First Report of Paralytic Shellfish Toxins in Marine Invertebrates and Fish in Spain

A paralytic shellfish poisoning (PSP) episode developed in summer 2018 in the Rías Baixas (Galicia, NW Spain). The outbreak was associated with an unprecedentedly intense and long-lasting harmful algal bloom (HAB) (~one month) caused by the dinoflagellate Alexandrium minutum. Paralytic shellfish toxins (PSTs) were analyzed in extracts of 45 A. minutum strains isolated from the bloom by high-performance liquid chromatography with post-column oxidation and fluorescence detection (HPLC-PCOX-FLD). PSTs were also evaluated in tissues from marine fauna (invertebrates and fish) collected during the episode and in dolphin samples. The analysis of 45 A. minutum strains revealed a toxic profile including GTX1, GTX2, GTX3 and GTX4 toxins. With regard to the marine fauna samples, the highest PSTs levels were quantified in bivalve mollusks, but the toxins were also found in mullets, mackerels, starfish, squids and ascidians. This study reveals the potential accumulation of PSTs in marine invertebrates other than shellfish that could act as vectors in the trophic chain or pose a risk for human consumption. To our knowledge, this is the first time that PSTs are reported in ascidians and starfish from Spain. Moreover, it is the first time that evidence of PSTs in squids is described in Europe.     

Investigation of extraction and analysis techniques for Lyngbya wollei derived Paralytic Shellfish Toxins

Paralytic Shellfish Toxins (PSTs) are highly toxic metabolic by-products of cyanobacteria and dinoflagellates. The filamentous cyanobacterium Lyngbya wollei produces a unique set of PSTs, including L. wollei toxins (LWT) 1-6. The accurate identification and quantification of PSTs from Lyngbya filaments is challenging, but critical for understanding toxin production and associated risk, as well as for providing baseline information regarding the potential for trophic transfer. This study evaluated several approaches for the extraction and analysis of PSTs from field-collected L. wollei dominated algal mats. Extraction of PSTs from lyophilized Lyngbya biomass was assessed utilizing hydrochloric acid and acetic acid at concentrations of 0.001-0.1 M. Toxin profiles were then compared utilizing two analysis techniques: pre-column oxidation (peroxide and periodate) High Performance Liquid Chromatography (HPLC) with Fluorescence (FL) detection and LC coupled with Mass Spectrometry (MS). While both acid approaches efficiently extracted PSTs, hydrochloric acid was found to convert the less toxic LWT into the more toxic decarbamoylgonyautoxins 2&3 (dcGTX2&3) and decarbamoylsaxitoxin (dcSTX). In comparison, extraction with 0.1 M acetic acid preserved the original toxin profile and limited the presence of interfering co-extractants. Although pre-chromatographic oxidation with HPLC/FL was relatively easy to setup and utilize, the method did not resolve the individual constituents of the L. wollei derived PST profile. The LC/MS method allowed characterization of the PSTs derived from L. wollei, but without commercially available LWT 1-6 standards, quantitation was not possible for the LWT. In future work, evaluation of the risk associated with L. wollei derived PSTs will require commercially available standards of LWT 1-6 for accurate determinations of total PST content and potency.     

Response patterns of cells in the feline caudal nucleus reticularis gigantocellularis after noxious trigeminal and spinal stimulation

Nucleus reticularis gigantocellularis has been shown, using both behavioral and physiological techniques, to be involved in the processing of nociceptive information in spinal systems. This investigation was designed to characterize the response patterns of nucleus reticularis gigantocellularis neurons to both spinal (superficial radial and sciatic nerve) and trigeminal (tooth pulp) noxious stimuli. One hundred and sixty-two neurons were studied using a poststimulus-time histogram analysis. Neurons in nucleus reticularis gigantocellularis were classified in four categories based on their responses to noxious stimuli: (i) 51% of the neurons responded to noxious stimuli delivered to all stimulus sites noted above with a short-latency, short-duration excitatory period, followed by a long-duration period of suppressed activity relative to control levels (“E-S cells”); (ii) 25% of the neurons studied responded to all noxious stimuli tested only with an excitatory response (“E cells”); (iii) 6% of the neurons responded to all noxious stimuli only with a period of suppressed activity (“S cells”) (some S cells had a period of increased activity after the period of suppression); (iv) 18% of the neurons had mixed responses, with the response depending on the site of stimulation (“M cells”). Except for M cells, each cell tended to respond with a characteristic response pattern, regardless of the site of stimulation.