Diet Analysis of Hawai‘i Island’s Blackburnia hawaiiensis (Coleoptera: Carabidae) using Stable Isotopes and High-Throughput Sequencing1 K. Roy,2,3,6 C. P. Ewing,2,4 and D. K. Price2,5 Abstract: Determining the diet of arthropods can be difficult due to their small size and complex food webs, especially in Hawai‘i, where knowledge of arthropod predator–prey interactions is sparse. The diet of the Hawai‘i Island-endemic carabid beetle, Blackburnia hawaiiensis Sharp (Coleoptera: Carabidae) is of particular interest because of its peculiar arboreal behavior and metathoracic flight wings. Our study objective was to determine the diet of B. hawaiiensis in replicated, geographically separated locations by using two different yet complementary laboratory techniques: natural abundance stable isotope analysis (SIA) and high-throughput sequencing (HTS). Overall, B. hawaiiensis had a greater average d15N and similar d13C compared to the other arthropods sampled in this study and HTS data revealed Diptera and Lepidoptera sequences in the beetle’s gut contents. These results are consistent with B. hawaiiensis being classified as a generalist predator. The combination of SIA and HTS are important methods for determining the diet of species within complex food webs, particularly for species that are difficult to observe in nature. Keywords: food web, metabarcoding, Hawai#i, carabid, B. hawaiiensis UNDERSTANDING THE DIET OF ORGANISMS can considering small, rare, or cryptic species elucidate conservation needs and inform (Gomez-Polo et al. 2015). Traditional diet management. However, trophic relationships studies often include direct observation of scat are often difficult to observe, especially when or gut contents, although these techniques are limited to organisms that consume indiges- tible structures leaving solid, identifiable 1This study was funded by the Hau‘oli Mau Loa remains (Hoogendoorn and Heimpel 2001). Foundation. Manuscript accepted 27 May 2020. 2 Specifically, arthropod diets and trophic Tropical Conservation Biology and Environmental positions are difficult to assign not only Science, University of Hawai‘i, Hilo, USA. 3U.S. Geological Survey, Pacific Island Ecosystems because of their small size but also because Research Center, Kīlauea Field Station, Hawai#i National many are generalists, therefore creating Park, Hawai#i, USA. complex food webs (Polis et al. 1989). 4 California Department of Forestry and Fire Protec- Additionally, generalist predators can partake tion, 5800 Chiles Road, Davis, CA, USA. 5School of Life Sciences, University of Nevada, Las in cannibalism and intraguild predation, Vegas, 4505 S Maryland Pkwy, Las Vegas, NV 89154, USA. eating not only herbivores but also other 6Corresponding author (e-mail: [email protected]). predators competing for the same resources Kylle Roy https://orcid.org/0000-0002-7993-9031 (Sabelis 1992, McNabb et al. 2001, Mestre et al. 2013). Pacific Science (2020), vol. 74, no. 3:245–256 With complex food webs, stable isotope doi:10.2984/74.3.3 analysis (SIA) can be a helpful tool for This article was created by a U.S. government employee characterizing trophic relationships among and is in the Public Domain. Public Domain information organisms (Mestre et al. 2013). Particularly, may be freely distributed and copied, but it is requested d15N and d13C are well documented to be that in any subsequent use the U.S. Department of Interior U.S. Geological Survey and the journal, Pacific useful for assessing trophic position, where Science, be given appropriate acknowledgment. the heavier isotope accumulates with 245 246 PACIFIC SCIENCE • July 2020 successive trophic levels (Cabana and measurements are the result of a natural Rasmussen 1996, Post 2002, Johnston et al. accumulation over time, while HTS identifies 2018, Kennedy et al. 2018, Kennedy et al. prey eaten within a few days or hours prior to 2019). In order to properly estimate trophic collection. positions in a given food web the establishment Blackburnia hawaiiensis Sharp (Coleoptera: of an isotopic baseline in that given system is Carabidae) is a Hawai#i Island endemic, necessary but often difficult to discern (Post numerically dominant predatory insect of 2002, Kristensen et al. 2016). In addition, the the Hawaiian montane forest (Liebherr and variability in measurements in species-rich, Zimmerman 2000); therefore understanding complex food webs can make it difficult to the beetle’s diet could have conservation and assign trophic levels (Mestre et al. 2013). management implications. The Hawaiian Therefore, it can be advantageous to employ carabids are a particularly diverse fauna with a complementary technique such as the use of 402 described endemic species descended genetic metabarcoding of gut contents, which