Phycological Research 2017
Quantitative real-time PCR detection of a harmful unarmoured dinoﬂagellate, Karlodinium australe (Dinophyceae) Nyuk Fong Kon,1 Winnie L. S. Lau,2 Kieng Soon Hii,1,2 Ing Kuo Law,2 Sing Tung Teng,3 Hong Chang Lim,4 Kazuya Takahashi,5 Haifeng Gu,6 Po Teen Lim2* and Chui Pin Leaw 2* 1
Institute of Biodiversity and Environmental Conservation, Universiti Malaysia Sarawak, Kota Samarahan, Malaysia, Institute of Ocean and Earth Sciences, University of Malaya, Bachok, Malaysia, 3Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, Kota Samarahan, Malaysia, 4Faculty of Applied Sciences and Computing, Tunku Abdul Rahman University College, Segamat, Malaysia, 5Asian Natural Environmental Science Center, The University of Tokyo, Tokyo, Japan and 6Third Oceanography Institute, Xiamen, China 2
SUMMARY We investigated a harmful algal bloom (HAB) associated with the massive ﬁsh kills in Johor Strait, Malaysia, which recurred a year after the ﬁrst incident in 2014. This incident has urged for the need to have a rapid and precise method in HAB monitoring. In this study, we develop a SYBR green-based realtime PCR (qPCR) to detect the culpable dinoﬂagellate species, Karlodinium australe. Species-speciﬁc qPCR primers were designed in the gene region of the second internal transcribed spacer of the ribosomal RNA gene (rDNA). The species speciﬁcity of the primers designed was evaluated by screening on the non-target species (Karlodinium veneﬁcum, Takayama spp., and Karenia spp.) and no cross-detection was observed. The extractable gene copies per cell of K. australe determined in this study were 19 998 505 (P < 0.0001). Estimation of cell densities by qPCR in the experimental spiked samples showed high correlation with data determined microscopically (R2 = 0.93). Using the qPCR assay developed in this study, we successfully detected the 2015 bloom species as K. australe. Single-cell PCR and rDNA sequencing from the ﬁeld samples further conﬁrmed the ﬁnding. With the sensitivity as low as ﬁve cells, the qPCR assay developed in this study could effectively and rapidly detect cells of K. australe in the environmental samples for monitoring purpose. Key words: ﬁsh kill, Malaysia, ribosomal RNA gene, second internal transcribed spacer. ........................................................................................
INTRODUCTION Rapidly expanding aquaculture activities and industrialization in coastal regions may have contributed to an increase in nutrient loadings, and resulted in localized eutrophication. Together with favorable physical conditions, this will promote rapid growth of phytoplankton, and algal bloom is likely to occur (Hall et al. 2008). Some of these algal blooms are known to cause harm by producing biotoxins that can intoxicate humans or kill marine organisms. Thus, rapid harmful algal detection tools are needed for regular monitoring and early prediction of these harmful algal blooms (HABs).
