Comparison of paralytic shellfish toxin (PST) production by the dinoflagellates Alexandrium lusitanicum NEPCC 253 and Alexandrium tamarense NEPCC 407 in the presence and absence of bacteria

FEMS Microbiology Ecology 36 (2001) 223^234

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Comparison of paralytic shell¢sh toxin (PST) production by the dino£agellates Alexandrium lusitanicum NEPCC 253 and Alexandrium tamarense NEPCC 407 in the presence and absence of bacteria Georgina L. Hold a b

a;

*, Elizabeth A. Smith a , T. Harry Birkbeck b , Susan Gallacher

a

Fisheries Research Services, Marine Laboratory, P.O. Box 101, Victoria Road, Aberdeen AB11 9DB, UK Division of Infection and Immunity, University of Glasgow, Joseph Black Building, Glasgow G12 8QQ, UK Received 14 December 2000; received in revised form 23 April 2001; accepted 9 May 2001 First published online 7 June 2001

Abstract The ability of two Alexandrium species to produce paralytic shellfish toxins (PST) in laboratory culture following the generation of bacteria-free cultures was investigated. The dinoflagellates Alexandrium lusitanicum NEPCC 253 and Alexandrium tamarense NEPCC 407 were cultured in the presence of antibiotics and tested for residual bacteria. After treatment with a cocktail of streptomycin, ciprofloxacin, gentamicin and penicillin G, bacteria could not be detected in either of the treated Alexandrium cultures using 17 different solid and broth bacterial growth media, by epifluorescence microscopy with the dye Sybr green 1, or polymerase chain reaction amplification using universal eubacterial primers designed to target the 16S rRNA gene. Subsequent analysis of A. lusitanicum for PST using high performance liquid chromatography demonstrated that the growth rate and toxin profile remained similar in both bacteria-free and control cultures, although the quantity of toxins produced differed with the bacteria-free culture producing generally more of each compound and also having a greater toxin content in terms of saxitoxin equivalents. A. tamarense also retained similarities between the bacteria-free and control cultures in terms of growth rates and toxin profile, although in this instance, depending on the growth stage and the toxin, the control culture produced more of some toxins than the bacteria-free culture. The control culture was also more toxic in terms of saxitoxin equivalents than the axenic culture. These results suggest that bacteria can influence toxin production in laboratory cultures of Alexandrium species although the mechanisms remain unknown. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. Keywords : Dino£agellate ; Axenic culture ; Paralytic shell¢sh toxin (PST) ; Epi£uorescence microscopy; High performance liquid chromatography (HPLC); Polymerase chain reaction (PCR)

1. Introduction The production of paralytic shell¢sh toxins (PST) has been attributed to dino£agellates, with members of the Alexandrium, Gymnodinium and Pyrodinium genera reported to produce PST [1]. Cyanobacteria are also recognised as producers of PST [2^4], and there is some evidence for production of these toxins by heterotrophic bacteria, which is reviewed in [5]. * Corresponding author. Present address: Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen, AB21 9SB, UK. Tel. : +44 (1224) 712751; Fax: +44 (1224) 716687; E-mail : [email protected]

PST consist of saxitoxin (STX) and at least 20 chemically related derivatives which vary in toxicity, hence the total toxicity of dino£agellates may alter depending on the combination and concentration of toxins present [5]. It has been suggested that bacteria associated with dino£agellates either in the natural environment or in laboratory cultures, may in£uence the production of PST by dino£agellates [5,6]. Several authors have shown that bacteria-free dino£agellate cultures produce PST [7^9], although con£icting evidence has been presented on the e¡ect of bacteria on dino£agellate toxicity. Some authors report higher toxicity in bacteria-free cultures [10,11] while others show the reverse [6]. However, there is no evidence for a species-speci¢c di¡erence.

0168-6496 / 01 / $20.00 ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. PII: S 0 1 6 8 - 6 4 9 6 ( 0 1 ) 0 0 1 3 6 - 2

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The above studies are dependent on the production and maintenance of axenic cultures. Several methods have been used to produce these cultures including washing with sterile diluents, using dilution series, ultrasonic treatment and di¡erential centrifugation [10,12^15]. In recent years these methods have been used either in conjunction with, or have been superseded by treatment with antibiotics [16,17] or other anti-bacterial agents, e.g. lysozyme [6]. The advantage of bactericidal treatments over physical dissociation methods is the ease of application, coupled with their e¡ectiveness in removing bacteria from mucilaginous algal species which cannot easily be physically separated from their accompanying micro£ora [16]. In many of the older published works assessment of the bacterial status of dino£agellate cultures was limited to determining bacterial growth on one or two di¡erent bacteriological media and in some cases, observations with light microscopy [18,19]. Often, the media formulations used were unsuitable for detection of culturable marine bacterial species [20^22] and did not take into account that 95% of marine strains are currently considered unculturable [23]. Therefore, more stringent methods for testing axenic cultures have been implemented, generally involving epi£uorescence microscopy [6,8,14,15] using dyes such as 4P,6-diamidino-2-phenylindole (DAPI) and acridine orange [24^26]. Recently, more e¡ective £uorescent stains such as Sybr green 1 have become available [27]. The aim of this investigation was twofold, ¢rstly to produce bacteria-free dino£agellate cultures and rigorously assess their bacterial status using a combination of culture methods, epi£uorescence microscopy and molecular techniques. Secondly, to determine if PST production in Alexandrium cultures was in£uenced by the presence of bacteria as determined by changes in toxin pro¢le measured by high performance liquid chromatography (HPLC). The cultures examined were Alexandrium lusitanicum NEPCC 253 and Alexandrium tamarense NEPCC 407. A. lusitanicum was originally isolated from Portuguese waters in 1962 and reported to remain toxic with the same toxin composition during 30 years in culture [28]. A. tamarense was also reported to remain toxic in culture after its isolation from Canadian waters in 1981 [29]. 2. Materials and methods

