Susana Franca

Paralytic shellfish toxins in the freshwater cyanobacterium Aphanizomenon flos-aquae, isolated from Montargil reservoir, Portugal

Montargil reservoir, located in a dry flat area in the centre of Portugal, was filled in 1958 to fulfil agricultural, electric and industrial requirements. In May 1996, an intensive bloom of phytoplankton was detected. The algal community was strongly dominated by cyanobacteria with predominance of Aphanizomenon flos-aquae from May to June and Microcystis aeruginosa from July to August. Extracts of samples collected during the bloom period showed high toxicity by mouse bioassay. During the M. aeruginosa predominance period, the toxicity was ascribed to the presence of hepatotoxins, but clear symptoms of paralytic shellfish poison were observed when A. flos-aquae was the dominant species. In order to confirm the production of neurotoxins a strain of A. flos-aquae was isolated and established in culture. In this manuscript, we show the morphological characteristics and confirm paralytic shellfish toxins production by the strain isolated and maintained in culture. Identification of the saxitoxin analogs was achieved using high performance liquid chromatography with postcolumn fluorescence derivatization (HPLC-FLD) and liquid chromatographic mass spectrometry technique (LC-MS). The toxins found in the culture extract were GTX5 (64.5 mol%), neoSTX (23.0 mol%), dcSTX (6.1 mol%), STX (5.4 mol%) and GTX6 (1.1 mol%). This is, to our knowledge, the first report of unambiguous evidence of paralytic shellfish toxins produced by freshwater cyanobacteria in Portugal. The toxin profile is rather different from the previously reported PSP producing A. flos-aquae and demonstrates its diversity in terms of toxin production.

PRODUCTION OF PARALYTIC SHELLFISH TOXINS BY APHANIZOMENON SP. LMECYA 31 (CYANOBACTERIA)1

We examined intracellular and extracellular paralytic shellfish toxins (PST) in a strain of Aphanizomenon sp. (LMECYA31) isolated from a Portuguese freshwater reservoir throughout the growth cycle and under different conditions affected by temperature and nitrate and phosphate availability. PST concentrations and compositions were greatly influenced by cell density, growth stage, and temperature and nutrients conditions. On a per‐cell basis results showed (1) the enhancement of PST cell quota after the end of exponential growth phase in nutrient replete batch cultures, (2) the absence of a PST increment at late growth stages under phosphate limitation, (3) a rise in PST maximum cell quota under nitrate depletion, and (4) the enhancement of toxin production at higher temperatures. The relative proportion of the four toxins detected, neoSTX, dcSTX, STX and GTX5, also changed within and between culture settings. While growing under phosphate rich media cells produced mainly GTX5 and neoSTX, whereas under phosphate limitation the proportion of STX and dcSTX increased substantially with culture age. Large amounts of extracellular toxins were found in the culture medium, increasing during culture time. Extracellular toxin composition in each culture was fairly constant and always similar to the intracellular composition found at late stages of growth. This further supported other research that indicates that PSTs are released to the water through cell lysis, and a significant concentration of PST may be expected to remain in the water upon the collapse of a toxic bloom or after cells removal by water treatment.

Taxonomy and production of paralytic shellfish toxins by the freshwater cyanobacterium Aphanizomenon gracile LMECYA40

Detection of Paralytic Shellfish Toxins (PST) in an algal bloom in a Portuguese drinking water reservoir (Lake Crato) was followed by isolation of a strain of Aphanizomenon gracile. The strain, coded as LMECYA40, was cultured and identified by combining a morphological study with 16S rRNA gene sequencing. The toxin profile of this isolate, as revealed by HPLC-FLD analysis, was similar to that of other Aphanizomenon strains (e.g. NH-5) isolated from North America, consisting of two PST analogues: neoSTX (0.27 fmol cell−1) and STX (0.05 fmol cell−1). Based on these toxin levels, we estimated that a culture with a cell density of 107 cells ml−1 should contain approximately 910 μg of STX equivalents per litre. Small volumes of water containing PST concentrations similar to those estimated from the cultured A. gracile would therefore contain PST amounts comparable to those used as limit enforcement for harvesting and consumption of shellfish in marine environments (80 μg of STX equivalents per 100 g of shellfish).

Toxic cyanobacteria strains isolated from blooms in the Guadiana river (southwestern Spain).

This paper describes the occurrence of toxic cyanobacteria along the Guadiana River over its course between Mérida and Badajoz (Extremadura, Spain). Water sampling for phytoplankton quantification and toxin analysis was carried out regularly between 1999 and 2001 in six different locations, including two shallow, slow-flowing river sites, two streamed river sites and two drinking water reservoirs. The cyanobacterial community differed significantly between these locations, especially during the summer. The predominant genera were Microcystis, Oscillatoria, Aphanizomenon and Anabaena. Using an ELISA assay the total microcystin contents of natural water samples from the most eutrophic locations ranged from 0.10 - 21.86 microg mcyst-LR equivalent x L(-1) in Valdelacalzada and 0.10-11.3 microg mcyst-LR equivalent x L(-1) in Vitonogales, and a seasonal variation of toxin content was observed. The amount of microcystins produced by each strain was determined by ELISA assay and the detection and identification of microcystin variants of three toxic strains of Microcystis aeruginosa was performed by high performance liquid chromatography (HPLC). The analysis of microcystins of the cultured strains revealed that toxin production was variable among different strains of M. aeruginosa isolated either from different blooms or from the same bloom.

