Development of a real-time PCR assay (SYBR Green I) for rapid identification and quantification of scyphomedusae Aurelia sp.1 planulae

Jianyan Wang; Yu Zhen; Tiezhu Mi; Zhigang Yu; Guoshan Wang

doi:10.1007/s00343-015-4091-0

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Development of a real-time PCR assay (SYBR Green I) for rapid identification and quantification of scyphomedusae Aurelia sp.1 planulae

Updated：2023-05-16

- Development of a real-time PCR assay (SYBR Green I) for rapid identification and quantification of scyphomedusae Aurelia sp.1 planulae
- Journal of Oceanology and Limnology Vol. 33, Issue 4, Pages: 974-987(2015)
- Affiliations：
  
  1. College of Marine Life Sciences Ocean University of China
  2. College of Environmental Science and Engineering Ocean University of China
  3. Key Laboratory of Marine Environment and Ecology Ministry of Education Ocean University of China
  4. College of Chemistry and Chemical Engineering Ocean University of China
  5. Key Laboratory of Marine Chemistry Theory and Technology Ministry of Education Ocean University of China
- Author bio：
- Funds：
- DOI：10.1007/s00343-015-4091-0
  CLC：
- Online First：10 April 2015，
  
  Published：2015
- Accepted：
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Wang, J., Zhen, Y., Mi, T. et al. Development of a real-time PCR assay (SYBR Green I) for rapid identification and quantification of scyphomedusae Aurelia sp.1 planulae., Chin. J. Ocean. Limnol. 33, 974–987 (2015). https://doi.org/10.1007/s00343-015-4091-0
DOI：

Wang, J., Zhen, Y., Mi, T. et al. Development of a real-time PCR assay (SYBR Green I) for rapid identification and quantification of scyphomedusae Aurelia sp.1 planulae., Chin. J. Ocean. Limnol. 33, 974–987 (2015). https://doi.org/10.1007/s00343-015-4091-0 DOI：

摘要

Abstract

The complicated life cycle of Aurelia spp.

comprising benthic asexually-reproducing polyps and sexually-reproducing medusae

makes it hard for researchers to identify and track them

especially for early stage individuals

such as planulae. To solve this problem

we developed a real-time PCR assay (SYBR Green I) to identify planulae in both cultured and natural seawater samples. Species-specific primers targeting Aurelia sp.1 mitochondrial 16S rDNA (mt 16S rDNA) regions were designed. Using a calibration curve constructed with plasmids containing the Aurelia sp.1 mt 16S rDNA fragment and a standard curve for planulae

the absolute number of mt 16S rDNA copies per planula was determined and from that the total number of planulae per sample was estimated. For the field samples

a 100-fold dilution of the sample DNA combined with a final concentration of 0.2 μg/μL BSA in the PCR reaction mixture was used to remove real-time PCR inhibitors. Samples collected in Jiaozhou Bay from July to September 2012 were subsequently analyzed using this assay. Peak Aurelia sp.1 planula abundance occurred in July 2012 at stations near Hongdao Island and Qingdao offshore; abundances were very low in August and September. The real-time PCR assay (SYBR Green I) developed here negates the need for traditional microscopic identification

which is laborious and time-consuming

and can detect and quantify jellyfish planulae in field plankton samples rapidly and specifically.

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references

Ahmed W, Huygens F, Goonetilleke A, Gardner T. 2008. Real-time PCR detection of pathogenic microorganisms in roof-harvested rainwater in Southeast Queensland, Australia. Appl. Environ. Microbiol., 74(17): 5 490–5 496.

Attrill M J, Wright J, Edwards M. 2007. Climate-related increases in jellyfish frequency suggest a more gelatinous future for the North Sea. Limnol. Oceanogr., 52(1): 480–485.

Barz K, Hirche H J. 2007. Abundance, distribution and prey composition of scyphomedusae in the southern North Sea. Mar. Biol., 151(3): 1 021–1 033.

Baxter E J, Walne A W, Purcell J E, McAllen R, Doyle T K. 2010. Identification of jellyfish from continuous plankton recorder samples. Hydrobiologia, 645(1): 193–201.

