Hybridization between the peat mosses, Sphagnum capillifolium and S. quinquefarium (Sphagnaceae, Bryophyta) as inferred by morphological characters and isozyme markers

 Isozyme markers and morphological characters were studied in four populations of Sphagnum capillifolium and S. quinquefarium. Recombinant plants were found in three populations, where the two species occur sympatrically. All recombinants possessed different haplotypes and combinations of morphological characters, which show that they are results of independent hybridization events. Strongly male-biased sex ratios were found for Sphagnum capillifolium in all populations where it grew sympatrically with S. quinquefarium. Most of the recombinants were also male fertile. These observations suggest that S. quinquefarium is the female parent in the primary crosses and in subsequent backcrosses. Received September 3, 2001; accepted March 16, 2002 Published online: November 7, 2002 Addresses of the authors: Nils Cronberg (e-mail: Nils.Cronberg@sysbot.lu.se), Department of Systematic Botany, Lund University, S?lvegatan 37, SE-223 62 Lund, Sweden. Rayna Natcheva (e-mail: renimoss@iph.bio.bas.bg), Institute of Botany, Bulgarian Academy of Sciences, 23 Acad. G. Bonchev Str., 1113 Sofia, Bulgaria.     

Distribution of Cottus poecilopus Heckel and C. gobio L (Pisces) in Scandinavia

Andreasson. S. (Department of Animal Ecology, Lund University, Lund. Sweden.) Distribution of Cottus poecilopus Heckel and C. gobio L. (Pisces) in Scandinavia. Zool. Scripta 1(2): 69–78, 1972. –The distribution of the freshwater sculpins Cottus poecilopus Heckel and C. gobio L. in Scandinavia is presented. In southern Scandinavia there are rather defined limits between the species with C. poecilopus in the upper parts of the water courses and C. gobio in the lower reaches. There are few cases of sympatry. In northern Scandinavia there is a broad overlap and coexistence is common in the large rivers. The distribution pattern seems to be partly a result of an interaction between the species. In the South, the sharp limits are set by falls blocking the upstream dispersal of C. gobio thus indicating an exclusion of C. poecilopus in the lower reaches below the falls by the presence of C. gobio there. Where the species meet or overlap there is a habitat segregation.     

AN UNUSUAL POLYUNSATURATED C-27 HYDROCARBON FROM THE MARINE DINOFLAGELLATE PYROCYSTIS LUNULA

Leblond, J. D.¹ & Chapman, P. J.^{2 1}Department of Biology, Middle Tennessee State University, Murfreesboro, TN 37132. ²US EPA (NHEERL), Gulf Ecology Division, 1 Sabine Island Dr., Gulf Breeze, FL 32561 Studies of the lipids of different algal species have revealed a diversity of fatty acids, sterols, and hydrocarbons, of which several are considered useful biomarkers, with potential for characterizing phytoplankton community composition. To extend this approach and characterize the lipids and lipid classes of laboratory-cultured marine dinoflagellates, a silicic acid fractionation system was developed to obtain compositional data for sterols and hydrocarbons of over forty species. In the course of this work, a neutral fraction obtained from a lipid extract of Pyrocystis lunula was found to contain an abundant quantity of a long-chain polyunsaturated hydrocarbon, along with previously reported keto-steranes. The hydrocarbon molecular weight (364) and retention time obtained by gas chromatography/mass spectrometry analysis suggested a C₂₇ compound, which was confirmed by reduction (Adams catalyst) to give the straight chain alkane, n-heptacosane. The presence of eight double bonds was established by deuteration to give a product with molecular weight 396. While the positions of double bonds have not been established, the carbon number of this hydrocarbon and the number of double bonds strongly suggest formation by decarboxylation of the recently described, long-chain polyunsaturated C₂₈ fatty acid shown to be a constituent of phospholipids. This hydrocarbon was not found in any other genus of the examined dinoflagellates, and appears to be one of the first identifications of a hydrocarbon in this class of algae. The function(s) of this compound in P. lunula is currently unclear.     

