First Report of Domoic Acid Production from Pseudo-nitzschia multistriata in Paracas Bay (Peru).

The Peruvian sea is one of the most productive ecosystems in the world. Phytoplankton production provides food for fish, mammals, mollusks and birds. This trophic network is affected by the presence of toxic phytoplankton species. In July 2017, samples of phytoplankton were obtained from Paracas Bay, an important zone for scallop (Argopecten purpuratus) aquaculture in Peru. Morphological analysis revealed the presence of the genus Pseudo-nitzschia, which was isolated and cultivated in laboratory conditions. Subsequently, the monoclonal cultures were observed by scanning electron microscopy (SEM), and identified as P. multistriata, based on both the morphological characteristics, and internal transcribed spacers region (ITS2) sequence phylogenetic analysis. Toxin analysis using liquid chromatography (LC) with high-resolution mass spectrometry (HRMS) revealed the presence of domoic acid (DA) with an estimated amount of 0.004 to 0.010 pg cell-1. This is the first report of DA from the coastal waters of Peru and its detection in P. multistriata indicates that it is a potential risk. Based on our results, routine monitoring of this genus should be considered in order to ensure public health.

Allelopathic effects of Margalefidinium polykrikoides and Gymnodinium impudicum in the growth of Gymnodinium catenatum.

Harmful algae blooms (HABs) are characterized for the coexistence of phytoplankton species with dynamic and complex biotic interactions (e.g., competition, symbiosis, predation, parasitism, allelopathy), that occur at fine temporal and spatial scales, and are relevant to understand the role that different species of phytoplankton play in the regulation of HABs. In this work the allelopathic effects of Margalefidinium polykrikoides (=Cochlodinium polykrikoides) and Gymnodinium impudicum on Gymnodinium catenatum were evaluated. The allelopathic abilities of M. polykrikoides and G. impudicum were investigated in bi-algal culture experiments and in trials in which target species were co-cultured, separated by a 10 µm membrane to prevent a direct cell-to-cell contact; and also by the addition of different volumes of culture media without cells. For all trials, cells of each species were harvested during exponential phase and cultured together by triplicate at three relative abundances: 1:1 (200 Cells mL-1 of each species, G. catenatum and M. polykrikoides or G. impudicum), 2:1 (400 Cells mL-1 of G. catenatum and 200 Cells mL-1 of M. polykrikoides or G. impudicum), and 1:2 (200 cells mL-1 of G. catenatum and 400 Cells mL-1 of M. polykrikoides or G. impudicum). All bioassays were carried out by triplicate in 250 mL Erlenmeyer flasks with 150 mL of modified GSe medium with an initial inoculum of 200 or 400 Cells mL-1. During experiments G. catenatum abundances were enumerated daily. In bi-algal culture experiments mortalities of G. catenatum were from 50% to 100% after 48 h of cell contact with M. polykrikoides or G. impudicum. In the case of culture media without cells, only M. polykrikoides caused a decrease in the cell abundance and growth rate of G. catenatum. Morphological changes occurred in G. catenatum when in contact with M. polykrikoides and G. impudicum, such as membrane shedding, prominent nucleus, loss of flagella, cell lysis, as well as the separation of long chains into individual cells. These results suggest that in the natural environment M. polykrikoides and G. impudicum have allelopathic interactions in G. catenatum, which could negatively affect its growth and survival, indicating that these species could displace blooms of G. catenatum.

Phylogenetic relationships of Pseudo-nitzschia subpacifica (Bacillariophyceae) from the Mexican Pacific, and its production of domoic acid in culture.

