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Dinoflagellates of the genera Gambierdiscus and Fukuyoa are known to produce several bioactive compounds including the potent neurotoxic ciguatoxins (CTXs) which are able to accumulate in fish and through the food web. When humans ingest fish contaminated with CTXs, it can result in an intoxication named ciguatera. Although not all the currently recognized species are able to produce toxins, G. australes and G. excentricus have been highlighted to be the most abundant and toxic among the species present in the Atlantic. Even though the genera Gambierdiscus and Fukuyoa are endemic to tropical areas, recently their presence was recorded in subtropical and temperate regions. In this work, the development of three molecular assays for the detection of the Gambierdiscus and Fukuyoa genera and for G. australes and G. excentricus species, based on the combination of recombinase polymerase amplification with detection via hybridization, is successfully described. Furthermore, a remarkable limit of detection of a single cell was achieved. Additionally, six different species have been used to check the ability of each primer set to give an amplified product, even in presence of potentially interfering non-target DNAs. Therefore, these developments provide a rapid and cost-effective strategy for detection of both genera and two of the most toxic species, which will undoubtedly contribute to reliable screening of samples and ciguatera risk assessment, guaranteeing seafood safety and protection of human health. info:eu-repo/semantics/acceptedVersion

The assessment of population structure and genetic diversity is crucial for the management and conservation of threatened species. Natural and artificial barriers to dispersal (i.e., gene flow) increase populations’ differentiation and isolation by reducing genetic exchange and diversity. Freshwater ecosystems are highly fragmented because of human activities. Threatened species with small population sizes are more sensitive to habitat fragmentation effects. Here, we investigate the genetic population structure and gene flow among seven populations of Aphanius sophiae in the Kor Basin by using sequences of the complete Cyt b gene and otolith morphometry. The Cyt b gene showed low level of genetic variation, only 4.12% of the identified sites were variable, and 2.42% were parsimony informative. Overall, haplotype diversity was low to moderate and nucleotide diversity was low to extremely low. Fish populations exhibited high levels of genetic differentiation, suggesting limited gene flow among them. These differences were obtained not only among geographically distant populations, but also among neighboring localities. Genetic population structure was supported by the AMOVA analysis and by the haplotype network (only one of 21 haplotypes were shared by two localities). Otolith morphometric analysis was in agreement with genetic results, the two most distant and isolated populations were clearly separated, and genetically close populations showed less differences in morphometry. A significant pattern of isolation by distance was also detected among A. sophiae populations, with genetic distance more correlated with hydrological distance than with geographic distance. Results suggested that limited gene flow due to habitat fragmentation is an important factor contributing to genetic structuring and to the loss of genetic variation of A. sophiae populations. Aphanius sophiae population structure seems to be the result of habitat fragmentation and water pollution, but other factors such as introduced species should be considered. Given the high degree of genetic structuring, the definition of conservation groups is of particular importance for A. sophiae, which should be considered endangered according to the IUCN criteria. Conservation plans must recognize the genetic independence of populations and manage separately preventing the loss of locally adapted genotypes. info:eu-repo/semantics/acceptedVersion

Ciguatera fish poisoning (CFP) is one of the most relevant seafood-borne diseases worldwide. It is caused by the ingestion of fish containing ciguatoxins (CTXs), lipophilic marine toxins produced by microalgae of the genera Gambierdiscus and Fukuyoa that accumulate into fish flesh and through the food webs. CFP is characterized by severe neurological, gastrointestinal, and cardiovascular disorders and affects approximately between 50,000 and 500,000 consumers annually worldwide. Real incidence of CFP is difficult to ascertain, due to under-reporting and misdiagnosis. Here, the first electrochemical immunosensor for the detection of CTXs is presented. Three different monoclonal antibodies (mAbs), two capture (3G8, 10C9) and a detector (8H4), were merged in a sandwich configuration for the combined detection of two main groups of CTX congeners (CTX1B and CTX3C ). Initially, the applicability of the immunosensor has been demonstrated with the analysis of fish samples coming from La Reunion island, providing results that correlate with mouse bioassay and cell-based assay. Then, fish coming from Mediterranean waters were analysed, giving promising results. Finally, extracts from Gambierdiscus and Fukuyoa were screened, allowing the separate detection of the two groups of CTX congeners, and giving new information regarding the toxin production of the genera. The developed bioanalytical tool is user-friendly, and can help to mitigate ciguatera risk, contributing to the protection of consumers health.

