Development and field assessment of a quantitative PCR for the detection and enumeration of the noxious bloom-former Anabaena planktonica

Anabaena planktonica is a harmful, bloom-forming freshwater cyanobacterium, which has arrived recently in New Zealand. In the short time since its incursion (<10 yr), A. planktonica has spread rapidly throughout lakes in the North Island. To date, the identification and enumeration of A. planktonica has been undertaken using light microscopy. There is an urgent demand for a highly sensitive and specific quantitative detection method that can be combined with a high sample processing capability in order to increase sampling frequency. In this study, we sequenced 36 cyanobacterial 16S rRNA genes (partial), complete intergenic transcribed spacers (ITS), and 23S rRNA genes (partial) of fresh-water cyanobacteria found in New Zealand. The sequences were used to develop an A. planktonica specific TaqMan QPCR assay targeting the long ITS1-L and the 5 ́ terminus of the 23S rRNA gene. The QPCR method was linear (R2 = 0.999) over seven orders of magnitude with a lower end sensitivity of approximately five A. planktonica cells in the presence of exogenous DNA. The quantitative PCR (QPCR) method was used to assess the spatial distribution and seasonal population dynamics of A. planktonica from the Lower Karori Reservoir (Wellington, New Zealand) over a five-month period. The QPCR results were compared directly to microscopic cell counts and found to correlate significantly (95% confidence level) under both bloom and non-bloom conditions. The current QPCR assay will be an invaluable tool for routine monitoring programs and in research investigating environmental factors that regulate the population dynamics and the blooming of A. planktonica. Thermophile Research Unit, Department of Biological Sciences, University of Waikato, Private Bag 3105, Hamilton, 2001, New Zealand, Phone: + 64-7-838-4593, Fax: + 64-7-838-4324, e-mail: c.cary@waikato. ac.nz Acknowledgments The research described in this paper is part of the Lakes Ecosystem Restoration New Zealand (LERNZ) program of the University of Waikato funded by the Foundation for Research Science and Technology (UOWX0505). We thank the Karori Wildlife Sanctuary staff for sample collection. SAW thanks the New Zealand Foundation for Research, Science and Technology for post-doctoral fellowship funding (CAWX0501). Limnol. Oceanogr.: Methods 5, 2007, 474–483 © 2007, by the American Society of Limnology and Oceanography, Inc. LIMNOLOGY and OCEANOGRAPHY: METHODS compounds in the water supply of more than 960,000 households (Kouzminov et al. 2007). A. planktonica appears to have a number of adaptive traits that may account for its dominance and spread throughout the North Island. It is highly buoyant, can “overwinter” in a planktonic vegetative state and moderate populations are maintained even when a water column is well mixed with light availability and temperatures being low (<8°C) (Hicks et al. 2007). To date, little is known about the invasive nature and environmental requirements of A. planktonica. Thus, the ability to rapidly and accurately identify and enumerate this species at low (≤10 cells mL–1) and high (≥15,000 cells mL–1) cell concentrations is essential to aid in bloom prediction and management. Blooms arise because of a complex interaction of chemical, physical, and biological variables including lake stratification and circulation, lake shape and depth, nutrient input, light supply, lake catchment use, and zooplankton densities (Oliver and Ganf 2000). Recently, computer models have been developed, which assist in understanding the complex processes that control lake dynamics (e.g., Hamilton and Schladow 1997; Schladow and Hamilton 1997). Such models can be used in early warning of imminent bloom development and also can help to investigate the responses of cyanobacterial populations to different management strategies. However, these models require validation with chemical, physical, and biological data from the waterbody being investigated. Development of in situ technologies and reporting equipment, e.g., telemetry, has allowed collection of data on numerous chemical and physical variables at frequent intervals. However, the technology to analyze biological data (e.g., differentiation and enumeration of phytoplankton) has not advanced as rapidly and commonly