A genetic technique to identify the diet of cownose rays, Rhinoptera bonasus: analysis of shellfish prey items from North Carolina and Virginia

Environmental Biology of Fishes volume 97, pages999–1012(2014)Cite this article

Abstract

Cownose rays are implicated in the consumption of commercially important shellfish on the U.S. East Coast. We tested this assumption by developing a molecular technique for species identification from cownose ray gut contents. Digestive tracts sampled from 33 rays in Pamlico Sound, NC and Chesapeake Bay, VA contained pieces of partially-digested tissue, well-digested tissue, fluid, and minute shell fragments which made visual identification to the species level nearly impossible. We sequenced the cytochrome oxidase subunit I (COI) for seven locally acquired bivalve species, chosen for their commercial and ecological importance in NC and VA. Sequences were used to design species-specific primers for each bivalve species to amplify polymerase chain reaction (PCR) products. We designed primers such that PCR products were sufficiently different in size to be distinguishable from one another when resolved on an agarose gel, and multiplexing of several species in one reaction was possible. Digestive tract sample testing revealed that cownose rays in Chesapeake Bay ate stout tagelus and soft shell clams. There was no evidence of the rays in the study consuming commercially important oysters, hard clams, and bay scallops. Further sampling over an extended period of time and additional locations is required to confirm these results. Our diagnostic tests could easily be expanded to elucidate the impact of cownose ray predation on prey populations.

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Notes

  1. 1.

    Baltic macoma primers were found to have amplified digestive tract samples that also tested positive with stout tagelus clam primers. The cross-amplification rate of the Baltic macoma primers with stout tagelus positives was 86 %, making those positives uncertain, and require further testing. To test for species identification of those uncertain samples, we sequenced a subsample (n = 8) of the uncertains and all samples were a match for stout tagelus sequence. BLAST searches of the Baltic macoma primers revealed significant matching to stout tagelus DNA sequences, so this cross-amplification is not unexpected.

References

  1. Ajemian M, Powers S (2011) Habitat-specific feeding by cownose rays (Rhinoptera bonasus) of the northern Gulf of Mexico. Environ Biol Fish 95(1):79–97

    Article  Google Scholar 

  2. Bade LM (2013) Use of genetic techniques to identify the diet of cownose rays, Rhinoptera bonasus, in North Carolina and Virginia: an analysis of shellfish prey items. East Carolina University, Greenville

    Google Scholar 

  3. Barnes RD (1974) Invertebrate Zoology 3. Ed. WB Saunders Co, Philadelphia, Pennsylvania

  4. Barnett A, Redd KS, Frusher SD, Stevens JD, Semmens JM (2010) Non-lethal method to obtain stomach samples from a large marine predator and the use of DNA analysis to improve dietary information. J Exp Mar Biol Ecol 393(1):188–192

    Article  Google Scholar 

  5. Bigelow HB, Schroeder WC (2002) Bigelow and Schroeder’s fishes of the gulf of Maine, 3rd edn. Smithsonian Institution Press, Washington

    Google Scholar 

  6. Blaylock RA (1989) A massive school of cownose rays, rhinoptera bonasus (rhinopteridae), in lower chesapeake Bay, Virginia. Copeia 1989(3):744–748

    Article  Google Scholar 

  7. Blaylock R (1993) Distribution and abundance of the cownose ray, Rhinoptera bonasus, in lower Chesapeake Bay. Estuar Coasts 16(2):255–263. doi:10.2307/1352498

    Article  Google Scholar 

  8. Collins AB, Heupel MR, Hueter RE, Motta PJ (2007) Hard prey specialists or opportunistic generalists? An examination of the diet of the cownose ray, Rhinoptera bonasus. Mar Freshw Res 58(1):135–144. doi:10.1071/MF05227

    Article  Google Scholar 

  9. Collins A, Heupel M, Simpfendorfer C (2008) Spatial distribution and long-term movement patterns of cownose rays Rhinoptera bonasus within an estuarine river. Estuar Coasts 31(6):1174–1183. doi:10.1007/s12237-008-9100-5

    Article  Google Scholar 

  10. Craig JK, Gillikin PC, Magelnicki MA, May LN (2010) Habitat use of cownose rays (Rhinoptera bonasus) in a highly productive, hypoxic continental shelf ecosystem. Fish Oceanogr 19(4):301–317. doi:10.1111/j.1365-2419.2010.00545.x

    Article  Google Scholar 

  11. Deagle BE, Kirkwood R, Jarman SN (2009) Analysis of Australian fur seal diet by pyrosequencing prey DNA in faeces. Mol Ecol 18(9):2022–2038

    CAS  PubMed  Article  Google Scholar 

  12. Dunn MR, Szabo A, McVeagh MS, Smith PJ (2010) The diet of deepwater sharks and the benefits of using DNA identification of prey. Deep-Sea Res I 157:923–930

    Article  Google Scholar 

  13. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32(5):1792–1797

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  14. Fisher R (2010) Life History, Trophic Ecology, and Prey Handling by Cownose Ray, Rhinoptera bonasus, from Chesapeake Bay. National Oceanic and Atmospheric Administration

  15. Fisher R, Garrett C, Grubbs RD (2011) Cownose ray (Rhioptera bonasus) predation relative to bivalve ontogeny. J Shellfish Res 30(1):187–196

    Article  Google Scholar 

  16. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3(5):294–299

    CAS  PubMed  Google Scholar 

  17. Goodman M, Conn P, Fitzpatrick E (2010) Seasonal occurrence of cownose rays (rhinoptera bonasus) in north Carolina’s estuarine and coastal waters. Estuar Coasts 34(3):640–651. doi:10.1007/s12237-010-9355-5

