ABSTRACT Some Neoparamoeba strains are pathogens of fish and invertebrates and were implicated in lobster mortality in Long Island Sound in recent years. To better understand the dynamics of these pathogens and their potential linkages to the mortality of marine animals, the capability to specifically detect and accurately quantify these parasites in the environment and in affected organisms is essential. Molecular markers such as mitochondrial cytochrome b (cob) can be powerful tools. In this study, we isolated cob from N. aestuarina and developed species-specific primers. Sequence analysis showed that this gene was A/T rich. Variable regions were identified and used to develop a species-specific primer set for real-time quantitative PCR (RTQ-PCR). This primer set was demonstrated to be specific (with no cross-reaction to N. pemaquidensis and other protists) and sensitive (with a detection limit of 1 cell/30 mL). Further, the quantitative capability of the RTQ-PCR system was verified by a strong correlation between the threshold cycle number and the logarithmic-transformed cob copy number used in the amplification. Using the established RTQ-PCR assay, we estimated that each N. aestuarina cell contained approximately 707 [+ or -] 63 copies of cob. This assay was applied to Lugol-fixed water samples collected from Long Island Sound during August 2002 to September 2003, which showed no detectable N. aestuarina. The cob primer set developed will be useful for future environmental detection of N. aestuarina and provides a basis from which cob gene probes for other Neoparamoeba and Paramoeba species can be developed.
KEY WORDS: cob, mitochondrial cytochrome b, Neoparamoeba aestuarina, Real-Time quantitative PCR (RTQ-PCR), species-specific gene probe
Paramoebid protists (Sarcomastigophora: Paramoebidae) are common marine protozoa (Page 1973), some of which, especially Paramoeba Schaudinn, 1896 and Neoparamoeba Page, 1987, have been linked to diseases and death in fish and invertebrates. Paramoeba perniciosa was first identified in association with "gray crab" disease in blue crabs (Callinectes sapidus) in Maryland, Virginia, North Carolina and South Carolina (Sprague & Beckett 1966, Sprague & Beckett 1968, Sprague et al. 1969, Newman & Ward 1973, Johnson 1977). In blue crabs, P. perniciosa is localized in hemal spaces and connective tissues and spreads to blood vessels and eventually to the heart during the terminal stage of infection (Johnson 1977). This parasite also invades muscle tissue and rarely, hepatopancreas, and in heavy infection can be found in central nervous system. Paramoeba invadens has been linked to massive mortality of sea urchin (Strongylocentrotus droebachiensis) in Nova Scotia, in which the parasite is widespread in all tissues but it is consistently localized in the radial nerve/water vascular canal tissue (Jones & Scheibling 1985, Jellett et al. 1988). More recently, N. pemaquidensis was also recognized as a pathogen of salmon and rainbow trout (Kent et al. 1988). In contrast to the Paramoeba species as mentioned earlier, N. pemaquidensis mostly infects the gills of salmon and other fish (Kent et al. 1988, Dykova et al. 2000). N. aestuarina has also been suggested as a potential agent of amoebic gill disease (Dykova et al. 2000). In addition, Neoparamoeba spp. have been shown to raise immune response in fish (Gross et al. 2004).
Protozoa in the Neoparamoeba/Paramoeba complex of species have been implicated in the massive mortality of the American lobster (Homarus americanus) in Long Island Sound in the fall of 1999 (CT DEP 1999, Tedesco & Van Patten 2000). In diseased and dead lobsters, paramoebid parasites have been identified most frequently in ganglia of the central nervous system (Russell et al. 2000). Although it remains unclear whether paramoebiasis is the primary cause of the die-off, it is believed to be at least involved in some stages of the sickening process (e.g., Russell et al. 2000, Mullen et al. 2004). Furthermore, paramoebid parasites may act synergistically with other stressors such as elevated temperature (Scheibling & Hennigar 1997), which occurred in Long Island Sound bottom water in 1999 (R. Wilson, pers. comm.). To correlate the shellfish disease and lobster mortality with Neoparamoeba/Paramoeba, a capability to detect and quantify these pathogens specifically and efficiently is essential.
