identified to individual species but lowland river as a test case. On 1 July 2013, a pulse of the instead are lumped taxonomically, for example by family, as used in organophosphate chlorpyrifos in the River Kennet, the largest tribu- RIVPACS and AUSIVAS systems (Wright, Sutcliffe, & Furse, 2000), tary of the River Thames in southern England, led to population or, less frequently, into trait-based groupings, such as “riverflies.” crashes and localized extinctions of many invertebrate taxa (see The taxonomically difficult groups in which most of the aquatic bio- Thompson et al., 2016 for details). We used samples collected diversity resides (e.g., chironomid midges) are typically either ignored upstream and downstream from the spill site over several km of the or treated as a single entity (Jones, 2008; Schmidt-Kloiber & Nijboer, river’s length to explore the effectiveness of metabarcoding, and to 2004). trial new environmental diagnostic protocols for identifying differen- These labour-saving shortcuts can nonetheless provide a broad tial responses of invertebrate communities to a profound environ- assessment of the ecological state of a water body, despite the huge mental perturbation. For example, some components of the local amounts of environmental-status information that are inevitably jet- community such as the dominant detritivore, the amphipod Gam- tisoned in the process, and have been successfully used for the marus pulex, were greatly reduced in number downstream from the assessment of habitat and water quality for many decades (Camargo, spill, whereas other taxa, especially those with an aerial adult life 1993; Marshall, Steward, & Harch, 2006; Sanchez-Montoya et al., stage, were far less affected, and at a later sampling time returned 2007). However, population responses to changes in water quality to postspill levels, possibly due to their ability to recolonize rapidly. can differ between even closely related species, and so The Chironomidae (nonbiting midges) as a group greatly increased in taxonomically coarse inventories may miss the full impact of impor- abundance after the spill. However, because community composition tant environmental stressors (Chessman, Trayler, & Davis, 2002; was only measured at higher taxonomic levels, rather than with spe- Gutierrez-C anovas, Velasco, & Millan, 2008; Stubauer & Moog, cies-level resolution, it is not possible to gain further insight into the 2000). Species-level identification can establish the link to known mechanisms of ecological resilience and recovery after the spill. ecological, physiological and behavioural traits, which may reflect dif- Specifically, the species composition of the postimpact chironomid ferential responses to environmental conditions, and also may reveal community might be largely unchanged from the pre-impact commu- the membership in feeding groups and position in trophic networks nity, or, despite the increased abundance, it might be composed of a (Bohan et al., 2017). These distinctions are lost if the assessment is subset of that community (nestedness) or of a new set of species at the level of genera or families, or other such coarse groupings dispersed from elsewhere (turnover). (Leese et al., 2016; Schmidt-Kloiber & Hering, 2015). The cox1 gene is the obvious choice of a marker for metabarcod- Recent protocols for metabarcoding, that is the sequencing of ing of aquatic invertebrates, but due to the constraints on read PCR amplicons from environmental specimen mixtures, could provide length, the widely used Illumina platform is not suited for sequencing faster and more highly resolved taxonomic identification of complex the full-length amplicon of the barcode region (658 bp). We have assemblages (Taberlet, Coissac, Pompanon, Brochmann, & Willerslev, metabarcoded two gene fragments covering the entire cox1 barcode 2012). This methodology applies Hebert, Cywinska, Ball, and region using two primer pairs shown to have broad target ranges DeWaard’s (2003) idea of species identification through short (Arribas et al., 2016), which here were applied for the first time to diagnostic DNA barcodes (a fragment of the cox1 gene) to the freshwater invertebrate communities. The parallel use of two bar- community level, and thanks to new high-throughput sequencing code fragments provides a test of amplification breadth and poten- (HTS) technology, the effort required for DNA barcoding