, Volume 14, Issue 3, pp 269–282 | Cite as

Finding copepod footprints: a protocol for molecular identification of diapausing eggs in lake sediments

  • Wataru Makino
  • Hajime Ohtsuki
  • Jotaro Urabe
Research paper


Even though calanoid copepods produce diapausing eggs that stay alive in lake sediments, these eggs have rarely been used paleolimnologically, as they lack diagnostic morphological features. In this study, we developed a method to identify copepod diapausing eggs in Japan as a clue toward reconstructing past plankton populations. We first determined a 28S ribosomal DNA (rDNA) (i.e., nc28S) regional sequence library (240 bp) of various calanoid copepod species using ethanol-fixed plankton samples collected from across the Japanese archipelago. Then we applied the UltraSHOT method to extract DNA from an individual diapausing egg. Finally, the nc28S region of diapausing eggs collected from various lakes was sequenced and compared with the regional library for species identification. In total, 21 haplotypes of the nc28S region were recovered from planktonic samples of 11 Japanese freshwater calanoid copepod species. Despite the short length of this region, no identical haplotypes were shared among the species analyzed, including the Acanthodiaptomus pacificus complex treated as a species. Even different lineages of A. pacificus could be separated. These results indicate that the nc28S region can be used as a barcode in Japan. A total of 112 diapausing eggs collected from various lakes and ponds was processed, and the nc28S region of each was successfully sequenced. All of these egg sequences matched one or the other of the nc28S haplotypes in the regional library mentioned above. The set of protocols we applied (i.e., preparing a comprehensive regional sequence library and sequencing egg DNA) is thus useful for involving copepod diapausing eggs in paleolimnological studies in lakes. The nc28S region treated in this study has a strong potential to uncover the paleodiversity of copepods, at least in Japan.


Japanese lakes Biodiversity Calanoid copepods Diapausing eggs 28S ribosomal DNA Molecular identification 



We thank anonymous reviewers for providing useful comments and references. This research was supported by grants from the Ministry of the Environment, Japan (the Environment Research and Technology Development Fund, No. D-1002) to JU and from the Japan Society for the Promotion of Science (KAKENHI, Nos. 16770011, 19770010, and 23570015) to WM. We are grateful to Syuhei Ban, Ryoma Hayashi, Ryuta Himori, Fujio Hyodo, Seiji Ishida, Michinobu Kuwae, Naoko Sasaki, Kohei Omoto, and Narumi K. Tsugeki for their support with some of our samplings. All works in this study comply with the current laws of Japan.

Supplementary material

10201_2013_404_MOESM1_ESM.pdf (384 kb)
Supplementary Fig. S1 (PDF 384 kb)


