Catarina Churro

Diversity and abundance of potentially toxic Pseudo-nitzschia peragallo in Aveiro coastal lagoon, Portugal and description of a new variety, P. pungens var. aveirensis var. nov.

The syndrome Amnesic Shellfish Poisoning (ASP) results from ingesting sea food contaminated by a neurotoxin, domoic acid. Diatoms of the genera Pseudo-nitzschia and Nitzschia are responsible for the production of this toxin. A total of eleven species of Pseudo-nitzschia were identified during two plankton surveys at the entrance and in the southern channels of Ria de Aveiro, the largest lagoon on the west coast of Portugal. During the first survey, conducted from February to October 2000, the following species were identified: P. australis, P. cuspidata, P. delicatissima, P. dolorosa, P. fraudulenta, P. pseudodelicatissima, P. subpacifica and a new variety of P. pungens. The second survey took place from September 2003 to April 2004, except for a break between December 2003-January 2004, along with one complementary sample taken in October 2004; it revealed three additional species, P. americana, P. calliantha and P. multistriata. In contrast, P. cuspidata, P. dolorosa and P. subpacifica were not found during the second survey. Five species: P. subpacifica, P. americana, P. calliantha, P. multistriata and P. cuspidata were reported for the first time in Portuguese waters. Overall, P. australis, P. pseudodelicatissima and a new variety of P. pungens were the most common species, the latter being present in all samples except October 2000. The highest concentrations of Pseudo-nitzschia cells registered were 90, 000 cells L–1 in August 2000 and 106, 000 cells L−1 in March 2004, with P. australis accounting for 65–75% of the cells. Cell concentrations were highest near the entrance of the lagoon and decreased along with salinity in the channels. Cultures of P. australis, P. americana, P. fraudulenta, P. multistriata and a new variety of P. pungens were established and tested for the production of domoic acid, but the results were negative. Morphological differences were found between our strains of P. pungens and two other previously described varieties of P. pungens regarding valve width, densities of striae, fibulae, poroids and band striae as well as differences in the morphology of the band striae. These differences were supported by phylogenetic analyses of ITS rDNA sequences, existence of a compensatory base change and mating studies indicate the presence of a separate taxon, which was described as a new variety, P. pungens var. aveirensis var. nov.

Evaluating the influence of light intensity in mcyA gene expression and microcystin production in toxic strains of Planktothrix agardhii and Microcystis aeruginosa.

Cyanobacteria are phytoplanktonic organisms widely occurring in freshwaters, being frequently associated with the production of toxins, namely microcystins (MCs). MCs are produced non-ribosomally by a multienzyme complex (mcy genes). It has been reported that environmental factors, such as light intensity, can influence toxin production. The aim of this study was to assess the influence of light intensity in the transcription of the mcyA gene and corresponding production of microcystins in toxic isolates of Planktothrix agardhii, where little is known, and compare them to Microcystis aeruginosa. For that purpose, cultures were exposed to three different light intensities (4, 20 and 30 μmol photons m(-2) s(-1)) for 18 days at 20 ± 1 °C. The growth was followed daily using absorbance readings. Samples were collected at each growth stage for cell counting, microcystins quantification and RNA extraction. The level of transcripts was quantified by RT-qPCR and the relative expression determined using 16S rDNA, gltA and rpoC1 as reference genes. The most stable reference genes in M. aeruginosa were rpoC1 and gltA, whereas in P. agardhii were 16S rDNA and gltA. There was a correspondence between the growth rate and light intensity in M. aeruginosa and P. agardhii. The growth rates for both species were lower at 4 and higher at 30 μmol photons m(-2) s(-1). Microcystin concentration per cell was similar between light intensities in M. aeruginosa and over time, while in P. agardhii it was higher in the stationary phase at 4 μmol photons m(-2) s(-1). There were differences in the expression of mcyA between the two species. In M. aeruginosa, the highest levels of expression occurred at 4 μmol photons m(-2) s(-1) in the adaptation phase, whereas for P. agardhii it was at 4μmol photons m(-2) s(-1) in the exponential growth phase. Our results indicate that the light intensities tested had distinct influences on the growth, microcystin production and mcyA expression levels, presenting considerable differences in M. aeruginosa and P. agardhii.

Risk Levels of Toxic Cyanobacteria in Portuguese Recreational Freshwaters

Portuguese freshwater reservoirs are important socio-economic resources, namely for recreational use. National legislation concerning bathing waters does not include mandatory levels or guidelines for cyanobacteria and cyanotoxins. This is an issue of concern since cyanotoxin-based evidence is insufficient to change the law, and the collection of scientific evidence has been hampered by the lack of regulatory levels for cyanotoxins in bathing waters. In this work, we evaluate the profile of cyanobacteria and microcystins (MC) in eight freshwater reservoirs from the center of Portugal, used for bathing/recreation, in order to determine the risk levels concerning toxic cyanobacteria occurrence. Three of the reservoirs did not pose a risk of MC contamination. However, two reservoirs presented a high risk in 7% of the samples according to the World Health Organization (WHO) guidelines for MC in bathing waters (above 20 µg/L). In the remaining three reservoirs, the risk concerning microcystins occurrence was low. However, they exhibited recurrent blooms and persistent contamination with MC up to 4 µg/L. Thus, the risk of exposure to MC and potential acute and/or chronic health outcomes should not be disregarded in these reservoirs. These results contribute to characterize the cyanobacterial blooms profile and to map the risk of toxic cyanobacteria and microcystins occurrence in Portuguese inland waters.

Detection of a Planktothrix agardhii Bloom in Portuguese Marine Coastal Waters

Cyanobacteria blooms are frequent in freshwaters and are responsible for water quality deterioration and human intoxication. Although, not a new phenomenon, concern exists on the increasing persistence, scale, and toxicity of these blooms. There is evidence, in recent years, of the transfer of these toxins from inland to marine waters through freshwater outflow. However, the true impact of these blooms in marine habitats has been overlooked. In the present work, we describe the detection of Planktothrix agardhii, which is a common microcystin producer, in the Portuguese marine coastal waters nearby a river outfall in an area used for shellfish harvesting and recreational activities. P. agardhii was first observed in November of 2016 in seawater samples that are in the scope of the national shellfish monitoring system. This occurrence was followed closely between November and December of 2016 by a weekly sampling of mussels and water from the sea pier and adjacent river mouth with salinity ranging from 35 to 3. High cell densities were found in the water from both sea pier and river outfall, reaching concentrations of 4,960,608 cells·L−1 and 6810.3 × 106 cells·L−1 respectively. Cultures were also established with success from the environment and microplate salinity growth assays showed that the isolates grew at salinity 10. HPLC-PDA analysis of total microcystin content in mussel tissue, water biomass, and P. agardhii cultures did not retrieve a positive result. In addition, microcystin related genes were not detected in the water nor cultures. So, the P. agardhii present in the environment was probably a non-toxic strain. This is, to our knowledge, the first report on a P. agardhii bloom reaching the sea and points to the relevance to also monitoring freshwater harmful phytoplankton and related toxins in seafood harvesting and recreational coastal areas, particularly under the influence of river plumes.