Identification of Neoparamoeba/Paramoeba is challenging. Although morphologic and histologic characteristics may allow separation of the documented species (e.g., Perkins & Castagna 1971), it is not practical in environmental or epidemiologic studies to process large number of samples for those characteristics. Molecular markers have proven to be powerful tools for specifically detecting and accurately quantifying microorganisms on large sample scales. Among potentially useful genes, mitochondrial (mt) genes are good candidates because they are generally conserved and yet their higher variability than some nuclear genes (Arise 1994, Saccone et al. 2000) renders these genes preferable for development of species-specific probes. Mt genes also appear to be more clock-like (i.e., of relatively even mutation rate across taxa), characteristics essential for unbiased phylogenetic separation of different taxa (Saccone et al. 2000). Cytochrome b (cob) is one of the mt genes that is widely used for phylogenetic and population genetic analyses (e.g., Callejas & Ochando 2000, Taylor & Hellberg 2003, Zhang et al. 2005). Employment of cob for detecting harmful marine protists has just begun (e.g., Zhang & Lin 2002) and has not been documented for amoeboid species.
In this study, we attempt to isolate cob from N. aestuarina and N. pemaquidensis and developed specific probes for each species. They were chosen because paramoebid parasites detected from infected Long Island Sound lobsters were most closely related to N. pemaquidensis (Mullen et al. 2005) and N. aestuarina is phylogenetically close to N. pemaquidensis (Peglar et al. 2003, Wong et al. 2004). We had success only with N. aestuarina and hence developed a real-time quantitative PCR (RTQ-PCR) assay only for this species at this time.
MATERIALS AND METHODS
Neoparamoeba aestuarina (ATCC 50744) and N. pemaquidensis (ATCC 30735) were provided by American Type Culture Center (ATCC, Manassas, VA) as nonaxenic cultures fed with polyclonal bacteria dominated by Klebsiella pneumoniae (ATCC 700831). Once the cultures, shipped on dry ice, arrived in the laboratory, cells were thawed, transferred to 1.5 mL microcentrifuge tubes, and harvested by centrifugation at x3000g at 4[degrees]C for 5 min. The cell density in the cultures was approximately 1 million cells/mL
DNA was extracted according to Zhang and Lin (2002). Briefly, cells were harvested by centrifugation at x3000g for 20 min at 4[degrees]C. Cell pellets were resuspended in 400 [micro]L DNA extraction buffer (0.1 M EDTA, 1% SDS, 0.2 mg/mL Proteinase K) and incubated overnight at 55[degrees]C. The crude DNA solutions were purified by phenol/chloroform (1:1 vol/vol) extraction. DNA was then precipitated by adding 20 [micro]L of 3M sodium acetate (pH 7) and 800 [micro]L of 99.9% ethyl ethanol, incubating at -20[degrees]C for 1 h. After centrifugation at x16000g for 10 min at 4[degrees]C, DNA pellets were washed with 75% ethanol, dried and dissolved in 10 mM Tris-HCl (pH 8.0) at a concentration equivalent to 4 x [10.sup.5] cells/ mL. Difficulties were encountered in separation of the N. pemaquidensis cells from agar, some of which coprecipitated with DNA. Therefore, more extensive phenol extraction was carried out to achieve DNA that was PCR-amplifiable as demonstrated with 18S rDNA primers.
