Hidden in the depths, discovery of a new spiny sucker eel of the genus Lipogenys Goode and Bean 1895 (Teleostei, Notacanthiformes, Notacanthidae) in the eastern North Atlantic Ocean.

This study describes Lipogenys hyalinumvelum, a new species of the genus Lipogenys found on the Portuguese coast on the northeastern Atlantic during a crustacean survey. Information on the classification history and known distribution of the genus Lipogenys is provided. Dichotomous keys to the genera of Notacanthidae and the species of Lipogenys, based on morphology, are provided. The specimens were analysed using both morphological and molecular methods, including DNA sequencing of the COI and 16S genes. The distinct genetic characteristics support the recognition of the present specimens as a new species. The hyaline color of the flap at the posterior edge of the operculum is a characteristic that differentiates L. hyalinumvelum from Lipogenys gillii and provides the etymology of the species name.

A 15-Year Retrospective Review of Ciguatera in the Madeira Islands (North-East Atlantic, Portugal).

The first ciguatera fish poisoning (CFP) in Portugal dates from 2008 when 11 people reported CFP symptoms after consuming a 30 kg amberjack caught around the Selvagens Islands (Madeira Archipelago). Since then, 49 human poisonings have been reported. The emergence of a new threat challenged scientists and regulators, as methods for toxic microalgae analyses and ciguatoxin (CTX) detection were not implemented. To minimise the risk of ciguatera, the Madeira Archipelago authorities interdicted fisheries in Selvagens Islands and banned the capture of amberjacks weighing more than 10 kg in the entire region of Madeira Archipelago. The accurate identification and quantification of the benthic toxin-producing algae species spreading to new areas require efforts in terms of both microscopy and molecular techniques. Two ciguatera-causing dinoflagellates, Gambierdiscus excentricus and Gambierdiscus australes, were identified in the Madeira Island and Selvagens sub-archipelago, respectively. Regarding the CTX analysis (N2a cell-based assay and LC-MS) in fish, the results indicate that the Selvagens Islands are a ciguatera risk area and that fish vectoring CTX are not limited to top predator species. Nevertheless, advances and improvements in screening methods for the fast detection of toxicity in seafood along with certified reference material and sensitive and selective targeted analytical methods for the determination of CTX content are still pending. This study aims to revise the occurrence of ciguatera cases in the Madeira Archipelago since its first detection in 2008, to discuss the risk management strategy that was implemented, and to provide a summary of the available data on the bioaccumulation of CTX in marine fish throughout the marine food web, taking into consideration their ecological significance, ecosystem dynamics, and fisheries relevance.

A review on aquatic toxins - Do we really know it all regarding the environmental risk posed by phytoplankton neurotoxins?

Aquatic toxins are potent natural toxins produced by certain cyanobacteria and marine algae species during harmful cyanobacterial and algal blooms (CyanoHABs and HABs, respectively). These harmful bloom events and the toxins produced during these events are a human and environmental health concern worldwide, with occurrence, frequency and severity of CyanoHABs and HABs being predicted to keep increasing due to ongoing climate change scenarios. These contexts, as well as human health consequences of some toxins produced during bloom events have been thoroughly reviewed before. Conversely, the wider picture that includes the non-human biota in the assessment of noxious effects of toxins is much less covered in the literature and barely covered by review works. Despite direct human exposure to aquatic toxins and related deleterious effects being responsible for the majority of the public attention to the blooms' problematic, it constitutes a very limited fraction of the real environmental risk posed by these toxins. The disruption of ecological and trophic interactions caused by these toxins in the aquatic biota building on deleterious effects they may induce in different species is paramount as a modulator of the overall magnitude of the environmental risk potentially involved, thus necessarily constraining the quality and efficiency of the management strategies that should be placed. In this way, this review aims at updating and consolidating current knowledge regarding the adverse effects of aquatic toxins, attempting to going beyond their main toxicity pathways in human and related models' health, i.e., also focusing on ecologically relevant model organisms. For conciseness and considering the severity in terms of documented human health risks as a reference, we restricted the detailed revision work to neurotoxic cyanotoxins and marine toxins. This comprehensive revision of the systemic effects of aquatic neurotoxins provides a broad overview of the exposure and the hazard that these compounds pose to human and environmental health. Regulatory approaches they are given worldwide, as well as (eco)toxicity data available were hence thoroughly reviewed. Critical research gaps were identified particularly regarding (i) the toxic effects other than those typical of the recognized disease/disorder each toxin causes following acute exposure in humans and also in other biota; and (ii) alternative detection tools capable of being early-warning signals for aquatic toxins occurrence and therefore provide better human and environmental safety insurance. Future directions on aquatic toxins research are discussed in face of the existent knowledge, with particular emphasis on the much-needed development and implementation of effective alternative (eco)toxicological biomarkers for these toxins. The wide-spanning approach followed herein will hopefully stimulate future research more broadly addressing the environmental hazardous potential of aquatic toxins.

