A report on the outbreak of Zika virus on Easter Island, South Pacific, 2014.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus circulating in Asia and Africa. In 2013, a large outbreak was reported on the archipelago of French Polynesia. In this study, we report the detection and molecular characterization of Zika virus for the first time in Chile from an outbreak among the inhabitants of Easter Island. A total of 89 samples from patients suspected of having ZIKV infection were collected between the period from January to May, 2014. Molecular diagnosis of the virus was performed by RT-PCR followed by the sequencing of the region containing the NS5 gene. A comparison of the viral nucleic acid sequence with those of other strains of ZIKA virus was performed using the MEGA software. Fifty-one samples were found positive for ZIKV by RT-PCR analysis. Further analysis of the NS5 gene revealed that the ZIKV strains identified in Easter Island were most closely related to those found in French Polynesia (99.8 to 99.9% nt and 100% aa sequence identity). These results strongly suggest that the transmission pathway leading to the introduction of Zika virus on Easter Island has its origin in French Polynesia.

Exhaled breath condensate analysis after long distance races.

The impact of an endurance race on pulmonary pro-oxidative formation and lipoperoxidation was evaluated using exhaled breath condensate (EBC). 3 groups of 12, 12 and 17 healthy recreational runners of both sexes ran 10, 21.1 and 42.2 km, respectively. EBC samples were obtained before the run and at 20 and 80 min post run. Concentrations of H2O2, NO2 - , malondialdehyde and pH were determined. The 10 km group showed no post-run variations for H2O2 and NO2 - concentrations. The 21.1 km group showed significant increments for NO2 - , and H2O2 concentrations in 20 min and 80 min samples. The 42.2 km group, showed increased NO2 - concentration in 20 min and 80 min samples, while H2O2 concentration increased only in the 20 min sample. In the 10 and 42.2 km groups neither malondialdehyde concentration nor pH showed differences. The 42.2 km group exhibited ΔH2O2 and ΔNO2 - medians higher than the 10 km group. ΔpH median decreased in 21.1 and 42.2 km groups, exhibiting values significantly lower than the 10 km group. ΔH2O2 y ΔNO2 - correlated directly with race time, while ΔpH, correlated inversely. In conclusion, intense prolonged exercise favors the increase in pulmonary pro-oxidative levels, with no modifications on lipoperoxidation. Running time relates to the magnitude of acute post exercise pro-oxidative formation.

Environmental refuges increase performance of juvenile mussels Mytilus chilensis: Implications for mussel seedling and farming strategies.

Estuarine ecosystems are characterized by a wide physical-chemical variation that in the context of global change scenarios may be exacerbated in the future. The fitness of resident organisms is expected to be influenced by such variation and, hence, its study is a priority. Some of that variation relates to water vertical stratification, which may create "environmental refuges" or distinct layers of water with conditions favoring the fitness of some individuals and species. This study explored the performance of juvenile mussels (M. chilensis) settled in two distinctive water depths (1 m and 4 m) of the Reloncaví fjord (southern Chile) by conducting a reciprocal transplants experiment. Salinity, saturation state and the contents of CO3 in seawater were among the factors that best explained the differences between the two layers. In such environmental conditions, the mussel traits that responded to such variation were growth and calcification rates, with significantly higher values at 4 m deep, whereas the opposite, increased metabolic stress, was higher in mussels raised and transplanted to the surface waters (1 m). Such differences support the notion of an environmental refuge, where species like mussels can find better growth conditions and achieve higher performance levels. These results are relevant considering the importance of M. chilensis as a shellfish resource for aquaculture and a habitat forming species. In addition, these results shed light on the variable responses exhibited by estuarine organisms to small-scale changes in the characteristics of the water column, which in turn will help to better understand the responses of the organisms to the projected scenarios of climate global change.

Context-Dependence in parasite effects on keyhole limpets.