In situ identification and localization of bacteria associated with Gyrodinium instriatum (Gymnodiniales, Dinophyceae) by electron and confocal microscopy

The presence of intracellular bacteria in the dinoflagellate Gyrodinium instriatum Freudenthal & Lee has previously been described but the bacterial flora associated with this species has not been characterized. In this study, new results of transmission electron microscopy (TEM) and in situ hybridization using several bacterial group-specific oligonucleotide probes are presented. The long-term association of endocytoplasmic and endonuclear bacteria with G. instriatum has been confirmed. All endonuclear and most of the endocytoplasmic bacteria labelled were identified as belonging to the betaproteobacteria. Large clusters of Cytophaga-Flavobacterium-Bacteroides (CFB) were labelled and observed in the cytoplasm of the dinoflagellate cells, but were absent from the nucleus. Gammaproteobacteria were only observed outside the dinoflagellates. No alphaproteobacteria were detected either free-living or intracellular. Empirical observation of intracellular CFB reflected a degradation process of moribund dinoflagellate cells, whereas the systematic colonization of dinoflagellate nucleoplasm by betaproteobacteria suggested a true symbiotic relationship. Natural colonization may have occurred, perpetuated by vertical transmission of intracellular bacteria to the dinoflagellate daughter cells, via a pool of bacteria sequestered within the nucleus. Dividing bacteria were observed in the nucleus and equilibrium may be maintained by release of endonuclear bacteria to the cytoplasm through nuclear envelope constrictions.

Reevaluation of Production of Paralytic Shellfish Toxin by Bacteria Associated with Dinoflagellates of the Portuguese Coast

ABSTRACT Paralytic shellfish toxins (PSTs) are potent neurotoxins produced by certain dinoflagellate and cyanobacterial species. The autonomous production of PSTs by bacteria remains controversial. In this study, PST production by two bacterial strains, isolated previously from toxic dinoflagellates, was evaluated using biological and analytical methods. Analyses were performed under conditions determined previously to be optimal for toxin production and detection. Our data are inconsistent with autonomous bacterial PST production under these conditions, thereby challenging previous findings for the same strains.

Bacterium-dinoflagellate interactions: investigative microscopy of Alexandrium spp. (Gonyaulacales, Dinophyceae)

Abstract The association of bacteria with dinoflagellates has been a neglected field of study, which has gained prominence in recent years because of the possible role of bacteria in toxin synthesis. A number of dinoflagellates undergo sexual reproduction, passing through various life-cycle stages in addition to the vegetative form. The presence of bacteria within dinoflagellates has been well established, but their presence throughout the dinoflagellate life-cycle has not been investigated. Using cultures of Alexandrium (A. tamarense, A. fundyense), we investigated the association of bacteria with various vegetative growth phases (lag, log, stationary) and sexual life-cycle stages (planozygote, planomeiocyte, hypnozygote), using scanning electron microscopy, transmission electron microscopy (TEM) and epifluorescence microscopy. Bacteria were found to be associated with the surfaces of vegetative cells, planozygotes, hypnozygotes and planomeiocytes. TEM showed bacteria to be present within all vegetative growth phases, as well as in the sexual planozygote, cyst and planomeiocyte. The presence of intra-cellular bacteria in vegetative cells was also confirmed using DAPI staining combined with epifluorescence microscopy, and lipopolysaccharide staining combined with TEM.

Telomeric DNA localization on dinoflagellate chromosomes: structural and evolutionary implications

Dinoflagellates are eukaryotic microalgae with distinct chromosomes throughout the cell cycle which lack histones and nucleosomes. The molecular organization of these chromosomes is still poorly understood. We have analysed the presence of telomeres in two evolutionarily distant and heterogeneous dinoflagellate species (Prorocentrum micans and Amphidinium carterae) by FISH with a probecontaining the Arabidopsis consensus telomeric sequence. Telomere structures were identified at the chromosome ends of both species during interphase and mitosis and were frequently associated with the nuclear envelope. These results identify for the first time telomere structures in dinoflagellate chromosomes, which are formed in the absence of histones. The presence of telomeres supports the linear nature of dinoflagellate chromosomes.

Topology of splicing and snRNP biogenesis in dinoflagellate nuclei

Background information. Dinoflagellates are protists that are hypothesized to have experienced a secondary loss of histones. Amongst eukaryotes, they are unique in lacking these proteins. To date, information on the mechanisms involving remodelling, transcription and splicing of their chromatin is limited. Dinoflagellate genes lack TATA boxes and downstream polyadenylation sites and particular linear arrangements. They have an α‐amanitin‐sensitive RNA polymerase, specific transcription factors and regulators, and both transcriptional and post‐transcriptional regulation of gene expression. Dinoflagellates produce either polycistronic or discrete mRNAs, and have conserved snRNAs (small nuclear RNAs), indicating that their genes are spliced.

Toxicity of Cyanobacteria Isolated from the Guadiana River

The Guadiana River flows through the Southwest area of the Iberian Peninsula crossing from the Spanish provinces of Extremadura and Andalucia to the southern region of Portugal. Preliminary studies on the presence and distribution of toxic cyanobacteria led to the identification of several cyanobacterial species in the Guadiana River itself as well as in the freshwater reservoirs established along its course. The objectives of this paper were the identification and quantification of planktonic cyanobacteria in freshwater reservoirs from the Guadiana River, with different applications (human consumption, agriculture purposes and recreational uses), and determination of their toxicity. After the isolation of cyanobacterial strains, the characterisation of their toxicological profile was carried out using immunoadsorbent assays (ELISA) and chromatographic techniques (HPLC-UV). Monitorization studies revealed that predominant species were Microcystis aeruginosa, Anabaena spp. and Aphanizomenon flos-aquae. Some strains of M. aeruginosa were responsible for microcystins production. The toxicity observed in ISACYA 10B was low. The strain ISACYA 2C produces high amounts of microcystins, and probably these toxins were responsible for the toxicity of the cyanobacterial bloom detected in these waters.