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Johor Strait, located between the southern Peninsular Malaysia and Singapore, is an important site of mariculture industries for both countries. Fish farms and the number of cages in the Strait have been reported to have increased compared to a decade ago, with the production of milkﬁsh (Chanos chanos) at US $ 6755/metric tons for Singapore, the second highest ﬁsh mariculture production among Southeast Asian countries in 2014 (SEAFDEC 2017). This is accompanied by the increasingly reported cases of HABs in the strait. The ﬁrst HAB event was recorded in 2002, where a massive bloom of Prorocentrum minimum (Pavillard) Schiller occurred in the strait (Usup et al. 2003). In a ﬁeld survey along the strait, Tan et al. (2013) encountered a low cell abundance of Karlodinium species, identiﬁed as K. veneﬁcum (Ballantine) Larsen at that time based on the morphological observations. The presence of Karlodinium species in the strait has gained serious attention as some species from the genus are known to kill ﬁsh (e.g. Deeds et al. 2002; Kempton et al. 2002). The genus Karlodinium was raised from polyphyletic Gymnodinium sensu lato after several taxonomic amendments based on the morphological ultrastructure features, the composition of photosynthetic pigments and the nuclear-encoded large subunit ribosomal RNA gene (LSU rDNA) sequences (Daugbjerg et al. 2000; Bergholtz et al. 2005). Before 2008, the genus comprised of four species: K. armiger Bergholtz, Daugbjerg & Moestrup, K. australe de Salas, Bolch & Hallegraeff, K. veneﬁcum and K. vitiligo (Ballantine) Larsen (Bergholtz et al. 2005; de Salas et al. 2005). Meanwhile, K. micrum has been synonymized as K. veneﬁcum (Bergholtz et al. 2005). The number of described Karlodinium species has grown to 11, with the addition of species: K. antarcticum de Sales, K. ballantinum de Salas, K. conicum de Salas, K. corrugatum de Salas, K. decipiens de Salas & Laza-Martinez (de Salas et al. 2008), K. corsicum (Paulmier) Siano & Zingone, and K. gentienii Nézan, Chomérat & Siano (Siano et al. 2009). Species of Karlodinium have a broad global distribution and have been reported from both the Paciﬁc
......................................................................................... *To whom correspondence should be addressed. Email: [email protected]
; [email protected]
Communicating Editor: Mitsunori Iwataki Received 15 August 2016; accepted 12 April 2017.
N. F. Kon et al.
and Atlantic oceans, spanning from the cold temperate waters to the warm tropical waters (summarized in Appendix S1 in the Supporting Information, with references herein). Among the Karlodinium species, K. veneﬁcum is known to produce ichthyotoxins, karlotoxins (KmTx), a family of amphidinol-like compound (reviewed in Wagoner et al. 2010). The species K. australe has never been related to any ﬁsh kill event until February 2014, where a bloom of K. australe occurred in the West Johor Strait, Malaysia and caused massive ﬁsh kills (Lim et al. 2014); this was the ﬁrst report where the species killed ﬁsh. In the subsequent year, the aquaculture operators in the West Johor Strait had been struck again with the losses of hundreds of tons of caged ﬁshes (Teng et al. 2016). It had been suspected that the bloom was attributed to the same species K. australe. A ﬁeld investigation has been undertaken and the bloom samples were used for the evaluation of the quantitative real-time PCR (qPCR) assay developed in the present study. In this study, a species-speciﬁc primer pair targeting K. australe was designed to target the second internal transcribed spacer (ITS2) of rDNA for the qPCR detection. Assay speciﬁcity and ampliﬁcation efﬁciency (AE) were evaluated, and further validated using the spiked samples for quantiﬁcation of cell number. The qPCR assay was successfully applied to the 2015 bloom samples and positively detected the species. The bloom species was also conﬁrmed by single-cell PCR and rDNA sequencing as K. australe.
MATERIALS AND METHODS Algal cultures Clonal cultures of Karlodinium australe were established from the western (site A; 1.38 N, 103.64 E) and central parts of Johor Strait (site B; 1.46 N, 103.75 E) during a massive bloom of K. australe in 2015 (Appendix S2 in the Supporting Information). The species identity was conﬁrmed by microscopic
observations and DNA sequence veriﬁcation of the D1–D3 LSU and ITS rDNA (Lim et al. 2014). The cultures were grown in ESDK medium (Kokinos & Anderson 1995) with a pH of 7.8–8.0, and a salinity of 30. Cultures were maintained at 25 0.5 C under a 12 : 12 h LD photoperiod in a MLR-352 versatile environmental test chamber (Panasonic, Gunma, Japan), illuminated by cool-white ﬂuorescent bulbs with light intensity of ~100 μmol photons m−2 s−1. Six strains of K. australe (KaJB05, KaJB08, KaJB10, KaJB11, KaJB12 and KaJB15) and other dinoﬂagellate strains were used to evaluate the speciﬁcity of the assay (Table 1).