Bayha K M, Dawson M N, Collins A G, Barbeitos M S, Haddock S H D. 2010. Evolutionary relationships among scyphozoan jellyfish families based on complete taxon sampling and phylogenetic analyses of 18S and 28S ribosomal DNA. Integr. Comp. Biol., 50(3): 436–455.

Bayha K M, Graham W M. 2009. A new Taqman© PCR-based method for the detection and identification of scyphozoan jellyfish polyps. Hydrobiologia, 616(1): 217–228.

Bolton T F, Graham W M. 2006. Jellyfish on the rocks: bioinvasion threat of the international trade in aquarium live rock. Biol. Invasions, 8(4): 651–653.

Brewer R H. 1976. Larval settling behavior in Cyanea capillata (Cnidaria: Scyphozoa). Biol. Bull., 150(2): 183–199.

Brewer R H. 1978. Larval settlement behavior in the jellyfish Aurelia aurita (Linnaeus) (Scyphozoa: Semaeostomeae). Estuaries, 1(2): 120–122.

Brewer R H. 1984. The influence of the orientation, roughness, and wettability of solid surfaces on the behavior and attachment of planulae of Cyanea (Cnidaria: Scyphozoa). Biol. Bull., 166(1): 11–21.

Bridge D, Cunningham C W, Schierwater B, DeSalle R, Buss L W. 1992. Class-level relationships in the phylum Cnidaria: evidence from mitochondrial genome structure. Proc. Natl. Acad. Sci. USA, 89(18): 8 750–8 753.

Bucklin A, Steinke D, Blanco-Bercial L. 2011. DNA barcoding of marine metazoa. Annu. Rev. Mar. Sci., 3(1): 471–508.

Calder D R. 1977. Nematocysts of the ephyra stages of Aurelia, Chrysaora, Cyanea, and Rhopilema (Cnidaria, Scyphozoa). Trans. Amer. Microsc. Soc., 96(1): 13–19.

Chandler D P, Wagnon C A, Bolton H. 1998. Reverse transcriptase (RT) inhibition of PCR at low concentrations of template and its implications for quantitative RT-PCR. Appl. Environ. Microbiol., 64(2): 669–677.

Cheng F P, Wang M X, Wang Y T, Zhang F, Li C L, Sun S. 2012. DNA barcoding of common medusozoa in Northern China based on mtCOI sequence. Oceanologia Et Limnologia Sinica, 43(3): 451–459. (in Chinese with English abstract)

Chia F S, Buckland-Nicks J, Young C M. 1984. Locomotion of marine invertebrate larvae: a review. Can. J. Zool., 62(7): 1 205–1 222.

Collins A G, Daly M. 2005. A new deepwater species of Stauromedusae, Lucernaria janetae (Cnidaria, Staurozoa, Lucernariidae), and a preliminary investigation of stauromedusan phylogeny based on nuclear and mitochondrial rDNA data. Biol. Bull., 208(3): 221–230.

Collins A G, Schuchert P, Marques A C, Jankowski T, Medina M, Schierwater B. 2006. Medusozoan phylogeny and character evolution clarified by new large and small subunit rDNA data and an assessment of the utility of phylogenetic mixture models. Syst. Biol., 55(1): 97–115.

Collins A G. 2002. Phylogeny of Medusozoa and the evolution of cnidarian life cycles. J. Evolution. Biol., 15(3): 418–432.

Daskalov G M, Grishin A N, Rodionov S, Mihneva V. 2007. Trophic cascades triggered by overfishing reveal possible mechanisms of ecosystem regime shifts. Proc. Natl. Acad. Sci. USA, 104(25): 10 518–10 523.

Dawson M N, Gupta A S, England M H. 2005. Coupled biophysical global ocean model and molecular genetic analyses identify multiple introductions of cryptogenic species. Proc. Natl. Acad. Sci. USA, 102(34): 11 968–11 973.

Demeke T, Adams R P. 1992. The effects of plant polysaccharides and buffer additives on PCR. Biotechniques, 12(3): 332–334.