Parasites alter the reproductive performance of their hosts, limit their growth, and thereby modify the energy budget of these hosts. Experimental studies and theoretical models suggest that the outcome of the host-parasite interactions could be determined by ecological factors such as food availability levels in the local habitats. Nutrient inputs may affect the host's food resource availability with positive or negative effects on parasite infection rates and tolerance of infection, however this has not been specifically evaluated in natural systems. In this study, we evaluate the effects of parasitism by Proctoeces humboldti on body size, gonadosomatic index (GSI), and metabolic rate (oxygen consumption) of their second intermediate host Fissurella crassa limpets, under contrasting natural conditions of productivity (upwelling center vs upwelling shadow sites). Our results evidenced that parasitized limpets collected from the intertidal habitat influenced by coastal upwelling site showed greater shell length, muscular foot biomass and GSI as compared to non-parasitized limpets collected in the same site, and compared to parasitized and non-parasitized limpets collected from the sites under the influence of upwelling shadow conditions. Oxygen consumption was lower in parasitized limpets collected from the upwelling-influenced site than in the other groups, independent of age, suggesting reduced metabolic stress in infected individuals inhabiting these productive sites. Our results suggest that increased productivity in upwelling sites could mitigate the conflict for resources in the P. humboldti - F. crassa system, influencing where such interaction is found in the continuum between parasitism and mutualism. Since parasitism is ubiquitous in natural systems, and play important roles in ecological and evolutionary processes, it is important to analyze host-parasite interaction across a variety of ecological conditions, especially in biological conservation.

Local infiltration of gonyautoxin is safe and effective in treatment of chronic tension-type headache.

BACKGROUND: Gonyautoxin are phycotoxins, whose molecular mechanism of action is a reversible block of the voltage-gated sodium channels at axonal level, impeding nerve impulse propagation. OBJECTIVE: To evaluate clinical efficacy of gonyautoxin in the treatment of patients with chronic tensional-type headache. METHODS: Open trial from September 2004 to 2005 in Hospital Clínico Universidad de Chile. Twenty-seven patients with chronic tension-type headache were locally infiltrated with gonyautoxins (50 micrograms) in ten sites considered as pain trigger points in a fixed infiltration protocol. In each site, a volume of 200 microlitres was injected. EMG recording was performed before and immediately after infiltrations. Main outcome measures are where a significantly drop-off in acute headache pain score occurs and number of days without headache pain. RESULTS: No side effects were detected in the follow-up period. From base line of 2 weeks, 19 patients of 27 (70%) are the successfully responders to the treatment. They showed the remarkable immediate effect after infiltration demonstrated by trapezium EMG recording. Patients reported a fall in pain score 5 minutes post-injection from 5.0 +/- 2.8 to 1.6 +/- 1.6 (mean +/- SD). The responder showed an average of 8.1 +/- 9.9 weeks of headache pain-free, all of them without a second infiltration or use of any additional analgesic medication. DISCUSSION: The therapeutic properties of gonyautoxin local infiltration in chronic tension-type headache patients are shown to be safe and effective. This report describes a new therapy for chronic tension-type headache involving local infiltrations of gonyautoxins. The immediate headache pain relief effect shown only minutes after toxin infiltrations were the most remarkable feature of this protocol. This is the first gonyautoxins testing report in the treatment of chronic tension-type headache.

Role of temperature and carbonate system variability on a host-parasite system: Implications for the gigantism hypothesis.

Biological interactions and environmental constraints alter life-history traits, modifying organismal performances. Trematode parasites often impact their hosts by inducing parasitic castration, frequently correlated with increased body size in the host (i.e., gigantism hypothesis), which is postulated to reflect the re-allocation of energy released by the reduction in the reproductive process. In this study, we compared the effect of a trematode species on shell size and morphology in adult individuals of the intertidal mussels Perumytilus purpuratus (>20 mm) collected from two local populations of contrasting environmental regimes experienced in central-southern Chile. Our field data indicates that in both study locations, parasitized mussels evidenced higher body sizes (shell length, total weight and volume) as compared with non-parasitized. In addition, parasitized mussels from the southern location evidenced thinner shells than non-parasitized ones and those collected from central Chile, suggesting geographical variation in shell carbonate precipitation across intertidal habitats of the Chilean coast. In laboratory conditions, mussels collected from a local population in central Chile were exposed to two temperature treatments (12 and 18 °C). Parasitized mussels showed higher growth rates than non-parasitized, regardless of the seawater temperature treatments. However, the metabolic rate was not influenced by the parasite condition or the temperature treatments. Our field and laboratory results support the parasite-induced gigantism hypothesis, and suggest that both the thermal environment and geographic location explain only a portion of the increased body size, while the parasitic condition is the most plausible factor modulating the outcome of this host-parasite interaction.

Long-lasting, reversible and non-neurotoxic inactivation of hippocampus activity induced by neosaxitoxin.