Pseudo-nitzschia is a cosmopolitan genus, some species of which can produce domoic acid (DA), a neurotoxin responsible for the Amnesic Shellfish Poisoning (ASP). In this study, we identified P. subpacifica for the first time in Todos Santos Bay and Manzanillo Bay, in the Mexican Pacific using SEM and molecular methods. Isolates from Todos Santos Bay were cultivated under conditions of phosphate sufficiency and deficiency at 16°C and 22°C to evaluate the production of DA. This toxin was detected in the particulate (DAp) and dissolved (DAd) fractions of the cultures during the exponential and stationary phases of growth of the cultures. The highest DA concentration was detected during the exponential phase grown in cells maintained in P-deficient medium at 16°C (1.14 ± 0.08 ng mL-1 DAd and 4.71 ± 1.11 × 10-5 ng cell-1 of DAp). In P-sufficient cultures DA was higher in cells maintained at 16°C (0.25 ± 0.05 ng mL-1 DAd and 9.41 ± 1.23 × 10-7 ng cell-1 of DAp) than in cells cultured at 22°C. Therefore, we confirm that P. subpacifica can produce DA, especially under P-limited conditions that could be associated with extraordinary oceanographic events such as the 2013-2016 "Blob" in the northeastern Pacific Ocean. This event altered local oceanographic conditions and possibly generated the presence of potential harmful species in areas with economic importance on the Mexican Pacific coast.

Phylogeny and species delineation in the marine diatom Pseudo-nitzschia (Bacillariophyta) using cox1, LSU, and ITS2 rRNA genes: A perspective in character evolution.

Analyses of the mitochondrial cox1, the nuclear-encoded large subunit (LSU), and the internal transcribed spacer 2 (ITS2) RNA coding region of Pseudo-nitzschia revealed that the P. pseudodelicatissima complex can be phylogenetically grouped into three distinct clades (Groups I-III), while the P. delicatissima complex forms another distinct clade (Group IV) in both the LSU and ITS2 phylogenetic trees. It was elucidated that comprehensive taxon sampling (sampling of sequences), selection of appropriate target genes and outgroup, and alignment strategies influenced the phylogenetic accuracy. Based on the genetic divergence, ITS2 resulted in the most resolved trees, followed by cox1 and LSU. The morphological characters available for Pseudo-nitzschia, although limited in number, were overall in agreement with the phylogenies when mapped onto the ITS2 tree. Information on the presence/absence of a central nodule, number of rows of poroids in each stria, and of sectors dividing the poroids mapped onto the ITS2 tree revealed the evolution of the recently diverged species. The morphologically based species complexes showed evolutionary relevance in agreement with molecular phylogeny inferred from ITS2 sequence-structure data. The data set of the hypervariable region of ITS2 improved the phylogenetic inference compared to the cox1 and LSU data sets. The taxonomic status of P. cuspidata and P. pseudodelicatissima requires further elucidation.

Effects of terrestrial runoff on the coral communities in Santiago Bay, Colima, Mexican Pacific Coast

Abstract:Coral reef ecosystems are under stress of different origins, from factors including sedimentation, fragmentation, overfishing, and tourism, depending on their geographical location, depth, and proximity to recreation areas. In this study of Juluapan Lagoon, we examined the relationship between various water-quality attributes and the status indicators of the coral community at La Boquita reef. During 2011 (12 months of sampling), six monitoring stations in the Juluapan lagoon were established in order to observe the gradient of the distribution of the physicochemical parameters: three stations on the upper part, or BI, (S4 to S6) and three more in the lower part, or BII, (S1 to S3). A control station (CS) was located in the coral reef close to the lagoon channel, and where dissolved inorganic nutrients and cellular carbon content were determined. Additionally, we considered the monitoring of three of the eight largest coral structures/headlands of this community: the first was the station closest to the channel communicating with Juluapan lagoon (C1), the second was in the intermediate region with respect to that lagoon (C2), and the third was farthest from the channel (C3). Three line intercept transects (LIT) 30 m in length and perpendicular to the coast provenance were established in each station, and the parameters indicative of the status of corals were evaluated in an area of 60 m2 on each transect (180 m2 by the station). Turbidity, evidence of fishing, signs of settling, algal coverage, abundance of fish, rate of sediment, and coral health records (as for CoralWach chart) were determined in situ and from digital photographs and videos. Considering various community status indicators used in the reef area, we could recognize a state of general deterioration, which was reflected in the loss of 17% of coral coverage. The main anthropogenic disturbances in adjacent areas to La Boquita reef included wastewater discharges into the lagoon, tourist developments in the coastal zone, deforestation and erosion resulting from inappropriate development, and the runoff of nutrients from agricultural lands nearby the lagoon. All these significantly contributed to the nutrient-enriched waters of the lagoon, especially in summer, with negative effects on the coral community. Continued exposure to these factors, coupled with the lack of control over other anthropogenic components, has promoted the maintenance of a chronic stress state in the studied coral community. Our findings highlight the need for the development of appropriate coastal management and conservation policies for the coral reefs of the Mexican Pacific Coast. Rev. Biol. Trop. 64 (3): 1185-1200. Epub 2016 September 01.