Primer Design and PCR
Lack of cob sequence information for amoeboid organisms made the primer design a challenge. Primers recently designed and demonstrated to be effective for a wide range of dinoflagellates (Zhang et al. 2005) were first tested but did not generate results. Therefore, new primers were designed based on a multi-alignment including several dinoflagellates and Acanthamoeba castellanii (strain ATCC 30010), the only amoebid protozoa in which a cob sequence (derived from complete mt DNA sequence) is available in GenBank. Two highly conserved regions were identified from the amino acid sequence alignment (Fig. 1). Six "Universal" degenerate primers (Table 1) were designed using consensusdegenerate hybrid oligonucleotide primers (CODEHOP) software (http://blocks.fhcrc.org/codehop.html). Combinations of these primers with dinoflagellate primers were used in PCR with genomic DNA as the template. Temperature-gradient PCR was carried with a PCR express thermocycler (Hybaid, Action Court, Middlesex, UK) with one single cycle of 3 min at 94[degrees]C followed by 35 cycles of 94[degrees]C for 30 sec, 42[degrees]C to 58[degrees]C for 30 sec (with 1[degrees]C increments), 72[degrees]C for 30 sec, and ended with one additional step of 72[degrees]C for 7 min. Results were analyzed on agarose gel electrophoresis.
[FIGURE 1 OMITTED]
Cloning and Sequencing
PCR products were excised from agarose gel, purified using Zymoclean Gel DNA Recovery Kit (Zymo Research, Orange, CA), and then ligated to a TA vector (Invitrogen). The recombinant DNA was transformed into XL1-Blue E. coli strain. Twenty clones were randomly picked for each PCR product and subjected to sequencing on ABI Prism 377 automated sequencer using Big-Dye Terminator Cycle Sequencing Kit (PE Applied Biosystems, Foster City, CA).
The nucleotide sequences were aligned using the CLUSTAL W (1.8) server at DDBJ (http://www.ddbj.nig.ac.jp/Welcome-e.html) on default values, and adjusted using the SeaView program to maintain codon integrity prior to phylogenetic analysis in the Phylo_Win package (Galtier et al. 1996). Phylogenetic trees were constructed from the nt data by the neighbor-joining method (Saitou & Nei 1987) and corrected using Kimura 2 parameter model (Kimura 1980).
Development of Species-specific Primers and Real-Time PCR
N. aestuarina cob sequences were aligned with orthologs from other unicellular organisms. Regions unique in N. aestuarina were identified and used to design species-specific primers using Beacon Designer version 2.1 software (Premier Biosoft International, Palo Alto, CA). Real-Time PCR was carried out with SYBR Green Supermix on an iCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA). Amplification conditions were 45 cycles of 20 sec of denaturation at 95[degrees]C, 30 sec of annealing at 58[degrees]C, and 20 sec of elongation at 72[degrees]C. Data on DNA amount were acquired at the end of extension step in each PCR cycle. The software that controlled the system analyzed the results automatically.
Field Sample Analysis
Samples were collected from four stations (A4, 09, H4 and K2) in Long Island Sound along a west-east axis (Fig. 2). The lobster mortality was most severe in the western Sound, where episodic hypoxia has recurred. Water samples were taken, mostly monthly-bimonthly (Table 2), onboard R/V Dempsey at 2 m depth with Niskin bottles mounted on a CTD rosette. Subsamples of 200 mL were taken and preserved immediately with Lugol iodine solution (Utermohl 1958) and transported to our laboratory within a week. Samples were then stored at 4[degrees]C until analysis. For each sample, 30 mL was removed from the sample bottle, and cells were concentrated by centrifugation at x3000g in two 15-mL tubes with a swing bucket for 20 min at 4[degrees]C. The cell pellet was subjected to DNA extraction as described earlier and dissolved in 30-[micro]L autoclaved distilled water. From each sample, 1 [micro]L of the 30 [micro]L DNA solution was used in Real-Time PCR using the specific primers.