BACKGROUND: Neosaxitoxin (NeoSTX) and related paralytics shellfish toxins has been successfully used as local anesthetic and muscle relaxants to treat a variety of ailments. The primary mechanism of action of these toxins occurs by blocking voltage-gated sodium channels with compounds such as TTX, lidocaine, or derivatives. However, most of these non-classical sodium channel blockers act with a reduced time effect as well as ensuing neurotoxicity. NEW METHOD: In this report, we show that the use of local NeoSTX injections inactivates the hippocampal neuronal activity reversibly with a by long-term dynamics, without neuronal damage. RESULTS: A single 10 ng/µl injection of NeoSTX in the dorsal CA1 region abolished for up to 48 h memory capacities and neuronal activity measured by the neuronal marker c-fos. After 72 h of toxin injection, the animals fully recover their memory capacities and hippocampal neuronal activity. The histological inspection of NeoSTX injected brain regions revealed no damage to the tissue or reactive gliosis, similar to vehicle injection. Acute electrophysiological recording in vivo shows, also, minimal spreading of the NeoSTX in the cerebral tissue. COMPARISON WITH EXISTING METHODS: Intracerebral NeoSTX injection showed longer effects than other voltage sodium channel blocker, with minimal spreading and no neuronal damage. CONCLUSION: NeoSTX is a new useful tool that reversibly inactivates different brains region for a long time, with minimal diffusion and without neuronal damage. Moreover, NeoSTX can be used as a valuable sodium channel blocker for many studies in vivo and with potential therapeutic uses.

The energetic physiology of juvenile mussels, Mytilus chilensis (Hupe): The prevalent role of salinity under current and predicted pCO2 scenarios.

As a result of human activities, climate forecasts predict changes in the oceans pCO2 and salinity levels with unknown impacts on marine organisms. As a consequence, an increasing number of studies have begun to address the individual influence of pCO2 and salinity but much remains to be done to understand their combined effects on the physiology and ecology of marine species. Our study addressed this knowledge gap by measuring the influence of current and predicted levels of pCO2 (380 and 1200 ppm, respectively) and salinity (20, 25 and 30 psµ) on the energetic physiology of juvenile mussels (Mytilus chilensis) from the south-eastern Pacific region. Our results indicate that a reduced salinity caused a significant reduction in clearance rate, absorption efficiency and scope for growth of this species. Meanwhile, an increase in pCO2 levels caused a reduction in excretion rates and interacted significantly with salinity in the rate of oxygen uptake measured in the mussel. These results suggest that potential changes in salinity might have a direct role on the physiology of M. chilensis. The effect of pCO2, although less prevalent among the variables measured here, did interact with salinity and is also likely to alter the physiology of this species. Given the ecological and economic importance of M. chilensis, we call for further studies exploring the influence of pCO2 across a wider range of salinities.

Intertidal pool fish Girella laevifrons (Kyphosidae) shown strong physiological homeostasis but shy personality: The cost of living in hypercapnic habitats.

Tide pools habitats are naturally exposed to a high degree of environmental variability. The consequences of living in these extreme habitats are not well established. In particular, little it is known about of the effects of hypercanic seawater (i.e. high pCO2 levels) on marine vertebrates such as intertidal pool fish. The aim of this study was to evaluate the effects of increased pCO2 on the physiology and behavior in juveniles of the intertidal pool fish Girella laevifrons. Two nominal pCO2 concentrations (400 and 1600µatm) were used. We found that exposure to hypercapnic conditions did not affect oxygen consumption and absorption efficiency. However, the lateralization and boldness behavior was significantly disrupted in high pCO2 conditions. In general, a predator-risk cost of boldness is assumed, thus the increased occurrence of shy personality in juvenile fishes may result in a change in the balance of this biological interaction, with significant ecological consequences.

Phosphoinositides in frog skeletal muscle: a quantitative analysis.