cotton-wool bungs and cooled to room temperature before the addition of 20 ml l31 f/2 nutrients. All cultures were maintained at 15³C with a 14:10 h light:dark cycle (irradiance level 0.5^1.5U1016 quanta s31 cm32 ). Nine identical £asks containing 1.25 l of f/2-enriched seawater were prepared for each dino£agellate culture with approximately 1000 dino£agellate cells from mid-exponential growth into each £ask introduced using aseptic transfer. This allowed an adequate sample volume to continuously monitor growth and also to sample for HPLC and PCR analyses at each growth point. 2.2. Dino£agellate growth curves 5 ml volumes of dino£agellate culture were aseptically removed every second day following gentle swirling of £asks to generate a uniform suspension. Lugol's iodine (10 Wl) prepared as described by Cowan and Steel [31] was added to each sample to ¢x the cells. Cells were counted using a Sedgewick-rafter slide following the procedure described by McAlice [32]. Twelve random ¢elds of vision per ml of ¢xed sample were counted, with this procedure carried out in triplicate for each growth curve sample to give statistically valid results. Dense cultures were serially diluted in sterile seawater to give between 10 and 20 cells per Wl before counting. Growth rates were calculated as described by Pirt [33]. 2.3. Isolation of bacteria from dino£agellate cultures Samples (1 ml) of dino£agellate cultures taken from early and late exponential phase, and stationary phase corresponding to days 4, 14 and 28 in A. lusitanicum and days 6, 21 and 28 in A. tamarense, and serially diluted (10-fold dilutions from neat ^ 1039 ) in sterile seawater. Samples (100 Wl) of each dilution were spread in triplicate onto marine agar plates (Difco 2216, MI, USA), and subsequently incubated for 14 days at 20³C. Bacteria from the dilution generating between 50 and 100 colonies were isolated from one of the triplicate plates, and replated individually onto marine agar to obtain pure cultures. Resultant pure bacterial isolates were categorised by colony morphology before being stored in marine broth plus 10% (v/v) glycerol at 370³C, prior to further investigation.

2.1. Dino£agellate strains and maintenance

2.4. Antibiotic resistance pro¢ling of bacteria isolated from dino£agellate cultures

Dino£agellate strains A. lusitanicum NEPCC 253, also known as AL 18 [30], and A. tamarense NEPCC 407 were supplied by the North East Paci¢c Culture Collection, Canada. Dino£agellate cultures were aseptically subcultured into seawater enriched with f/2 media without silica (Sigma, Dorset, UK) as described in [17]. Prior to use, aged (3 months) seawater was autoclaved at 110³C for 30 min in borosilicate wide-neck £asks with non-absorbent

Bacteria isolated from dino£agellate cultures were inoculated into 10 ml marine broth (Difco, MI, USA), and incubated for 24 h in a shaking incubator (20³C, 120 osc. min31 ). Each bacterial suspension (200 Wl), was spread evenly onto marine agar plates and allowed to air dry to form a bacterial lawn. Sterile individual antibiotic sensitivity discs (5 mm) containing : ^ kanamycin 30 and 5 Wg; streptomycin 25 and 10 Wg; penicillin G 10 unit ;

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G.L. Hold et al. / FEMS Microbiology Ecology 36 (2001) 223^234 Table 1 Bacterial growth media formulations Medium name a

Marine ST 10b Peptone A Peptone Bc Malt extract Yeast extractc Seawater Casein Peptone glucose Peptone yeastc f/2

1 month, with control, untreated cultures included for comparison.