Molecular characterization of three Mhc class II B haplotypes in the ring-necked pheasant

We investigated the class II B genes in free-ranging population of the ring-necked pheasant Phasianus colchicus by a combination of restriction fragment length polymorphism (RFLP), polymerase chain reaction (PCR), and DNA sequencing. Special attention was paid to the variation in the second exon, which encodes the peptide-binding ₁-domain. The population was introduced, but it still exhibited major histocompatibility complex polymorphism with at least three segregating class II B haplotypes and consequently six genotypes. We found two class II B genes associated with each haplotype. The class II B genes of birds had until then only been molecularly characterized in the domestic chicken. the pheasant genes were highly variable, although one of the amplified sequences was found in two different haplotypes. Taken together, the most polymorphic positions (residues 37 and 38) were not identical in any of the predicted protein sequences, but all except one of the motifs had already been foud in the domestic chicken. Structurally important features in mammalian class II B genes were generally conserved also in the pheasant sequences, but the loss of a potential salt bridge constituent (Arg₇₂) in several sequences may suggest a slightly different structure of the adjacent parts of the peptide-binding groove. The pheasant genes are most closely related to the so called B-LBII family in the chicken, indicating that this represents a major line of development among avian class II B genes.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession numbers X75403-X75407. Correspondence to: H. Wittzell, Department of Theoretical Ecology, Ecology Building, Lund University, S-223 62 Lund, Sweden.     

Bacillus thuringiensis Cry4Aa insecticidal protein: Functional importance of the intrinsic stability of the unique α4–α5 loop comprising the Pro-rich sequence

The long loop connecting transmembrane α4 and α5 of the Bacillus thuringiensis Cry4Aa toxin possesses a unique feature with Pro-rich sequence (Pro¹⁹³Pro¹⁹⁴_Pro¹⁹⁶) which was shown to be crucial for toxicity. Here, the structural role in the intrinsic stability of the Pro-rich sequence toward toxin activity was investigated. Three Val-substituted mutants (P193V, P194V and P196V) and one Phe-substituted mutant (P193F) were generated and over-expressed in Escherichia coli as inclusions at levels equal to the wild-type. Bioassays demonstrated that all mutants, particularly P193V and P193F whose inclusions were hardly soluble in carbonate buffer (pH 9.0), exhibited reduced toxicity, suggesting an essential role in toxin function by the specific cyclic structure of individual Pro residues. Analysis of the 65-kDa Cry4Aa structure from 10-ns molecular dynamics (MD) simulations revealed that the α4–α5 loop is substantially stable as it showed low structural fluctuation with a 1.2-Å RMSF value. When the flexibility of the α4–α5 loop was increased through P193G, P194G and P196G substitutions, decreased toxicity was also observed for all mutants, mostly for the P193G mutant with low alkali-solubility, suggesting a functional importance of loop-rigidity attributed by individual Pro-cyclic side-chains, particularly Pro¹⁹³. Further MD simulations revealed that the most critical residue−Pro¹⁹³ for which mutations vastly affect toxin solubility and larval toxicity is in close contact with several surrounding residues, thus playing an additional role in the structural arrangement of the Cry4Aa toxin molecule. Altogether, our data signify that the intrinsic stability of the unique Cry4Aa α4–α5 loop structure comprising the Pro-rich sequence plays an important role in toxin activity.     

WINTER PHYTOPLANKTON BLOOMS UNDER ICE ASSOCIATED WITH ELEVATED OXYGEN LEVELS1

Many shallow lakes in north temperate zones experience reduced dissolved oxygen concentration under ice. However, some shallow lakes display supersaturated dissolved oxygen concentrations (>20 mg·L ^{? 1}) in late winter under conditions of maximum ice thickness. During the winters of 1996, 1997, and 1999, we collected phytoplankton samples from Arrowwood Lake near Pingree, North Dakota to determine whether a specific alga was involved in dissolved oxygen supersaturation in this lake. Although dissolved oxygen supersaturation was not observed during this period, we did observe an increase in dissolved oxygen concentration that was associated with a phytoplankton bloom during late February and early March in both 1996 and 1997. In 1996, the bloom was composed of the dinoflagellate, Peridinium aciculiferum (Lemm.) Lemm. and several species of cryptomonads. A similar bloom of P. aciculiferum was followed by a bloom of several species of euglenoids in 1997. In contrast, P. aciculiferum was only a minor component of the winter phytoplankton, dissolved oxygen concentrations remained low, and no bloom event was observed in 1999. Statistical analyses indicated a significant relationship (r_s = 0.57, P = 0.019) between dissolved oxygen levels and the density of the dinoflagellate, P. aciculiferum, but no significant relationship between dissolved oxygen levels and densities of other phytoplankton. These results suggest that the elevated levels of dissolved oxygen are associated with the dinoflagellate, P. aciculiferum. This bloom was most likely the result of an excystment event rather than a general growth response.     