  1. Adamowicz SJ, Menu-Marque S, Halse SA, Topan JC, Zemlak TS, Hebert PDN, Witt JDS (2010) The evolutionary diversification of the Centropagidae (Crustacea, Calanoida): a history of habitat shifts. Mol Phylogenet Evol 55:418–430PubMedCrossRefGoogle Scholar
  2. Albert MR, Chen G, MacDonald GK, Vermaire JC, Bennett M, Gregory-Eaves I (2010) Phosphorus and land-use changes are significant drivers of cladoceran community composition and diversity: an analysis over spatial and temporal scales. Can J Fish Aquat Sci 67:1262–1273CrossRefGoogle Scholar
  3. Asmyhr MG, Cooper SJB (2012) Difficulties barcoding in the dark: the case of crustacean stygofauna from eastern Australia. Invertebr Syst 26:583–591CrossRefGoogle Scholar
  4. Auclair JC, Frenette JJ, Dodson J (1993) Zooplankton community structure in southwestern Québec lakes: the rôles of acidity and predation. J Plankton Res 15:1103–1128CrossRefGoogle Scholar
  5. Ban S (1992) Seasonal distribution, abundance and viability of dapause eggs of Eurytemora affinis (Copepoda: Calanoida) in the sediment of Lake Ohnuma, Hokkaido. Bull Plankton Soc Jpn 39:41–48Google Scholar
  6. Ban S, Minoda T (1991) The effect of temperature on the development and hatching of diapausing and subitaneous eggs in Eurytemora affinis (Copepoda: Calanoida) in Lake Ohnuma, Hokkaido, Japan. Bull Plankton Soc Jpn Spec Vol:299–308Google Scholar
  7. Ban S, Minoda T (1992) Hatching of diapause eggs of Eurytemora affinis (Copepoda: Calanoida) collected from lake-bottom sediments. J Crust Biol 12:51–56CrossRefGoogle Scholar
  8. Bennike O (1998) Fossil egg sacs of Diaptomus (Crustacea: Copepoda) in Late Quaternary lake sediments. J Paleolimnol 19:77–79CrossRefGoogle Scholar
  9. Bissett A, Gibson JAE, Jarman SN, Swadling KM, Cromer L (2005) Isolation, amplification, and identification of ancient copepod DNA from lake sediments. Limnol Oceanogr Methods 3:533–542CrossRefGoogle Scholar
  10. Blanco-Bercial L, Bradford-Grieve J, Bucklin A (2011) Molecular phylogeny of the Calanoida (Crustacea: Copepoda). Mol Phylogenet Evol 59:103–113PubMedCrossRefGoogle Scholar
  11. Briski E, Cristescu ME, Bailey SA, MacIssac HJ (2011) Use of DNA barcode to detect invertebrate invasive species from diapausing eggs. Biol Invasion 13:1325–1340CrossRefGoogle Scholar
  12. Brooks JL, Dodson SI (1965) Predation, body size, and composition of plankton: the effect of a marine planktivore on lake plankton illustrates theory of size, composition, and predation. Science 150:28–35PubMedCrossRefGoogle Scholar
  13. Bucklin A, Allen LD (2004) MtDNA sequencing from zooplankton after long-term preservation in buffered formalin. Mol Phylogenet Evol 30:879–882PubMedCrossRefGoogle Scholar
  14. Bucklin A, Steinke D, Blanco-Bercial L (2011) DNA barcoding of marine metazoa. Annu Rev Mar Sci 3:471–508CrossRefGoogle Scholar
  15. Carpenter SR, Kitchell JF (1985) Cascading trophic interactions and lake productivity. BioScience 35:634–639CrossRefGoogle Scholar
  16. Cepada GD, Blanco-Bercial L, Bucklin A, Beron CM, Vinas MD (2012) Molecular systematic of three species of Oithona (Copepoda, Cyclopoida) from the Atlantic Ocean: comparative analysis using 28S rDNA. PLoS ONE 7:e35861CrossRefGoogle Scholar
  17. Chan EM, Derry AM, Watson LA, Arnott SE (2008) Variation in calanoid copepod resting egg abundance among lakes with different acidification histories. Hydrobiologia 614:275–284CrossRefGoogle Scholar
  18. Costa FO, deWaard FR, Boutillier F, Ratnasingham S, Dooh ST, Hajibabael M, Hebert PDN (2007) Biological identifications through DNA barcodes: the case of the Crustacea. Can J Fish Aquat Sci 64:272–295CrossRefGoogle Scholar
  19. De Stasio BT Jr (1989) The seed bank of a freshwater crustacean: copepodology for the plant ecologist. Ecology 70:1377–1389CrossRefGoogle Scholar
  20. deWaard JR, Hebert PDN, Humble LM (2011) A comprehensive DNA barcode library for the looper moss (Lepidoptera: Geometridae) of British Columbia, Canada. PLoS ONE 6:e18290PubMedCrossRefGoogle Scholar
  21. Dussart BH, Defaye D (1983) Répertoire mondial des crustacés copépodes eaux intérieures. I. Calanoïdes. CNRS, ParisGoogle Scholar
  22. Dussart BH, Defaye D (1995) Copepoda. Introduction to the Copepoda. SPB Academic Publishing, AmsterdamGoogle Scholar
  23. Fell JW, Scorzetti G, Gonnell L, Graig S (2006) Biodiversity of micro-eukaryotes in Antarctic Dry Valley soils with < 5% soil moisture. Soil Biol Boichem 38:3107–3119CrossRefGoogle Scholar
  24. 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 Biotech 3:294–299Google Scholar
  25. Frey DG (1964) Remains of animals in quaternary lake and bog sediments and their interpretation. Ergebn Limnol 2:1–116Google Scholar
  26. Hairston N Jr, Olds EJ (1984) Population differences in the timing of diapause: adaptation in a spatially heterogeneous environment. Oecologia 61:42–48CrossRefGoogle Scholar
  27. Hairston N Jr, Van Brunt RA, Kearns CM, Engstrom DR (1995) Age and survivorship of diapausing eggs in a sediment egg bank. Ecology 76:1706–1711CrossRefGoogle Scholar