Reference

2.7. Epi£uorescence microscopy

[46] [47] [46] [46] [48] [20] [49] [50] [50] [50] [48]

a

Medium was also used at a 1/100 dilution. Medium was also used at a 1/10 and 1/10 000 dilution. c Media were also prepared without the addition of iron. b

cipro£oxacin 5 Wg; gentamicin 120 Wg; novobiocin 30 Wg (Oxoid, Basingstoke, UK), were pressed onto the agar surface. Plates were subsequently inverted and incubated at 20³C for 48 h. Antibiotic sensitivity was identi¢ed by production of a zone of inhibition of 5 mm or greater diameter around the disc. 2.5. Antibiotic treatment of dino£agellate cultures Antibiotic cocktails containing either cipro£oxacin (23 Wg ml31 ), gentamicin (120 Wg ml31 ) and streptomycin (12.5 Wg ml31 ) or cipro£oxacin (46 Wg ml31 ), gentamicin (240 Wg ml31 ) and streptomycin (25 Wg ml31 ) or cipro£oxacin (46 Wg ml31 ), gentamicin (240 Wg ml31 ), streptomycin (25 Wg ml31 ) and penicillin G (20 units ml31 ), were added to mid-exponential phase dino£agellate cultures and incubated for 12 days. Dino£agellates were subcultured through three growth cycles to dilute out the antibiotics prior to assessment of their bacteriological status, growth rates and toxin pro¢les. Untreated cultures i.e. without addition of antibiotics, were maintained concurrently. All cultures were incubated under conditions as previously described. 2.6. Use of di¡erent marine culture media to assess the presence of bacteria in antibiotic-treated dino£agellate cultures To detect the presence of remaining culturable bacteria in antibiotic-treated cultures, 17 di¡erent media, including both general and selective formulations, were used (see Table 1). Samples of antibiotic-treated cultures were inoculated into broths and onto agar plates of the 17 media formulations. Five tubes containing 9 ml of broth from each of the 17 media formulations were inoculated with 1 ml of the antibiotic-treated culture, whilst three agar plates of each media formulation were inoculated with 100 Wl of culture. These were incubated at 20³C for

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Sybr green 1 nucleic acid stain (Molecular Probes Inc., The Netherlands), was prepared and stored following the method described by Marie et al. [27]. Samples (10 ml) from dino£agellate cultures were taken aseptically and diluted in sterile seawater to give approximately 105 cells ml31 . Sybr green 1 stain at a ¢nal dilution of 1034 of the commercial solution was added to samples, which were subsequently incubated in the dark at 20³C for 5 min before placing in a ¢lter column and slowly drawing ( 6 150 mm Hg vacuum) the cell suspension onto a black polycarbonate membrane (0.2 Wm, Porvair Filtronics, Middlesex, UK). The membrane was ¢xed onto a microscope slide by addition of a drop of immersion oil above and below the membrane, and a coverslip added. Slides were immediately examined by epi£uorescence microscopy (Zeiss, Axiovert 10) using oil immersion at an excitation wavelength of 460 nm. 2.8. Bacterial 16S rRNA PCR ampli¢cation of dino£agellate cultures. Antibiotic-treated dino£agellate cultures of A. lusitanicum NEPCC 253 and A. tamarense NEPCC 407 from early, late exponential and stationary growth phases (1000 ml), were collected by centrifugation (10 000Ug, 10 min) and DNA extracted following the method of Scholin et al. [34]. Each sample was subjected to two sets of PCR reactions: set 1 eubacterial primers described by Muyzer et al. [35] and set 2 by Suzuki et al. [36]. These universal primer sets ampli¢ed di¡erent length fragments of the 16S rRNA gene, with primer set 1 targeting the hypervariable V3 region corresponding to nucleotide positions 341^534 in Escherichia coli, and generating an approximately 200-bp PCR product. Primer set 2 eubacterial primers 27F and 1522R ampli¢ed almost the whole 16S rRNA gene (approximately 1500 bp). Ampli¢cations were performed using a Techne Genius thermocycler, with each 100-Wl reaction volume containing 100 ng genomic DNA, 2 mM MgCl2 and 0.3 WM of each primer. Thermocycling conditions for primer set 1 following initial denaturation of 94³C for 5 min were, 30 cycles of 94³C for 30 s, 55³C for 45 s and 72³C for 30 s, followed by a ¢nal extension for 10 min at 72³C. Thermocycling conditions for primer set 2 were as described by Suzuki et al. [36]. Prior to visualisation of PCR products, the 100-Wl PCR reaction volumes were concentrated to 10 Wl using Prep-a-Gene (Bio-Rad, Hemel Hempstead, UK). Concentrated PCR products were subjected to electrophoresis in a 2% (w/v) agarose gel in 1UTAE containing ethidium bromide (0.5 Wg ml31 ), with markers (100 bp and 1 kb ; Gibco, Paisley, UK) included on the gel for reference.

