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: kylleroy@hawaii.edu). 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), ease of gut material in the samples (McNabb et al. access, and local inspection to control for 2001). Taxa were stored at 20 °Cfora abiotic factors: elevation range of 1120–1210 maximum of one month before further proces- m, a substrate age of 200–750 years, and a sing. To prepare the specimens for SIA, they mean annual temperature of 15.5–16 °C. The were oven-dried at 60 °Cfor48 hrs, pulver- three sites were also characterized as tropical ized, then placed in 8 5mm tin capsules. montane forest with a Metrosideros polymorpha Individuals were pooled until 0.5–1mg dry- canopy, tree fern (Cibotium spp.) subcanopy, weight of arthropod and 1–2mg of plant and active populations of B. hawaiiensis material was sampled (typically 2 Nabis,2 (Figure 1). Annual rainfall was slightly higher Pagiopalus, 20 Collembola, 3 Leptogryllus,4 at one of the sites; the mean annual Laupala,and4Cibotium leaflets and 5 Metrosi- precipitation of Ka‘iholena and Na-huku is deros leaves). Isotopic compositions and d15N 2,500–3,000 mm/yr while Pu‘u Maka‘ala is and d13C concentrations of each sample were 4,500–5,700 mm/yr (Giambelluca et al. 2013). measured using an elemental analyzer coupled Sites were ground-truthed to select areas with to an isotope ratio mass spectrometer (EA- ® low human and feral ungulate disturbance and IRMS; PN-150 Costech Auto Sampler and ® predominantly native vegetation. Elemental Analyzer with Finnegan Conflow III ® Arthropods for both molecular and stable regulator and Isodat 3.0 software; Thermo isotope techniques were collected along fence Electron Corporation, San Jose, CA, USA) by lines for approximately 600 m in order to the first author at the University of Hawai‘iat minimize damage to delicate native vegetation Hilo Analytical Laboratory (http://www.uhh. during the study. Most arthropod collections hawaii.edu/∼analab/). Peach leaf standard took place at dusk and dawn during several (NIST 1547) was also run with the samples trips May–November 2014, which is the dry for quality control. season when B. hawaiiensis is most prevalent and active in the forest (Liebherr pers. com. Blackburnia Collection for Genetic Analyses of 2014). Supplemental SIA collection occurred Gut Contents in Summer 2015 to increase sample sizes. All arthropods were collected at ∼1.3 m above Blackburnia hawaiiensis were transferred ground in the lower forest arthropod within 30 min after collection to 2 ml screw- community. Arthropods were collected by top micro stand tubes containing 95% ethanol 248 PACIFIC SCIENCE • July 2020

FIGURE 1. (A) Study sites on Hawai‘i Island including substrate ages and rainfall isohyets. (B)Pu‘u Maka‘ala. (C)Na-huku. (D)Ka‘iholena. Substrate layers were created by USGS (Sherrod et al. 2007) and precipitation layer created by Rainfall Atlas Hawai‘i(Giambelluca et al. 2013). Ka‘iholena and Pu‘uMaka‘ala were formed from a Mauna Loa volcanic eruption and Na-huku was that of Kīlauea.

(to prevent digestion of prey in the gut of the flanking the 157-bp region of the Zeale primers beetles) and then stored at 80 °C until (ZBJ-ArtF1c and ZBJ-ArtR2c) for gut content further processing. Blackburnia hawaiiensis metabarcoding (Zeale et al. 2010, Bohmann were identified and sexed under a Leica et al. 2011)ofthe68availablesequenceswere MZ-2500 binocular dissecting scope accord- considered for potential primer synthesis. The ing to Liebherr and Zimmerman (2000). Platynini primers were designed to amplify a 230-bp barcode region of carabid mitochondrial Molecular Techniques DNA (F: AGATATTGGAACWTTATATTT- TATTTTTGG, R: ATCAAAAACTTATAT- Primer design and PCR — A reference TATTTATTCGAGG). Blackburnia hawaiiensis sequence for B. hawaiiensis was created by samples were amplified using: initial denatura- first designing Platynini carabid-specific pri- tion at 95 °C (3 min); 30 cycles of 95 °C(30s), mers using mitochondrial cytochrome-oxi- 53 °C (45 s); and 72 °C(45s),withafinal dase I (COI) barcode sequences obtained from extension at 72 °C (5 min). Platynini primers GenBank for 68 Platynini species. Sequences were tested on 3 individuals from each site, were aligned in Geneious v. 8.1.6 (Biomatters, yielding identicalsequences (Genbank accession Aukland, NZ). Conserved regions of DNA No. MH733243). Blackburnia hawaiiensis Diet Analysis • Roy et al. 249