Design of species-speciﬁc primers The development of qPCR assay in this study was conformed to the MIQE guidelines (Minimum Information for Publication of qPCR Experiments; Bustin et al. 2009). Nucleotide sequences of the ITS rDNA of 51 related taxa including Karlodinium veneﬁcum, K. armiger, Karenia sp., Takayama spp. and Gymnodinium sp. were retrieved from GenBank nucleotide database (Appendix S3 in the Supporting Information). The sequences were aligned with ITS rDNA sequences of K. australe (GenBank accession: KJ670418, KJ670419, KJ670420, KJ670421) using Clustal X (Thompson et al. 1997), and edited by BioEdit Sequence Alignment Editor v184.108.40.206 (Hall 1999). Multiple sequence alignment was performed to search for variable sites for potential speciesspeciﬁc primer regions. A phylogenetic analysis was performed for ITS2 rDNA of Karlodinium species inferred by sequence-structure information. A total of 44 ITS2 secondary structures of Karlodinium species (Appendix S3 in the Supporting Information) were modeled following the procedures as detailed in Lim et al. (2014). Three outgroup taxa of Takayama species were chosen. Multiple sequence-structure alignment was generated in 4SALE v1.7 using a 12 × 12 speciﬁc scoring matrix (Seibel et al. 2006, 2008; Wolf et al. 2014). The aligned dataset was saved in the format of one-letter encoded for maximum
Table 1. Dinoﬂagellate strains used in this study Species
Date of collection
Alexandrium afﬁne A. andersonii A. tamutum A. minutum A. tamiyavanichii A. cf. tamarense Coolia malayensis Karlodinium australe K. australe K. australe K. australe K. australe K. australe K. veneﬁcum K. veneﬁcum K. veneﬁcum Karenia brevis Karenia mikimotoi Protoceratium sp. Takayama xiamenensis
AaPa AnPa01 AuKa01 AmKB02 AcSM01 AtPA01 CmKd01 KaJB05 KaJB08 KaJB10 KaJB11 KaJB12 KaJB15 CCMA144 LAMB090611 LAMB090601 CCMA165 CCMA83 GgSM01 CCMA186
PulauAman, Penang, Malaysia PulauAman, Penang, Malaysia Pulau Abai, Sabah, Malaysia Tumpat, Kelantan, Malaysia SamariangBatu, Sarawak, Malaysia PulauAman, Penang, Malaysia Kota Kinabalu, Sabah, Malaysia Johor Strait, Malaysia Johor Strait, Malaysia Johor Strait, Malaysia Johor Strait, Malaysia Johor Strait, Malaysia Johor Strait, Malaysia East China Sea, China East China Sea, China North Sea, Germany USA East China Sea, China Samariang Batu, Sarawak, Malaysia East China Sea, China
May 2002 — — September 2001 March 2011 May 2002 September 2010 March 2015 March 2015 March 2015 March 2015 March 2015 March 2015 September 2015 June 2009 June 2009 — July 2012 August 2010 July 2003
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qPCR detection of Karlodinium australe parsimony (MP) and maximum likelihood (ML) analyses, while another aligned dataset was saved in sequence-only for Bayesian inference (BI). The MP was performed in PAUP v4.0b.10 (Swofford 2002) and bootstrap support was estimated based on 1000 bootstrap replicates. ML tree was constructed using phangorn (Schliep 2011) implemented in R (R Core Team 2011), with 500 replicates for bootstrap support. BI analysis was performed using MrBayes v3.2.5 (Huelsenbeck & Ronquist 2001), and posterior probabilities were estimated with 25% burn-in from four simultaneous MCMC chains of 5 000 000 generations. The primer sites were selected within the ITS2 region, with the amplicon length limited to <150 bp. The potential primer sequences were then analyzed by using IDT OligoAnalyzer 3.1 (http://sg.idtdna.com/calc/analyzer). The primers for K. australe were designed based on the same criteria as in Kon et al. (2015). The primer speciﬁcity was analyzed in silico by using Primer-BLAST (Ye et al. 2012) to determine potential crossreactivity with non-target Karlodinium species. The speciﬁc K. australe primers that designed in this study were Karloits2F (50 -ACATATGCCAAGTGTTTTCGCGCTTG-30 , GC content of 46.2%, Tm = 60.5 C) and Karloits2R (50 -GCAACGAAAGAGACACACAAGGTGC-30 , GC content of 52.0%, Tm = 60.6 C) with an amplicon size of 143 bp. The primers were then synthesized for further qPCR assay (First Base, Selangor, Malaysia).