relies on traditional microscopic methods, which are laborious and often cannot differentiate to species level. Detection of microbial species using molecular techniques allows specificity and sensitivity combined with speed and high sample processing capability (e.g., Coyne et al. 2001). For instance, the ability of quantitative PCR (QPCR) to determine cell abundance by measuring the input copy number of a specific DNA target sequence has resulted in the recognition and acceptance of this technique as a tool for environmental monitoring of a range of cyanobacteria and micro-algae taxa (Kurmayer and Kutzenberger 2003; Popels et al. 2003; Vaitomaa et al. 2003; Coyne et al 2005a; Rinta-Kanto et al. 2005). In this study, a QPCR assay targeting the hyper-variable regions of long intergenic transcribed spacer (ITS1-L) and the 5 ́-terminus of the 23S rRNA gene was developed in order to identify and enumerate A. planktonica rapidly and accurately in environmental water samples. The QPCR has been optimized and validated for data analysis using the comparative CT method for relative quantification (Livak and Schmittgen 2001). An internal reference standard QPCR was incorporated into the data analysis by adding a known concentration of exogenous plasmid DNA to each sample (Coyne et al. 2005b). Unlike absolute quantification, the relative quantification approach has been demonstrated to compensate for methodological bias and sample-to-sample variation in extraction and amplification efficiencies and thus provides more accurate and realistic quantification results (Lebuhn et al. 2004; Coyne et al. 2005b). The QPCR for A. planktonica was applied to environmental water samples from the Lower Karori Reservoir in Wellington, New Zealand over a five-month period to assess the spatial distribution and seasonal population dynamics during bloom and non-bloom conditions and to compare cell abundances by QPCR to microscopic cell counts. This method will improve the ability to predict and understand the factors contributing to the dominance of A. planktonica, and will be valuable in understanding the physiological requirements of this invasive organism. Materials and procedures Isolation, culturing, and cell harvesting—Single filaments were isolated by micro-pipetting from lake water samples and were transferred to 24-well plates containing 500 μL MLA medium (Bolch and Blackburn, 1996) per well. The samples were incubated under the following conditions; 100 μEin × m–2 × s–1; 12: 12 hour light : dark at 18 ± 1°C (Contherm, BioSyn 6000CP). Successfully isolated strains were maintained in 50 mL plastic bottles (Biolab) under the same conditions as described. Cultures were harvested by centrifugation for 10 min at 16.1g with a benchtop centrifuge (Eppendorf 5415R) and the pellet stored at –20°C until processed. Collection of environmental samples—Twenty-eight phytoplankton samples (500 mL) were collected from the water column of the Lower Karori Reservoir (41°17’S, 174°45′E) between 22 May 2006 and 13 September 2006. Samples were taken from the reservoir surface and three different depths (5, 10, and 15 m) using a Van Dorn sampler. A 40 mL aliquot of each sample was filtered onto GF/C glass microfiber filter (47 mm ∅, Whatman, England) using low-pressure vacuum filtration. Filters were folded once enclosing the filtered cells and stored separately in a sterile plastic bags at –20°C until processed. A 100 mL aliquot of each sample was preserved with Lugol’s iodine (10% [w/v] potassium iodide, 5% [w/v] iodine, 10% [v/v] acetic acid) for microscopic cell enumeration. Microscopic cell enumeration—Microscopic enumeration of environmental samples was carried out using an inverted microscope (CKX41, Olympus) and Utermöhl settling chambers (Utermöhl 1958). After mixing, a sub-sample of 10 mL was pipetted into each Utermöhl chambers and allowed to settle for at least 4 h. A. planktonica cells in one central transect were counted at ×400 magnification. If fewer than 150 cells were observed, then transect counts were undertaken at ×200 magnification or cells on the whole chamber floor were counted. Each sample was counted in triplicate. The counting error of this method is approximately ±20% (Hötzel and Croome 1999). DNA extraction—DNA from environmental samples was extracted using a modified protocol of Kurmayer et al. (2003). Cary et al. QPCR development for Anabaena planktonica