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The lower detection limit of the PCR ampli¢cation using the two primer sets was estimated by assessing their ability to generate ampli¢cation products with di¡ering concentrations of various bacterial morphotypes isolated from A. lusitanicum. Dilutions of representatives of the previously isolated morphotypes were prepared to provide 10^107 colony forming units (cfu) per 100-Wl PCR reaction. These ampli¢cation products were visualised following concentration as previously described. 2.9. PST extraction from dino£agellates Dino£agellates from three points in the growth cycle were sampled for toxin analysis. Following determination of cell concentrations, cells from 1000 ml were collected by centrifugation (2000Ug, 10 min), the supernate decanted and the resulting cell pellet resuspended in 1 ml acetic acid (0.05 M), before storage at 320³C overnight. Following thawing of samples, cells were disrupted by the addition of 25% w/v glass beads (150^212 Wm diameter, Sigma, Dorset, UK), and vortex-mixed for 3 min. Microscopic examination of cell debris revealed that the cells had been completely disrupted. All samples were brie£y centrifuged (13 000Ug, 30 s), with the supernatant ¢ltered through 0.45-Wm syringe ¢lters (Nalgene, Rochester, USA) prior to storage at 320³C for subsequent toxin analysis. 2.10. HPLC analysis of dino£agellate samples HPLC analysis of dino£agellate samples followed the method described by Franco et al. [37], with the following amendments : a silica-based reverse-phase column was used for toxin separation (C18 ; 250U4 mm internal diameter, Purospher, Merck, Glasgow, UK), with a mobile

phase £ow rate of 1.0 ml min31 , held at constant temperature (35³C). External calibration standards gonyautoxins 1/4 and 2/3 (GTX 1, 2, 3 and 4), STX and neo-STX (NRC, Canada) were included before sample analysis and after every fourth injection to monitor the performance of the system. Toxin composition pro¢les were determined from triplicate analyses and expressed as toxicity per cell. Con¢rmation of retention times of sample peaks with those of known toxins was determined by inclusion of internal toxin standards within samples. Total toxicity values were determined as described by Parkhill and Cembella [37], with conversion ¢gures used as detailed in Franco et al. [38]. HPLC was performed using an autosampler and Spectra System P4000 pumps (Thermo Separation Products, Hemel Hempstead, UK), and a RF-535 £uorescence detector (Shimadzu, Milton Keynes, UK) with computer integration (PC1000, V 3.5; Thermo Separation Products). The signal to noise ratio allowed toxin peaks which were three times the size of the background noise level to be determined, with minimum limits for each toxin on the column determined as: GTX 1, 0.28 ng; GTX 2, 0.33 ng; GTX 3, 0.08 ng; GTX 4, 0.12 ng; STX, 0.57 ng and neo-STX, 3.77 ng. 3. Results 3.1. Assessment of the bacteriological status of antibiotic-treated Alexandrium cultures using a range of marine media Bacteria isolated on marine agar from the A. lusitanicum culture were categorised into four distinct morphotypes, as were bacteria isolated from A. tamarense, with some sim-

Table 2 Sensitivity of the di¡erent bacterial morphotypes to antibiotics and percentage removal of bacterial isolates following antibiotic treatment Antibiotic sensitivity

Streptomycin

Bacteria removed (%) with antibiotic combination Novobiocin

Colony description ^ A. lusitanicum NEPCC 253 Yellow/orange +d 3e Large cream + + Small rose + 3 Flat beige 3 3 Colony description ^ A. tamarense NEPCC 407 Small rose + 3 Large cream 3 3 Small cream 3 3 Rough beige + +

Penicillin

Gentamicin

Cipro£oxacin

Kanamycin

1a

2b

3c

+ + + 3

+ 3 + 3

+ 3 + +

+ 3 3 3

50 20 45 75

100 85 100 100

100 100 100 100

+ + + 3

+ + + 3

+ + + +

3 3 3 3

55 30 15 35

100 100 80 100

100 100 100 100

a

Cipro£oxacin (23 Wg ml31 ), gentamicin (120 Wg ml31 ) and streptomycin (12.5 Wg ml31 ). Cipro£oxacin (46 Wg ml31 ), gentamicin (240 Wg ml31 ) and streptomycin (25 Wg ml31 ). c Cipro£oxacin (46 Wg ml31 ), gentamicin (240 Wg ml31 ), streptomycin (25 Wg ml31 ) and penicillin G (20 units ml31 ). d + = antibiotic sensitive. e 3 = antibiotic resistant. b