3 included in each qPCR run. The qPCR cycling conditions are: a hold stage at 95 C for 20 s, followed by 40 cycles of 95 C for 3 s, and 60 C for 30 s. A melting curve analysis (MCA) was performed following the ampliﬁcation cycles. gBlocks gene fragment of the ITS2 rDNA of K. australe ﬂanked by both forward and reversed primers, with an extra ten base pair (bp) long at both ends, was synthesized (Integrated DNA Technologies, Coralville, IA, USA). The synthetic gene fragments were used as a standard DNA for calibration curve construction. The standard DNA was included in every assay of the environmental samples, for assessing AE. The lyophilized, synthetic gBlocks gene fragment was ﬁrst re-suspended in 200 μL TE buffer (Tris 1 M, EDTA 0.5 M, pH 8) to obtain a stock concentration of 1 ng μL−1. The gene fragment copy numbers in 1 ng μL−1 was then calculated as described in Kon et al. (2015). The stock solution was diluted to 1 × 108 gene copies, followed by 10-fold serial dilutions in triplicate, ranging from 1 × 102–107 copies. The serially diluted synthetic gene fragments were used as template in qPCR reactions in triplicate for the calibration curve construction. Calibration curve was constructed by plotting triplicate Cq values against the log-transformed gene copy numbers using GraphPad Prism v. 5.03 (GraphPad Inc., La Jolla, CA, USA). Linear regression analysis was performed on the calibration curve to determine R2 value and the slope. The AE was calculated as AE = [10(−1/slope) − 1] × 100%.
Evaluation of primers speciﬁcity Primer cross-reactivity test was performed by qPCR using the genomic DNAs (gDNAs) from cultures of K. australe and other closely related species (K. veneﬁcum, Karenia spp. and Takayama xiamenensis Gu) (Table 1). Positive gDNAs were conﬁrmed by amplifying the LSU rDNA using the primer pair D1R (Lenaers et al. 1989) and D3Ca (Scholin & Anderson 1996). PCR was performed on a peqSTAR 96X Universal Gradient thermocycler (Peqlab, Erlangen, Germany) as described in Kon et al. (2015). The PCR products were visualized on a 2% agarose gel stained with SYBR® Safe DNA gel stain (Invitrogen, Life Technologies, Carlsbab, CA, USA), and captured using an Inﬁnity ST5 Gel Imaging System (Vilber Lourmat, Marne-la-Vallée, France). Primers’ speciﬁcity was also tested against gDNAs of other non-target phytoplankton species by conventional PCR (Alexandrium tamiyavanichii Balech, A. afﬁne (Inoue & Fukuyo) Balech, A. minutum Halim, A. tamutum Montresor, Beran & John, A. andersonii Balech, A. cf. tamarense (Lebour) Balech, Protoceratium sp. and Coolia malayensis Leaw, P.T. Lim & Usup; Table 1). Conventional PCR and cross-reactivity test by qPCR were performed as described above using Karloits2F and Karloits2R.
