Canadian Journal of Fisheries and Aquatic Sciences Utilizing DNA metabarcoding to characterize the diet of marine-phase Arctic lamprey (Lethenteron camtschaticum) in the eastern Bering Sea Journal: Canadian Journal of Fisheries and Aquatic Sciences Manuscript ID cjfas-2018-0299.R1 Manuscript Type: Article Date Submitted by the 18-Nov-2018 Author: Complete List of Authors: Shink, Katie; University of Alaska Fairbanks, College of Fisheries and Ocean Sciences Sutton, Trent; University of Alaska, College of Fisheries and Ocean Sciences, FisheriesDraft Division Murphy, James; National Marine Fisheries Service - NOAA López, J. Andrés; University of Alaska, College of Fisheries and Ocean Sciences, Fisheries Division; University of Alaska Museum of the North LAMPREYS < Organisms, DIET < General, FEEDING < General, DNA < Keyword: General Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? : https://mc06.manuscriptcentral.com/cjfas-pubs Page 1 of 36 Canadian Journal of Fisheries and Aquatic Sciences 1 Utilizing DNA metabarcoding to characterize the diet of marine-phase Arctic lamprey (Lethenteron 2 camtschaticum) in the eastern Bering Sea. 3 4 KATIE G. SHINK1,*, TRENT M. SUTTON1, JAMES M. MURPHY2, and J. ANDRÉS LÓPEZ1,3 5 6 1College of Fisheries and Ocean Sciences, University of Alaska Fairbanks, 2150 Koyukuk Street, 7 Fairbanks, Alaska 99775; email: [email protected], [email protected] 8 9 2Ted Stevens Marine Research Institute, Auke Bay Laboratories, Alaska Fisheries Science Center, 10 NOAA Fisheries, 17109 Point Lena LoopDraft Road, Juneau, Alaska 99801; email: 11 [email protected] 12 13 3University of Alaska Museum of the North, 1962 Yukon Drive, Fairbanks, Alaska 99775: email: 14 [email protected] 15 16 *Corresponding author 17 Katie G Shink 18 University of Alaska Fairbanks 19 College of Fisheries and Ocean Sciences 20 Email: [email protected] 21 Phone: 1 509 995 3389 22 23 24 1 https://mc06.manuscriptcentral.com/cjfas-pubs Canadian Journal of Fisheries and Aquatic Sciences Page 2 of 36 25 Abstract 26 To understand the marine feeding ecology of Arctic lamprey (Lethenteron camtschaticum) in the 27 eastern Bering Sea, visual observations and DNA metabarcoding of gut contents (N = 250) were 28 used to characterize Arctic lamprey diet composition in 2014 and 2015. Differences among 29 individual diets were evaluated by collection year, capture site, and fish size. Hard structures and 30 tissues were observed during visual examinations of gut contents, and 10 ray-finned fish taxa were 31 identified by DNA metabarcoding. The most frequent taxa included capelin (Mallotus villosus), 32 Pacific herring (Clupea pallasii), Pacific sand lance (Ammodytes hexapterus), and gadids. Six taxa 33 were reported for the first time as prey for Arctic lamprey. Individual diets differed between 34 collection years, among capture sites, and among size classes; however, both collection year and Draft 35 size explained only a small portion of diet variability (R2 = 0.01 and 0.04, respectively) relative to 36 station site (R2 = 0.49). These study results indicate that Arctic lamprey are flesh-feeding species, 37 and highlighted the value of DNA metabarcoding to characterize the diet of a poorly understood 38 lamprey species. 39 Introduction 40 Characterizing the diets of marine-phase lamprey poses a special challenge to researchers. 41 Diet analysis of fishes has largely relied upon morphological identification of prey remains and/or 42 undigested hard structures within digestive tracts (Madenjian et al 1998; Creque and Czesny 2012; 43 Whitney et al. 2017). However, digested blood and/or tissue masses consumed by lamprey 44 generally yield limited details on prey composition. Observations of lamprey wounds on teleost 45 fishes and occurrences of hard, undigested structures within lamprey intestinal contents have been 46 routinely used to identify prey (Beamish 1980; Maitland et al. 1984; Novomodnyy and Belyaev 2 https://mc06.manuscriptcentral.com/cjfas-pubs Page 3 of 36 Canadian Journal of Fisheries and Aquatic Sciences 47 2002; Renaud et al. 2009). While observations of lamprey wounds provide insights into lamprey 48 feeding interactions, identified prey may be biased toward highly valued and frequently encountered 49 commercial fishes (Hardisty and Potter 1971). In addition, undigested hard structures, while 50 taxonomically informative, often have variable recovery and digestion rates and may not be 51 regularly ingested during predation, which can lead to biased or misleading dietary inferences 52 (Tollit et al. 1997; Bowen 2000; Cottrell and Trites 2002). As a result, trophic interactions of 53 marine-phase lamprey remain poorly understood (Mesa and Copeland 2009). 