Analysis of Christmas Shearwater Puffinus nativitatis from Kure - PowerPoint PPT Presentation

Is it Better to be Seen or Sequenced? A Comparison of Methods in the Prey Analysis of Christmas Shearwater Puffinus nativitatis from Kure Atoll. JOHN BACZENAS MARS 4040 DR. HYRENBACH HAWAII PACIFIC UNIVERSITY

Background  By investigating ecological relationships between seabirds and their communities (Harrison et al. 1983), knowledge of their diets can lead to an understanding of the integrity of the ecosystem  Through morphological methods, it is difficult to get a high resolution of taxonomic data from diet samples due to high levels of digestion (McInnes et al. 2017)  Metabarcoding uses the “barcode of life” region of DNA and allows for more accurate identification of prey items (Hirai et al. 2018)

Goals and Hypothesis  Purpose of study is to determine if the metagenetic method is comparable to morphological method or if they refute each other  Hypothesis: The metagenetic method will be confirmed viable and comparable to the morphological method.

Sampling  95 samples were collected during DLNR summer population surveys conducted on Kure Atoll  Stored in a plastic bag and frozen in water to preserve samples Screenshot of google maps

Sampling  95 samples were collected during DLNR summer population surveys conducted on Kure Atoll  Stored in a plastic bag and frozen in water to preserve samples  Transported from Kure Atoll to Pelagicos lab located at HPU Oceanic Institute

Morphological method  From September 2017- June 2018, 91 samples were visually sorted, weighed, and classified into food classes.  Ashmole & Ashmole (1967) method of processing  Mass was taken in grams for the individual prey items  200-mL of sample water collected for future metagenetic processing  Sorted prey items stored in freezer for future analysis CHSH regurgitation sample, sorted into individual prey items and food classes.

Metagenetic Method  DNA was extracted from diet samples using a Phenol-Chloroform-Isoamyl extraction method established by Renshaw et al. (2015)  Extracted DNA was cleaned using a magnetic bead procedure to remove unwanted lengths of fragments  Due to limited amount of space during the MiSeq-Illumina sequencing run, 31 samples were selected at random to be further processed.  The 31 samples were processed through methods of PCR and specific Cytochrome c Oxidase subunit I gene (CO1) markers were selected to attach to the desired fragments of DNA  These markers are described by Leray et al. (2013) and attach to the “barcode of life” region of DNA

Metagenetic Method  After DNA was duplicated, the samples were sent to the University of Notre Dame to be sequenced using MiSeq-Illumina methods (Hirai et al. 2017)  After DNA was sequenced, the data was processed back at OI via bioinformatics and comparing to the database GENBANK to obtain the taxonomic information of the samples

Morphological Results Squid Fish Total % Number 27.27 72.73 100.00 % Mass 49.05 50.95 100.00 POO 50.00 50.00 100.00 Table 2. Metric calculations of the diet composition of 31 CHSH regurgitations collected on Kure Atoll between 2009 and 2017. Samples were processed using the morphological method of Ashmole & Ashmole (1967).

Morphological Results 100 80 60 % 40 20 0 % Number % Mass POO Squid Fish Figure 1. Metric calculations of the diet composition of 31 CHSH regurgitations collected on Kure Atoll between 2009 and 2017. Samples were processed using the morphological method of Ashmole & Ashmole (1967).

Metagenetic Results CO1 Markers Food Class Leray 1 Leray 2 B12 X 2 P Squid 1156 158782 404428 109904.78 0 252283 1460242 1687752 Fish Table 4. Contingency results comparing the association of three CO1 markers (Leray1, Leray 2, and B12) and the food classes analyzed from 31 CHSH regurgitations collected from Kure Atoll between 2009 and 2017.

Metagenetic Results 100 80 60 POO 40 20 0 Leray 1 Leray 2 B12 Squid Fish Figure 2. Metric calculations of the diet composition of 31 CHSH regurgitations collected on Kure Atoll between 2009 and 2017. Samples were processed using the metagenetic method with three different CO1 markers (Leray 1, Leray 2, and B12).