qPCR assay and ampliﬁcation efﬁciency The qPCR assay was performed on an Applied Biosystems 7500 Fast Real-time PCR System (Applied Biosystems, Life Technologies, Austin, TX, USA). The 20 μL-reaction contained 10 μL of SYBR Select Master Mix (Applied Biosystems) and 2 μL of DNA template. The primer concentrations were optimized by qPCR (the range of 250–400 nM were tested) and incorporated an optimum primer concentration of 300 nM in each qPCR reaction. No template control (NTC) and positive control using the synthetic gene fragments were
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Determination of extractable gene copies per cell The extractable ITS2 gene copy numbers per cell of K. australe was determined from samples containing spiked cultured cells of K. australe ranging from 20–300 cells mL−1. One milliliter of cultured cells was spiked into the environmental sample. The environment sample was collected by a 20-μm plankton net from Melawi, Malaysia (6.00 N, 102.42 E). We conﬁrmed no Karlodinium cells in the sample microscopically, and rDNA ampliﬁcation and sequencing using the ITS primer pair, ITS1F and ITS1R (Leaw et al. 2001). Spiked samples were then concentrated by gentle sieving through a 5 μm-mesh sieve, and then transferred to a microfuge tube. The cell density of Karlodinium in each tube was counted in triplicate using a Sedgewick-Rafter counting slide under a microscope. The gDNAs of the samples were extracted by using a commercial DNA extraction kit, DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and eluted in 30 μL elution buffer. The samples were then qPCR analyzed in triplicate as described above. The gene copies of each sample determined from qPCR based on the calibration curve were then plotted against the cell densities determined microscopically. The regression slope was deﬁned as extractable gene copies per cell.
Assay sensitivity and accuracy Culture strains KaJB11 and KaJB15 at exponential growth phase were used in these experiments. The assay sensitivity in terms of cell numbers was tested with minimum number of cells. Samples containing one, ﬁve, and ten cells per reaction were prepared by singly isolation using micropipetting
4 technique. The gDNAs were extracted using the same DNA extraction kit as mentioned above, and qPCR analyzed using the same method as described above. Random-spiked cell samples were used to test the assay accuracy in quantifying the target cells by comparing the actual cell densities that were based on microscopic cell counts and that of those based on qPCR cell estimates. Spiked cell samples were prepared by sieving as described above. The cell densities in each sample range from 5 to 600 cells mL−1 (5, 14, 28, 56, 74, 139, 278 and 556 cells mL−1) were conﬁrmed by microscopic cell counts in one or two replicates. The gDNA extraction was performed as mentioned above and then qPCR was performed in triplicate. The potential cell densities estimated by qPCR assay were then compared with the actual cell densities.
Sample collection during the 2015 bloom event In March 2015, a recurrent algal bloom in the Johor Strait has caused massive ﬁsh kill and severe aquaculture losses to the operators. A ﬁeld investigation was conducted to identify the species responsible for the event. Two-liter surface water samples were collected from the bloom site at the central part of the Johor Strait (site B; Appendix S2 in the Supporting Information). The samples were then preserved with 2% formalin and kept at −20 C. Aliquots of 20, 100, and 200 μL preserved samples (in triplicate) were sieved through a 5 μm-mesh sieve and rinsed with TE buffer (Tris 1 M, EDTA 0.5 M, pH 8) to wash off the formalin preservative prior to DNA extraction. The environmental DNA (eDNA) was extracted using the same DNA extraction kit as above. The eDNA was then analyzed by SYBR Green I-based qPCR assay. MCA was performed following the SYBR Green-based qPCR ampliﬁcation. Positive controls using the gBlock gene fragments were included in each run to assess if Cq delayed.