The contents of major phospholipids per g of wet wt. in frog skeletal muscle are: 5.3 mumol PC; 1.4 mumol PE; 1 mumol SM; 0.4 mumol PtdIns; 0.3 mumol CL; and 0.13 mumol PS. The quantities of polyphosphoinositides per g of wet wt. are: 181 nmol PtInsP; 28 nmol PtdInsP2; and 8 nmol lyso-PtdInsP2. The specific activity of labelling of the total muscle ATP attained by external incubation with [32P]Pi was found to be 57 dpm/nmol x g muscle wet wt. PtdInsP2, the highest labelled polyphosphoinositide, showed a specific activity of 64,000 dpm/nmol per g muscle wet wt., suggesting that high specific activity ATP may be compartmentalized in the local environment of the triads and used as a substrate by the PtdIns and PtInsP kinase in that region. PtdInsP2 which is the immediate precursor for the release of InsP3, is found at a significant concentration and strategically located for its postulated role as a substrate for the action of phosphoinositidase C. The presence of a novel endogenous polyphosphoinositide, lyso-PtdInsP2, in animal tissues is reported for the first time. Electrical stimulation leads towards a rapid catabolization of polyphosphoinositides revealed by reductions in the 3H- and 32P-labelling, suggesting that muscle excitation is associated with the activation of breaking down of polyphosphoinositides.

Synthesis of polyphosphoinositides in transverse tubule and sarcoplasmic reticulum membranes of frog skeletal muscle.

Synthesis of polyphosphoinositides has been studied in transverse (T-) tubule and sarcoplasmic reticulum (SR) membrane fractions of frog skeletal muscle, following 32P-labeling with [gamma-32P]ATP. Purified SR and T-tubule fractions respectively synthesize 9.4 +/- 0.8 and 71.9 +/- 9.8 pmol PtdInsP/mg per min, indicating nearly 8-fold higher activity of PtdIns kinase in the T-tubules than in the SR. The activity of this enzyme in both membrane systems is maximum at pH 7 and pCa 6. PtdInsP2 is synthesized from the endogenous PtdInsP, only in T-tubule membranes by the action of PtdInsP kinase. This lipid is the most intensely 32P-labeled phosphoinositide (181.7 +/- 9.2 pmol/mg per min) in these membranes. PtdIns kinase in the T-tubule and SR membranes, and PtdInsP kinase in the former are modulated by the free [Mg2+]. Loss of radiolabel from transiently maximal 32P-incorporation in polyphosphoinositides in T-tubule membranes, concomitant with a decrease in the ATP concentration in the incubation buffer, shows the occurrence of phosphoinositidases in these membranes. Under the conditions used, no such activities were evident in SR membranes. Compound 48/80, a mixture of condensation products of N-methyl-p-methoxyphenethylamine with formaldehyde, known to block phosphoinositidase C and phospholipase A2, causes a dose-dependent increase in the 32P-label of PtdInsP, in T-tubule membranes. The synthesis of lyso PtdInsP2, a deacylated form of PtdInsP2 which occurs in nearly equal quantities in both T-tubule and SR membranes, may result from a mechanism independent of phospholipase A2.

Phosphoinositides in giant barnacle muscle fibers: a quantitative analysis at rest and following electrical stimulation.

Quantitative data are presented on the composition of the major phospholipids in isolated giant barnacle muscle fibers. It is shown, using internal perfusion techniques, that the high specific activity of labeling of polyphosphoinositides in vivo is attained by the activities of specific kinases. Electrical stimulation causes a reduction in the specific activity of labeling of PtdInsP2. This phospholipid, which is the immediate precursor for the release of InsP3, is found at a significant concentration (40 nmol/g wet weight) in single barnacle muscle fibers, sufficient to support a role as precursors of second messengers. The rapid catabolization of PtdInsP2 in the absence of external Ca2+ suggests that E-C coupling in barnacle muscle may be associated with a voltage-dependent, Ca(2+)-independent, activation of the breakdown of polyphosphoinositides.

Biodiversity of rocky intertidal benthic communities associated with copper mine tailing discharges in northern Chile.

Copper mine tailings have been discharged around the city of Chanaral, in northern Chile, for more than 60 years. This report summarizes a 17-month long monitoring study of species richness and biodiversity at five intertidal sites around the point of the tailing discharge. Total dissolved copper in sites close to the point of discharge varied between 8.72 microg/l and 34.15 microg/l, showing that there has not been a significant reduction since 1994. However, species richness has increased, suggesting a possible recovery of the system. While diversity of sessile organisms correlates negatively with dissolved copper, diversity of mobile invertebrates did not correlate with the metal concentration. To explain the observed results we discuss the role of algal turf interference on the distribution of mobile invertebrates at reference sites, a top-down effect caused by the absence of carnivores at impacted sites, and an avoidance strategy by some species to reduce their contact with contaminated seawater.