Susceptibility of two fishes (Oreochromis niloticus and Cyprinodon variegatus) to Pfiesteria shumwayae and its associated toxin: Influence of salinity

Researchers examining the mechanisms of ichthyotoxicity of Pfiesteria shumwayae have come to different conclusions about the role of toxin in this process. Some attribute fish mortality solely to direct attack by these pedunculate dinoflagellates on exposed fish tissue while others have provided evidence for a role of a soluble toxin. Detection of toxin, especially in low concentrations, is a function of the sensitivity of the selected bioassay methods and the various groups addressing this question have utilized different methods. One notable difference in fish bioassay methods utilized to detect Pfiesteria-associated toxin (PfTx) is the species of fish tested. Studies that have not detected PfTx in bioassays generally have used Cyprinodon variegatus (sheepshead minnow) as the test fish while those that have detected toxin generally used Oreochromis spp. (Tilapia). In this study response of these two fish species was compared to determine their relative sensitivity to physical attack by P. shumwayae and to PfTx. The results indicate that Oreochromis niloticus is more susceptible to P. shumwayae and its associated toxin than C. variegatus and implicate differences in the ability these species to osmoregulate as a contributing factor for this phenomenon. Salinity stress enhanced susceptibility of O. niloticus to PfTx and thus improved the sensitivity of the bioassay. The observation that salinity stress enhances toxicity to O. niloticus provides additional information regarding the mechanism of PfTx toxicity although the conditions utilized are not representative of the natural habitat of these freshwater fish.     

Variation in paralytic shellfish toxin composition within the Protogonyaulax tamaronsis/catenella species complex; red tide dinoflagellates

Unialgal isolates of the Protogonyaulax (—Gonyaulax) tamarensis/catenella species complex, a group of dinoflagellates which causes paralytic shellfish poisoning (PSP), were subjected to toxin analysis by HPLC. Protogonyaulax isolates from widely separated geographical locations were compared, including the northeastern Pacific (British Columbia and Washington State), eastern Canada, Portugal, the United Kingdom and New Zealand. Two distantly related gonyaulacoid species were also analyzed, but the presence of PSP toxins was not detected. Although Protogonyaulax isolates varied markedly in total toxin concentration and toxicity, even through the culture cycle, the toxin ratios of individual isolates were distinctive and relatively constant. No toxins were detected in the Plymouth (U.K.) isolate of P. tamarensis, from the species type locality. Two isolates from Vancouver Island (British Columbia), which were previously considered to be non-toxic according to the mouse bioassay, revealed weak toxin spectra by HPLC. Within populations from English Bay (British Columbia) the toxin profiles of tamarensoid isolates tended to be conservative. However, this was not the case for the catenelloid forms from Washington State, which displayed a greater degree of toxin heterogeneity. Significantly, there was no identifiable relationship between toxicity or toxin profiles and the morphological characteristics conventionally used to separate the two dominant morphotypes into species within this species complex.     

Responses of Phyto- and Zooplankton Communities to Prymnesium polylepis (Prymnesiales) Bloom in the Baltic Sea

A large bloom of Prymnesium polylepis occurred in the Baltic Sea during the winter 2007 – spring 2008. Based on numerous reports of strong allelopathic effects on phytoplankton exerted by P. polylepis and its toxicity to grazers, we hypothesized that during this period negative correlations will be observed between P. polylepis and (1) main phytoplankton groups contributing to the spring bloom (i.e., diatoms and dinoflagellates), and (2) zooplankton growth and abundance. To test these hypotheses, we analyzed inter-annual variability in phytoplankton and zooplankton dynamics as well as growth indices (RNA∶DNA ratio) in dominant zooplankton in relation to the Prymnesium abundance and biomass. Contrary to the hypothesized relationships, no measurable negative responses to P. polylepis were observed for either the total phytoplankton stocks or the zooplankton community. The only negative response, possibly associated with P. polylepis occurrence, was significantly lower abundance of dinoflagellates both during and after the bloom in 2008. Moreover, contrary to the expected negative effects, there were significantly higher total phytoplankton abundance as well as significantly higher winter abundance and winter-spring RNA∶DNA ratio in dominant zooplankton species in 2008, indicating that P. polylepis bloom coincided with favourable feeding conditions for zooplankton. Thus, primary consumers, and consequently also zooplanktivores (e.g., larval fish and mysids), may benefit from haptophyte blooms, particularly in winter, when phytoplankton is scarce.     

