A Pilot Study Examining the Diet of Introduced Alaska Blackfish

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A pilot study examining the diet of introduced Alaska blackﬁsh (Dallia pectoralis T. H. Bean, 1880) in Kenai, Alaska, by metabarcoding doi:10.7299/X78052XK by Matt Bowser1and Apphia Bowser

Figure 1: Panoramic montage of a pond off of the Kenai Spur Highway and Candlelight Drive, the locality from which blackﬁsh specimens were collected. A full resolution image is available on Arctos (doi:10.7299/X7ZP46FP).

Introduction iron bacterial scum. Only one other fish species, a single specimen of a nine-spined stickleback (Pungitius pun- Last year we wrote about some food items of the Alaska gitius (Linnaeus, 1758), https://www.inaturalist.org/ blackfish, Dallia pectoralis T. H. Bean, 1880 (Bowser et al., observations/31561030), was observed in this pond. 2019), a fish species that is native to most of Alaska, but We attempted to collect blackfish from other reaches of not the Kenai Peninsula (Eidam et al., 2016; Bowser, 2018). the stream below this pond where there would have been We wanted to learn more about how these introduced fish more potential for interaction between blackfish and other may alter the ecology of Kenai Peninsula waters, especially fish species, but found only small, juvenile blackfish down- how blackfish may affect native fish species through com- stream. petition for invertebrate prey. The collected blackfish were placed on ice in a cooler, transported to the lab, and frozen. Later we thawed Methods five adult blackfish (Arctos records KNWRObs:Fish:12– KNWRObs:Fish:16), measured their lengths, dissected out We collected blackfish under Alaska Department of Fish & their entire guts, and squeezed gut contents into vials of Game permit Number SF2019-111. UniGard -100 propylene glycol antifreeze. On 23 August 2019 we collected blackfish from a small, Vials of gut contents were shipped to RTL Genomics shallow pond in Kenai, Alaska (60.5681 °N, -151.1901 °W in Lubbock, Texas (https://rtlgenomics.com/) for RTL ± 40 m) (Bowser, 2019), the same pond from which we Genomics’ standard microbial diversity assay using the ml- had obtained blackfish the previous year (Bowser et al., COIint/jgHCO2198 (GGWACWGGWTGAACWGTWTAY- 2019). This pond (Figure 1) is fed by a small inlet stream CCYCC/TAIACYTCIGGRTGICCRAARAAYCA) primer and its level is maintained by a dam at the outlet, from set. which the stream flows through the Kenai Golf Course and Extraction methods, sequencing methods, and result- into the Kenai River. There is little open water; most of ing raw sequence data are provided in Bowser and Bowser the pond is thickly filled with Potamogeton and flocculent (2020). 1US Fish & Wildlife Service, Kenai National Wildlife Refuge, Soldotna, Alaska, [email protected]

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Tree scale: 1

bfdZotu207 bfdZotu205Pisidium sp Pisidium bfdZotu207 casertanum

bfdZotu113 Pisidium casertanum

bfdZotu120 Chydorus sp bfdZotu120 bfdZotu45 Pisidium casertanum

bfdZotu62 Chydorus sphaericus

bfdZotu190 Leucorrhinia hudsonica bfdZotu134 Leucorrhinia glacialis bfdZotu171 Aeshna sp bfdZotu171

bfdZotu29 Leucorrhinia hudsonica bfdZotu21 Leucorrhinia proxima bfdZotu48 Gyraulus sp clade B TS 2018 BOLD AEB5660

bfdZotu80 Libellula quadrimaculata bfdZotu166 Trichocerca sp bfdZotu166

bfdZotu4 Aeshna eremita

bfdZotu2 Aeshna juncea bfdZotu188 Ploima sp bfdZotu188 bfdZotu153 Ploima sp bfdZotu153 bfdZotu68 Trichocerca sp bfdZotu68 bfdZotu72 Rotifera sp bfdZotu72 bfdZotu151 Ploima sp bfdZotu151

bfdZotu147 Ploima sp bfdZotu147

bfdZotu55 Pleuroxus aduncus bfdZotu128 Rotifera sp bfdZotu128 bfdZotu185 Chydoridae sp bfdZotu185