Ciguatera fish poisoning is one of the most relevant seafood-borne illnesses worldwide. It is caused by the ingestion of fish contaminated by ciguatoxins (CTXs). Primary producers of CTXs are dinoflagellates of the genera Gambierdiscus and Fukuyoa. This study is focused on the development of bioanalytical tools for the detection of Gambierdiscus and Fukuyoa. To achieve this objective, Recombinase Polymerase Amplification (RPA), which consists of an isothermal DNA amplification during a short period (30 minutes), was combined with an enzyme-linked oligonucleotide assay (ELONA). To evaluate the specificity of the RPA-ELONA, firstly primers for the genera Gambierdiscus/Fukuyoa were exposed to genomic DNA of different species (G. australes, G. excentricus, G. belizeanus, G. balechi and F. paulensis) and other microalgae used as controls (O. cf. ovata, P. lima and C. monotis). The same genomic DNA pools were also tested with species-specific primers for Gambierdiscus australes and Gambierdiscus excentricus. Finally, DNA was extracted from single cells of the previously mentioned genera and species, and tested with all the primer sets. For both the experiments, detection was achieved only when combining capture probes with their target RPA product, and no significant responses were observed in the presence of non-target DNA. Obtained results demonstrate the ability of the system to discriminate not only the genus Gambierdiscus/Fukuyoa from other microalgae, but also G. australes and G. excentricus species from their congeners. Furthermore, the limit of detection is as low as a single cell. Acknowledgments: The research has received funding from the Ministerio de Ciencia, Innovacion y Universidades through the CIGUASENSING (BIO2017-87946-C2-2-R) project. The authors acknowledge support from CERCA Programme/Generalitat de Catalunya. G. Gaiani acknowledges IRTA-Universitat Rovira i Virgili-Banco Santander for her PhD grant (2018PMF-PIPF-19).

Seafood contamination with marine toxins due to harmful algal blooms (HAB’s) is a global public health issue on the rise. Most Countries have monitoring programs for the detection of toxins in shellfish of toxic phytoplankton in seawater to prevent consumer intoxifications. The use of solid phase adsorbent and toxin tracking (SPATT) technology as toxin detection straight from the aquatic environment could complement the labour-intensive traditional monitoring methods. In this work, several types of cyclodextrins (cyclic oligomers with a conical structure and an internal cavity) have been evaluated as novel materials for SPATT. Cyclodextrins were tested at Masnou harbour (Catalonia, NW Mediterranean) during a Dinophysis sp. bloom. The cyclodextrins and the commercial Diaion (HP-20) were deployed twice for a 1-week period at five different locations of Masnou harbour. At the time of the experiment, Dinophysis sp. reached abundances as high as 91 341 cells /L. Successful accumulations of the lipophilic marine toxins; okadaic acid (OA) and pectenotoxin-2 (PTX2) were quantified by Liquid Chromatography- tandem Mass Spectrometry (LC-MS/MS). Higher levels of PTX2 were found in all cyclodextrins whereas OA and PTX2 contents were similar in the commercial resin. Accumulation of OA was higher in the commercial resin than in cyclodextrins, but these proved best for PTX2 adsorption. A clear correlation between cell abundance and toxin accumulation was observed. The research has received funding from the Ministerio de Ciencia, Innovacion y Universidades (MICINN), the Agencia Estatal de Investigacion (AEI) and the Fondo Europeo de Desarrollo Regional (FEDER) through the CIGUASENSING project (BIO2017-87946-C2-1-R and BIO2017-87946-C2-2-R). The authors acknowledge support from CERCA Programme/Generalitat de Catalunya.

The effects of dietary phospholipids (PL) on the performance of juvenile stellate sturgeon (Acipenser stellatus) was evaluated in terms of growth and feed efficiency parameters, muscle and liver fatty acid profiles, activity of digestive and antioxidative stress enzymes, and expression of lipoprotein lipase (lpl) and insulin-like growth factor (igf1). For this purpose, seven isoproteic (44% crude protein) and isolipidic (17% crude fat) diets containing graded levels of soybean lecithin (0, 1, 2, 4, 6, 8 and 10%) were prepared, resulting in 0.3, 0.9, 1.6, 2.7, 3.9, 5.3 and 5.4% of dietary PLs, respectively. At the end of the nutritional study (75 days), we found that there was a positive quadratic polynomial response between growth performance parameters and dietary PLs; somatic growth increased with increasing dietary PL levels up to 3.9% when growth parameters remained stable. Dietary PLs reduced the accumulation of fat stores in the liver and up-regulated the expression of the lipoprotein lipase gene, confirming the important role of this enzyme in incorporating plasma lipids into tissues, whereas the activities of CAT and SOD showed a positive linear increase with dietary PL levels. Increasing dietary PLs from 0.9 to 3.9% promoted the activity of gastric (pepsin) and pancreatic (trypsin, chymotrypsin, bile salt-activated lipase and α-amylase) enzymes, whereas higher inclusion levels of PLs did not provide any advantage in terms of A. stellatus digestive capacities. The analysis of fatty acid profiles in diets and selected tissues (liver and muscle) showed the capacity of A. stellatus juveniles to desaturate and elongate linoleic (C18:2n–6) and alpha-linolenic (C18:3n–3) acids to arachidonic (C20:4n-6), eicosapentaenoic (C20:5n-3) and docosahexaenoic (C22:6n-3) acids. Based on the results of growth performance, feed efficiency and physiological parameters, the inclusion of 3.9% of PLs in compound diets for juvenile stellate sturgeon are recommended. info:eu-repo/semantics/acceptedVersion

