Use of DNA Metabarcoding for Stomach Content Analysis in the Invasive Lionfish Pterois Volitans in Puerto Rico
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Vol. 558: 181–191, 2016 MARINE ECOLOGY PROGRESS SERIES Published October 25 doi: 10.3354/meps11738 Mar Ecol Prog Ser Contribution to the Theme Section ‘Invasion of Atlantic coastal ecosystems by Pacific lionfish’ OPENPEN ACCESSCCESS Use of DNA metabarcoding for stomach content analysis in the invasive lionfish Pterois volitans in Puerto Rico Chelsea A. Harms-Tuohy*, Nikolaos V. Schizas, Richard S. Appeldoorn Department of Marine Sciences, University of Puerto Rico at Mayagüez, Call Box 9000, Mayagüez, PR 00681, USA ABSTRACT: Studies of lionfish feeding ecology seek to document the ecological impact of this invasive predatory species and determine which native prey species are at greatest risk. There are 2 common approaches to feeding ecology through gut content analysis: morphological identifica- tion to the lowest possible taxonomic rank and/or DNA barcoding of individual prey components in the stomach. The major disadvantage of both techniques is their inability to use advanced digested material. This study introduces next-generation sequencing to lionfish feeding ecology, employing DNA metabarcoding to analyze all components of the gut contents, including the pre- viously unidentifiable portion. Sixty-three lionfish were caught from the inshore and offshore reefs of La Parguera, Puerto Rico. Stomach contents were separated into 2 sample components — a liquid (i.e. digested) and undigested tissue. A 313 bp region of the cytochrome oxidase subunit I (COI) gene was amplified from extracted DNA using specific primers for Caribbean reef fish. Samples were sequenced with an Illumina MiSeq platform, and the resulting 950+ sequences were compared against GenBank and the Barcode of Life Database to identify specimens at the lowest taxonomic level. Thirty-nine fish species from 16 families were identified (35 each in the digested and tissue fractions), including members of Pomacentridae, Acanthuridae, Gobiidae, Apogonidae, and Scaridae. Using the digested liquiform material proved efficient in detecting prey species, especially those that would have been missed with traditional methods. KEY WORDS: Reef fish · Feeding ecology · Invasive species · Caribbean · Cytochrome oxidase subunit I · COI · Next-generation sequencing · NGS INTRODUCTION tem, whereby complete eradication is unfeasible (Thresher & Kuris 2004). This scenario is exacerbated Invasive species are capable of altering ecosys- when their presence extends to areas that remain tems, evolving with their new environment (Mooney inaccessible to management, such as mesophotic & Cleland 2001) and driving native species extinc- depths, or in cases where the spread of the invasive tions (Pimm 1987, Fritts & Rodda 1998). In response, species is driven by larval dispersal. Aside from management of invasive species attempts to mitigate investigating management strategies, invasion ecol- their ecological and economic impacts (Buckley ogists must simultaneously seek to identify which 2008). However, marine invasive species present a native communities may be at greatest risk, either difficult management scenario where vectors pro- ecologically or economically. moting their spread and establishment may be Invasive species alter ecosystems through com - known (i.e. ballast transport, aquarium trade) but petition, niche displacement, hybridization, and pre- cannot be easily regulated or avoided without strict dation, among other processes (Mooney & Cleland enforcement (Bax et al. 2003). Marine invaders, once 2001). In particular, predation in the marine environ- established, often become integrated into the ecosys- ment is a driving force structuring the fish communi- © The authors 2016. Open Access under Creative Commons by *Corresponding author: [email protected] Attribution Licence. Use, distribution and reproduction are un - restricted. Authors and original publication must be credited. Publisher: Inter-Research · www.int-res.com 182 Mar Ecol Prog Ser 558: 181–191, 2016 ties on coral reefs (Hixon 1991). Aside from observing fishes. Researchers have sought to address what lion- this predator−prey interaction in situ, predation can fish consume, in terms of species and size classes, in also be documented using visual inspection or, more an effort to document which species may suffer the recently, DNA barcoding to assess biodiversity in greatest level of mortality. Feeding ecology has been diet from gut contents or feces (Sheppard & Harwood a key component in many lionfish studies, resulting 2005). Over a decade has passed since DNA bar - in our current understanding of site specificity in coding first proved useful in biodiversity applications dietary preferences (Côté & Maljkovic´ 2010, Muñoz (Hebert et al. 2003), and has recently been promoted et al. 2011, Layman & Allgeier 2012) and overall as an ecological tool for addressing issues including a diversity of diet (Albins & Hixon 2008, Morris & species’ invasion potential, trophic interactions, and Akins 2009, Green et al. 2011). food webs (Joly et al. 2014). With the advancement There are 2 common approaches to lionfish feeding and lower cost of DNA sequencing and massive ecology through gut content analysis: morphological growth of reference databases, a metabarcoding ap - identification to the lowest possible taxon (i.e. using proach using next-generation sequencing (NGS) has morphological characters to identify whole or only quickly emerged as a promising method for higher- partially digested specimens) or a DNA barcoding ap- resolution diet analysis (Pompanon et al. 2012, Ta - proach, which involves sequencing of the mitochon- berlet et al. 2012, de Barba et al. 2014, Deagle et al. drial 16S rRNA or cytochrome oxidase subunit I (COI) 2013). Metabarcoding is the combination of DNA- genes from all distinct prey components of the stom- based identification and high-throughput DNA se - ach. Morphological identification relies heavily on the quencing that reduces sampling effort and maxi- ability to identify digested organisms to the species mizes species-level identification of tissue remnants level, which is not possible in many cases (Baker et al. that were previously undetected or underused by 2014). This technique discards useful information that traditional methods. could be obtained in the digested portion of the stom- There are known constraints on metabarcoding, ach contents (the liquids or digested pulp). However, including the inability to quantify the species infor- the traditional morphological method is widely ap- mation obtained (Deagle et al. 2010, 2013, Bowles et plied (Albins & Hixon 2008, Morris & Akins 2009, al. 2011, Murray et al. 2011). Results are limited or Alexander & Haynes 2011, Jud et al. 2011, Muñoz et biased to the frequency of occurrence, which still al. 2011, Green et al. 2012, Frazer et al. 2012, Layman provides useful information when seeking to under- & Allgeier 2012, Green & Côté 2014), while the more stand localized effects of an invasive predator. How- accurate DNA barcoding approach has been less fre- ever, the underlying variability in DNA quality, dif- quently used (Barbour et al. 2010, Valdez-Moreno et ferential breakdown of that DNA during digestion, al. 2012, Côté et al. 2013). Despite the higher resolu- and differences in digestion stages (Deagle & Tollit tion attained with this approach, traditional DNA bar- 2007, Troedsson et al. 2009, Valentini et al. 2009b), as coding also has disadvantages. This technique does well as the objective of identifying several different not reduce sampling effort (Coissac et al. 2012) and organisms within the same sample (i.e. the gut) can be applied only to items in the stomach contents (Valentini et al. 2009a), still hinder the quantification for which barcode information is available either in aspect in metabarcoding of gut contents. Despite databases or can be generated during concomitant se- these disadvantages, metabarcoding is quickly gain- quencing of pos sible prey from the area. However, as ing popularity as a tool for assessing biodiversity in opposed to morphological identification, analyzed animal diets (Leray et al. 2013, de Barba et al. 2014). items can include unrecognizable specimens, liquids, NGS allows for the highest degree of confidence in or pulp (Saitoh et al. 2003), but this approach requires gut content analysis (Pompanon et al. 2012) with sig- mol ecular cloning and is therefore labor intensive and nificantly reduced sampling effort (Taberlet et al. costly. These digested products may contain under- 2012), but has only recently been applied to fish represented prey items, or prey items that have yet to feeding ecology (Leray et al. 2013, 2015). be acknowledged within the diet. Understanding the extent and possible ecological In this study, we used metabarcoding analysis of all impact of the lionfish Pterois volitans invasion of the lionfish stomach contents, regardless of their diges- Western Atlantic, Gulf of Mexico and Caribbean is tive stage, to provide a more accurate profile of the an issue that employs all facets of lionfish biology lionfish prey in Puerto Rico while demonstrating that and ecology. Of particular interest is how this Indo- the methodological approach is applicable to all Pacific fish will affect native coral reef fauna, espe- other regions of the invasion. Metabarcoding resolu- cially commercially and ecologically important reef tion of lionfish stomach contents is supported by the Harms-Tuohy et al.: DNA metabarcoding of lionfish stomach contents 183 a priori knowledge, albeit site specific, of the