bfdZotu199 Eurycercus longirostris bfdZotu1 Eurycercus longirostris bfdZotu91 Gyraulus parvus bfdZotu168 Eurycercus sp bfdZotu167 bfdZotu79 SimocephalusbfdZotu37 exspinosus Simocephalus exspinosus bfdZotu201 Eurycercus sp bfdZotu201 bfdZotu22 Chydoridae sp bfdZotu22 bfdZotu170bfdZotu95 Eurycercus Eurycercus sp bfdZotu170 sp bfdZotu95 bfdZotu58 Eurycercidae sp bfdZotu58 bfdZotu169 Eurycercus sp bfdZotu168 bfdZotu164 Eurycercus sp bfdZotu164 bfdZotu197 Eurycercus sp bfdZotu197 bfdZotu67 Rotifera sp bfdZotu67 bfdZotu119 Eurycercus sp bfdZotu119 bfdZotu187 Ploima sp bfdZotu187

bfdZotu103 Rhamphomyia sp BOLD ACG3627 bfdZotu40 Rotifera sp bfdZotu40 bfdZotu81 HybomitrabfdZotu35 itasca Psectrocladius conjungens bfdZotu30 Rotifera sp bfdZotu30 bfdZotu104 Hybomitra itasca bfdZotu136 Cricotopus sp BOLD ACF8041 bfdZotu179 Rotifera sp bfdZotu179 bfdZotu132 Psectrocladius sordidellus bfdZotu64bfdZotu66 Psectrocladius Chironomidae obvius sp bfdZotu66 bfdZotu90 Rotifera sp bfdZotu90 bfdZotu98 Tanytarsus mendax bfdZotu34 Tanytarsus mendax bfdZotu158 Bdelloidea sp bfdZotu158 bfdZotu15bfdZotu16 Cladopelma Chironomus bicarinatum sp BOLD ACR4689 bfdZotu198 Dicrotendipes tritomus bfdZotu115 Rotaria sp bfdZotu115 bfdZotu106 Dicrotendipes tritomus bfdZotu28 Chironomus bifurcatus bfdZotu215 Rotaria sp bfdZotu215 bfdZotu97 Chironomus bifurcatus bfdZotu51 Chironomus bifurcatus bfdZotu63 Hydra utahensis strain AK12b bfdZotu87 Cricotopus trifasciatus bfdZotu50 Orthocladius smolandicus bfdZotu36 Psectrotanypus sp ES01 BOLD AAG0314 bfdZotu33 Psectrotanypus sp ES01 BOLD AAG0314 bfdZotu163bfdZotu167 Corynoneura Corynoneura sp BOLD sp BOLDACI4506 ABY3407 bfdZotu53 Psectrocladius sordidellus bfdZotu27 Angarotipula illustris bfdZotu99 Chironomidae sp bfdZotu99 bfdZotu107 Procladius sp cf flavifrons BOLD AAN5387 bfdZotu124 Procladius sp BOLD ADM0059 bfdZotu52 Procladius nigriventris bfdZotu6bfdZotu18 Procladius Procladius sp BOLD sp AAP3007 cf fuscus BOLD AAQ0606 bfdZotu160 Macrocyclops sp BOLD AAG9780 bfdZotu114 Procladius sp BOLD AAP3007

bfdZotu47 Macrocyclops sp BOLD AAG9780

bfdZotu19 Dasyhelea modesta bfdZotu13 Macrocyclops sp BOLD AAG9780 bfdZotu181 Ceratopogonidae sp bfdZotu181 bfdZotu7 CeratopogonidaebfdZotu76 Bezziasp bfdZotu7 sp BOLD ACB0747 bfdZotu180bfdZotu65 Bezzia sp Bezzia BOLD ACB0747 sp bfdZotu65 bfdZotu182 Macrocyclops sp BOLD AAG9778 bfdZotu60bfdZotu118 Bezzia Ceratopogonidaesp BOLD ADL5519 sp bfdZotu118 bfdZotu61 Dasyhelea sp BOLD ADL5519 bfdZotu46 Bezzia sp BOLD ACR1960 bfdZotu9 Macrocyclops sp BOLD AAG9778 bfdZotu71 Bezzia sp BOLD ACR1960 bfdZotu74 Trichoptera sp SlikokOtu592 bfdZotu111 Cyclopidae sp bfdZotu111 bfdZotu161 Bezzia sp BOLD ACR1960 bfdZotu78 Cyclopidae sp bfdZotu78

bfdZotu83 Cyclopidae sp bfdZotu83 bfdZotu100 Acanthocyclops capillatus

bfdZotu125 Cypridopsis vidua bfdZotu43 Cypridopsis vidua bfdZotu133 Naididae sp BOLD ADJ8525 bfdZotu20 Cypridopsis vidua

bfdZotu57 Slavina appendiculata bfdZotu206 Candonidae sp bfdZotu206 bfdZotu17 Candona candida bfdZotu211 Hexanauplia sp bfdZotu211 bfdZotu3 Cyprididae sp bfdZotu3 bfdZotu165bfdZotu157 ChaetogasterbfdZotu70 Naididae Naididae diaphanus sp bfdZotu157 sp bfdZotu70

