Molecular Detection of the Harmful Raphidophyte Chattonella subsalsa Biecheler by Whole-Cell Fluorescence in-situ Hybridisation Assay

Species of the genus Chattonella (Raphidophyceae) are a group of marine protists that are commonly found in coastal waters. Some are known as harmful microalgae that form noxious blooms and cause massive fish mortality in finfish aquaculture. In Malaysia, blooms of Chattonella have been recorded since the 1980s in the Johor Strait. In this study, two strains of Chattonella were established from the strait, and morphological examination revealed characteristics resembling Chattonella subsalsa. The molecular characterization further confirmed the species’ identity as C. subsalsa. To precisely detect the cells of C. subsalsa in the environment, a whole-cell fluorescence in-situ hybridisation (FISH) assay was developed. The species-specific oligonucleotide probes were designed in silico based on the nucleotide sequences of the large subunit (LSU) and internal transcribed spacer 2 (ITS2) of the ribosomal DNA (rDNA). The best candidate signature regions in the LSU-rRNA and ITS2-rDNA were selected based on hybridisation efficiency and probe parameters. The probes were synthesised as biotinylated probes and tested by tyramide signal amplification with FISH (FISH-TSA). The results showed the specificity of the probes toward the target cells. FISH-TSA has been proven to be a potential tool in the detection of harmful algae in the environment and could be applied to the harmful algal monitoring program.


INTRODUCTION
Harmful algal bloom (HAB), also known as "red tide", occurs when harmful microalgae grow in high biomass in the water column, causing severe consequences such as food poisoning syndromes in humans who consume algal toxinscontaminated seafood and massive mortality of marine organisms (Hoagland et al. 2002). Paralytic shellfish poisoning has been the focus of attention in Malaysia, as it has been linked to the majority of human intoxication cases Usup et al. 2012;Yñiguez et al. 2021). Several causative dinoflagellates, including Pryodinium bahamense Plate, Alexandrium tamiyavanichii Balech, A. minutum Halim, and Gymnodinium catenatum Graham, have been documented throughout the Malaysian waters (Leaw et al. 2005;Lim et al. 2007). Nonetheless, other algalrelated incidents have been documented in Malaysia, such as massive fish kills in aquaculture farms 2014;Teng et al. 2016;Yñiguez et al. 2021;Lum et al. 2021). The majority of these events have been linked to marine harmful dinoflagellates, such as Margalefidinium polykrikoides (Margalef) Gómez, Richlen, and Anderson, Noctiluca scintillans (Macartney), Kofoid & Swezy, and Karlodinium australe Salas, Bolch, and Hallegraeff (Lim et al. 2014;Teng et al. 2016).
Among the harmful microalgae, several groups of raphidophytes have been recognized as harmful to marine organisms (Lum et al. 2021). Members of the genus Chattonella Biecheler are among those that have caused severe damage to the aquaculture industries in many coastal countries, for instance Japan (Okaichi 2003;Imai & Yamaguchi 2012). The first record of Chattonella bloom has been reported on the Malabar Coast, India, while the most severe fish kill event was recorded in Harima-Nada, the Seto Inland Sea, Japan, in the summer of 1972 (Imai & Yamaguchi 2012). In Malaysia, the occurrence was first documented in 1983 along the Johor strait (Maclean 1989).
Conventionally, light microscopy has been used to identify the morphological characteristics of Chattonella species. The species are unicellular, bi-flagellated, and pigmented, ranging from golden brown to greenish in some species depending on the fucoxanthin content (Klöpper et al. 2013). In general, species of Chattonella are differentiated based on cell size, cell shape, presence of a hyaline posterior tail, and mucocysts (Hara & Chihara 1982;Hara et al. 1994;Bowers et al. 2006). However, diversity in the morphology of Chattonella is high, even within the same species. Often, molecular characterization using gene markers such as ribosomal RNA genes (rDNA) is required to aid species recognition (Bowers et al. 2006;Demura et al. 2009). Among the species of Chattonella, C. antiqua (Hada) Ono, C. marina (Subrahmanyan) Hara and Chihara, C. ovata Hara and Chihara (also referred to as C. marina complex sensu Demura et al. 2009), and C. subsalsa Biecheler have been reported to cause HABs that are associated with massive farmed-fish mortality and impact the economies of affected countries worldwide (Hiroishi et al. 2005;Edvardsen & Imai 2006;Imai et al. 2006;Imai & Yamaguchi 2012;Lum et al. 2021).
In the Johor Strait shared between Malaysia and Singapore, the occurrence of Chattonella has often been reported from the monitoring and research studies of both countries (Khoo & Wee 1997;Leong et al. 2015;Tan et al. 2016;Kok et al. 2019;Liow et al. 2019). Morphological plasticity in the species, however, has hampered precise species recognition, particularly in the preserved environmental samples, where the cells tend to deform and the morphology deteriorates after fixation. Morphological plasticity in the species, however, has hampered precise species recognition, particularly in the preserved environmental samples, where the cells tend to deform and the morphology deteriorates after fixation (Katano et al. 2009). This often leads to species misidentification. Alternative approaches, such as molecular techniques (Bower et al. 2006;Stacca et al. 2016), could therefore be explored to overcome the limitation. In this study, whole-cell tyramide signal amplification-fluorescence in situ hybridisation (FISH-TSA) was developed to detect the harmful raphidophyte Chattonella subsalsa. The ribosomal RNAtargeted species-specific probes were designed in silico and applied in the assay.