The Zeale primers amplify a wide range of to determine the ideal concentration of arthropod orders (Zeale et al. 2010, Bohmann template needed by quantitative real-time et al. 2011, Piñol et al. 2014a). Preliminary PCR. The samples were diluted, amplified testing of these primers on various Hawaiian (Template Hi-Q amplification, Ion One taxa common to the environment in which Touch 2 system), and sequenced on the B. hawaiiensis was collected, was conducted in Personal Genome Machine (PGM) using order to find the optimal annealing tempera- the Hi-Q Kit with the Ion Torrent 318v2 ture for a wide range of Hawaiian arthropods chip (Life Technologies) as described by the including Drosophila heteroneura (Diptera: manufacturer (Life Technologies). The Drosophilidae), Laupala spp., Lepidoptera sequencing chemistry for 200-bp read length spp., Leptogryllus spp., Nabis spp., Eupetinus and version 4.4.3 of the TorrentSuite software and Prosopeus spp. (Coleoptera: Nitidulidae), was used for base calling (Life Technologies). and Triginidium spp (Orthoptera: Trigonidii- All library preparation and Ion Torrent dae). Optimal annealing temperature for all sequencing were performed by the first author taxa was determined to be 51 °C and used for at the University of Hawai‘i at Hilo Evolu- further HTS. tionary Core Genomics Laboratory. DNA extraction — For HTS, B. hawaiiensis foregut, midgut, and hindgut were dissected, Data Analysis homogenized, and extracted with the Qiagen DNeasy Blood and Animal Tissue Kit Analysis of stable isotopes — The stable (Qiagen Ltd, Crawley, UK) according to isotopic composition of element X is expressed the manufacturer’s protocol. Five gut content as a difference in ratios and in parts per d ‰ extractions from each site were used for HTS, thousand: X( )=(Rsample/Rstandard 1) including 8 males and 7 females (Ka‘iholena 1,000 where Rsample and Rstandard are the ratio 13 12 15 14 2 males and 3 females; Na-huku 3 males and of heavy to light isotope ( C: Cand N: N) 2 females; Pu‘u Maka‘ala 3 males and of the sample and standard (Craig 1953). 2 females). DNA quality was checked on a Carbon isotope data were normalized to USGS 13 1.5% agarose gel. 40 (d C vs VPDB = 26.4) and USGS 41 (d13C vs VPDB 37.6) (SD = 0.2‰). Nitrogen High-throughput sequencing — Samples were isotope data were normalized to USGS 40 prepared using the Ion Torrentrecommenda- (d15NvsAir=4.5) and USGS 41 (d15NvsAir tions for preparing amplicon libraries without 47.6) (SD = 0.2‰). fragmentation. The Ion Plus Fragment R version 3.6.3 was used for all statistical Library Sequencing Kit (Life Technologies) analyses (R Core Team 2020). We fit linear was used with minor modifications. All 15 mixed-effects models using the lmer function samples were pooled into an equal-molar provided by the lme4 package for R (Bates concentration of 15 ng/ml, with a 20-cycle et al. 2015) using site as a random effect with initial PCR amplification to reduce the taxa as the predictor, and d13C and d15N as the amplification bias. Additionally, the end repair response variables in their respective models. reagent volumes were reduced by 75% and Post hoc tests for differences between model the adapter ligation and nick repair reagent coefficients were done using the mltcomp volumes by 50% to compensate for the low package (Hothorn et al. 2008), comparing throughput of DNA yields from gut extrac- linear combinations of d13C and d15N tions. B. hawaiiensis values to all other taxa. Logically Sample purity was evaluated (DNA High constrained multiplicity adjustments were Sensitivity kit, Bioanalyzer 2100, Agilent used to control multiple comparison P-values Technologies) and quantified (Qubit dsDNA (Westfall et al. 1999). HS Assay Kit, Qubit 3.0 Fluorometer, Life Technologies) between all library steps. Prior High-throughput sequencing bioinformatic to sequencing, the Kappa Biosystems Ion analysis — Torrent suite software sorted all Torrent Library Quantification Kit was used sequences by barcode into separate BAM files, 250 PACIFIC SCIENCE • July 2020 which were then converted into FASTQ files TABLE 1 using a simple stream editor (sed) script. Average 13C and 15N Isotope Values (±1 SE) of Study Sequences were then filtered and clustered Taxa. Number of Samples Analyzed (N) of Each Taxa is 15 using USEARCH (Edgar 2010) according to Reported Below. Taxa are Listed in Decreasing N from Top to Bottom (Krehenwinkel et al. 2017b). BLASTn was used to assign Operational Taxonomic Unit Taxa d15N d13C (OTU) clusters to order (McGinnis and Madden 2004) using a reference library B. hawaiiensis (N = 13) 1.7 ± 0.30 26.5 ± 0.30 containing NCBI COI databases concate- Nabis (N = 11) 2.6 ± 0.25 26.0 ± 0.16 nated with the COI B. hawaiiensis sequence, Pagiopalus (N = 13) 3.0 ± 0.42 26.9 ± 0.27 using a minimum similarity of 95%. Only Laupala (N = 14) 4.0 ± 0.29 26.9 ± 0.25 order classification information was retained Leptogryllus (N =8) 5.3 ± 0.35 25.6 ± 0.20 because of the limits of HTS such as Collembola (N =9) 6.0 ± 0.19 26.6 ± 0.19 sequencing accuracy and the limited reference Metrosideros (N =9) 6.7 ± 1.79 29.6 ± 0.12 database available. Cibotium (N =9) 6.2 ± 0.38 29.9 ± 0.44

