Feeding Habits and Novel Prey of Larval Fishes in the Northern San

Feeding habits and novel prey Larval longfin smelt and Pacific herring had Table 1. PCR and blocking primers used in this study. Primer ID Sequence (5' --- 3') Role mjHCO2198 TAA ACY TC W GGR TGW CCR AAR AAY CA Metabarcoding of larval fishes in the northern overlapping diets in the northern Estuary. mlCOIintF1 GGW ACW GGW TGA ACW GTW TAY CCY CC Metabarcoding & barcoding San Francisco Estuary Lfs_COIBlk_668R GTG ACC GAA GAA TCA GAA TAG ATG CTG G* Longfin smelt blocker /3SpC3/ Clp_COIBlk_668R GTG ACC GAA GAA TCA GAA TAG GTG TTG G* Clupeid blocker PRESENTER: New prey were detected in both species, and we /3SpC3/ 1 jgHCO21982 TAI ACY TCI GGR TGI CCR AAR AAY CA Individual barcoding Michelle Jungbluth obtained species-level ID of cryptic prey by using DNA LCO14905 GGT CAA CAA ATC ATA AAG ATA TTG G Individual barcoding [email protected] * denotes phosphorothioate bond to prevent exonuclease degradation by the high-fidelity polymerase Jillian Burns1, Lenny metabarcoding Grimaldo2, Anne Slaughter1, Aspen Katla3, Results: Longfin smelt Pacific herring Zooplankton Bosmina longirostris 1 100% Ceriodaphnia laticaudata Wim Kimmerer Ceriodaphnia sp. Daphnia sp. 75% Eurytemora carolleeae Longfin smelt Pacific herring 1 San Francisco State University, Estuary & Acanthocyclops americanus Acanthocyclops robustus Ocean Science Center, San Francisco, CA Limnoithona tetraspina Cyclopoida C undance (%) 50%

2 b Harpacticoida A ICF Inc, Richmond, CA Liposcelis rufa 3 Clupea pallasii North Seattle College, Seattle, WA 25% Spirinchus thaleichthys Cottus asper

e Read A Hydra oligactis v Non−metazoans 0% * Other (<5%) Background: What food resources support Unknown 13 14 15 16 24 26 20 21 22 23 13 14 15 16 24 26 20 21 22 23 13 14 15 16 24 26 20 21 22 23 Relati declining fish populations in the San Francisco San Pablo Bay Suisun Bay San Pablo Bay Suisun Bay San Pablo Bay Suisun Bay Estuary? Traditional diet analysis methods can be Tow #, Location Figure 2. Mean relative read abundance in longfin smelt, Pacific herring, and biased to identify hard-bodied prey and the zooplankton samples. “Other” = prey IDs contributing <5% to the sample. resolution of prey items depends on condition and the ease of identification. Here we applied dietary Phylum: Class Best ID Annelida: Capitellida Dasybranchus sp. DNA (dDNA) metabarcoding to study the diets of Ceriodaphnia laticaudata Phylum Class Best ID Morph. Molecular Ceriodaphnia sp. larval longfin smelt and Pacific herring in the Chydorus brevilabris Annelida Polychaeta Dasybranchus sp. Chydorus sp. Branchiopoda Daphnia magna Daphnia sp. northern San Francisco Estuary in spring 2017 (wet Daphnia magna Figure 5. NMDS plot of sequence diversity in each sample, colors represent Daphnia sp. Acartiella sinensis year) to get a higher resolution view of what defines Branchiopoda Acartiella sinensis Eurytemora carolleeae different types of samples: longfin smelt, Pacific herring, zooplankton with fish, Eurytemora carolleeae Sinocalanus doerrii and zooplankton without fish. Ellipses represent 95% confidence groupings of Tortanus dextrilobatus prey for larval fishes in the Estuary. We also Osphranticum labronectum each sample type Pseudodiaptomus forbesi Unid. Calanoida Arthropoda Acanthocyclops americanus Acanthocyclops americanus

compared dDNA to morphological diet analysis in a thropoda Acanthocyclops robustus r Acanthocyclops robustus A Copepoda FO% Copepoda subset of samples. Acanthocyclops sp. Acanthocyclops sp. Limnoithona tetraspina 100 Unid. Cyclopoida Results:

y ID Mesocyclops pehpeiensis 75 Limnoithona tetraspina e Cyclopoida C Unid. Harpacticoida • Eurytemora carolleeae (affinis) most common