Determination of intestinal absorption of the paralytic shellfish toxin GTX-5 using the Caco-2 human cell model.

Contributing to the human health risk assessment, the present study aims to evaluate the ability of paralytic shellfish toxins (PSTs) to cross the human intestinal epithelium by using the Caco-2 permeability assay. A crude extract prepared from the PST producer dinoflagellate Gymnodinium catenatum strain, GCAT1_L2_16, and the PST analogue gonyautoxin-5 (GTX-5) prepared from a certified reference material (CRM) were tested. In the conditions of the assay, none of the compounds altered Caco-2 viability, or the integrity of cell monolayers. The GTX-5 apparent permeability coefficients are 0.9×10-7 and 0.6×10-7 cm s-1 for the crude extract and CRM, respectively, thus, <10-6 cm s-1, which indicates that humans absorb this PST analogue poorly. The present study also reveals that, during a 90-min exposure, GTX-5 is not metabolised to a high extent by Caco-2 or retained in the Caco-2 cytoplasm. Since it is known that GTX-5 can be found in the spleen, liver or kidney of the victims, as well as in the urine samples of patients who consumed contaminated seafood, further research is needed to clarify the transport mechanisms involved, permeation time and dose-dependence, and the possible role of intestinal microflora.

A novel cyanobacterial geosmin producer, revising GeoA distribution and dispersion patterns in Bacteria.

Cyanobacteria are ubiquitous organisms with a relevant contribution to primary production in all range of habitats. Cyanobacteria are well known for their part in worldwide occurrence of aquatic blooms while producing a myriad of natural compounds, some with toxic potential, but others of high economical impact, as geosmin. We performed an environmental survey of cyanobacterial soil colonies to identify interesting metabolic pathways and adaptation strategies used by these microorganisms and isolated, sequenced and assembled the genome of a cyanobacterium that displayed a distinctive earthy/musty smell, typical of geosmin, confirmed by GC-MS analysis of the culture's volatile extract. Morphological studies pointed to a new Oscillatoriales soil ecotype confirmed by phylogenetic analysis, which we named Microcoleus asticus sp. nov. Our studies of geosmin gene presence in Bacteria, revealed a scattered distribution among Cyanobacteria, Actinobacteria, Delta and Gammaproteobacteria, covering different niches. Careful analysis of the bacterial geosmin gene and gene tree suggests an ancient bacterial origin of the gene, that was probably successively lost in different time frames. The high sequence similarities in the cyanobacterial geosmin gene amidst freshwater and soil strains, reinforce the idea of an evolutionary history of geosmin, that is intimately connected to niche adaptation.

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.

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.

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.

Species-specific real-time PCR cell number quantification of the bloom-forming cyanobacterium Planktothrix agardhii.

A species-specific method to detect and quantify Planktothrix agardhii was developed by combining the SYBR Green I real-time polymerase chain reaction technique with a simplified DNA extraction procedure for standard curve preparation. Newly designed PCR primers were used to amplify a specific fragment within the rpoC1 gene. Since this gene exists in single copy in the genome, it allows the direct achievement of cell concentrations. The cell concentration determined by real-time PCR showed a linear correlation with the cell concentration determined from direct microscopic counts. The detection limit for cell quantification of the method was 8 cells µL(-1), corresponding to 32 cells per reaction. Furthermore, the real-time qPCR method described in this study allowed a successful quantification of P. agardhii from environmental water samples, showing that this protocol is an accurate and economic tool for a rapid absolute quantification of the potentially toxic cyanobacterium P. agardhii.