RESULTS Stable Isotope Analysis the linear mixed-effects models with taxa as the predictors were preferred over the null d15 Blackburnia hawaiiensis had the highest N model (Table 2, Table 3). Blackburnia d13 15 of all taxa and similar C to all arthropods hawaiiensis d N was not significantly different d15 d13 (Figure 2, Table 1). For both N and C, from that of Nabis (P = .13) and significantly

15 13 FIGURE 2. d N and d C isotope ratios of B. hawaiiensis, Pagiopalus, Nabis, Laupala, Leptogryllus, Collembola, Metrosideros, and Cibotium. Colored dots are mean ± 1 SE, and open dots are raw data points. Taxa are listed in descending d15N values. Blackburnia hawaiiensis Diet Analysis • Roy et al. 251

TABLE 2 TABLE 3 Multiple Comparisons of B. hawaiiensis to All Other Taxa Multiple Comparisons of B. hawaiiensis to All Other Taxa Output from the Linear Mixed Effects Model Using Output from the Linear Mixed Effects Model Using Taxa as the Predictor and 15N as the Response with Site Taxa as the Predictor and 13C as the Response with Site as a Random Effect (15N ∼ Taxa+ 1+(1j Site)). Null as a Random Effect (13C ∼ Taxa+ 1+(1 j Site)). Null Deviance 364.7 on 83 df Residuals, AIC 370.7. Alter- Deviance 326.7 on 83 df Residuals, AIC 332.7. Alter- native Deviance 241.0 on 76 df residuals, AIC 261.0. native Deviance 206.1 on 76 df Residuals, AIC 226.1. Random Effects Standard Deviation of 0.404, 86 Random Effects Standard Deviation of 0.287, 86 Observations on 3 Groups (Sites) Observations on 3 Groups (Sites).

B. hawaiiensis Estimate Standard Z-Value P-Value B. hawaiiensis vs. Estimate Standard Z-Value P-Value vs. Taxa Error Taxa Error Nabis 0.876 0.390 2.245 .132 Nabis 0.464 0.320 1.451 .567 Pagiopalus 1.220 0.374 3.265 .007 Pagiopalus 0.430 0.306 1.404 .603 Laupala 2.247 0.367 6.127 <.001 Laupala 0.338 0.301 1.125 .803 Leptogryllus 3.531 0.429 8.222 <.001 Leptogryllus 0.919 0.352 2.612 .053 Collembola 4.275 0.413 10.354 <.001 Collembola 0.091 0.338 0.268 1.000 Metrosideros 4.964 0.413 12.022 <.001 Metrosideros 3.102 0.338 9.168 <.001 Cibotium 4.475 0.413 10.838 <.001 Cibotium 3.39 0.338 10.022 <.001