Pr Unid. Insecta 50 Unid. Copepoda Insecta y ID Insecta and abundant prey sequence in both species Liposcelis rufa e Liposcelis rufa 25 Malacostraca Neomysis japonica Palaemon modestus Pr Malacostraca Neomysis japonica Unid. Arthropoda • Acanthocyclops americanus and A. robustus Unid. Arthropoda Clupea pallasii Thecostraca Balanus crenatus Acanthogobius flavimanus also common Clupea pallasii Gasterosteus aculeatus

Acanthogobius flavimanus Chordata Unid. Actinopterygii

Spirinchus thaleichthys Actinopterygii Unid.animal Cottus asper Rotifera Unid. Rotifera • General agreement with morphological diet Chordata

Actinopteri Gasterosteus aculeatus Unid.plant Cnidaria: Hydrozoa Hydra oligactis Unknown non−metazoans analysis Echinodermata: Asteroidea Leptasterias hexactis Unknown Mollusca: Bivalvia Potamocorbula amurensis • Higher resolution ID 5 0 5

Non−metazoans 80 50 25 25 50 80 Unknown 100 100 Frequency of Occurrence (%) Figure 1. Map of study location: San Pablo Bay and • Four fish species found in longfin guts Suisun Bay in the northern San Francisco Estuary, Figure 4. The mean frequency of occurrence (FO%) of prey California (eggs likely) items in longfin smelt guts comparing morphological analysis Zoo (Fish) (Morph.) to molecular analysis in a subset of samples where Longfin smelt Pacific herring Methods: Zoo (Non-fish) both analysis methods were performed. • Soft-bodied prey: polychaete and cnidarian 1) Larval fish trawls (505 µm net) and zooplankton tows Figure 3. Mean frequency of occurrence (FO%) in longfin smelt, Pacific herring, zooplankton samples (with fish) and zooplankton (with no fish). present but uncommon (110 µm net) preserved in 95% Ethanol; 2) Processed for dietary DNA (dDNA) metabarcoding and zooplankton community DNA metabarcoding Conclusions: Acknowledgements: • Fish digestive tract removed and DNA extracted • When longfin and herring larvae overlap, they depend on the same primary food Sea Grant Delta Science Postdoctoral Fellowship and State and • Fish-DNA blocking primers and universal mtCOI Federal Water Contractors Inc., Award #18-02. primers (Table 1) to generate DNA libraries; resources Fish were collected under the California Fish and Wildlife Prop 1 Grant • Zooplankton subsampled and bulk DNA extracted • Generally eat what is abundant: no strong prey selection* study 2081; CESA Scientific Collecting Permit #4086 to ICF Inc. SFSU GTAC facility Illumina MiSeq Sequencer NSF Award #1427772, • Universal primers (as above) applied to generate • *Longfin did not consume many L. tetraspina and UC Davis Genome Center MiSeq Sequencer NIH Award DNA libraries #1S10OD010786-01 • Illumina MiSeq paired-end sequenced • Herring did: can exploit this additional abundant food resource NSF REU Program Award #1659175 for DNA barcoding. 3) Classified sequences using NCBI blastn3 combined with • Longfin fed on more fish species (eggs), and herring eggs are attached to substrates UC Davis Fish Conservation and Culturing Laboratory facility for 4 providing longfin smelt adults to aid in primer design. RDP Classifier • Substrates are beneficial habitat: protection and food 4) DNA barcodes generated for species missing from C. Brennan (Tenera Inc., ICF) for expertise in fish taxonomy database; References: • Adult female egg-bearing cyclopoid and harpacticoid 1Leray, M., et al., (2013). Front. Zool., 10(1), 34. copepods, voucher photographed, extracted; 2Geller, J., et al., (2013). Molec. Ecol. Res., 13(5), 851–861. 3 1,2,5 Altschul, S.F., et al., (1990). J. Mole. Biol., 215. • PCR for mtCOI gene using universal primers 4Wang, Q., et al., (2007). Appl Environ. Microbiol, 73(16), 5261-5267. 5Folmer, O., et. al., (1994). Molec. Mar. Biol. Biotech., 3, 294–299.