Duarte C M, Pitt K A, Lucas C H, Purcell J E, Uye S I I, Robinson K, Brotz L, Decker M B, Sutherland K R, Malej A, Madin L, Mianzan H, Gili JM, Fuentes V, Atienza D, Pages F, Breitburg D, Malek J, Graham W M, Condon R H. 2012. Is global ocean sprawl a cause of jellyfish blooms? Front. Ecol. Environ., 11(2): 91–97.

Galluzzi L, Penna A, Bertozzini E, Vila M, Garces E, Magnani M. 2004. Development of a real-time PCR assay for rapid detection and quantification of Alexandrium minutum (a Dinoflagellate). Appl. Environ. Microbiol., 70(2): 1 199–1 206.

Graham W M, Bayha K M. 2007. Biological invasions by marine jellyfish. In: Nentwig W ed. Biological Invasions. 1st edn. Springer, Heidelberg, Germany. p.239–255.

Graham W M, Martin D L, Felder D L, Asper V L, Perry H M. 2003. Ecological and economic implications of a tropical jellyfish invader in the Gulf of Mexico. Biol. Invasions, 5(1–2): 53–69.

Holst S, Jarms G. 2007. Substrate choice and settlement preferences of planula larvae of five scyphozoa (cnidaria) from german bight, north sea. Mar. Biol., 151(3): 863–871.

Holst S. 2012. Morphology and development of benthic and pelagic life stages of North Sea jellyfish (Scyphozoa, Cnidaria) with special emphasis on the identification of ephyra stages. Mar. Biol., 159(12): 2 707–2 722.

Hoover R A, Purcell J E. 2009. Substrate preferences of scyphozoan Aurelia labiata polyps among common dock-building materials. Hydrobiologia, 616(1): 259–267.

Hunter E, Taylor N, Fox C J, Maillard M, Taylor M I. 2012. Effectiveness of TaqMan probes for detection of fish eggs and larvae in the stomach contents of a teleost predator. J. Fish. Biol., 81(1): 320–328.

Ishii H, Ohba T, Kobayashi T. 2008. Effects of low dissolved oxygen on planula settlement, polyp growth and asexual reproduction of Aurelia aurita. Plankton. Benthos Res., 3(Supplement): 107–113.

Ishii H. 2003. Development time of planula larvae on the oral arms of the scyphomedusa Aurelia aurita. J. Plankton Res., 25(11): 1 447–1 450.

Jiang J L, Alderisio K A, Singh A, Xiao L H. 2005. Development of procedures for direct extraction of Cryptosporidium DNA from water concentrates and for relief of PCR inhibitors. Appl. Environ. Microbiol., 71(3): 1 135–1 141.

Kalinina O, Lebedeva I, Brown J, Silver J. 1997. Nanoliter scale PCR with TaqMan detection. Nucl eic Acids Res., 25(10): 1 999–2 004.

Keen S L. 1987. Recruitment of Aurelia aurita (Cnidaria: Scyphozoa) larvae is position-dependent, and independent of conspecific density, within a settling surface. Mar. Ecol. Prog. Ser., 38: 151–160.

Ki J S, Hwang D S, Lee J S. 2010. Simultaneous detection of Aurelia and Chrysaora scyphozoan jellyfish on a DNA microarray. J. Mar. Biol. Assn. UK, 90(6): 1 111–1 117.

Ki J S, Hwang D S, Shin K, Yoon W D, Lim D, Kang Y S, Lee Y, Lee J S. 2008. Recent moon jelly (Aurelia sp.1) blooms in Korean coastal waters suggest global expansion: examples inferred from mitochondrial COI and nuclear ITS-5.8 S rDNA sequences. ICES J. Mar. Sci., 65(3): 443–452.

Ki J S, Kim I C, Lee J S. 2009. Comparative analysis of nuclear ribosomal DNA from the moon jelly Aurelia sp.1 (Cnidaria: Scyphozoa) with characterizations of the 18S, 28S genes, and the intergenic spacer (IGS). Hydrobiologia, 616(1): 229–239.

Kramp P L. 1961. Synopsis of the Medusae of the World. Cambridge University Press, New York. 469p.