Consumption of seafood contaminated with ciguatoxins (CTXs) leads to a foodborne disease known as ciguatera. Primary producers of CTXs are epibenthic dinoflagellates of the genera Gambierdiscus and Fukuyoa. In this study, thirteen Gambierdiscus and Fukuyoa strains were cultured, harvested at exponential phase, and CTXs were extracted with an implemented rapid protocol. Microalgal extracts were obtained from pellets with a low cell abundance (20,000 cell/mL) and were then analyzed with magnetic bead (MB)-based immunosensing tools (colorimetric immunoassay and electrochemical immunosensor). It is the first time that these approaches are used to screen Gambierdiscus and Fukuyoa strains, providing not only a global indication of the presence of CTXs, but also the ability to discriminate between two series of congeners (CTX1B and CTX3C). Analysis of the microalgal extracts revealed the presence of CTXs in 11 out of 13 strains and provided new information about Gambierdiscus and Fukuyoa toxin profiles. The use of immunosensing tools in the analysis of microalgal extracts facilitates the elucidation of further knowledge regarding these dinoflagellate genera and can contribute to improved ciguatera risk assessment and management. info:eu-repo/semantics/acceptedVersion

Summary We report the first description of the ontogenic development of the axial skeleton and unpaired fins of hatchery reared bay snook (Petenia splendida, Perciformes: Cichlidae) from hatching (5.3–5.5 mm in standard length, SL) to the juvenile stage (16.1–17.5 mm SL) by means of alcian blue-alizarin red staining. The study of axial skeleton development was complemented by digital analysis of its level of ossification (number of red pixels) in order to identify major changes in vertebral column and caudal fin complex development. The main events of the skeletogenesis of the vertebral column and unpaired fins in P. splendida larvae were comprised between the onset of notochord segmentation and completion of the caudal fin complex at 6 (171 degree days post hatch, ddph; 6.4–6.5 mm SL) and 24 days post hatching (dph) (684 ddph, 8.9–9.4 mm SL), respectively; whereas the anal and dorsal fins completed morphogenesis at older ages (36 dph, 1026 ddph, 11.0–11.8 mm SL). In this sense, the development of a subcarangiform swimming mode in early juveniles was achieved with complete ossification of vertebral bodies and caudal lepidotrichia, as well as partial ossification of the caudal-fin complex elements (hypurals, epurals, uroneural and parahypural). This information can be considered as a reference for other studies evaluating the quality of larvae and the influence of rearing biotic and abiotic factors in the skeletogenesis of this cichlid species and the occurrence of skeletal deformities.

The zebra mussel (Dreissena polymorpha Pallas, 1771) and the quagga mussel (D. rostriformis Deshayes, 1838) are successful invasive bivalves with substantial ecological and economic impacts in freshwater systems once they become established. Since their eradication is extremely difficult, their detection at an early stage is crucial to prevent spread. In this study, we optimized and validated a qPCR detection method based on the histone H2B gene to quantify combined infestation levels of zebra and quagga mussels in environmental DNA samples. Our results show specific dreissenid DNA present in filtered water samples for which microscopic diagnostic identification for larvae failed. Monitoring a large number of locations for invasive dreissenid species based on a highly specific environmental DNA qPCR assay may prove to be an essential tool for management and control plans focused on prevention of establishment of dreissenid mussels in new locations We are indebted to different colleagues for zebra mussel sample collections: Concha Durán (Ebro Hydrografic Confederation, Spain) for the logistic advice; Anna Terrats and Carolina Solà (Catalan Water Agency from the Government of Catalonia, Spain) for the filtered-water environmental samples. We would thank to Mariona Palacios for her collaboration in the analysis into her Final Master Project. This research was carried out within the objectives of the research project CGL200909407 of the Spanish Ministerio de Ciencia e Innovación (MICINN)