Algal Cultures and Morphological Observation
Live plankton samples were collected from the Johor Strait using a 20 µm-mesh plankton net and vertically hauled into subsurface seawater (< 5 m) during high tide. The micropipette technique was used to isolate the targeted cells. Cultures were established and grown in f/2 medium. Live plankton samples were collected from the Johor Strait using a 20 µm-mesh plankton net and vertically hauled into subsurface seawater (< 5 m) during high tide. The micropipette technique was used to isolate the targeted cells. Cultures were established and grown in f/2 medium (Guillard & Ryther 1962) with a salinity of 30, 25 ± 0.5°C, under a light intensity of 100 µmol photons m -2 s -1 , with a 12: 12 h light: dark photoperiod.
Morphological observation of cell shape and chloroplast was performed using an Olympus IX51 research microscope (Olympus, Tokyo, Japan). To observe the nuclear position, cells were first stained with the DAPI-nuclei stain and then examined under ultraviolet light with a UV filter set. Digital images were captured with an Olympus DP72 digital camera (Olympus, Tokyo, Japan).

Genomic DNA Extraction, rDNA Amplification and Sequencing
The genomic DNA of Chattonella cultures was extracted as described by Leaw et al. (2010). In brief, the mid-exponential cells from 200 mL of cultures were harvested by centrifugation (1100 ×g, one min). The cell pellets were rinsed with ddH 2 O and resuspended in 10× NET lysis buffer (5 M NaCl, 0.5 M EDTA, 1 M Tris-HCl, pH 8) and 1% sodium dodecyl sulphate. The mixture was incubated at 65°C and subsequently extracted with chloroform: isoamyl alcohol (24:1) and phenol: chloroform: isoamyl alcohol (25:24:1). The genomic DNA was then precipitated by adding absolute ethanol and 3 M sodium acetate (pH 5). The DNA pellet was then rinsed with cold 70% ethanol. Finally, the DNA pellet was dissolved in 30 µL of TE buffer (10 mM Tris-HCl, pH 7.4; and 1 mM EDTA, pH 8) and stored at -20°C until further analysis.

Phylogenetic Analyses
Taxon sampling was performed by retrieving the LSU and ITS-rDNA nucleotide sequences of Chattonella species from the NCBI GenBank nucleotide database ( Table 1). The sequences of Heterosigma akashiwo were used as an outgroup. The newly obtained C. subsalsa sequences in this study and the retrieved sequences were multiple aligned using the program MUSCLE (https://www.ebi.ac.uk/Tools/ msa/muscle/). The aligned datasets were phylogenetic inferred using Phylogenetic Analysis Using Parsimony* (PAUP*) v4.0 b10 (Swofford 2003) and MrBayes v3.1.2 (Huelsenbeck & Ronquist 2001), as described in Leaw et al. (2016).