RESULTS AND DISCUSSION Primer speciﬁcity The ITS rDNA has been widely used in phylogenetic studies owing to its sequence heterogeneity at lower taxonomic levels. The ITS2 phylogenetic tree inferred from the sequencestructure information based on MP, ML, and BI yielded identical tree topologies; only BI tree is shown with MP/ML bootstrap supports and BI clade credibility values presented (Appendix S4 in the Supporting Information). The trees revealed a highly supported sister clade comprised of K. autrale and K. armiger (MP/ML/BI, 92/100/1.00), which is congruent to that of the LSU tree in Lim et al. (2014). Karlodinium veneﬁcum formed a highly supported clade, consisted of two subclades: subclade I comprised of K. veneﬁcum strains from Balearic Sea of the North eastern Atlantic Ocean, while subclade II were found widely distributed in the north western and north eastern Atlantic and Western Paciﬁc (Appendix S4 in the Supporting Information). Lim et al. (2014) has demonstrated the presence of four hemi-
N. F. Kon et al. compensatory base changes (HCBCs) in the ITS2 transcript when compared to that of K. armiger; 3–7 HCBCs when compared to K. veneﬁcum. In this study, a 143-bp length sequence signature of K. australe in the ITS2 rDNA were selected; the region was veriﬁed in silico to be uniquely distinctive to K. australe when compared to the ITS2 transcripts of other Karlodinium species. Furthermore, the K. australe species-speciﬁc primer sites (Karloits2F and Karloits2R) are readily variable from its closely related species, Karlodinium spp., Karenia spp. and Takayama sp. Testing of primers speciﬁcity in silico showed that the primers only hit on sequences of K. australe in the blastn search, but no match to K. amiger and K. veneﬁcum in the GenBank nucleotide database (NCBI). The primers demonstrated 100% identity to sequences of K. australe in a 100% query coverage when blasted in NCBI GenBank nucleotide database; the blastn search hit all ITS sequences of K. australe (accessions: KJ670418, KJ670419, KJ670420 and KJ670421). The in situ speciﬁcity of the primer pair was evaluated using gDNAs of the closely related species, K. veneﬁcum, Karenia spp. and Takayama xiamenensis (Table 1). The gDNAs were ﬁrst conﬁrmed by showing positive LSU rDNA ampliﬁcation in the conventional PCR (Fig. 1a). The assay speciﬁcity evaluated by conventional PCR using various gDNAs from other non-target species showed that only gDNA from K. australe (KaJB15) produced positive ampliﬁcation, but other dinoﬂagellates (Alexandrium tamiyavanichii, A. afﬁne, A. minutum, A. tamutum, A. andersonii, A. tamarense, Protoceratium cf. reticulatum and Coolia malayensis) did not (Appendix S5 in the Supporting Information). The qPCR speciﬁcity test performed using the K. australe-speciﬁc primer pair also showed no positive ampliﬁcation other than K. australe (KaJB05) (Fig. 1b,c), conﬁrming no cross reactivity to the non-target species.
qPCR assay speciﬁcity The gene fragment calibration curve constructed with the optimized primer concentration of 300 nM showed AE of 94.6%. Figure 2 shows the calibration curve with a linear dynamic range over a six-order of magnitude (102–107 copies, R2 = 1.00, slope = −3.46). The standard DNAs (positive controls) using gBlock gene fragments ampliﬁed together with the unknown samples showed AEs >90%. The NTC included in each qPCR run showed no ampliﬁcation throughout the study. The qPCR assay in this study had a limit of detection of as low as 102 gene copies, as shown in the linear detection range (Fig. 2). The assay tested using samples containing only one target cell showed positive ampliﬁcation which produced positive melt peak in the MCA; however, the Cq obtained for single cells was inconsistent, probably due to variable gene copy number in one cell. It has been reported that different species has different number of gene copies, and even vary slightly at different growth phases between cells of the same species (e.g. Galluzzi et al. 2010). Alternative possible explanation in this deviation is the efﬁciency of commercial DNA extraction kit in recovering the DNA. Nishimura et al. (2016) has demonstrated that the DNA recovery efﬁciency (RE) of DNeasy Plant Mini Kit (Qiagen) for Gambierdiscus spp. could range from 6 to 88%. Furthermore, Kon et al. (2015) has shown that RE of the same kit on Alexandrium tamiyavanichii
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qPCR detection of Karlodinium australe
Derivative Reporter (-Rn’)
1.0 2 0.5 0 0
20 24 Cycle
75 80 85 Temperature (oC)
Fig. 1. qPCR cross-reactivity test of the Karlodinium australe species-speciﬁc primers performed using gDNAs of closely related species. (a) Gel image showing positive LSU rDNA ampliﬁcation in the conventional PCR using gDNAs of: 1, K. veneﬁcum LAMB090611; 2, K. veneﬁcum LAMB090601; 3, K. veneﬁcum CCMA144; 4, Karenia mikimotoi CCMA83; 5, K. brevis CCMA165; 6, Takayama xiamenensis CCMA186; 7, K. australe KaJB05; NTC, no template control; and L, 1kb DNA ladder. (b) The corresponding ampliﬁcation curves. (c) Melting curves showing only templates from K. australe produced positive ampliﬁcation.