DISCUSSION differed from all other taxa (P = .007 for Blackburnia hawaiiensis Isotopic Differences Pagiopalus, P < .001 for all other taxa) (Table2). Blackburnia hawaiiensis d13C was significantly Because d15N often indicates trophic level different from both plant taxa, Metrosideros (Post 2002, Shiels et al. 2018), our d15N and Cibotium (P < .001), but was not signifi- mixed-effects model suggests B. hawaiiensis is cantly different from all other arthropods in a higher trophic category than all tested (Table 3). taxa except for Nabis, or damsel bugs. These data indicate B. hawaiiensis is on a similar trophic level to that of Nabis, which are High-Throughput Sequencing considered generalist predators that feed on a The Ion Torrent run produced 4,477,826 variety of other insects (Lattin 1989, Stasek et DNA sequencing reads with 63% loading. al. 2018). On average, B. hawaiiensis d15Nis The mean read length was 211 bp. Each more than 1.3 values higher than the other barcode returned on average 254,811 ± significantly different taxa (Table 1). Interest- 5,754.22 reads per barcode with a minimum ingly, d15NofB. hawaiiensis was significantly number of reads at 199,987 and a maximum of higher than Pagiopalus, a genus of running 270,275. After USEARCH filtering, we crab spider or Philodromidae, which have also recovered an average of 29,279 ± 871.841 been documented to consume a variety of SE reads per specimen, ranging from arthropods (Huseynov 2008). In Hawai#i, 21,912 to 34,077 reads. 0.05% of the usable stable isotopic studies have been conducted sequences were prey; therefore, an average of on native spiders, and Kennedy et al. (2018) in 200 predator reads was generated for each particular conducted research in close proxi- prey sequence. mity to Pu‘u Maka‘ala. Kennedy et al.’s (2018) Prey items were amplified in eight of the 15 data suggest similar d15N for Tetragnathid gut content samples, five females, and three and Ariamnes spiders and B. hawaiiensis. males. The majority of prey reads belonged to Signatures of d13C tend to reflect the food Diptera (98%, 215 reads). Lepidoptera was source of an animal (Cabana and Rasmussen found only in the gut contents of a single 1996, Post 2002, Hobson et al. 2010). Our individual (2%, 5 reads). linear mixed-effects model showed that 252 PACIFIC SCIENCE • July 2020

B. hawaiiensis d13C values were similar to all data difficult to interpret. For example, the the arthropod taxa used in this study and absence of site-specific reference databases therefore are all potential prey items for B. and the potential for sequencing errors in hawaiiensis. Blackburnia hawaiiensis d13C values HTS prevent the assigning of reads to species were different from the tested plant material level. Blocking primers or host-specific pri- indicating that the beetle is unlikely to be mers modified with a C3 spacer at the 30 end consuming plant material. Additionally, our of the forward universal primer can also be d13C data suggest that Collembola is a likely used to reduce the number of predator base for the Hawaiian montane forest arthro- sequences (Vestheim and Jarman 2008, Dea- pod food web, and better suited than gle et al. 2013, Gomez-Polo et al. 2015). Metrosideros and Cibotium for future SIA However, blocking primers have not proven studies using mixing models. to be useful for arthropod predators that Future research using Bayesian mixing consume closely related organisms (Gomez- models such as MixSIR (Moore and Semmens Polo et al. 2015, Piñol et al. 2014b). For 2008) or SIAR (Parnell et al. 2010) would aid example, Piñol et al. (2014b) found that the in determining the trophic position and use of blocking oligonucleotides reduced spatial diet contributions of arthropod predator sequence by less than one order of prey. In order to use these mixing models, magnitude. After the alignment of the COI feeding studies and larger sample sizes are arthropod sequences available in Genbank, it necessary. Controlled feeding experiments of became clear that this approach could not be B. hawaiiensis for determining the trophic used for B. hawaiiensis because high conserva- enrichment factor could be possible, as tion of the COI target sequence would have Liebherr (2000) successfully raised a closely led to significant underestimation of insect related species, although a self-perpetuating orders within the samples. The COI region is colony was not established. highly conserved and thus significant propor- tions of all insect orders examined may have been inappropriately blocked by blocking High-Throughput Sequencing Analysis primers, as seen in Toju and Baba (2018). Our HTS analyses suggest that B. hawaiiensis The addition of other barcoding genes such as is consuming at least two orders of arthro- 16S ribosomal RNA and ITS (Krehenwinkel pods: Diptera and Lepidoptera. However, et al. 2019), a Hawaiian arthropod reference these orders may not reflect the full spectrum library, and a larger sample size may improve of diet due to sample size (N = 15), noting that genetic diet analyses and perhaps identify prey prey items were amplified in only eight of the items that were not detected in this study. individuals’ gut contents. Interestingly, host amplicons were abundant despite reducing Blackburnia hawaiiensis, A Generalist Predator the amount of available host material through dissection and limiting the initial PCR to 20 Knowledge of feeding ecology can ultimately cycles. Our results were similar to that of improve the effectiveness of land management Piñol et al. (2014a) and Gomez-Polo et al. and conservation practices for species and (2015), who both used the brute-force ecosystems. In this study, we found evidence approach with 40 initial PCR cycles resulting that B. hawaiiensis is a generalist predator that in over 99% predator sequences. Interest- consumes at least two orders of arthropods, ingly, in Piñol et al. (2014b), reducing PCR and has a trophic position similar to that of cycles only increased the proportion of the damsel bugs. Unfortunately, Diptera and less well-amplified taxa, not the presence of Lepidoptera larvae were not abundantly the taxa themselves. collected through the beating method and High-throughput sequencing is a powerful were not included as potential prey items in and rapidly evolving technique but there are the SIA. Liebherr and Zimmerman (2000) still many shortcomings that can make the suggested that the foraging activity of Blackburnia hawaiiensis Diet Analysis • Roy et al. 253