Results Leray 1 Squid Fish Total % Number 0.46 99.54 100 POO 24.24 75.76 100 % FOO 25.81 80.65 106.45 Leray 2 % Number 9.81 90.19 100 POO 47.46 52.54 100 % FOO 90.32 100 190.32 B12 % Number 19.33 80.67 100 POO 44.44 55.56 100 % FOO 77.42 96.77 174.19 Table 5. Metric calculations of the diet composition of 31 CHSH regurgitations collected on Kure Atoll between 2009 and 2017. Samples were processed using the metagenetic method with three different CO1 markers (Leray 1, Leray 2, and B12).

Comparison Fish Method Present Absent X 2 P Metagenetic 25 6 0.48 0.49 Leray 1 Morphological 27 4 Metagenetic 31 0 4.28 0.04 Leray 2 27 4 Morphological Metagenetic 30 1 1.96 0.16 B12 27 4 Morphological Squid Metagenetic 8 23 23.68 0.00 Leray 1 27 4 Morphological Metagenetic 28 3 0.16 0.69 Leray 2 27 4 Morphological Metagenetic 24 7 0.99 0.32 B12 27 4 Morphological Table 6. Contingency results the association of the presence/ absence of two food classes (fish/squid) and the method (morphological/metagenetic) used to process the samples.

Comparison Fish Method Present Absent X 2 P Metagenetic 25 6 0.48 0.49 Leray 1 Morphological 27 4 Metagenetic 31 0 4.28 0.04 Leray 2 27 4 Morphological Metagenetic 30 1 1.96 0.16 B12 27 4 Morphological Squid Metagenetic 8 23 23.68 0.00 Leray 1 27 4 Morphological Metagenetic 28 3 0.16 0.69 Leray 2 27 4 Morphological Metagenetic 24 7 0.99 0.32 B12 27 4 Morphological Table 6. Contingency results the association of the presence/ absence of two food classes (fish/squid) and the method (morphological/metagenetic) used to process the samples.

Conclusion  Both methods produced similar results with respects to POO  Based on the contingency tests, there was a lack of association between the two food classes and which method was used.  Confirmed that metagenetics is a viable method for analyzing seabird diet composition  For future studies, try and expand the database to obtain more accurate taxonomic information  See if there is any additional methods of setting a threshold to further “clean up” the metagenetic data

References Ashmole, N. P., & Ashmole, M. J. (1967). Comparative feeding ecology of sea birds of a tropical oceanic island.  Peabody Museum of Natural History Bulletin, Yale University, 24, 1-139. Harrison, C. S., Hida, T. S., & Seki, M. P. (1983). Hawaiian Seabird Feeding Ecology. Wildlife Monographs, 85, 3 – 71.  Hirai, J., Nagai, S., & Hidaka, K. (2017). Evaluation of metagenetic community analysis of planktonic copepods using  illumina miseq: Comparisons with morphological classification and metagenetic analysis using roche 454. [PDF]. PLoS ONE, 12(7), 1-19. Hirai, J., Hamamoto, Y., Honda, D., & Hidaka, K. (2018). Possible aplanochytrid (Labyrinthulea) prey detected using  18S metagenetic diet analysis in the key copepod species calanus sinicus in the coastal waters of the subtropical western north pacific. Plankton & Benthos Research, 13(2), 75-82. Leray, M., Yang, J. Y., Meyer, C. P., Mills, S. C., Agudelo, N., Ranwez, V., . . . Machida, R. J. (2013). A new versatile  primer set targeting a short fragment of the mitochondrial COI region for metabarcoding metazoan diversity: Application for characterizing coral reef fish gut contents [PDF]. Frontiers in Zoology, 10(34), 1-14. McInnes, J. C., Alderman, R., Lea, M.-A., Raymond, B., Deagle, B. E., Phillips, R. A., . . . Jarman, S. N. (2017). High  occurrence of jellyfish predation by black-browed and Campbell albatross identified by DNA metabarcoding. Molecular Ecology, 26, 4831-4845. Renshaw, M. A., Olds, B. P., Jerde, C. L., Mcveigh, M. M., & Lodge, D. M. (2015). The room temperature  preservation of filtered environmental DNA samples and assimilation into a phenol- chloroform-isoamyl alcohol DNA extraction. Molecular Ecology Resources, 15(1), 168 – 176.

Acknowledgements  David Hyrenbach, Ph.D.  Ilana Nimz  Mark Renshaw  Matt Iacchei, Ph.D.  And the Staff at the OI

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