In silico rRNA-Targeted Oligonucleotide Probe Design
The rDNA sequences of Chattonella species retrieved from GenBank and SILVA (http://www.arb-silva.de/) public databases were used to identify potential signature regions by using the PROBE_DESIGN tool of the ARB programme package (Ludwig et al. 2004). The parameters for probe design included probe length, percentage of GC content, melting temperature (T m ), and self-complementary (Kumar et al. 2005). The probe candidates were selected for both target and probe sequences and were displayed in a result list (Kumar et al. 2005; Tables 2 and 3). The selected probe candidates were then evaluated using the PROBE Match Tool (PMT) of the ARB. The oligonucleotide sequences were then subjected to extensive specificity tests through BLAST comparisons against nucleotide databases of non-target sequences. The candidate sequences that complemented the region of target sequences with at least one mismatch in other non-target sequences were chosen (Hugenholtz et al. 2002). BLAST was also used to confirm that the sequences were transcribed in the correct orientation (Hugenholtz et al. 2002). The selected probes satisfying the in silico experimental constraints were then synthesised as a biotinylated probe (IDT Inc., Singapore).

Tyramide Signal Amplification-fluorescence in situ Hybridisation
Cells were fixed with Lugol iodine solution (~1%) and transferred to a glass slide that was pre-fixed with 2% HistoGrip TM (Invitrogen, Life Technologies, USA) (Breininger & Baskin 2000). The fixed cells were air-dried and later rinsed twice with 5× SET hybridisation buffer (10% Nonidet) and allowed to stand in the buffer for 3 min (Chen et al. 2008). Then, the probe was added to the slide containing the cells. The slide was incubated in a dry bath at 58°C for 30 min. The slide was washed twice with a 5 SET buffer after incubation.
Following that, 1% blocking reagent was added and incubated at room temperature for 30 min, horseradish peroxidase (HRP) solution was added to the slide and incubated at room temperature for 30 min. The glass slide was then washed with phosphate buffered saline (PBS) that was pre-heated to 37°C. The tyramide working solution (TSA kit with Alexa Fluor® 488 Tyramide; Molecular Probe®, Life Technologies, USA) was then added to the slide in the dark and incubated at room temperature for 10 min. The slide was rinsed again in PBS to remove any excess tyramide working solution. The universal UniC probe (positive control) (5´-/5Biosg/ GWA TTA CCG CGG CKG CTG-3´) and UniR probe (negative control) (5´-/5Biosg/ CAG CMG CCG CGG TAA TWG-3´) were used as controls (Lebaron et al. 1997).
The slides were then observed under an Olympus IX51 microscope equipped with a filter set (470 nm-490 nm excitation and 510 nm-550 nm emission) under UV light. Digital images were captured with an Olympus DP72 digital camera (Olympus).

Species Identification
Two strains of C. subsalsa from the Johor Strait were established and used in this study. Cells of the two strains showed similar morphology, with cell dimensions of 36.6 ± 2.9 µm long and 20.5 ± 4.5 µm wide (n = 50). Under LM, cells are oval, pear-like in shape, which is similar to other C. subsalsa reported previously (Fig. 1). There are two sub-equal, hetero-dynamic flagella at the anterior of the cells (Fig. 1A). The flagella can only be observed in the living cells. The cells contain many golden-brown chloroplasts, which appear barrel-shaped (Fig. 1B). The nucleus is large and appears oval in shape; it is located in the middle of the cell (Fig. 1D).

