Abstract
Genetic studies play a great role for determining the biology of bivalves, particularly those covering population genetics, phylogeny, breeding, stock management, and conservation. However, DNA sampling methods that require removal of bivalves from the water and/or opening of their shells often cause stress and damage to bivalves, which can be lethal. The invasiveness of DNA sampling has made it difficult to conduct genetic studies in threatened species, rare species, and/or breeding lineages. In the present study, we developed a non-invasive method for bivalve DNA sampling using the water-filled mantle cavity (WMC). Our method can extract DNA from a small WMC sample (about 100 µl), collected using a fine needle and syringe without opening the shell. We demonstrated that the WMC sample contains intact mitochondrial and nuclear DNA. DNA contamination from other organisms, such as adjacent bivalve individuals, did not affect the resulting PCR and DNA sequencing analyses. Finally, the individuals from whom WMC was collected remained alive for more than 2 months after the experiments. This non-invasive method will be of great assistance in investigating the genetics of bivalves.
Similar content being viewed by others
References
Claremont, M., D. G. Reid & S. T. Williams, 2008. A molecular phylogeny of the Rapaninae and Ergalataxinae (Neogastropoda: Muricidae). Journal of Molluscan Studies 74: 215–221.
Clusa, L., L. Miralles, A. Basanta, C. Escot & E. García-Vázquez, 2017. eDNA for detection of five highly invasive molluscs. A case study in urban rivers from the Iberian Peninsula. PloS ONE 12: e0188126.
Chiesa, S., L. Lucentini, R. Freitas, F. N. Marzano, F. Minello, C. Ferrari, L. Filonzi, E. Figueira, S. Breda, G. Baccarani & E. Argese, 2014. Genetic diversity of introduced Manila clam Ruditapes philippinarum populations inferred by 16S rDNA. Biochemical Systematics and Ecology 57: 52–59.
Colgan, D. J., W. F. Ponder, E. Beacham & J. Macaranas, 2007. Molecular phylogenetics of Caenogastropoda (Gastropoda: Mollusca). Molecular Phylogenetics and Evolution 42: 717–737.
Combosch, D. J. & G. Giribet, 2016. Clarifying phylogenetic relationships and the evolutionary history of the bivalve order Arcida (Mollusca: Bivalvia: Pteriomorphia). Molecular Phylogenetics and Evolution 94: 298–312.
Cordero, D., M. Delgado, B. Liu, J. Ruesink & C. Saavedra, 2017. Population genetics of the Manila clam (Ruditapes philippinarum) introduced in North America and Europe. Scientific Reports 7: 39745.
Cowart, D. A., M. A. Renshaw, C. A. Gantz, J. Umek, S. Chandra, S. P. Egan, D. M. Lodge & E. R. Larson, 2018. Development and field validation of an environmental DNA (eDNA) assay for invasive clams of the genus Corbicula. Management of Biological Invasions 9: 27–37.
da Cruz Santos-Neto, G., I. S. da Silva Nunes, C. R. Beasley, A. R. B. Silva, C. P. Gomes & C. H. Tagliaro, 2018. Evolution in action: allopatry, variable diversity and a stepping-stone model of migration among populations of the freshwater bivalve Triplodon corrugatus from the north-eastern Amazon. Hydrobiologia 810: 227–237.
Davies, M. S. & S. J. Hawkins, 1998. Mucus from marine molluscs. Advances in Marine Biology 34: 1–71.
de Oliveira, M. J. S., C. R. Beasley, N. G. V. Barros, N. do Socorro Marques-Silva, L. R. L. de Simone, E. S. Lima & C. H. Tagliaro, 2017. Two African origins of naturalized brown mussel (Perna perna) in Brazil: past and present bioinvasions. Hydrobiologia 794: 59–72.
Folmer, O., M. Black, W. Hoeh, R. Lutz & R. Vrijenhoek, 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3: 294–299.
Furuta, H. & A. Kajita, 1983. Dimeric hemoglobin of the bivalve mollusc Anadara broughtonii: complete amino acid sequence of the globin chain. Biochemistry 22: 917–922.
Gardner, J. P., C. Boesche, J. M. Meyer & A. R. Wood, 2012. Analyses of DNA obtained from shells and brine-preserved meat of the giant clam Tridacna maxima from the central Pacific Ocean. Marine Ecology Progress Series 453: 297–301.
Geist, J. & R. Kuehn, 2005. Genetic diversity and differentiation of central European freshwater pearl mussel (Margaritifera margaritifera L.) populations: implications for conservation and management. Molecular Ecology 14: 239–425.
Kurita, Y., N. Hashimoto & H. Wada, 2016. Evolution of the molluscan body plan: the case of the anterior adductor muscle of bivalves. Biological Journal of the Linnean Society 119: 420–429.
Liu, Y. G., T. Kurokawa, M. Sekino, T. Tanabe & K. Watanabe, 2013. Complete mitochondrial DNA sequence of the ark shell Scapharca broughtonii: an ultra-large metazoan mitochondrial genome. Comparative Biochemistry and Physiology Part D: Genomics and Proteomics 8: 72–81.
Lopes-Lima, M., L. E. Burlakova, A. Y. Karatayev, K. Mehler, M. Seddon & R. Sousa, 2018. Conservation of freshwater bivalves at the global scale: diversity, threats and research needs. Hydrobiologia 810(1): 1–14.
Masaoka, T. & T. Kobayashi, 2005. Species identification of Pinctada imbricata using intergenic spacer of nuclear ribosomal RNA genes and mitochondrial 16S ribosomal RNA gene regions. Fisheries Science 71: 837–846.
Meyer, J. B., L. E. Cartier, E. A. Pinto-Figueroa, M. S. Krzemnicki, H. A. Hänni & B. A. McDonald, 2013. DNA fingerprinting of pearls to determine their origins. PloS ONE 8: e75606.
Morino, Y., N. Hashimoto & H. Wada, 2017. Expansion of TALE homeobox genes and the evolution of spiralian development. Nature Ecology & Evolution 1: 1942.
Sassoubre, L. M., K. M. Yamahara, L. D. Gardner, B. A. Block & A. B. Boehm, 2016. Quantification of environmental DNA (eDNA) shedding and decay rates for three marine fish. Environmental Science & Technology 50: 10456–10464.
Zhang, X., Q. Li, L. Kong & H. Yu, 2018. Epigenetic variation of wild populations of the Pacific oyster Crassostrea gigas determined by methylation-sensitive amplified polymorphism analysis. Fisheries Science 84: 61–70.
Acknowledgements
This study was funded from Kurita Water and Environment Foundation (17B019) and partly supported by Tohoku Ecosystem-Associated Marine Sciences and JPSP KAKENHI (15K14561). We deeply thank Dr. Minoru Ikeda for his helpful comments on genetic analysis. We also thank anonymous reviewers for their helpful comments.
Author information
Authors and Affiliations
Corresponding author
Additional information
Handling editor: Christian Sturmbauer
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Kurita, Y., Kijima, A. A noninvasive method for extracting bivalve DNA from the water-filled mantle cavity. Hydrobiologia 829, 237–243 (2019). https://doi.org/10.1007/s10750-018-3835-1
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10750-018-3835-1