bfdZotu210 Podocopida sp bfdZotu210

bfdZotu116 Lumbriculis sp BOLD AAG4731 bfdZotu176bfdZotu26 Lumbriculida Lumbriculida sp BOLD sp BOLD ADR8620 ADR8620 bfdZotu129 Lumbriculus sp BOLD AAG5080

bfdZotu105 Candonidae sp BOLD AAN8521

bfdZotu127 Candonidae sp BOLD AAN8521 bfdZotu12 Podocopida sp bfdZotu12 bfdZotu112 Candonidae sp bfdZotu112 bfdZotu126 Candonidae sp BOLD AAN8521

bfdZotu85 Candonidae sp BOLD AAN8521 bfdZotu175 Podocopida sp bfdZotu175

bfdZotu88 Candonidae sp BOLD AAN8521 bfdZotu149 Candonidae sp BOLD AAN8521 bfdZotu135 Candonidae sp BOLD AAN8521

Figure 2: Phylogram of retained ESV sequences. Colors representing major groups are the same as in Figure 3. The tree can be viewed interactively or downloaded from https://itol.embl.de/tree/16415961276811585237767.

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Raw reads were processed using the SCVUC COI uniquely identified food items and 137 occurrence records metabarcode pipeline version 4.3.0 (https://github.com/ of these food items (Arctos records UAMObs:Ento:244406– Hajibabaei-Lab/SCVUC_COI_metabarcode_pipeline). UAMObs:Ento:244542). Arthropods represented by 62,166 This pipline runs SeqPrep (St. John, 2016), CUTADAPT (98%) of the reads, followed by rotifers (431 reads, 0.7%), (Martin, 2011), VSEARCH (Rognes et al., 2016), UNOISE annelid worms (384 reads, 0.6%), molluscs (160 reads, (Edgar, 2016), and the RDP classifier (Wang et al., 2007) us- 0.3%), and one species of hydra (Hydra utahensis Hyman, ing the COI Classifier v4 reference dataset (Porter and Ha- 1931, strain AK12b sensu Martínez et al. (2010), 31 reads, jibabaei, 2018). Processing steps were run via Snakemake 0.05%). The most abundant groups in terms of read abun- (Köster and Rahmann, 2012). Our SCVUC configuration file dances were odonates (32%), dipterans (24%), cladocerans (Bowser, 2020b) and snakefile (Bowser, 2020c) are available (20%), ostracods (16%), and copepods (7%) (Figure 3). on Arctos. The resulting exact sequence variants (ESVs) were ESV abundances by group also compared to ESVs obtained by Bowser et al. (2020) (dataset: Bowser, 2020d), sequences from an Alaska terres- Odonata 32% trial arthropod DNA barcode COI reference library (https: //github.com/mlbowser/AKTerrInvCOILib), and a FASTA Diptera 24% file of sequences from the authors’ LifeScanner (http: //lifescanner.net/) records (http://www.boldsystems. other 1% org/index.php/Public_SearchTerms?query=DS-BOWSER) Copepoda 7% using vsearch --usearch_global. We also submitted our ESVs to NCBI BLAST (Johnson et al., 2008) and the Cladocera 20% Ostracoda 16% BOLD ID Engine (Ratnasingham and Hebert, 2007) searches and scrutinized the results. We followed the guidlines of Sigovini et al. (2016) when assigning provisional names. Figure 3: Percentages of ESV abundances in blackfish diet We removed all reads identified as Dallia pectoralis; Bos by taxonomic group. taurus Linnaeus, 1758; and all non-animals. The small numbers of Bos taurus reads likely came from bovine serum Of the 103 unique identifications, 13 were compara- albumin added during DNA amplification. As a final check tively abundunt, each representing ≥ 1% of the total num- of identifications, we generated a phylogeny of the fil- ber of reads (Figure 4). All of the reads of Aeshna eremita tered ESVs using NGPhylogeny.fr, “NGPhylogeny Analyse Scudder, 1866 (Odonata: Aeshnidae), the most abundant - FastME/OneClick” option (Desper and Gascuel, 2002; species identified, came from a single blackfish. We de- Criscuolo and Gribaldo, 2010; Junier and Zdobnov, 2010; tected Aeshna juncea Linnaeus, 1758, the second most abun- Katoh and Standley, 2013; Lefort et al., 2015; Lemoine et al., dant species in our samples, from three fish. Ceratopogo- 2019) and examined the tree using iTOL (Letunic and Bork, nidae sp. bfdZotu7 was both abundant and frequent in our 2019) (Figure 2). The FASTA file of retained ESV sequences samples, detected in gut contents of four out of five black- is available from Arctos (Bowser, 2020a). fish. To prevent reporting false postive occurrences, we removed occurrences represented by ≤ 0.05% of the total The relative abundance of each food item in terms of number of reads of an ESV. Complete analysis details are read abundances varied widely among the five blackfish provided in Bowser (2020e). individuals. For each fish, a different prey species was the We tried to follow the guidelines of Penev et al. (2017) most abundant food item. by publishing occurrence data on Arctos, which supplies Three of the most abundant ESVs could be associated occurrence data to GBIF. Specimen records, images, and with niether described species nor BOLD Barcode Index other related files have been made available via an Arc- Numbers (Ratnasingham and Hebert, 2013). The ESV iden- tos project at http://arctos.database.museum/project/ tified as Ceratopogonidae sp. bfdZotu7 was 98.71% similar 10003367. (p-dist) to a private record on BOLD. The ESV tentatively identified as Cyprididae sp. bfZotu3 had no close matches in BOLD or BASTn search results, but the closest matches Results (83.99% similarity) were Cyprididae. The ESV identified as Podocopida sp. bfdZotu12 was closest (95.44% simi- The retained 131 Exact Sequence Variants (Figure 2 ) were lar) to a sequence from an ostracod specimen identified represented by 63,172 reads. The ESVs were assigned to 103 as Podocopida (BOLD processid: OZFWC245-11).

