GUO Hao, LIU Yongjian, ZHANG Qi, YUAN Xiutang, ZHANG Weiwei, ZHANG Zhifeng. A quantitative polymerase chain reaction assay for the enumeration of brown tide algae Aureococcus anophagefferens in coastal waters of Qinhuangdao[J]. Acta Oceanologica Sinica, 2015, 34(2): 132-136. doi: 10.1007/s13131-015-0615-x
Citation: GUO Hao, LIU Yongjian, ZHANG Qi, YUAN Xiutang, ZHANG Weiwei, ZHANG Zhifeng. A quantitative polymerase chain reaction assay for the enumeration of brown tide algae Aureococcus anophagefferens in coastal waters of Qinhuangdao[J]. Acta Oceanologica Sinica, 2015, 34(2): 132-136. doi: 10.1007/s13131-015-0615-x

A quantitative polymerase chain reaction assay for the enumeration of brown tide algae Aureococcus anophagefferens in coastal waters of Qinhuangdao

doi: 10.1007/s13131-015-0615-x
  • Received Date: 2014-01-06
  • Rev Recd Date: 2014-06-20
  • Aureococcus anophagefferens, a small pelagophyte algae, has caused brown tide blooms in coastal waters of Qinhua-ngdao in recent years, presenting significant negative impacts on the shellfish mariculture industry. Under standard light microscopy, it is visually indistinguishable from other small algae in field samples due to its extremely small size. In this study, quantitative polymerase chain reaction (qPCR) based on 18S rDNA sequences was developed and used to detect and enumerate A. anophagefferens. A linear regression (R2= 0.91) was generated based on cycle thr-esholds value (Ct) versus known concentrations of A. anophagefferens. Twenty-two field samples collected in coastal waters of Qinhuangdao were subjected to DNA extraction and then analyzed using qPCR. Results showed that A. anophagefferens had a wide distribution in coastal waters along Qinhuangdao. Elevated A. anophagefferens abun-dance, category 3 brown tide blooms (>200 000 cells/mL) occurred at Dongshan Beach and Tiger-stone Beach in August in 2013. In shellfish mariculture areas along coastal waters of Qinhuangdao, 4 stations had category 3 blooms, and 6 stations had category 2 blooms (35 000-200 000 cells/mL) in August and all stations had category 1 blooms (>0 to ≤35 000 cells/mL) in October. Quantitative PCR allows for detection of A. anophagefferens cells at low levels in filed samples, which is essential to effective management and prediction of brown tide blooms.
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  • Anderson D M, Keafer B A, Kulis D M, et al. 1993. An immunofluores-cent survey of the brown tide chrysophyte Aureococcus anophag-efferens along the Northeast coast of the United-States. J Plankton Res, 15: 563-580
    Anderson D M, Kulis D M, Cosper E M. 1989. Immunofluorescent detection of the brown tide organism, Aureococcus anophageffer-ens. In: Cosper E M, Bricelj V M, Carpenter E J. eds. Novel Phytop-lankton Blooms: Causes and Impacts of Recurrent Brown Tides and Other Unusual Blooms, Lecture Notes on Coastal and Estuar-ine Studies. Springer-Verlag, 213-228
    Antonella P, Luca G. 2013. The quantitative real-time PCR applica-tions in the monitoring of marine harmful algal bloom (HAB) spe-cies. Environ Sci Pollut Res, 20: 6851-6862
    Bowers H A, Tengs T, Glasgow H B, et al. 2000. Development of real-time PCR assays for rapid detection of Pfiesteria piscicida and related dinoflagellates. Appl Environ Microbiol, 66: 4641-4648
    Bricelj V M, Mac-Quarrie S P, Schaffner R A. 2001. Differential effects of Aureococcus anophagefferens isolates ("brown tide") in uni-algal and mixed suspensions on bivalve feeding. Mar Biol, 139: 605-615