Kreader C A. 1996. Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Appl. Environ. Microbiol., 62(3): 1 102–1 106.

Kroiher M, Berking S. 1999. On natural metamorphosis inducers of the cnidarians Hydractinia echinata (Hydrozoa) and Aurelia aurita (Scyphozoa). Helgoland Mar. Res., 53(2): 118–121.

Lo W T, Purcell J E, Hung J J, Su H M, Hsu P K. 2008. Enhancement of jellyfish (Aurelia aurita) populations by extensive aquaculture rafts in a coastal lagoon in Taiwan. ICES J. Mar. Sci., 65(3): 453–461.

Lucas C H. 1996. Population dynamics of Aurelia aurita (Scyphozoa) from an isolated brackish lake, with particular reference to sexual reproduction. J. Plankton Res., 18(6): 987–1 007.

Lucas C H. 2001. Reproduction and life history strategies of the common jellyfish, Aurelia aurita, in relation to its ambient environment. Hydrobiologia, 451(1–3): 229–246.

Madani M, Subbotin S A, Moens M. 2005. Quantitative detection of the potato cyst nematode, Globodera pallida, and the beet cyst nematode, Heterodera schachtii, using Real-Time PCR with SYBR green I dye. Mol. Cell. Probe., 19(2): 81–86.

Mayer A G. 1910. Medusae of the World: The Hydromedusae. Carnegie institution of Washington, Washington D. C. 230p.

McManus G B, Katz L A. 2009. Molecular and morphological methods for identifying plankton: what makes a successful marriage? J. Plankton Res., 31(10): 1 119–1 129.

Miglietta M P, Schuchert P, Cunningham C W. 2009. Reconciling genealogical and morphological species in a worldwide study of the Family Hydractiniidae (Cnidaria, Hydrozoa). Zoo. Scr., 38(4): 403–430.

Mills C E. 2001. Jellyfish blooms: are populations increasing globally in response to changing ocean conditions? Hydrobiologia, 451(1–3): 55–68.

Miyake H. Terazaki M, Kakinuma Y. 2002. On the polyps of the common jellyfish Aurelia aurita in Kagoshima Bay. J. Oceanogr., 58(3): 451–459.

Möller H. 1980. Population dynamics of Aurelia aurita medusae in Kiel Bight, Germany (FRG). Mar. Biol., 60(2–3): 123–128.

Moura C J, Cunha M R, Porteiro F M, Rogers A D. 2011. The use of the DNA barcode gene 16S mRNA for the clarification of taxonomic problems within the family Sertulariidae (Cnidaria, Hydrozoa). Zoo. Scr., 40(5): 520–537.

Müller W A, Leitz T. 2002. Metamorphosis in the Cnidaria. Can. J. Zool., 80(10): 1 755–1 771.

Nagai M, Yoshida A, Sato N. 1998. Additive effects of bovine serum albumin, dithiothreitol and glycerolon PCR. Biochem. Mol. Biol. Int., 44(1): 157–163.

Pan M, McBeath A J, Hay S J, Pierce G J, Cunningham C O. 2008. Real-time PCR assay for detection and relative quantification of Liocarcinus depurator larvae from plankton samples. Mar. Biol., 153(5): 859–870.

Purcell J E, Arai M N. 2001. Interactions of pelagic cnidarians and ctenophores with fish: a review. Hydrobiologia, 451(1–3): 27–44.

Purcell J E, Uye S, Lo W. 2007. Anthropogenic causes of jellyfish blooms and their direct consequences for humans: a review. Mar. Ecol. Prog. Ser., 350: 153–174.

Purcell J E. 2005. Climate effects on formation of jellyfish and ctenophore blooms: a review. J. Mar. Biol. Assn. UK, 85(3): 461–476.

Purcell J E. 2012. Jellyfish and ctenophore blooms coincide with human proliferations and environmental perturbations. Ann. Rev. Mar. Sci., 4(1): 209–235.

Richardson A J, Bakun A, Hays G C, Gibbons M J. 2009. The jellyfish joyride: causes, consequences and management responses to a more gelatinous future. Trends Ecol. Evol., 24(6): 312–322.