from three colonization events: 140 Black- can yield higher prey resolution. burnia (Platynini), 239 Mecyclothorax (Morio- The ability to elucidate food webs through morphini), and 23 Bembidion (Bembidiini) genetics has greatly improved with recent (Liebherr and Zimmerman 2000, Liebherr technological advancements (Mestre et al. 2008a, Liebherr 2008b). Of the 140 Black- 2013, Piñol et al. 2014b, Gomez-Polo et al. burnia, only 23 species in the subgenus 2015). Metabarcoding, the genetic study of Colpocaccus possess fully functioning flight environmental samples utilizing universal wings. Blackburnia hawaiiensis is of interest primers and high-throughput sequencing for a diet study because it is the only (HTS), facilitates the identification of complex Blackburnia on Hawai#i Island with fully mixtures of DNA sequences (Shokralla et al. functioning flight wings, allowing for 2012). HTS has become a powerful tool for increased foraging capabilities (Liebherr and ecologists, allowing the examination of dietary Zimmerman 2000). breadth without the use of species-specific Worldwide, carabids are commonly con- primers (Gomez-Polo et al. 2015). For exam- sidered to be opportunists, consuming a wide ple, Piñol et al. (2014a) used HTS to determine variety of prey items such as Diptera and the diet of linyphiid spiders, Gomez-Polo et al. Lepidoptera larvae, Collembola, and aphids (2015) determined that of a hemipteran, and (Borror and White 1970, Hagley et al. 1982, Krehenwinkel et al. (2017a) determined the Fawki and Toft 2005). Liebherr (pers. com. diet of grass spiders. Although effective for 2014) observed remnants of spiders, cater- determining potential prey items, HTS has pillars, and fruiting bodies of moss mats in the limitations such as sequencing error and gut contents of Blackburnia spp. Although incomplete reference libraries (Deagle et al. carabids are usually assumed to be generalist 2013, Piñol et al. 2014b, Thomas et al. 2014). predators (Allen 1979, Hagley et al. 1982, Because of the limitations of HTS, combining Ekbom 1992, Bilde and Toft 1997, Ball and SIA for a time-integrated trophic structure may Bousquet 2001, Fawki and Toft 2005) several be advantageous, particularly for complex are phytophagous (Honek et al. 2003, arthropod food webs (Vander Zanden et al. Menalled et al. 2007) and oligophagous 1997). (Hatteland et al. 2011, Brandmayr and The integrated approach of using both SIA Brandmayr 2013), such as Scaphinotus, which and HTS allows for a better understanding of feed exclusively on snails (Ober et al. 2011). the feeding ecology of organisms that are Our objective was to determine the diet of difficult to rear in the lab and observe in B. hawaiiensis, testing the hypothesis that this nature. Employing these two techniques beetle is a generalist predator, consuming provides complementary information with more than one arthropod order. Ultimately, two lines of diet evidence at different we intended to enhance basic knowledge of resolutions (Carreon-Martinez and Heath Hawaiian arthropods where conservation 2010, Whitaker et al. 2019). Stable isotope efforts are hindered by a lack of taxonomic Blackburnia hawaiiensis Diet Analysis • Roy et al. 247 and ecological data (Howarth and Mull 1992, gently shaking brown Cibotium fronds over a Medeiros et al. 2013). We tested this beating sheet for 10 s and aspirating the hypothesis by analyzing the d15N and d13C arthropods into snap-cap tubes. isotope values of these beetles and other taxa collected in the same microhabitat, and by Arthropod Collection for Stable Isotope Analyses using genetic metabarcoding on the dissected gut contents of B. hawaiiensis. Focal taxa selection for stable isotope analyses was based on the following criteria: observed in MATERIALS AND METHODS the same microhabitat as B. hawaiiensis (brown Cibotium leaves), common to all three sites, and Site Description easily identifiable in the field based on morpho- Three geographically separated, replicated logical characteristics. Taxa included adult Nabis sites on Hawai#i Island were chosen for this (Hemiptera: Nabidae), Pagiopalus (Araneae: study: Na-huku (N 19° 240; W 155° 140), Philodromidae), Collembola, Leptogryllus Ka‘iholena (N 19° 110; W 155° 350), and Pu‘u (Orthoptera: Gryllidae), Laupala (Orthoptera: Maka‘ala (N 19° 320; W 155° 130). Substrates Gryllidae), and Cibotium (Cyatheales: Cibotia- at Na-huku consist of Kīlauea volcanic erup- ceae) and Metrosideros (Myrtales: Myrtaceae) leaf tions and Ka‘iholena and Pu‘u Maka‘ala were litter. Specimens were sorted by taxon and kept formed from Mauna Loa eruptions. The alive for 2–4 days in order to allow their digestive selection of sites was guided by Light tracks to clear, reducing the contamination of Detection and Ranging (LIDAR),