[FIGURE 2 OMITTED]
RESULTS AND DISCUSSION
cob Gene in Neoparamoeba
PCR amplification of cob in these Neoparamoeba species proved difficult. Initial attempts using all possible combinations of the available primers (Table 1) at 55[degrees]C annealing temperature did not yield any positive results. Use of a lower annealing temperature (48[degrees]C) with primers cob5d and cob3a (Fig. 1, Table 1) generated a product of 433 bp for N. aestuarina (GenBank accession number AY743963) and a product of 436 for N. pemaquidensis (GenBank accession number AY743964). Sequencing of the cloned products followed by BLAST search showed that the former was a cob sequence closely related to eukaryotic homologs, but the latter was most similar to sequences of cytochrome bc1 complex genes in [alpha]-proteobacbacteria.
Phylogenetic analyses including the sequences yielded from the Neoparamoeba strains, Acanthamoeba, the only close relative of Neoparamoeba in which cob sequence was available, and some other eukaryotic and prokaryotic unicellular organisms showed that the N. aestuarina-derived cob clustered with eukaryotes and the N. pemaquidensis-derived sequence with proteobacteria (Fig. 3). These phylogenetic placements, with strong bootstrap support, reinforced the BLAST analysis result shown earlier. The PCR product for N. pemaquidensis was most likely derived from bacteria present in the N. pemaquidensis culture (e.g., Klebsiella pneumoniae [ATCC 700831] provided as food). The failure to isolate cob from N. pemaquidensis suggests difference in cob sequence between N. aestuarina and N. pemaquidensis (e.g., variation in intron structure). Hence, only the N. aestuarina-derived cob sequence was analyzed further.
[FIGURE 3 OMITTED]
As Neoparamoeba spp. are commonly associated endocytotically with Perkinsella sp. (Kinetoplastida), the so-called parasome (Dykova et al. 2003), the N. aestuarina-derived cob can theoretically be Neoparamoeba- or parasome-origin. However, it is highly unlikely that the primers used would have amplified cob of the parasome, because kinetoplastida cob genes are highly "cryptic" with numerous deletions or insertions of thymidine (Scott 1995, Stuart et al. 2001). Even if the parasome cob gene is amplifiable, its sequence would not likely be recognizable without addition or removal of thymidine. Therefore, the cloned cob gene fragment is considered a genuine mitochondrial cob from N. aestuarina.
When N. aestuarina cob sequence was aligned with homologs from other organisms, including Acanthamoeba, highly variable regions of the gene were identifiable (Fig. 4). Using Beacon Designer version 2.1, with G/C content and self-annealing taken into consideration, a pair of primers, ParacobF and ParacobR, were obtained (Table 1).
[FIGURE 4 OMITTED]
For the primer set to be useful for field studies, specificity, quantification capability and sensitivity need to be demonstrated. Specificity was first tested with DNA extracted from IV. aestuarina and N. pemaquidensis. Using annealing temperatures 50[degrees]C to 58[degrees]C, PCR with the primer set did not generate any product for N. pemaquidensis, whereas a single DNA product was consistently yielded from N. aestuarina (Fig. 5). No amplicon was obtained from other protist cultures such as dinoflagellates (not shown). The specificity of the primer set was also verified by the lack of PCR product from the DNA extracted from water samples collected from eastern Long Island Sound at the Avery Point campus of University of Connecticut, in which the plankton assemblage consists of numerous protist species.
[FIGURE 5 OMITTED]
Real-Time PCR was used to determine whether this primer set would allow quantification of N. aestuarina with a reasonable detection limit. First, a dilution series of the cloned cob plasmid was used in Real-Time PCR to construct a standard curve (Fig. 6). The amplification product increased with gene copy number (Fig. 5), and, therefore, threshold cycle number decreased exponentially with gene copy number (Fig. 6). A very good correlation was attained between the threshold cycle number and the logarithmic-transformed gene copy number (Fig. 6); thus, the quantitative capability of the Real-Time PCR system is demonstrated. Further analysis indicated that the system was able to detect cob plasmid at as low a dose as 10 copies.