was as low as 8.0 2.9% (mean SD) with the range values between 5.6 and 11.0%. Samples containing ﬁve and 10 target cells, however, were consistently ampliﬁed and yielded constant Cq values for a reliable quantiﬁcation. As such, the minimum number of cells for a reliable quantiﬁcation was determined to be ﬁve cells per reaction. The value of copy numbers per cell has been used to estimate the cell abundance in an unknown sample. The extractable gene copies per cell of K. australe determined in this study was 19 998 505 (slope SD) (R2 = 0.98, P < 0.0001; Fig. 3). Estimation of cell densities in random-spiked cell samples using this copy numbers per cell showed a positive correlation with cell densities determined microscopically (R2 = 0.93, P < 0.0001; Fig. 4). Comparison of qPCR cell estimates with the actual cell densities that were based on microscopic cell count revealed reliable quantiﬁcation of the qPCR assay developed in this study.
Species detection during the bloom The plankton samples collected during the recurrent bloom in 2015 that caused massive ﬁsh kills in the Strait showed extremely high density of Karlodinium-like monospeciﬁc dinoﬂagellate, with maximum cell densities up to 2 × 108 cells L−1 based on microscopic cell counts. Several studies have demonstrated that the main ﬁsh-killing Karlodinium species,
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K. veneﬁcum, has been associated with numerous ﬁsh kill events globally (reviewed in Place et al. 2012) after its ﬁrst bloom in Walvis Bay, Namibia causing massive ﬁsh kills (Bergholtz et al. 2005). The killing agent, KmTx, has been discovered (Kempton et al. 2002). Recently, K. australe has been attributed to massive ﬁsh mortality in Johor Strait, Malaysia (Lim et al. 2014); however, whether the killing agent was due to the same mechanism of KmTx has not yet been identiﬁed. Morphological discrimination between species of Karlodinium as well its closely related taxa, i.e. Karenia and Takayama, is challenging as the morphological features are ambiguous, particularly in ﬁxed samples (Bergholtz et al. 2005). Fixation of unarmored dinoﬂagellates can be sometimes problematic and make traditional microscopic identiﬁcation difﬁcult. Improper ﬁxation for unarmored dinoﬂagellates that lack the protective theca may cause cell shape distortion and shrinkage (Sherr & Sherr 1993; McCoy et al. 2014). The morphological characteristics such as cell size, position of nucleus, and the arrangement of chloroplasts that used to identify species of Karlodinium are also subtle and often misleading. Hence, molecular approach becomes an alternative diagnostic tool in detecting and identifying the species. In this study, the ﬁeld samples collected from the 2015 bloom were screened with the qPCR assay developed using the K. australe species-speciﬁc primer pair, and the results
N. F. Kon et al.
Calibration curve of the cycle threshold (Cq) constructed
using the K. australe ITS2 gBlock gene fragment. Error bars denote the standard deviation from triplicate ampliﬁcations; dashed lines represent 95% conﬁdence limit. AE, ampliﬁcation efﬁciency.
showed positive ampliﬁcation, and the MCA revealed identical melt peaks with the positive controls (Fig. 5), thus conﬁrmed the speciﬁc ampliﬁcation and species identity. Moreover, single-cell PCR and ITS rDNA sequencing from the ﬁeld samples yielded identical sequences of K. australe in the NCBI
Fig. 4. Assay accuracy testing on random-spiked samples by comparing the actual cell densities and the qPCR cell estimates. The graph shows a good ﬁt between the cell densities determined by manual cell counts and by qPCR. Dashed lines represent the 95% conﬁdence limits.