Five classes Euroscore modification improves accuracy of prediction of postoperative mortality and possibly the length of mechanical ventilation of cardiac surgery patients.

AIM: The additive EuroSCORE system for predicting operative mortality of cardiac patients tends to underestimate the mortality risk of high risk patients and concomitantly to overestimate that of low risk patients. We propose a modification of stratification groups aiming at improving its precision. We also tested its ability to predict the length of postoperative mechanical ventilation of our patients. METHODS: The high risk group of the EuroSCORE system (>6 points) was divided into three additional groups (group I: 0-2 points, group II: 3-5 points, group III: 6-8 points, group IV: 9-13 points, group V: >14 points) thus producing a 5 classes system. In a group of 301 cardiac surgery patients operated on in a low volume cardiac center, we calculated the expected mortality rate for each EuroSCORE class, the calibration of the modified scoring system, the ROC and the corresponding AUC values and the relative risk of each predisposing factor used by the original EuroSCORE sytem. RESULTS: The proposed modification increased the discrimination ability of EuroSCORE in predicting mortality (Hosmer-Lemeshow P=0.78, ROC size: 0.791) and marginally affected its accuracy in predicting length of postoperative mechanical ventilation (Hosmer-Lemeshow: 0.11, ROC size: 0.711). Combined operations of CABG and valve replacement were shown to exert a statistically significant effect on mortality (odds ratio 3.85, CI: 1.15-12.87, P=0.028). CONCLUSION: The proposed modification of additive EuroSCORE can presumably increase its discrimination ability in predicting mortality of cardiac patients handled in a low volume cardiac center. The need for prolonged mechanical ventilation could be predicted with acceptable accuracy, possibly providing support in resource management.

A search for endogenous modulators of the GABA receptor in different regions of the rat CNS.

The possible existence of endogenous substances other than ?-aminobutyric acid (GABA), that can also bind to rat brain GABA receptors, has been investigated in synaptic membranes derived from whole rat brain, or from cerebral cortex; as well as in isolated synaptic vesicles obtained from cerebral cortex, striatum, hypothalamus, cerebellum and spinal cord and in the superfusion fluid of electrically stimulated brain cortex slices, where a GABA-like substance is released by a calcium-dependent process. The detector used to study the presence of such presumed non-GABA endogenous ligands, were frozen and thawed rat brain synaptic membranes, that had been treated with 0.05% Triton X-100 and thoroughly washed. With this highly sensitive preparation, at least 5 pmol of GABA/ml could be detected. The extracts of the different preparations where these hypothetical ligands were looked for, were analyzed by means of gel filtration on Sephadez G-10, paper chromatography and high voltage electrophoresis. In a very great number of experiments performed, the only endogenous ligand detected was GABA itself. The possible influence of a number of peptides on binding of GABA to its receptor, was also looked for. No significant effect was found for substance P, neurotensin, cholecystokinin octapeptide sulfated, somatostatin, thyrotropin releasing hormone, luteinizing hormone releasing hormone, methionine enkephalin (all 10(?5) M), angiotensin II (10(?4) M), ACTH (3 x 10(?7)M), poly-l-lysine (30 ?g/ml) or poly-l-glutamate (30 ?g/ml).

Glutamate in rat brain cortex synaptic vesicles: influence of the vesicle isolation procedure.

Rat brain cortex synaptic vesicles have been isolated by 3 different procedures. The one of Hata et al. (J. Neurochem., 27 (1976) 139) gave synaptic vesicles with a high glutamate content, but also, as judged by [3H]ouabain binding and electron microscopy, with considerable contamination by plasma membrane vesicles. This did not allow a precise estimation of the glutamate content of each synaptic vesicle. The second procedure used (Life Sci., 21 (1977) 1075), in which the tissue is homogenized with an all glass homogenizer, yielded vesicles of higher purity, but with no glutamate. A slightly modified Kadota and Kadota procedure (J. Cell Biol., 58 (1973) 135) gave synaptic vesicles of a very high purity that were filtered on a Sepharose 4B column, and there, the synaptic vesicle fraction of highest purity was estimated to contain 3640 glutamate molecules in each glutamatergic vesicle. This is equivalent to an intravesicular concentration of 0.21 M, that is, at least 10 times higher than the glutamate concentration in the rat brain cortex.