AKES Newsletter http://www.akentsoc.org/newsletter.php Volume 13, Issue 1, June 2020 22 KNWRObs:Fish:12 KNWRObs:Fish:13 KNWRObs:Fish:14 KNWRObs:Fish:15 KNWRObs:Fish:16

12261 Aeshna eremita 7 282 11959 Aeshna juncea 7069 Candona candida 1056 959 293 4531 Ceratopogonidae sp. bfdZotu7 4808 Chironomus sp. BOLD:ACR4689 4718 Cladopelma bicarinatum 1557 112 955 Cyprididae sp. bfdZotu3 20 1613 59 Eurycercus longirostris 32 10 1649 Macrocyclops sp. BOLD:AAG9778 1249 Macrocyclops sp. BOLD:AAG9780 467 677 Podocopida sp. bfdZotu12 273 4 473 Procladius sp. BOLD:AAP3007 637 Procladius sp. cf. fuscus BOLD:AAQ0606

Figure 4: Read abundances of identified food items from each of five blackfish specimens. Only food items that represented ≥ 1% of the total number of reads were included. The area of each circle is proportional to read abundances. Discussion Some of the ESVs matched DNA barcode sequences from locally collected entities that had not been associated It appeared that the adult blackfish that we collected with a formally described species. These included Lumbri- had recently consumed exclusively invertebrates, mostly culida sp. BOLD:ADR8620, a lumbriculid worm collected arthropods. No DNA from other fish species was detected. previously from near Nordic Lake, Soldotna (BOLD proces- It should be noted, however, that other fish were compar- sid: MOBIL6661-18); Lumbriculus sp. BOLD:AAG4731, an- atively rare in this pond. A single nine-spined stickleback other lumbriculid worm documented from a temporary was the only other fish documented. It may have been pool in Soldotna (BOLD processid: MOBIL1270-16); and Tri- possible that juvenile blackfish were consumed by adult choptera sp. SlikokOtu592, an ESV from near Headquarters blackfish. These would not have been detected because all Lake documented by Bowser et al. (2020) (Arctos GUID: blackfish reads were removed from the analysis. UAMObs:Ento:239239). Overall, our results are consistent with other studies of Seven chironomid species identified from our samples blackfish diet (Ostdiek and Nardone, 1959; Chlupach, 1975; appeared to be new distribution records for Alaska. These Gudkov, 1998; Eidam, 2015; Eidam et al., 2016; Bowser were Chaetocladius conjugens Brundin, 1947; Chironomus bi- et al., 2019) which collectively show that the most im- furcatus Wuelker, Martin, Kiknadze, Sublette & Michiels, portant prey groups include cladocerans, ostracods, flies, 2009; Cladopelma bicarinata (Brundin, 1947); Cricotopus trifas- dragonflies, snails, caddisflies, and copepods. What sepa- ciatus (Meigen, 1813); Dicrotendipes tritomus (Thienemann & rates our results from previous studies is that metabarcod- Kieffer, 1916); Orthocladius smolandicus Brundin, 1947; and ing methods yielded much finer identifications, allowing Procladius nigriventris (Kieffer, 1924). us to document which species were consumed by blackfish.