blastn search. This study successfully detected and identiﬁed the bloom species in 2015 by our K. australe qPCR assay. However, the assay was not able to quantify the cell abundance during the bloom due to improper ﬁeld sample preservation. The only available bloom samples collected during the time were preserved in 1% formalin preservative. qPCR quantiﬁcation using the eDNA extracted from the formalin-ﬁxed ﬁeld samples had led to underestimation of cell numbers as compared to microscopic cell count. This could be accounted for the loss of cells via sieving. The formalin preserved samples used in this study have been kept for about 2 months prior to analysis; this might cause degradation of DNA in the formalin-preserved condition, or cells might burst under
Fig. 3. Veriﬁcation of the extractable gene copy number per cell determined as the slope of linear regression of cell densities
Fig. 5. Melting curve analysis showing same melt peaks between
enumerated microscopically vs. gene copies estimated from gene
the bloom-samples (solid lines) and the gene fragment-positive
fragment calibration curve.
controls (dash lines). NTC showed no ampliﬁcation (grey line).
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qPCR detection of Karlodinium australe freezing–thawing condition. Eckford-Soper and Daugbjerg (2015) have demonstrated that qPCR ampliﬁcation showed approximately seven cycles delay between the Lugol-ﬁxed cells that were kept at −20 C for 1 and 2 months. Besides that, Godhe et al. (2002) showed that even formalin-treated samples could preserve cells morphology and the gDNA, and claimed that it worked well with PCR ampliﬁcation, but they suggested that this preservation method might not be suitable for unarmoured dinoﬂagellates. Preserving Karlodinium in formalin preservative kept the cells observable, but the preserved cells of K. australe appeared smaller and transparent compared to the living cells. Saline ethanol has been demonstrated as the best preservative suggested for algae (Miller & Scholin 2000); however, Karlodinium cells lysed after the preservation (Kon, N.F., unpublised observation). Other alternative ﬁxation methods of unarmored dinoﬂagellates have been tested and validated for qPCR-based cell quantiﬁcation, for instance, Lugol’s iodine was suggested as the most ideal short-term ﬁxative (Eckford-Soper & Daugbjerg 2015), by which could be adopted in future studies. Cell densities of K. australe obtained from the bloom samples reported in this study was performed manually by microscopic cell count instead of qPCR quantiﬁcation, until a more suitable preservation method for unarmored dinoﬂagellates is developed. On-ﬁeld eDNA extraction and direct cold storage of the eDNA samples would be an alternative procedure to substitute sample preservation step in this purpose. It was also observed that the cells were easily broken if centrifuged under high speed. Proper sample processing steps were undertaken to avoid loss of cells. In this study, gentle sieving was used to concentrate the Karlodinium bloom samples rather than centrifugation, but a smaller mesh size (e.g. <5 μm-mesh) is recommended.
Conclusion Karlodinium australe was conﬁrmed to be responsible for the ﬁsh kill event in 2014 and 2015 in Johor Strait. There is an urgent need for routine monitoring and early warning systems to be in place to minimize the losses in aquaculture in the future. In conclusion, the K. australe species-speciﬁc primer pair designed in this study showed the speciﬁcity and applicability to detect K. australe in the environmental samples. The qPCR assay developed could be applied to the ﬁeld for routine monitoring of HAB especially in Johor Strait for recurrent blooms. The qPCR assay has shown its sensitive detection of as low as ﬁve cells, which is beneﬁcial for the detection of species at low cell numbers, especially at the initiation stage of bloom.
ACKNOWLEDGMENTS This work was funded by the Malaysian government through the Ministry of Education under HiCoE Fund [IOES-2014C]; Ministry of Science, Technology and Innovation under Sciencefund [SF018-2014]; and University of Malaya UMRP [RU009-2015]; and China-Indonesia Maritime Cooperation Fund to H Gu. N. F. Kon was supported by the Ministry of Education MyPhD Scholarship, and this work formed part of her PhD project.
© 2017 Japanese Society of Phycology
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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Appendix S1. Global distribution of Karlodinium species. Appendix S2. Map showing the Johor Strait bordering Malaysia and Singapore. Appendix S3. ITS rDNA sequence information of Karlodinium. Appendix S4. ITS2 Bayesian tree of Karlodinium species. Appendix S5. Cross-reactivity test of the Karlodinium australe.
© 2017 Japanese Society of Phycology