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ilarities noted between the morphotypes from the two dino£agellate cultures. The antibiotic sensitivity of the bacterial isolates was assessed using a range of antibiotics (Table 2), with all strains sensitive to a combination of cipro£oxacin, gentamicin and streptomycin (Table 2). This combination of antibiotics (combination 1 and 2; Table 2) was subsequently added to the two dino£agellate cultures and incubated for 12 days, after which time the dino£agellates were subcultured into fresh antibiotic-free media, and their bacterial status assessed by checking for bacterial growth on marine agar. Although antibiotic combination 2 was more e¡ective at reducing bacterial numbers than combination 1, one morphotype still remained in each dino£agellate culture (Table 2). Further antibiotic pro¢ling of the resistant bacteria demonstrated that they were sensitive to penicillin G. This antibiotic was subsequently added to the antibiotic cocktail (combination 3) and proved bactericidal to the micro£ora of the two dino£agellate cultures, as determined by lack of bacterial growth after 14 days at 20³C initially on marine agar and subsequently using 16 other di¡erent agar formulations detailed in Table 1. However, a fungus identi¢ed as a Botrytis sp. (Dr S. Moss, personal communication) was isolated from the antibiotic-treated A. tamarense culture from most of the agar preparations shown in Table 1, with the exception of the low nutrient formulations, namely ST1034 , f/2, seawater media and 1/100 strength marine media. The fungus was not isolated from the control A. tamarense culture. E¡orts to remove the fungi using a number of fungicidal reagents were unsuccessful. As the detection limit for bacteria by the plating method was 10 cfu ml31 , a more sensitive test for the presence of bacteria, with a detection limit of 2 cfu ml31 [39] was carried out by inoculating 1 ml of each dino£agellate culture into ¢ve replicate tubes containing 9 ml broth of each media listed in Table 1. After 30 days incubation, no turbidity was observed, although Botrytis was again detected in most of the broth cultures inoculated with the antibiotic-treated A. tamarense culture, with the exception of the low nutrient formulations mentioned above. 3.2. Epi£uorescence microscopy of dino£agellate cultures The bacterial status of the dino£agellate control and antibiotic-treated cultures was examined using epi£uorescence microscopy with Sybr green 1 stain. Fig. 1a shows an A. lusitanicum NEPCC 253 cell prior to antibiotic treatment. The nucleus of the cell was visible as a large green £uorescing mass with the rest of the dino£agellate cell faintly auto£uorescing red due to the presence of chlorophyll. The remainder of the green £uorescence, in the form of small particles, indicated the presence of bacterial cells. Following antibiotic treatment (Fig. 1b), bacteria were not visible either attached to the dino£agellate cell or free-living in the culture media.

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Fig. 1. Epi£uorescence microscopy pictures of A. lusitanicum NEPCC 253 before and after antibiotic treatment Sybr green 1 (40 000U magni¢cation). a: control culture. b: antibiotic-treated culture.

3.3. Use of PCR to con¢rm the axenic status of dino£agellate cultures Prior to determining the bacterial status of the dino£agellate cultures with PCR, the spectrum of bacteria targeted by each of the two primer sets was investigated. Searches of databases, namely GenBank and the ribosomal database project (RDP), indicated that some L-Proteobacteria and the Planctomycetes were not ampli¢ed by primer set 1 and certain Vibrio species were not ampli¢ed by primer set 2. However, use of both primer sets covered the range of Eubacteria deposited in the databases. The sensitivity of the method was also assessed. DNA was extracted from representatives of the four di¡erent bacterial morphotypes obtained from A. lusitanicum NEPCC 253 and PCR-ampli¢ed using the two primer sets. This indicated that the method could successfully amplify all bacterial morphotypes when 10 cfu were present in the PCR reaction. Although 1 cfu per reaction

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was enough to generate an ampli¢cation product on occasions. PCR products were not detected in antibiotic-treated dino£agellate cultures with either primer set but were detected, as expected, in control cultures. The bacteria-free cultures were subsequently passaged three times over a 90day period in antibiotic-free media and their bacterial status again checked as described above. These cultures remained bacteria-free. 3.4. Dino£agellate growth rates The growth of the dino£agellate cultures over a 30-day period was monitored using dino£agellate cell counts, with Fig. 2A,B showing mean log10 values over the 30-day period. Di¡erences were observed in the cell counts between the control and antibiotic-treated cultures of A. lusitanicum during the ¢rst eight days although growth rates of the cultures were identical (W = 0.1 day31 ). As shown in Fig. 2A, the control culture initially contained a lower cell concentration than the axenic culture (Fig. 2A). However, a steep increase in cell concentration was observed in the control culture over the ¢rst 8 days compared to the axenic culture, but from day 8 onwards concentrations were very similar between the two cultures. With A. tamarense, the growth rates of the two cultures were also

Fig. 3. HPLC chromatograms of (a) PST standards GTX 1^4, (b) A. lusitanicum NPECC 253 control culture ^ early exponential growth (sample 1) and (c) A. lusitanicum NEPCC 253 axenic culture ^ early exponential growth (sample 1). GTX standard spiked dino£agellate samples overlaid as coloured traces (blue and red respectively) on chromatograms (b) and (c).

identical (W = 0.14 day31 ), although di¡erences were observed in cell concentrations between days 7 and 15 (Fig. 2B). 3.5. Production of PST by control and axenic cultures of A. lusitanicum and A. tamarense Fig. 2. Growth curve (n = 3 þ S.E.M.) over 30 days for (A) A. lusitanicum NEPCC 253 and (B) A. tamarense NEPCC 407 in the presence/absence of bacteria. Arrows indicate cell sampling points for HPLC analysis. O = control culture; F = axenic culture.