PLEUROCLADIA LACUSTRIS IN ARCTIC AMERICA1

Several large populations of Pletirocladia lacustris A. Br. were found at Devon Island, N.W.T., Canada, in an intermittently marine habitat. The morphology, cytology, and ecology of this species were described from living material from two habitats, the limestone and granite shores. The genus Kolderupia Lund ivas found synonymous with Pleurocladia; consequently Kolderupia maritima and K. lucifuga were lectotypified, characterized, and transferred to Pleurocladia. The genus Pleurocladia should be regarded as a member of the Ectocarpaceae until life history studies show a need for reclassification. In the Ectocarpaceae, Pleurocladia is probably most closely related to Protectocarpus Kuckuck. The distribution of P. lacustris indicated that the species, if not extremely rare, is uncommon.     

THE MARINE DINOFLAGELLATE ALEXANDRIUM OSTENFELDII: PARALYTIC SHELLFISH TOXIN CONCENTRATION,COMPOSITION, AND TOXICITY TO A TINTINNID CILIATE1

The composition of paralytic shellfish toxins in the marine dinoflagellate Alexandrium ostenfeldii (Paulsen) Balech et Tangen grown in unialgal culture was determined by high-performance liquid chromatography. The toxin profile revealed that the low-potency sulfamate toxin B₂ was dominant (90 molar % of total toxins), but small amounts of the weakly toxic 21-N-sulfocarbamoyl derivatives C₁₊₂ and trace amounts of the carbamate toxins GTX₂ and GTX₃ were also present. The mammalian toxicity was confirmed by a modification of the conventional AOAC mouse bioassay (0.6–1.4 pg STX_eq· cell^-1). The acute toxicity to a potential predator, the tintinnid ciliate Favella ehrenbergi (Clap, et Lach.) Jörg., was also investigated. The ciliate was able to graze on A. ostenfeldii when the cell concentration of the dinoflagellate was low (<2000 cells · mL^-1). At higher concentrations the ciliate was affected by exudates (presumably PSP toxins) that induced backward swimming followed by swelling and lysis of the cell. Fluorescence microscopy of calcofluor-stained cells was employed as an easy and rapid method to identify this and other thecate dinoflagellates.     

Competitive abilities of Tn5 Tox- mutants of a rhizobacterium inhibitory to wheat growth

Antibiosis has been thought to impart a competitive advantage to soil microorganisms. A rhizobacterium of the genus Pseudomonas produces a toxin that inhibits the growth of other microorganisms and winter wheat (Triticum aestivum L.). The bacterium was mutagenized with the Tn5 transposon to obtain toxin-negative (Tox^-) mutants or was selected for its spontaneous resistance to rifampicin. Tox^- mutants were used to determine the role of the toxin in wheat root inhibition, root colonization, and rhizosphere competitiveness. Four Tox^- (loss of inhibition of both E. coli and wheat root growth) and four partial Tox⁺ (partial loss of inhibition of E. coli and wheat root growth) Tn5 mutants were isolated. Seven of the mutants had different Tn5 chromosomal insertions, which suggests that toxin production is the result of several gene loci. Competitive root-colonization abilities of the Tox^- isolates were studied in winter wheat rhizospheres using varied population levels in autoclaved and nonautoclaved soil. Toxin production did not affect the competitive abilities of these organisms with native soil microflora. Results here indicate that toxin production by these organisms is not the primary mechanism of their competitive advantage in root colonization. Thus, opportunities exist for biological control of plant-suppressive bacteria using these Tox^- strains.     

ENVIRONMENTAL AND ENDOGENOUS REGULATION OF CYST GERMINATION IN TWO FRESHWATER DINOFLAGELLATES

The role of excystment in relation to seasonal succession was investigated in two freshwater dinoflagellates, Ceratium hirundinella (O.F. Müller) Dujardin and Peridinium aciculiferum (Lemmermann). Field studies and laboratory experiments were performed to determine which factors regulate the timing of cyst germination. Environmental factors (temperature, light, nutrients, and anoxia) and endogenous factors (maturation period and biological clock) were investigated. Our main results indicate that temperature and internal maturation period determine when germination can occur. C. hirundinella had a maturation period of 4.5 months and germinated in the laboratory and in the field at temperatures above 6° C. P. aciculiferum had a maturation period of 2.5 months and germinated in the laboratory and in the field at temperatures below 7° C. In addition, our results indicated that both species were regulated by a biological clock. Furthermore, anoxia prevented the germination of C. hirundinella, contrary to results in earlier studies. To conclude, we could explain the appearance in plankton of the two dinoflagellate species through two main factors regulating excystment, that is, temperature and maturation period.     