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Acknowledgments In previous studies, almost all identifications were coarse, with identifications lumped by orders or even higher-level We thank Mike Baldwin for reviewing our list of potential Angarotip- groupings. new distribution records and pointing out that ula illustris (Doane, 1901) was already known to occur in The variation in abundances of food items across the Alaska (Brodo, 2018). Rob Massengill, Kristine Dunker, five blackfish individuals suggests that these fish are op- and Derek Sikes provided comments that substantially im- portunistic, consuming whatever invertebrates they find proved the article. and not seeking out any particular kind of prey item. It was surprising that we found none of the food items documented by Bowser et al. (2019) from blackfish from the References same pond. Some differences in diet might have been expected due to season variation. Bowser et al. (2019) had Bowser, M. 2018. Kenai blackfish came from collected blackfish on 18–19 October, two months later than Bethel. Refuge Notebook 20:95–96. URL https: our 23 August collecting date. Some of the food items doc- //www.fws.gov/uploadedFiles/Region_7/NWRS/Zone_ umented by Bowser et al. (2019) were terrestrial wetland 2/Kenai/Sections/What_We_Do/In_The_Community/ inhabitants that had likely become available to the black- Refuge_Notebooks/2018_Articles/Refuge_Notebook_ fish due to flooding at the time. The water level of the v20_n47.pdf. pond was much lower when we sampled in August 2019 due to a warm, dry summer. Even with these differences Bowser, M. L. 2019. Work Journal 2019. Ex- in sampling date and water levels, we had expected to doc- ported pdf version. U.S. Fish & Wildlife Service, ument at least some of the same species. The observed lack Kenai National Wildlife Refuge., Soldotna, Alaska. of overlap of observed prey items between the two studies doi:10.7299/X7RB74XZ. supports our conclusion that blackfish are highly oppor- tunistic. Bowser, M. L. 2020a. Exact sequence variants from black- The rotifer ESVs and other small-bodied invertebrates fish diet in FASTA format. doi:10.7299/X7V1253J. we observed may have been prey items of the blackfish or they may have been eaten by arthropods that were then Bowser, M. L. 2020b. SCVUC pipeline configuration file for eaten by blackfish. blackfish diet analysis. doi:10.7299/X7Q81DD8. It should be noted that, due to potential biases related Bowser, M. L. 2020c. SCVUC pipeline snakefile for black- to metabarcoding methods, the relative read abundances fish diet analysis. doi:10.7299/X7KH0NPJ. that we report may not be directly related to the relative proportions of food items in the diets of the blackfish that Bowser, M. L. 2020d. Supplementary material 5. ASV se- we collected (see Deagle et al., 2019, for an overview). Re- quences. doi:10.3897/BDJ.8.e50124.suppl5. gardless of potential metabarcoding biases due to differences in recovery and amplification of target DNA across Bowser, M. L. 2020e. Work Journal 2020. U.S. Fish & taxonomic groups, we believe that some of the differences Wildlife Service, Kenai National Wildlife Refuge., Sol- in the wide range of read abundances that we observed had dotna, Alaska. URL https://github.com/mlbowser/ to do with how recently prey items had been consumed. journal2020. Recent meals in blackfish stomachs would be expected to Bowser, M. L., and A. M. Bowser. 2020. Raw have more intact DNA than the remains of food items fur- metagenomic data from gut contents of Dallia pec- ther along in the intestines, where much of the DNA would toralis have been broken down. specimens collected in Kenai, Alaska, in 2019. doi:10.5281/zenodo.3731327. In conclusion, we documented trophic relationships between Alaska blackfish and their prey at a particular time Bowser, M. L., M. Bowser, E. Bowser, A. Bowser, and and place. To learn more about how blackfish interact E. Bowser. 2019. Some food items of introduced with other fish species, we would like to see similar work Alaska blackfish (Dallia pectoralis T. H. Bean, 1880) done in waterbodies where there may be more interactions in Kenai, Alaska. Newsletter of the Alaska Entomo- among fish species. It would also be good to examine di- logical Society 12:8–11. doi:10.7299/X7H995H7, URL ets from a wider range of sizes of blackfish as was done http://www.akentsoc.org/doc/AKES_newsletter_ by Chlupach (1975) and to compare blackfish diets with 2019_n1_a04.pdf. diets of other fish species in the same systems to learn more about potential competition and predation among Bowser, M. L., R. Brassfield, A. Dziergowski, T. Eske- fish species. lin, J. Hester, D. R. Magness, M. McInnis, T. Melvin,

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