Phylogenetic Inferences of LSU and ITS rDNA
A total of 23 LSU rDNA sequences and 30 sequences of the ITS of Chattonella were retrieved from the GenBank nucleotide database. Both LSU and ITS rDNA datasets yielded identical tree topologies for maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI); the BI tree is shown in Fig. 2. The trees revealed two monophyletic clades with strong support values (MP/ML/BI, 100/100/1); one clade comprised species in the C. marina complex: C. marina var. antiqua, C. marina var. marina, C. minima, and C. marina var. ovata, while the other clade comprised only taxa from C. subsalsa. The two C. subsalsa strains (CtSg01 and CtSg02) in this study were grouped with other C. subsalsa strains and formed a distinct clade that separated from the strains of C. marina complex, according to both LSU and ITS phylogenetic trees. The strains of C. subsalsa obtained in this study are in boldface.

Species-Specific Oligonucleotide Probes of Chattonella subsalsa LSU rRNA signature region and probe
In the first run, a total of 21 candidate sequences of the potential signature regions in the LSU rDNA of C. subsalsa were detected at a 730-nucleotide length (Table 2). At least one mismatch was found between the related species, such as C. marina var. antiqua and C. marina. The probes selected in silico by ARB contained 18 bases, with GC contents in the range of 50% to 70%. Several of them showed the Gibb energy (ΔG°) greater than -14 kcal/mol, indicative of secondary structure formation (Table 2). A confirmatory test of the probe specificity was performed by blasting in the nucleotide database. The blastn results showed that the probes selected were not specific to C. subsalsa, where the probes matched diatom species with 100% coverage and 100% identity. Therefore, a second attempt at in silico analysis was performed with a slight modification of the signature regions. A total of seven candidate sequences were chosen (Table 3). The length of the probes was in the range of 19 bases to 23 bases, longer than the first run, to ensure the presence of GC complementary pairs at the start and end of the probe sequences. Subsequently, the parameters of the probes were determined, and the specificity of the probes was evaluated through blastn search. Out of the seven probe candidates, Probe Set 7 (5´-GGG GAA UCC GGG UUG GUU UC-3´) was selected (Fig. 3) based on its high GC content (60%), lowest Gibb energy (∆G° = -20.2 kcal/mol), and lower melting point (58.6°C) in contrast to other probes (Table 3). The sequence was further synthesised as a biotinylated probe to perform the FISH assay in the later analysis. According to the Probe Nomenclature, the probe was designated as L-S-C.sub-0039-a-A-20 (Alm et al. 1996).

ITS2 rRNA signature region and probe
The ITS2 region of the rDNA was used to design a species-specific probe as it is more specific at the species level than the LSU rDNA. In this study, ten candidate sequences of C. subsalsa were determined from a 262-bp-long ITS2-rDNA complete sequence; the sequences that are expected to identify the target are listed in Table 4. The candidate sequence length was in the range of 18 bases to 21 bases. These candidate sequences were then subjected to specificity analysis by performing BLAST comparisons against the nucleotide databases, and the results showed that there was no match to other non-target species. Among the ten candidate sequences (Table 4), probe set 10 (5´-TGG AGA TCT GAA CAG TGA GG-3´) was chosen because it exhibited a lower ∆G°, which is -16.7 kcal/mol, comprised of 52.4% of the GC pair with a 100% hybridisation efficiency. Most importantly, the probe is unique to C. subsalsa, and a total of six mismatches were found in the sequence when compared to other non-target species (Fig. 3B). This ITS2 probe was designated as I-S-C.sub-0219-a-A-21 and synthesised as a biotinylated probe for later hybridisation experiments.

Tyramide signal amplification-fluorescence in situ hybridisation (FISH-TSA)
The FISH-TSA assay with the biotinylated-labelled probes was tested on the clonal cultures of C. subsalsa. The species Heterosigma akashiwo was used as the nontarget species. When treated with the positive-control eukaryotic-universal UniC probe, the hybridised cells of C. subsalsa and H. akashiwo showed bright green fluorescence signals (Fig. 4). Lime-green fluorescence signals were observed when C. subsalsa cells were hybridised with C. subsalsa LSU-rRNA and ITS-rDNA species-specific probes (Fig. 4). In contrast, when the cells were treated with the negative-control UniR probe, they showed chartreuse-yellow fluorescence with low intensity (Fig. 4). When the C. subsalsa species-specific probes were tested on H. akashiwo cells, chartreuse-yellow fluorescent signals were observed, indicating negative results (Fig. 5).