Production of goniodomin A by the planktonic, chain-forming dinoflagellate Alexandrium monilatum (Howell) Balech isolated from the Gulf Coast of the United States

The chain-forming dinoflagellate Alexandrium monilatum has been reported to be associated with widespread discolored water and increased fish mortality in the Mississippi Sound and off the eastern and western coasts of Florida. Previous studies over the last 60–70 years have determined that A. monilatum produces a harmful substance(s) that is predominantly contained in the cell mass as exhibited by apparent increased toxicity when the organism cytolyses. The current research in our lab corroborated earlier research demonstrating that A. monilatum produces a lipophilic toxin, unlike its Alexandrium relatives noted for their production of saxitoxin-like toxins. Using sophisticated chemical, chromatographic, and analytical techniques, we have successfully purified and identified the molecular structure of the toxin produced by A. monilatum. We utilized a 500 MHz NMR to carry out a number of experiments (i.e., ¹H, ¹³C, COSY, HSQC, and HMBC) to unambiguously determine the molecular structure of the toxin. In addition, we report mass analysis of the toxin utilizing electrospray ionization-mass spectrometry (ESI-MS), matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS), and Q-TOF mass spectral techniques. The toxin is representative of a polyether macrolide with an empirical formula of C₄₃H₆₀O₁₂. This toxic compound is shown to be identical to a Japanese tidepool toxin identified as goniodomin A, which is produced by another Alexandrium species.     

Production of amyloglucosidase by Aspergillus niger under different cultivation regimens

Amyloglucosidase (AMG) was produced by Aspergillus niger in solid-state fermentation (SSF), submerged fermentation (SmF) and an aqueous, two-phase system of polyethyleneglycol (PEG) and salt. In SSF, a fed-batch mode of operation gave a yield of 64 U/ml compared with 44 U/ml in batch mode. Similar trends were observed for SmF, where fed-batch cultivation gave a yield of 102 U/ml compared with 66 U/ml in batch. Shorter cultivation times (66 h) were required for SmF than for SSF (96 h). In the aqueous, two-phase cultivation, the productivity and yield of AMG were both twice those in the control fermentation.M. Ramadas is with the Department of Biochemistry, Faculty of Medicine, University of Jaffna, Kokuvil, Sri Lanka. O. Holst and B. Mattiasson are with the Department of Biotechnology, Chemical Center, Lund University, Box 124, S-221 00 Lund, Sweden     

Biological interactions in enclosed plankton communities including Alexandrium catenella and copepods: Role of phosphorus

A microcosm approach was used to test whether: a) growth under unbalanced nutrient conditions (varying N:P ratios) affected the susceptibility of a phytoplankton community including the dinoflagellate Alexandrium catenella (a paralytic shellfish toxin producer) to mesozooplankton grazing, and b) the potential effects of unbalanced nutrient conditions were mediated by changes in toxicity of A. catenella or by other mechanisms. The experimental setup consisted of fifteen 30 l microcosms, filled with water from the Barcelona Harbour and subjected to treatments combining nutrient inputs at three different N:P ratios (Redfield N:P ratio or nutrient-balanced, high N:P and low N:P), addition or omission of A. catenella (an estimated initial concentration of 38 A. catenella cells ml^− 1, a value typical for blooms in harbours of the Catalan coast), and selective addition of a cultured population of Acartia grani. P sufficiency had a strong positive effect on the growth of A. grani, both with or without A. catenella addition, presumably due to enhanced food quality of the prey community. The presence of this copepod resulted in lower concentrations of ciliates, A. catenella, and other dinoflagellates, suggesting active grazing by the copepods. No noxious effects of A. catenella on the copepods were detected at the relatively low cell concentrations of that dinoflagellate used in the experiment.     

Contribution of Ciliated Microprotozoans and Dinoflagellates to the diet of three Copepod species in the Bay of Biscay