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Control and axenic cultures of A. lusitanicum and A. tamarense were sampled at three di¡erent points in the dino£agellate growth cycle, namely early and late exponential phase, and stationary phase (as indicated by the

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Fig. 4. E¡ect of bacteria on PST production by A. lusitanicum NEPCC 253 grown in batch culture over 30 days. Results calculated from HPLC data (n = 3), with error bars indicating the maximum and minimum toxin concentration when minimum and maximum cell counts are considered. Sample points 1^3 refer to early exponential, late exponential and stationary phase respectively. E = control culture, l = axenic culture.

and 3 were more pronounced (Fig. 3c). The peak at V3.5 min was also present in axenic samples. The highest proportion of the toxin load in A. lusitanicum cells from both the control and axenic cultures, at all three sample points, was due to GTX 4 followed by GTX 1 (Table 3). In the early and late exponential growth phase (samples 1 and 2), the control culture contained a higher proportion of GTX 4 than the axenic culture but the reverse was true in the stationary phase of growth. The axenic culture contained the highest proportion of GTX 1 in early and late exponential phase whereas the control culture contained the highest proportion in the stationary phase (sample 3; Table 3). The axenic culture produced a higher quantity of each toxin compared to control cultures, with the exception of GTX 4 at sample point 1 (early exponential phase) for which equivalent quantities were produced by both the

sampling points on Fig. 2) and the bacteria-free status of the axenic cultures con¢rmed as described above. These samples were subsequently analysed by HPLC to assess production of PST. This was repeated twice more with results similar to those presented below. Fig. 3 is an example of the chromatograms obtained using toxin standards GTX 1^4 (Fig. 3a) and sample 1 of the control (Fig. 3b) and axenic (Fig. 3c) cultures of A. lusitanicum, before and after spiking with toxin standards. The major toxin peaks in the control dino£agellate culture co-eluted with standards of GTX 1 and 4 with traces of GTX 2 and 3 also observed (Fig. 3b). A further peak at V3.5 min was also apparent on sample chromatograms, but could not be attributed to a PST toxin standard. In the axenic culture, peaks co-eluting with standards of GTX 1 and 4 were again observed although at di¡erent ratios to the control culture, and the peaks co-eluting with standards of GTX 2

Table 3 Contribution of each toxin to the total toxin load and total toxicity, expressed as STX equivalents for A. lusitanicum NEPCC 253 Sample

Individual toxin contribution to total toxin load (%) culture treatment

1 ^ Early exponential 2 ^ Late exponential 3 ^ Stationary a b

control axenic control axenic control axenic

GTX 1 32.5 42.7 22.3 30.9 50.0 38.0

GTX 2 a

T 0.2 T T ndb nd

Total toxicity (STX equivalents per cell (pg)) GTX 3

GTX 4

T 5.8 T T nd nd

67.5 51.3 77.7 69.1 50.0 62.0

T = toxin present at 6 0.33 ng (on the column) for GTX 2, and 6 0.08 ng (on the column) for GTX 3. nd = toxin not detected.

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0.179 0.284 0.017 0.029 0.011 0.037

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Fig. 5. E¡ect of bacteria on PST production by A. tamarense NEPCC 407 grown in batch culture over 30 days. Results calculated from HPLC data (n = 3), with error bars indicating the maximum and minimum toxin concentration when minimum and maximum cell counts are considered. Sample points 1^3 refer to early exponential, late exponential and stationary phase respectively. E = control culture, l = axenic culture.

axenic and control cultures (Fig. 4). Measurable quantities of GTX 2 and GTX 3 were only detectable in early exponential phase from the axenic culture. A. tamarense demonstrated a more diverse toxin pro¢le than A. lusitanicum, with six toxin groups quanti¢ed including both carbamate and sulfocarbamoyl toxins (Table 4). The presence of a further toxin, the decarbamoyl toxin dcGTX 2, was suspected but could not be con¢rmed due to the lack of a calibrated standard. Again a peak at V3.5

min was observed in all sample chromatograms, but remained unidenti¢ed. GTX 4 was the dominant toxin in both the axenic and control cultures, with the exception of early exponential phase from the axenic culture in which C2 dominated (Table 4). It was also noted that the contribution of GTX 1 to the toxin loading of these cultures was much lower than that observed for A. lusitanicum (Tables 3 and 4). With A. tamarense, the axenic cultures produced a high-

Table 4 Contributions of each toxin detected to the total toxin load and total toxicity, expressed as STX equivalents for A. tamarense NEPCC 407 Sample

Individual toxin contribution to total toxin load (%) culture treatment

1 ^ Early exponential 2 ^ Late exponential 3 ^ Stationary a b

control axenic control axenic control axenic

GTX 1 a

T ndb 1.1 3.6 5.0 3.7

Total toxicity (STX equivalents per cell (pg))

GTX 4

neo-STX

C1

C2

C4

95.6 29.2 92.5 86.9 82.8 93.7

0.3 T 3.9 1.4 2.9 0.22

nd nd 0.2 T 1.4 0.1

4.1 70.8 2.3 8.1 7.9 0.85

nd nd nd nd nd 1.7

T = toxin present at 6 0.33 ng (on the column) for GTX 2, and 6 0.08 ng (on the column) for GTX 3. nd = toxin not detected.