DISCUSSION
In this study, two species-specific oligonucleotide probes in the LSU-rRNA and ITS2-rDNA were developed to detect the harmful raphidophyte Chattonella subsalsa. The probes were applied in the assay of whole-cell fluorescence in situ hybridisation (FISH) for species detection. The region of the LSU-rRNA gene was chosen to owe to have a universally conserved region while exhibiting some taxonspecific variable regions (Amann & Ludwig 2000). However, the results of the specificity analysis on the LSU-rRNA selected sequences showed cross-identity with other Chattonella species and diatom species. Therefore, a more taxonspecific rDNA region, the ITS2-rDNA, has been selected to design the speciesspecific probe of C. subsalsa. The biotinylated probes developed in this study have been tested on the C. subsalsa cells through the assay of FISH-TSA. The technique of FISH has been widely used in identifying HAB species such as Pseudo-nitzschia spp., Alexandrium spp., and Karenia brevis (Davis) Hansen & Moestrup (Miller & Scholin 1998, Chen et al. 2008. The method, however, has been shown to exhibit less sensitivity when observed under an epi-fluorescence microscope (Lecuyer et al. 2008). The efficiency of FISH, therefore, has been improved by tyramide signal amplification (TSA) to obtain a better resolution in the FISH application (Lecuyer et al. 2008). FISH-TSA is a protocol that enables detection with a very small probe by signal amplification (Schriml et al. 1999). The biotinylated probes have been designed to achieve the enzymatic action of HRP as they provided strong enzymatically amplified signals and improved the resolution (Kerstens et al. 1995).
In this study, both LSU-rRNA and ITS2-rDNA probes of C. subsalsa exhibited positive green fluorescent signals when hybridised into the cells of C. subsalsa. Generally, the ITS2-rDNA probe does not give whole-cell fluorescence as it was only hybridised to the nucleus of the cells. However, cells of C. subsalsa that were applied with the ITS2-rDNA probe showed almost whole-cell fluorescence owing to its large nucleus as shown in Fig. 1.
To confirm the specificity of the probes, both C. subsalsa species-specific probes were tested with the non-target species H. akashiwo. The results showed that H. akashiwo showed light-yellow fluorescence when tested with the ITS2-rDNA probe, like the negative control. This showed that the ITS2-rDNA probe was specific only to C. subsalsa. But when tested with the LSU-rRNA probe, it showed yellow-green fluorescence that made it difficult to evaluate if the result was positive or negative. It is thus suggested that the ITS2-rDNA probe is better than the LSU-rRNA probe in detecting C. subsalsa.
The assay of FISH-TSA was applied to microscope glass slides throughout the study. This method has been previously described by Chen et al. (2008) as applied to H. akashiwo cells. The cell harvesting procedures such as centrifugation and filtration that were previously applied to the armoured dinophyte Alexandrium and the diatom Pseudo-nitzschia (Miller & Scholin 1998) were less suitable in this case as cells tend to burst when undergoing centrifugation or filtration.
Several factors affect the efficiency of FISH-TSA. Physiological growth conditions of the cells are among the factors that affect FISH-TSA detection , Chen et al. 2008. Kim et al. (2004) discovered that exponentially growing cells have higher fluorescent intensities than stationary phase cells. The low fluorescent intensity of the cells was likely due to the decreasing rRNA content in stationary-phase cells (Anderson et al. 1999).

CONCLUSION
To conclude, the species-specific oligonucleotide probe of C. subsalsa was successfully designed in the ITS2-rDNA region. The results of this study revealed that the ITS2 probe was more specific as compared to the LSU probe. The strong fluorescent signal in FISH-TSA also proves its efficiency in detecting harmful algal species from environmental samples. Future field applications should be carried out to further evaluate the feasibility of this assay for HAB monitoring purposes.