Russell F S. 1970. The Medusae of the British Isles: 2. Pelagic Scyphozoa with a supplement to the first volume on hydromedusae. Cambridge University Press, New York. 283p.

Schroth W, Jarms G, Streit B, Schierwater B. 2002. Speciation and phylogeography in the cosmopolitan marine moon jelly, Aurelia sp. BMC Evol. Biol., 2(1): 1.

Svane I, Dolmer P. 1995. Perception of light at settlement: a comparative study of two invertebrate larvae, a scyphozoan planula and a simple ascidian tadpole. J. Exp. Mar. Biol. Ecol., 187(1): 51–61.

Teo I A, Choi J W, Morlese J, Taylor G, Shaunak S. 2002. LightCycler qPCR optimisation for low copy number target DNA. J. Immunol. Method., 270(1): 119–133.

Tsai Y L, Olson B H. 1992. Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction. Appl. Environ. Microbiol., 58(2): 754–757.

Vadopalas B, Bouma J V, Jackels C R, Friedman C S. 2006. Application of real-time PCR for simultaneous identification and quantification of larval abalone. J. Exp. Mar. Biol. Ecol., 334(2): 219–228.

Wan A Y, Zhang G T. 2012. Annual occurrence of moon jellyfish Aurelia sp.1 in the Jiaozhou Bay and its impacts on zooplankton community. Oceanologia et Limnologia S inica, 43(3): 494–501. (in Chinese with English abstract)

Wang J Y, Zhen Y, Wang G S, Mi T Z, Yu Z G. 2013. Molecular identification and detection of moon jellyfish (Aurelia sp.) based on partial sequencing of mitochondrial 16S rDNA and COI. Chinese Journal of Applied Ecology, 24(3): 847–852. (in Chinese with English abstract)

Wang S W, Zhang G T, Sun S, Wang Y T, Zhao Z X. 2012. Population dynamics of three scyphozoan jellyfish species during summer of 2011 in Jiaozhou Bay. Oceanologia et Limnologia S inica, 43(3): 471–479. (in Chinese with English abstract)

Wei T, Lu G J, Clover G. 2008. Novel approaches to mitigate primer interaction and eliminate inhibitors in multiplex PCR, demonstrated using an assay for detection of three strawberry viruses. J. Virol. Meth., 151(1): 132–139.

Wilde J, Eiden J, Yolken R. 1990. Removal of inhibitory substances from human fecal specimens for detection of group A rotaviruses by reverse transcriptase and polymerase chain reactions. J. Clin. Microbiol., 28(6): 1 300–1 307.

Wilson I G. 1997. Inhibition and facilitation of nucleic acid amplification. Appl. Environ. Microbiol., 63(10): 3 741–3 751.

Xia J Q, Yason C V, Kibenge F. 1995. Comparison of dot blot hybridization, polymerase chain reaction, and virus isolation for detection of bovine herpesvirus-1 (BHV-1) in artificially infected bovine semen. Can. J. Vet. Res., 59(2): 102–109.

Yasuda T. 1969. Ecological studies on the jelly-fish, Aurelia aurita in Urazoko Bay, Fukui Prefecture-I: Occurrence pattern of the medusa. Bull. Jap. Soc. Sci. Fish., 35(1): 1–6. (in Japanese with English abstract)

Yuan J, Mi T Z, Zhen Y, Yu Z G. 2012. Development of a rapid detection and quantification method of Karenia mikimotoi by real-time quantitative PCR. Harmful Algae, 17: 83–91.

Zhang H Y, Zhao L, Wei H. 2012. Origin of the giant jellyfish in Qingdao offshore in summer. Oceanologia et Limnologia Sinica, 43(3): 662–668. (in Chinese with English abstract)

Zhang T, Fang H H P. 2006. Applications of real-time polymerase chain reaction for quantification of microorganisms in environmental samples. Appl. Environ. Microbiol., 70(3): 281–289.

Zrzavý J, Mihulka S, Kepka P, Bezd<ek A, Tietz D. 1998. Phylogeny of the Metazoa based on morphological and 18S ribosomal DNA evidence. Cladistics, 14(3): 249–285.

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