    Cosper E M, Dennison W C, Carpenter E J, et al. 1987. Recurrent and persistent brown tide blooms perturb coastal marine ecosystem. Estuaries 10: 284-290
    Caron D A, Dennett M R, Moran D M, et al. 2003. Development and application of a monoclonal-antibody technique for counting Aureococcus anophagefferens, an alga causing recurrent brown tides in the Mid-Atlantic United States. Appl Environ Microbiol, 69: 5492-5502
    Coyne K J, Hutchins D A, Hare C E, et al. 2001. Assessing temporal and spatial variability in Pfiesteria piscicida distributions using molecular probing techniques. Aquat Microb Ecol, 24: 275-285
    Coyne K, Handy S M, Demir E. 2005. Improved quantitative real-time PCR assays for enumeration of harmful algal species in field sam-ples using an exogenous DNA reference standard. Limnol Ocea-nogr Methods, 3: 381-391
    Gastrich M D, Wazniak C E. 2002. A brown tide bloom index based on the potential harmful effects of the brown tide alga, Aureococcus anophagefferens. Aquat. Ecosyst. Health Manage, 5: 1-7
    Gibson U E, Heid C A, Williams P M. 1996. A novel method for real time quantitative RT-PCR. Genome Res, 6: 995-1001
    Heid C A, Stevens J, Livak K J, et al. 1996. Real time quantitative PCR. Genome Res, 6: 986-994
    KONG Fanzhou, YU Rencheng, ZHANG Qingchun, et al. 2012. Pigme-nt characterization for the 2011 bloom in Qinhuangdao implicat-ed "brown tide" events in China. Chinese Journal of Oceanology and Limnology, 30 (3): 361-370
    Medlin L, Elwood H J, Stickel S, et al. 1988. The characterization of enzymatically amplified eukaryotic 16S like rRNA-coding regions. Gene, 71: 491-499
    Naidoo A D. 1999. Brown tides in Saldanha Bay. Research Highlights 1998-1999, South African Department of Environmental Affairs and Tourism
    Popels L C, Cary S C, Hutchins D A, et al. 2003. The use of quantitative polymerase chain reaction for the detection and enumeration of the harmful alga Aureococcus anophagefferens in environmental samples along the United States East Coast. Limnol Oceanogr: Methods, 1: 92-102
    Popels L C, Hutchins D A. 2002. Factors affecting dark survival of the brown tide alga Aureococcus anophagefferens (Pelagophyceae). J Phycol, 38: 738-744
    Smayda T J, Villareal T A. 1989. The 1985 "brown-tide" and the open phytoplankton niche in Narragansett Bay during summer. In: Cosper E M, Bricelj V M, Carpenter E J, eds. Novel phytoplankton blooms: causes and impacts of recurrent brown tides and other unusual blooms, lecture notes on coastal and estuarine studies. Springer-Verlag, 159-187
    Suzuki M T, Taylor L T, DeLong E F. 2000. Quantitative analysis of small-subunit rRNA genes in mixed microbial populations via 5′-nuclease assays. Appl Environ Microbiol, 66: 4605-4614
    Tracey G A. 1988. Feeding reduction, reproductive failure, and morta-lity in Mytilus edulis during the 1985 brown tide in Narragansett-Bay, Rhode-Island. Mar Ecol Prog Ser, 50: 73-81
    Trice T M, Glibert P M, Lea C, et al. 2004. HPLC pigment records provide evidence of past blooms of Aureococcus anophagefferens in the Coastal Bays of Maryland and Virginia, USA. Harmful Algae, 3(4): 295-304
    Tyrrell J V, Bergquist P R, Saul D J, et al. 1997. Oligonucleotide probe technology as applied to the study of harmful algal blooms. N Z J Mar Freshw Res, 31: 551-560
    Zhang Qingchun, Qiu Limei, Yu Rencheng, et al. 2012. Emergence of brown tides caused by Aureococcus anophagefferens Hargraves et Sieburth in China. Harmful Algae, 19: 117-124
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