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0.520 0.179 1.453 0.017 2.466 0.011

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er concentration of GTX 1 and C4 (when detected) and a greater quantity of GTX 4 in late exponential and stationary phase samples (samples 2 and 3). However, control cultures produced more GTX 4 in early exponential phase, C1 in late exponential and stationary phase, C2 in stationary phase, and neo-STX in all samples (Fig. 5). Additionally, di¡erent trends were noted in the concentration of GTX 4 between the axenic and control cultures, with the control culture decreasing in concentration of GTX 4 over the growth cycle, while the axenic culture showed an increase in GTX 4 as the culture approached the stationary phase of growth (Fig. 5). Tables 3 and 4 also summarise the data in terms of the total toxicity of each culture in STX equivalents derived from the concentration of each toxin and its reported toxicity in the mouse bioassay [38]. Comparing control and bacteria-free cultures, total toxicity was consistently 1.5^3fold higher in axenic cultures of A. lusitanicum, while the reverse was true for A. tamarense with between 3- and 220fold higher levels of toxicity observed in control cultures. 4. Discussion Bacteria can be considered an integral part of the physical environment of toxic dino£agellates, alternating between being free-living in the medium, attached to the dino£agellate cell wall or existing internally within the dino£agellate cell [5]. In all cases there is limited information available on the e¡ect of such bacteria on dino£agellate production of PST [5]. However, some studies have shown that PST are still produced in bacteria-free cultures of Alexandrium species [7^9]. Although, a few studies [6,11] have compared the toxicity of axenic dino£agellate cultures to those of control dino£agellates containing their normal bacterial £ora, none have provided detailed information on the toxin pro¢le and toxin concentrations in these cultures. This study aimed to provide such information by initially producing axenic cultures and subsequently assessing di¡erences in the concentration of PST produced between dino£agellates containing their natural microbial population and those in which the bacterial £ora had been removed. In this investigation axenic dino£agellates were prepared after treatment with a bactericidal antibiotic cocktail which was shown to be e¡ective against all the isolated bacterial morphotypes present. A more stringent approach was taken than reported in other studies in assessing the axenic nature of the resultant dino£agellate cultures. This involved using 17 di¡erent bacterial growth media in both solid and broth form, epi£uorescent microscopy and PCR. By using a wide range of solid and broth media formulations, including both oligotrophic and nutrient rich formulas, the probability of detecting bacteria with di¡ering nutrient requirements was greatly increased. The possibility that non-culturable bacteria remained in the dino£agel-

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late cultures was also assessed using epi£uorescence microscopy. Other researchers have applied this technique in conjunction with axenic dino£agellates, generally with DAPI or acridine orange dyes [6,15]. Substituting Sybr green 1 [27] for these dyes in this study had the advantage of reducing auto£uorescence from background detritus and increasing the clarity of the images. Although molecular techniques have been used routinely to detect the presence of bacteria within a community from a range of environments, such as marine picoplankton [40], hot springs [41,42] and seawater [36], there are no published reports of the use of such techniques to examine the bacterial status of dino£agellate cultures. In this study, a PCR method with an ampli¢cation lower level of 10 cfu per PCR reaction was used to demonstrate that control dino£agellate cultures contained bacterial DNA. Use of two primer sets capable of amplifying DNA from a wide range of Eubacteria provided strong evidence that lack of ampli¢cation of DNA from axenic cultures was due to the absence of Eubacteria from the cultures. In obtaining bacteria-free dino£agellate cultures doubts can always arise as to whether bacteria are still present but are not detected by the techniques used. However, by using a combination of the three methods described in this study, this uncertainty is reduced and the possibility that endosymbiotic bacteria remain is vastly reduced compared to other investigations. The treated A. lusitanicum and A. tamarense cultures could reasonably be considered bacteria-free as determined by the above methods. The GTX 1, 2, 3 and 4, with the epimer pair GTX 1 and 4 dominant, were detected in both axenic and control cultures of A. lusitanicum which is consistent with data reported by other researchers for bacteria-containing cultures [28,29]. This implies that removal of bacteria did not inhibit toxin production by this dino£agellate nor change the toxin pro¢le. In the A. lusitanicum culture di¡erences were observed in cell concentrations at the beginning of the growth cycle and it is possible that the cultures were in asynchronous growth in the early part of the cycle, with convergence at day 8 representing the beginning of synchronous cell growth. The di¡erences in cell concentration between days 1^8 could be due to several reasons, including the use of the antibiotic cocktail. As the antibiotic-treated dino£agellate cultures had been subcultured several times into antibiotic-free media prior to the experiment, it is unlikely that this was responsible for the di¡erences between the cultures. Other possible reasons to explain the di¡erences in cell concentrations and toxicity between the cultures include di¡erences in the handling of the control and axenic cultures. However, all cultures were handled and maintained using identical procedures and environmental parameters, including light exposure times and light intensities. It was, therefore, concluded that the changes in toxin production and dino£agellate cell concen-