Toxic effects, pharmacokinetics and clearance of saxitoxin, a component of paralytic shellfish poison (PSP), in cats.

Saxitoxin (STX) was the first known and most studied toxic component of paralytic shellfish poisoning (PSP). This toxin blocks neuronal transmission by binding to the voltage-gated Na+ channel. Although the toxin's mechanism of action is well known at the molecular level, there are still many unresolved questions about its pharmacokinetics and the PSP intoxication syndrome in mammals. Some of these questions are addressed in the present paper, which describes an experimental design which allowed us to follow the dynamics of STX poisoning in vivo. Adult cats were anaesthetized and permanently coupled to artificial ventilation, they were then intravenously injected with Low (2.7 microg of STX/kg) and high doses (10 microg of STX/kg) of toxin. Cardiovascular parameters such as blood pressure and electrocardiograms were recorded, urine and blood samples were collected during the four hours of experimental time. In order to quantify mass amount of STX, we used the post-column derivatization HPLC method. Urine and blood samples were cleansed using a C-18 Sep-Pack cartridge and ultrafree microcentrifuge filters. At the end of each experiment, the animals were killed and tissue samples from brain, liver, spleen and medulla oblongata were extracted to measure the amount of STX. As compared to control period, Low doses of STX made no difference in hemodynamics parameters. In contrast, high doses drastically reduced blood pressure, produced myocardial failure and finally cardiac arrest. Administration of 2.5 microg/kg x min of dobutamine restored hemodynamics parameters and allowed the animal to overcome the shock. With high doses, the calculated STX renal clearance in cats is 0.81 ml/min x kg(-1). This valued corresponds to 20.25% of the reported inulin renal clearance. Nevertheless with Low doses the STX renal clearance is 3.99 ml/min x kg(-1). This data suggest that in cats with normal cardiovascular parameters and diuresis, the STX excretion mainly involves glomerular filtration. During experimental time, no PSP toxins other than STX was detected in the body fluids and tissue samples analyzed, indicating that the mammals can not metabolize this molecule. STX was found in intensely irrigated organs such as the liver and spleen but also in the central nervous system (brain and medulla oblongata), showing that STX was capable of crossing the blood brain barrier.

Inhibitory effects of Microcystis aeruginosa toxin on ion pumps of the gill of freshwater fish.

A microsomal fraction enriched in ion pump enzymes was isolated from the gill of the carp (Cyprinus carpio Linneo). Mg(2+)-dependent (Na+ + K+), Na+, HCO3- and Ca(2+)-stimulated ATPase activities were studied following treatment with microcystin-LR-like toxin, the major toxic component isolated from Microcystis aeruginosa culture. These enzyme activities were inhibited in a dose-dependent manner. The maximum inhibition of each enzyme, induced with nM concentration of the toxin, was similar to that produced by inhibitors specific for each ATPase activity. The Mg(2+)-ATPase activity and non-specific hydrolysis of ATP were unaffected. These results strongly suggest that the massive fish death during M. aeruginosa blooms may result from the loss of ion homeostatic processes produced by the inhibitory action of microcystin on the ion pumps of gill chloride cells.

The first evidence of paralytic shellfish toxins in the fresh water cyanobacterium Cylindrospermopsis raciborskii, isolated from Brazil.

The blooms of toxic cyanobacteria (blue-green algae) are causing problems in many countries. During a screening of toxic freshwater cyanobacteria in Brazil, three strains isolated from the State of Sao Paulo were found toxic by the mouse bioassay. They all were identified as Cylindrospermopsis raciborskii by a close morphological examination. Extracts of cultured cells caused acute death to mice when injected intraperitoneally after developing neurotoxic symptoms which resembled to those caused by paralytic shellfish toxins. The analysis of the sample by HPLC-FLD postcolumn derivatization method for paralytic shellfish toxins resulted in the detection of several saxitoxin analogs. To avoid being misled by false peaks, the sample was reanalyzed after purification and also under the different postcolumn derivatizing conditions. Finally, the newly developed LC-MS method for paralytic shellfish toxins was applied to unambiguously identify the toxins. One isolate produced neosaxitoxin predominantly with saxitoxin as a minor component. The other two showed identical toxin profiles containing saxitoxin and gonyautoxins 2/3 isomers in the ratio of 1:9. This is the first evidence of paralytic shellfish toxins in this species and also the occurrence of the toxin producing cyanobacterium in South American countries.

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.