    Article  Google Scholar 

  18. Hebert PDN, Cywinska A, Ball SL, deWaard JR (2003) Biological identifications through DNA barcodes. Proc R Soc Lond Ser B Biol Sci 270(1512):313–321. doi:10.1098/rspb.2002.2218

    CAS  Article  Google Scholar 

  19. King RA, Read DS, Traugott M, Symondson WOC (2008) Molecular analysis of predation: a review of best practice for DNA-based approaches. Mol Ecol 17:947–963

    CAS  PubMed  Article  Google Scholar 

  20. Orth RJ (1975) Destruction of eelgrass, Zostera marina, by the cownose ray, Rhinoptera bonasus, in the Chesapeake Bay. Chesap Sci 16(3):205–208. doi:10.2307/1350896

    Article  Google Scholar 

  21. Peterson C, Fodrie J, Summerson H, Powers S (2001) Site-specific and density-dependent extinction of prey by schooling rays: generation of a population sink in top-quality habitat for bay scallops. Oecologia 129(3):349–356. doi:10.1007/s004420100742

    Google Scholar 

  22. Pompanon F, Deagle BE, Symondson WOC, Brown DS, Jarman SN, Taberlet P (2011) Who is eating what: diet assessment using next generation sequencing. Mol Ecol 21(8):1931–1950

    PubMed  Article  Google Scholar 

  23. Rambaut A (1995) Se-Al: Sequence alignment program. Oxford University Press, Oxford

    Google Scholar 

  24. Ratnasingham S, Hebert PDN (2007) bold: The Barcode of Life Data System (http://www.barcodinglife.org). Molecular Ecology Notes 7 (3):355–364. doi:10.1111/j.1471-8286.2007.01678.x

  25. Ruppert EE, Fox RS (1988) Seashore animals of the Southeast: A guide to common shallow-water invertebrates of the southeastern Atlantic coast. Univ of South Carolina Press, Columbia

    Google Scholar 

  26. Saccone C, De Giorgi C, Gissi C, Pesole G, Reyes A (1999) Evolutionary genomics in Metazoa: the mitochondrial DNA as a model system. Gene 238(1):195–209

    CAS  PubMed  Article  Google Scholar 

  27. Sasko DE, Dean MN, Motta PJ, Hueter RE (2006) Prey capture behavior and kinematics of the Atlantic cownose ray, Rhinoptera bonasus. Zoology 109(3):171–181

    PubMed  Article  Google Scholar 

  28. Smith J, Merriner J (1985) Food habits and feeding behavior of the cownose ray, Rhinoptera bonasus, in lower Chesapeake Bay. Estuar Coasts 8(3):305–310. doi:10.2307/1351491

    Article  Google Scholar 

  29. Smith J, Merriner J (1986) Observations on the reproductive biology of the cownose ray, Rhinoptera bonasus, in Chesapeake Bay. Fish Bull 84(4):871–877

    Google Scholar 

  30. Smith J, Merriner J (1987) Age and growth, movements and distribution of the cownose ray, Rhinoptera bonasus, in Chesapeake Bay. Estuar Coasts 10(2):153–164. doi:10.2307/1352180

    Article  Google Scholar 

  31. Yoccoz NG (2012) The future of environmental DNA in ecology. Mol Ecol 21:2031–2038

    PubMed  Article  Google Scholar 

Download references

Acknowledgments

The authors wish to acknowledge the Institute for Coastal Science and Policy and the Department of Biology at East Carolina University for logistics and financial support. Animal collection and handling procedures were approved by the East Carolina University Animal Use and Care Committee, Animal Use Protocol #D268. The American Elasmobranch Society, Save Our Seas Foundation, and the ECU-American Fisheries Society subunit contributed symposium, publication, travel, and student funding support. We are grateful for the help of Robert Fisher, Joseph Smith, R. Dean Grubbs, John Morrissey, Denise Mayer, Captain George Beckwith, Jason Rock, NCDMF, Lena Keller, Shona Paterson, and for the generosity of numerous friends and colleagues who helped with shellfish collection and cownose ray sampling. The authors wish to thank Matthew Ajemian and Julie A. Neer, symposium organizers and special issue editors, for their contributions and guidance. The views expressed in this article are those of the authors and do not necessarily represent the views or policies of the U.S. Environmental Agency.

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Affiliations

  1. Department of Biology, East Carolina University, Greenville, NC, 27858, USA

    Lyndell M. Bade, Christopher N. Balakrishnan, Susan B. McRae & Joseph J. Luczkovich

  2. Ecological Exposure Research Division U.S. EPA, Cincinnati, OH, 45268, USA

    Erik M. Pilgrim

  3. Departments of Biology and Chemistry, Bates College, Lewiston, ME, 04240, USA

    Lyndell M. Bade

Authors
  1. Lyndell M. Bade
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  2. Christopher N. Balakrishnan
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  3. Erik M. Pilgrim
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  4. Susan B. McRae
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  5. Joseph J. Luczkovich
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Correspondence to Lyndell M. Bade.

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Bade, L.M., Balakrishnan, C.N., Pilgrim, E.M. et al. A genetic technique to identify the diet of cownose rays, Rhinoptera bonasus: analysis of shellfish prey items from North Carolina and Virginia. Environ Biol Fish 97, 999–1012 (2014). https://doi.org/10.1007/s10641-014-0290-3

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Keywords

  • Elasmobranch
  • Diet
  • Feeding ecology
  • Molecular techniques
  • COI