[FIGURE 6 OMITTED]
Second, the RTQ-PCR system was used to estimate cob copy number in N. aestuarina and then to recalculate detection limits in terms of the number of N. aestuarina cells. A dilution series of N. aestuarina DNA was subjected to Real-Time PCR as for the dilution series of cob plasmid described earlier. Proportional increases in PCR product (or decrease of threshold cycle number) with increases of DNA concentration was observed (Fig. 6). Using the standard curve constructed with the plasmid dilution series, the total number of cob copies from each DNA dilution was derived. By averaging the results from all DNA dilutions (n = 8), an estimate of 707 [+ or -] 63 copies per cell was obtained. Finally, based on this estimate, the theoretical detection limit was estimated as 10/707 = 0.014 cell/reaction. Because we normally isolate DNA from 30-mL field-collected water samples, dissolve DNA in 30 [micro]L autoclaved distilled water and then use 1 [micro]L for Real-Time PCR reaction, the practical detection limit of this system is 1 cell per 30 mL of water sample (or 0.03 cell/mL). Therefore, this PCR based cob detection system is in theory considerably more sensitive than whole-cell staining methods (e.g., Lin et al. 2003).
Detection of N. aestuarina in Long Island Sound
Using the RTQ-PCR system, no positive results were generated from any of the DNA samples extracted from water samples collected along the west-east axis of Long Island Sound during August 2002 to September 2003 (Fig. 2, Table 2). The same DNA samples have been used recently to analyze diversity of dinoflagellates and small-sized phytoplankton communities and shown to be of PCR-amplification quality (Zhang & Lin unpublished data). To further examine whether the negative results were because the primer set or PCR conditions used in this study were more susceptible to inhibitory chemicals that might exist in the samples, N. aestuarina DNA extracted from the culture (standard DNA) was spiked with DNA isolated from Long Island Sound water samples and amplified in parallel with the pure standard DNA. No difference was found between the spiked and nonspiked DNA. All these results indicate that the negative results from the field samples were not due to failure of PCR, rather, N. aestuarina was not present above, if at all, the detection limit in the samples. There is a possibility that the absence of N. aestuarina in the samples was a result of sample fixation and storage in Lugol solution. This fixative has been used widely to preserve plankton cells and extensive tests with dinoflagellates have shown that it preserves cells effectively (Zhang & Lin 2002 and unpubl.), but has not been examined for N. aestuarina. If Lugol solution proves effective in preserving N. aestuarina then this negative result would suggest that N. aestuarina is not likely one of the diverse and widespread Neoparamoeba species that have recently been detected in Long Island Sound using SSU rDNA (Gast 2004). It would follow that whereas the SSU rDNA-based analysis has shown that the paramoebid parasite infecting the diseased lobster from Long Island Sound is closely related to N. pemaquidensis (Mullen et al. 2004), it is not likely to be N. aestuarina.
This study demonstrates that (1) cob is highly A/T rich and challenging to clone in Neoparamoeba (as in protistan cob genes in general); (2) each N. aestuarina cell contains approximately 707 [+ or -] 63 copies of cob; (3) the cob-based RTQ-PCR system developed is useful for detecting and quantifying N. aestuarina.
Further research is required to clone cob from other Neoparamoeba species to establish capability to detect and quantify other paramoebid pathogens. Once more species are investigated, dualgene primers like cob-SSU rDNA primer set developed for Priesteria piscicida (Zhang & Lin 2002) may prove desirable for distinguishing some of the closely related species. These multiple genes would also be very useful for phylogenetically resolving these taxa.
The authors thank Connecticut Sea Grant for development funds to support this study under Grant No. LMP/D-1, through the US Department of Commerce, National Oceanic and Atmospheric Administration (NOAA) Award NA16RGI364, and the Water Quality Monitoring group of the Connecticut Department of Environmental Protection for the water samples.
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SENJIE LIN * AND HUAN ZHANG
Department of Marine Sciences, University of Connecticut, 1080 Shennecossett Road, Groton, Connecticut 06340
* Corresponding author: E-mail: firstname.lastname@example.org
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