Predation of three calanoid copepods (Calanus helgolandicus, Temora longicornis and Centropages chierchiae) on phytoplankton, dinoflagellates and ciliates was estimated in the Gironde estuarine plume (Bay of Biscay) during winter by means of in situ incubations. Both phytoplankton and ciliates were part of the diet of those three species, while only Centropages chierchiae also included a significant portion of dinoflagellates in its diet. The clearance rates of Calanus helgolandicus for ciliates and phytoplankton reached 2.8 and 4.0 ml copepod^–1 h^–1, respectively, those of Temora longicornis were 3.2 and 1.8 ml copepod^–1 h^–1, and those of Centropages chierchiae were 4.3 and 0.8 ml copepod^–1 h^–1.Neither Calanus helgolandicus nor Temora longicornis selected dinoflagellates, given the low clearance rates measured for this prey category (0.05 and 0.03 ml copepod^–1 h^–1, respectively). By contrast, Centropages chierchiae included dinoflagellates in its diet, with a clearance rate of 4.9 ml copepod^–1 h^–1. Within a given prey category (ciliates or dinoflagellates), all three copepods selected larger prey types (>40 m) over smaller ones (40 m). This implies a better detection and capture of larger motile prey compared to small ones. The results are discussed with regard to the omnivorous feeding behavior of these copepods observed here, during a late winter phytoplankton bloom.     

PROROCENTRUM LEVIS,A NEW BENTHIC SPECIES (DINOPHYCEAE) FROM A MANGROVE ISLAND,TWIN CAYS,BELIZE1

As part of a long‐term study of benthic dinoflagellates from the Belizean barrier reef system, we report a new species: Prorocentrum levis M. A. Faust, Kibler, Vandersea, P. A. Tester et Litaker sp. nov. P. levis cells are oval in valve view and range in size from 40 to 44 μm long and 37 to 40 μm wide. Each valve surface is smooth, with 221–238 valve pores and 99–130 marginal pores. These pores are uniformly small and range in diameter from 0.13 to 0.19 μm. Asexual reproduction in P. levis is atypical, occurring within a hyaline envelope, and produces long branching chains of adherent cells. A phylogenetic analysis of SSU rDNA indicated that of the Prorocentrum species sequenced so far, P. levis was most closely related to P. concavum. P. levis produces okadaic acid and dinophysis toxin‐2 (DTX2). Further, SEM observations and SSU rDNA sequence for P. belizeanum M. A. Faust, which was isolated at the same time, are also presented.     

Sequence elements essential for the rapid turnover of <Emphasis Type="Italic">Crithidia fasciculata</Emphasis> ornithine decarboxylase

Summary. Ornithine decarboxylase (ODC) has a very fast turnover in mammalian cells, but is a stable enzyme in T. brucei and other trypanosmatid parasites like Leishmania donovani. However, Crithidia fasciculata, which is a phylogenetically closely related trypanosomatid to L. donovani, has an ODC with a rapid turnover. Interestingly, C. fasciculata ODC, but not L. donovani ODC, is rapidly degraded also in mammalian systems. In order to obtain information on what sequences are important for the rapid degradation of C. fasciculata ODC, we produced a variety of C. fasciculata/L. donovani ODC hybrid proteins and characterized their turnover using two different mammalian expression systems. The results obtained indicate that C. fasciculata ODC contains several sequence elements essential for the rapid turnover of the protein and that these regions are mainly located in the central part of the enzyme. Present address: Department of Microbiology, Moyne Institute of Preventive Medicine, University of Dublin – Trinity College, Dublin, Ireland Authors’ address: Lo Persson, Department of Experimental Medical Science, Lund University, BMC D-12, S-221 84 Lund, Sweden     

Effect of Associated Bacteria on the Growth and Toxicity of Alexandrium catenella

Saprophytic bacteria in cultures of the marine dinoflagellate Alexandrium catenella were removed to assess their effect on growth and paralytic shellfish poisoning toxin production of this dinoflagellate. The actual axenic status was demonstrated by the lack of observable bacteria both immediately after treatment and following extended incubation in the absence of antibiotics. Bacteria were measured by counting CFU and also by epifluorescence microscopy and PCR amplification of bacterial 16S-23S spacer ribosomal DNA to detect noncultivable bacteria. Removal of bacteria did not have any effect on the growth of the dinoflagellate except for the inhibition of A. catenella disintegration after reaching the stationary phase. Toxicity was determined in dinoflagellate cell extracts by different methods: high-performance liquid chromatography (HPLC); an electrophysiological test called the Electrotest, which measures the inhibition of saxitoxin-sensitive Na⁺ channels expressed in a cell line; and a mouse bioassay, which measures the toxic effect on the whole mammal neuromuscular system. A lower toxicity of the dinoflagellates in axenic culture was observed by these three methods, though the difference was significant only by the mouse bioassay and HPLC methods. Altogether the results indicate that axenic cultures of A. catenella are able to produce toxin, though the total toxicity is probably diminished to about one-fifth of that in nonaxenic cultures.