Hawaiian carabids is not limited by spatial Tropical Conservation Biology and Environ- constraints. The beetles can be found in moss mental Sciences at the University of Hawai#i #ō # mats, under logs and rocks, and on hi a trees at Hilo. Any use of trade, firm, or product # # and ie ie vines (Pandanaceae: Freycinetia names is for descriptive purposes only and ) in addition to fronds where arborea Cibotium does not imply endorsement by the U.S. B. hawaiiensis was collected in this study, a nod to their generalist eating habits (Liebherr and Government. Zimmerman 2000). Indeed, larvae of both Diptera and Lepidoptera orders could be Literature Cited easily captured by a mobile predator in decaying woody tissue. Although further Allen, R. T. 1979. The occurrence and research is necessary to determine the full importance of ground beetles in agricul- tural and surrounding habitats. Pages scope of B. hawaiiensis diet, the results from – this study contribute to the limited but 485 505 in T. L. Erwin, G. E. Ball, and advancing Hawaiian arthropod diet ecology D. R. Whitehead, eds. Carabid beetles: literature. The methodological approaches their evolution, natural history and classi- used in this study will be useful to document fication. Dr. W. Junk Publishing, The the natural history and diet of arthropods and Hague-Boston-London. Ball, G. E., and Y. Bousquet. 2001. Carabidae could ultimately improve the study of trophic – ecology. Latreille. Pages 32 132 in R. H. Arnett Jr, and M. C. Thomas, eds. American beetles, Volume 1 (Archostemata, Myxophaga, AUTHOR CONTRIBUTIONS Adephaga, Polyphaga: Staphylinifornia). KR, CPE, and DKP conceived and designed CRC Press, Boca Raton, FL, USA. the experiments. KR and CPE conducted field Bates, D., M. Mächler, B. Bolker, and S. Walker. 2015. Fitting linear mixed-effects work and identification of arthropods. KR – performed the experiments, statistical analy- models using lme4. J. Stat. Softw. 67:1 48. sis, and wrote the manuscript. KR, CPE, and Bilde, T., and S. Toft. 1997. Consumption by DKP revised the manuscript. carabid beetles of three cereal aphid species relative to other prey types. Entomophaga 42:21–32. ACKNOWLEDGMENTS Bohmann, K., A. Monadjem, C. Lehmkuhl Noer, M. Rasmussen, M. R. K. Zeale, We would like to thank Dr. Rebecca Ostertag, E. Clare, G. Jones, E. Willerslev, Thomas Fezza, Anne Veillet, Tara Holitzki, M. Thomas, and P.Gilbert.2011. Molecular Dr. Renee Bellinger, and the Geib lab at dietary analysis of two African free-tailed USDA-ARS-DKI-PBARC (including Dr. bats (Molossidae) using high throughput Scott Geib, Brian Hall, and Theodore sequencing. PLoS One 6:e21441. DeRego) for making this project possible. Borror, D. J., and R. E. White. 1970. A field Dr. Henrick Krehenwinkel, Dr. Jun Y. Lim, guide to the insects of American North of and Dr. Susan R. Kennedy assisted in genetic Mexico. Houghton Mifflin Company, data analysis and provided valuable advice. Boston. We thank the DLNR, TNC, HAVO, and Brandmayr, P.,and T. Zetto Brandmayr. 2013. Food and feeding behaviour of DOFAW for site access and permitting. A Lincinus “ ” ‘ species (Coleoptera: Carabidae Lincinini). special mahalo to the Hau oli Mau Loa Ital. J. Zool. 20:171–181. DOI: 10.1080/ Foundation for financial support. We also 00269786.1986.10736497. thank Dr. Carter T. Atkinson, Dr. Dennis Cabana, G., and J. B. Rasmussen. 1996. Lapointe, and two anonymous reviewers for Comparison of aquatic food chains using review of this article. This project was nitrogen isotopes. Proc. Natl. Acad. Sci. conducted for the fulfillment of MSc in 93:10844–10847. 254 PACIFIC SCIENCE • July 2020

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