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trations were the direct result of removal of the bacterial micro£ora. The growth rate of the A. lusitanicum culture was una¡ected by removal of its bacterial micro£ora, although the concentration of individual toxins and total toxicity generally was greater in axenic cultures compared to control cultures. However, these results con£ict with those from the study of Doucette and Powell [6], in which the same dino£agellate strain was found to be approximately 50% less toxic in axenic cultures compared to a control. The lack of growth rate information in that study makes direct comparison di¤cult. Removal of bacterial had a di¡erent e¡ect on the toxicity of A. tamarense NEPCC 407, compared to A. lusitanicum. In most instances the toxin pro¢le, including the dominance of GTX 4, was similar in control and axenic cultures, although some di¡erences were found, unlike the A. lusitanicum culture. The most notable di¡erence between the two dino£agellate species is that removal of the bacterial micro£ora from A. tamarense NEPCC 407 resulted in a decrease in overall toxicity of the culture. Depending on the growth stage and the toxin, the control culture produced more of some compounds than the axenic culture ; this was particularly noticeable with neoSTX. However, the bacteria-free culture did contain a culturable fungus, Botrytis sp., which was not isolated from the control culture. Thus, it was di¤cult to conclude whether the di¡erences observed between the A. tamarense cultures were due to bacteria alone. It is possible that the fungus was present as a spore or in very low concentrations in the control culture. Bacteria have been shown to suppress fungal growth in other environments [43], which leads to the suggestion that removal of bacteria from the culture reduced competition for nutrients and enhanced growth of the fungus. Evidence to support this view, is the inability of the fungus to grow in low nutrient media including the dino£agellate growth media, and the observation that no other axenic dino£agellate cultures maintained in this laboratory, and subcultured using the same aseptic technique, contained this fungus. The study of Singh et al. [10] demonstrated that removing bacteria from cultures of A. tamarense resulted in changes in growth rate and higher toxicity in the axenic culture, the reverse of that observed here with the A. tamarense culture in which growth rates were una¡ected and toxicity was lower in axenic cultures. Also, Dantzer and Levin [11] showed higher toxicity in some axenic A. tamarense cultures on a per cell basis. However, as with the Doucette and Powell study [6], dino£agellate growth rate information was omitted, although cell counts for the sampling points would suggest that the growth rates di¡ered, indicating an indirect e¡ect on toxicity due to the removal of bacteria. This study is the ¢rst to provide detailed information on individual toxin pro¢les between axenic and control dino-

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£agellate cultures and shows that bacteria have a limited e¡ect, if any on the dino£agellate toxin pro¢le but can a¡ect the quantity of toxin produced. Summarising this data with that of previous studies [6,10,11] leads us to conclude that bacteria directly in£uence toxin production in some dino£agellate cultures while in others they may exert an indirect e¡ect through in£uencing the growth rate. The mechanisms by which this occurs are unknown but Gallacher and Smith [5] suggest that a number of factors could be involved including the production of unknown co-factors which inhibit or stimulate dino£agellate toxin synthesis, signalling molecules, nutrient availability and metabolism of the toxins by bacteria. Changes in nutrients and other parameters, e.g. salinity, in£uence dino£agellate growth and toxin production in laboratory cultures [1]. This study suggests that bacteria may have a quantitatively similar e¡ect to these other more easily measurable factors. Providing the above information on laboratory cultures is of interest but leads to questions on the role of bacteria in dino£agellate toxicity in the much more complex and dynamic marine environment. Therefore, future studies should also investigate the role of viruses in dino£agellate growth and toxin production given that phytoplankton are readily susceptible to these agents [44]. De¢ning the identity of bacteria which in£uence toxicity in dino£agellate cultures will aid in ¢eld investigations by allowing the development of bacterial oligonucleotide probes. Bacteria isolated from dino£agellates used in this study have subsequently been identi¢ed [45], with studies currently underway to develop oligonucleotide probes to isolates of interest for use in investigations into dino£agellate toxicity in the environment. Acknowledgements This research was supported by the Fisheries Research Services, Marine Laboratory, Aberdeen and the EU FAIR programme CT96-1558. Thanks are extended to Jennifer Graham for technical assistance and Sylvia Duncan for advice on growth curve analysis. References [1] Cembella, A.D. (1988) Ecophysiology and metabolism of paralytic shell¢sh toxins in marine microalgae. In: Physiological Ecology of Harmful Algal Blooms (Anderson, D.M., Cembella, A.D. and Hallegrae¡, G.M. Eds.), pp. 381^403. NATO ASI Series Vol. G4, Springer-Verlag, Berlin. [2] Mahmood, N.A. and Carmichael, W.W. (1986) Paralytic shell¢sh poisons produced by the freshwater cyanobacterium Aphanizomenon £os-aquae NH-5. Toxicon 24, 175^186. [3] Carmichael, W.W., Evans, W.R., Yin, Q.Q., Bell, P. and Moczydlowski, E. (1997) Evidence for paralytic shell¢sh poisons in the freshwater cyanobacterium Lyngbya wollei (Farlow ex Gomont) comb. nov.. Appl. Environ. Microbiol. 63, 3104^3110.

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