  28. Hajibabaei M, Smith MA, Janzen DH, Rodriguez JJ, Whitefield JB, Hebert PDN (2006) A minimalist barcode can identify a specimen whose DNA is degraded. Mol Ecol Notes 6:959–964CrossRefGoogle Scholar
  29. Hebert PDN, Cywinska A, Ball SL, deWaaard JR (2003a) Biological identifications through DNA barcodes. Proc R Soc Lond B 270:313–321CrossRefGoogle Scholar
  30. Hebert PDN, Ratnasingham S, deWaard DR (2003b) Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc R Soc London B 270(Suppl 1):S96–S99CrossRefGoogle Scholar
  31. Hutchinson GE (1967) A treatise on limnology, vol. 2. Introduction to lake biology and the limnoplankton. John Wiley and Sons, New YorkGoogle Scholar
  32. Ishida S, Otsuki H, Awano T, Makino W, Suyama Y, Urabe J (2012) DNA extraction and amplification methods for ephippial carapace of Daphnia resting eggs in lake sediments: a novel approach for reconstructing zooplankton population structure from the past. Limnology 13:261–267CrossRefGoogle Scholar
  33. Jeppesen E, Jensen JP, Søndergaard M, Lauridsen T, Landkildehus F (2000) Trophic structure, species richness and biodiversity in Danish lakes: changes along a phosphorus gradient. Freshw Biol 45:201–218CrossRefGoogle Scholar
  34. Jeppesen E, Leavitt PR, De Meester L, Jensen JP (2001) Functional ecology and palaeolimnology: using cladoceran remains to reconstruct anthropogenic impact. Trends Ecol Evol 16:191–198PubMedCrossRefGoogle Scholar
  35. Kiesling TL, Wilkinson E, Rabalais J, Ortner PB, McCabe MM, Fell JW (2002) Rapid identification of adult and naupliar stages of copepods using DNA hybridization methodology. Mar Biotech 4:30–39Google Scholar
  36. Kim S, Song K-H, Ree H-I, Kim W (2012) A DNA barcode library for Korean Chironomidae (Insecta: Diptera) and indexes for defining barcode gap. Mol Cells 33:9–17PubMedCrossRefGoogle Scholar
  37. Lampert W, Sommer U (1997) Limnoecology. The ecology of lakes and streams. Oxford University Press, OxfordGoogle Scholar
  38. Leavitt PR, Carpenter SR, Kitchell JF (1989) Whole-lake experiments: the annual records of fossil pigments and zooplankton. Limnol Oceanogr 34:700–717CrossRefGoogle Scholar
  39. Machida RJ, Tsuda A (2010) Dissimilarity of species and forms of planktonic Neocalanus copepod using mitochondrial COI, 12S, nuclear ITS, and 28S gene sequences. PLoS ONE 5:e10278PubMedCrossRefGoogle Scholar
  40. Makino W, Tanabe AS (2009) Extreme population genetic differentiation and secondary contact in the freshwater copepod Acanthodiaptomus pacificus in the Japanese Archipelago. Mol Ecol 18:3699–3713PubMedCrossRefGoogle Scholar
  41. Makino W, Knox MA, Duggan IC (2010) Invasion, genetic variation and species identity of the calanoid copepod Sinodiaptomus valkanovi. Freshw Biol 55:375–386CrossRefGoogle Scholar
  42. Marrone F, Lo Brutto S, Hundsdoerfer AK, Arculeo M (2013) Overlooked cryptic endemism in copepods: systematics and natural history of the calanoid subgenus Occidodiaptomus Borutzky 1991 (Copepoda, Calanoida, Diaptomidae). Mol Phylogenet Evol 66:190–202PubMedCrossRefGoogle Scholar
  43. Meusnier I, Singer GAC, Landry J-F, Hickey DA, Hebert PDN, Hajibabaei M (2008) A universal DNA mini-barcode for biodiversity analysis. BMC Genomics 9:214PubMedCrossRefGoogle Scholar
  44. Mizuno T, Takahashi E (eds) (2000) An illustrated guide to freshwater zooplankton in Japan (in Japanese). Tokai University Press, TokyoGoogle Scholar
  45. Montero-Pau J, Gómez A, Muños J (2008) Application of an inexpensive and high-throughput genomic DNA extraction method for the molecular ecology of zooplanktonic diapausing egg. Limnol Oceanogr Methods 6:218–222CrossRefGoogle Scholar
  46. Pace ML (1986) An empirical analysis of zooplankton community size structure across lake trophic gradients. Limnol Oceanogr 31:45–55CrossRefGoogle Scholar
  47. Parker BR, Wilhelm FM, Schindler DW (1996) Recovery of Hesperodiaptomus articus populations from diapausing eggs following elimination by stocked salmonids. Can J Zool 74:1292–1297CrossRefGoogle Scholar
  48. Schindler DW (1977) Evolution of phosphorus limitation in lakes. Science 195:260–262PubMedCrossRefGoogle Scholar
  49. Selden AP, Huys R, Stephenson MH, Heward AP, Taylor PN (2010) Crustaceans from bitumen clast in Carboniferous glacial diamictite extend fossil records of copepods. Nat Commun 1:50PubMedCrossRefGoogle Scholar
  50. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetic analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2371–2379CrossRefGoogle Scholar
  51. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24:4876–4882CrossRefGoogle Scholar
  52. Xu Z-H, Wang G-Z, Mu Q, Wu L-S, Li S-J (2011) An approach to the study of copepod egg banks based on efficient DNA extraction from individual copepod eggs. Mar Biol Res 7:592–598CrossRefGoogle Scholar

Copyright information

© The Japanese Society of Limnology 2013

Authors and Affiliations

  1. 1.Graduate School of Life SciencesTohoku UniversitySendaiJapan

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