54 The food habits of closely related species of lamprey can vary from blood to the flesh of 55 their prey (Potter and Hilliard 1987; Renaud et al. 2009). Flesh-feeding species are generally 56 characterized with having smaller buccalDraft glands, a smaller oral disc with fewer teeth, and having an 57 enlarged median cusp on the U-shaped transverse lingual lamina atop the ‘tongue-like piston’ 58 (Potter and Hilliard 1987; Renaud et al. 2009). Lampreys that exhibit flesh-feeding food habits 59 target smaller-bodied fishes and inflict serious damage that often results in the death of the prey 60 (Roos et al. 1973; Beamish 1980; Maitland et al. 1984; Renaud et al. 2009). Flesh-feeding species 61 are known to ingest large pieces of flesh (including, on some occasions, whole fish) and have been 62 shown to penetrate the prey’s body cavity to consume internal organs (Beamish and Williams 1976; 63 Beamish 1980; Maitland et al. 1984). In contrast, blood-feeding lampreys primarily target larger 64 fish species that are less susceptible to damage because wounds from blood feeders are 65 characterized by a single hole or slide where blood can continuously be extracted (Potter and 66 Hilliard 1987; Renaud et al. 2009; Patrick et al. 2009). Previous research has largely inferred the 67 food habits of different lamprey species based on morphological characteristics of the oral disc and 68 dentition (Potter and Hilliard 1987; Renaud et al. 2009). Despite food habits and feeding 3 https://mc06.manuscriptcentral.com/cjfas-pubs Canadian Journal of Fisheries and Aquatic Sciences Page 4 of 36 69 mechanisms ranging from blood-feeding (i.e., parasitic) to flesh-feeding (i.e., predatory), the term 70 ‘parasitic’ is regularly used to describe the feeding ecology of all lampreys. 71 The Arctic lamprey (Lethenteron camtschaticum) is hypothesized to be a flesh-feeding 72 lamprey species (Potter and Hilliard 1987; Renaud et al. 2009). This conclusion was based on 73 morphological similarities in dentition to known flesh-feeding species (e.g., the European river 74 lamprey [Lampetra fluviatilis] and Western river lamprey [Lampetra ayresii]) and not explicit 75 evaluations of the diet (Potter and Hilliard 1987; Renaud et al. 2009). Visual examination of the 76 intestinal contents of known flesh-feeding species revealed the presence of tissue masses, fins, 77 scales, eggs, and internal organs (Beamish and Williams 1976; Beamish 1980; Maitland et al. 1984; 78 Renaud et al. 2009). Although visual observationsDraft provided insights into the food habits and feeding 79 mode of these lamprey species, unidentifiable remains were reported in up to 56% of the lampreys 80 examined (Beamish 1980; Maitland et al. 1984; Beamish and Neville 1995). To date, no studies 81 have examined the intestinal contents of marine-phase Arctic lamprey, and the potential occurrence 82 and frequency of these structures in the diet is currently unknown. 83 Much of what is known about Arctic lamprey diets originated from visual observations of 84 lampreys attached to fish and incidences of lamprey wounds on fishes (Nikol’skii 1956; Gritzenko 85 1968; Heard 1966; McPhail and Lindsey 1970; Nursall and Buchwald 1972; Novomodnyy and 86 Belyaev 2002; Shevlyakov and Parensky 2010). Arctic lamprey have been found attached to 87 Chinook salmon (Oncorhynchus tshawytscha), sheefish (Stenodus nelma), starry flounder 88 (Platichthys stellatus), and smelt (Osmeridae) within watersheds in southwestern Alaska and in the 89 Beaufort Sea (McPhail and Lindsey 1970). Within Russian estuaries, Arctic lamprey wounds have 90 been observed on forage fishes (e.g., Clupeidae, Osmeridae) and juvenile Pacific salmon, 4 https://mc06.manuscriptcentral.com/cjfas-pubs Page 5 of 36 Canadian Journal of Fisheries and Aquatic Sciences 91 suggesting the importance of these taxa as prey (Nikol’skii 1956; Gritzenko 1968; Novomodnyy 92 and Belyaev 2002; Shevlyakov and Parensky 2010). 93 The application of molecular techniques to characterize prey species in predator diets 94 improves detection and taxonomic resolution of prey relative to traditional morphological methods 95 (Braley et al. 2010; Carreon-Martinez et al. 2011; Moran et al. 2016). Continued development and 96 refinement of ‘DNA metabarcoding’ approaches for accurate species identification has made it 97 possible to characterize diet components that lack taxonomic characteristics with little a priori 98 information on predator diets (Valentini et al. 2009; Pompanon et al. 2012; Taberlet et al. 2012). 99 Prey DNA can be isolated from fecal or gastrointestinal tract samples and used for targeted 100 sequencing of taxonomically informativeDraft genome regions (reviewed in Pompanon et al. 2012). 101 However, DNA metabarcoding has only been used in a limited number of studies