ResumenDependiendo de la ubicación geográfica, la profundidad, y la proximidad a zonas de recreo, algunos arrecifes de coral están bajo estrés por factores que incluyen la sedimentación, la fragmentación, la sobrepesca y el turismo. En la presente investigación, se estudian los diferentes atributos de calidad de agua de la Laguna Juluapan e indicadores del estatus de la comunidad coralina La Boquita. Durante 2011 (12 meses de muestreo), seis estaciones de monitoreo fueron establecidas dentro de la laguna de Juluapan, a fin de observar el gradiente de distribución de los parámetros fisicoquímicos: tres estaciones en la parte interna ó B1, (S4 a S6), y tres en la parte más externa, ó B2 (S1 a S3); una estación control (CS) fue ubicada en la zona coralina cercana a la comunicación con la Laguna de Juluapan, fueron determinados nutrientes inorgánicos disueltos y contenido de carbono celular. Adicionalmente se realizó el monitoreo de tres de los ocho cabezales coralinos que conforman esta comunidad. El primero de ellos fue localizado en la cercanía de la intercomunicación con la Laguna Juluapan (C1), el segundo en la región intermedia respecto a la proximidad de dicho cuerpo lagunar (C2), y el tercero totalmente alejado respeto a la intercomunicación (C3). En cada estación fueron colocadas, tres líneas de transectos de intersección (LIT) de 30 m de longitud de forma perpendicular al perfil de la costa, a partir de ello, fueron evaluados diversos parámetros de la condición coralina, cubriendo un área de 60 m2 en cada transecto (180 m2 por estación). En cada área de transecto fue evaluada la turbidez, evidencias de daños por pesca, daños por sedimentación, cobertura de algas, abundancia de peces, tasa de sedimentación, además de obtenerse registros de la salud coralina usando como indicador la tarjeta CoralWatch in situ, a partir de tomas fotográficas y videos. Considerando los diferentes indicadores del estatus de la comunidad coralina estudiada, nosotros pudimos reconocer un estado general de deterioro, reflejado en la pérdida del 17% de la cobertura de coral. Las principales alteraciones antropogénicas en las zonas adyacentes al arrecife La Boquita corresponde a descargas de aguas residuales en la laguna, desarrollos turísticos en la zona costera, la deforestación y efecto erosivo asociado al desarrollo urbano inadecuado, así como el escurrimiento de nutrientes de tierras agrícolas alrededor de la laguna. Estos factores contribuyen de manera significativa al enriquecimiento de nutrientes a la laguna, sobre todo en verano, con efectos negativos sobre la comunidad de coral. La exposición continua a estos factores, junto con la falta de control sobre otros componentes antropogénicos, promueve la existencia de un estado de estrés crónico en la comunidad coralina estudiada. Nuestros resultados destacan la necesidad de implementar políticas apropiadas de conservación para los arrecifes coralinos de la costa del Pacífico mexicano.

The inhibitory effect of a non-yessotoxin-producing dinoflagellate, Lingulodinium polyedrum (Stein) Dodge, towards Vibrio vulnificus and Staphylococcus aureus

ResumenEl aumento de la resistencia bacteriana a los antibióticos ha causado preocupación a nivel mundial, por lo que se ha promovido la búsqueda de nuevos compuestos. Debido a su abundancia y diversidad, el fitoplancton marino constituye una importante fuente potencial de tales compuestos. La investigación sobre dinoflagelados ha llevado al descubrimiento de inhibidores de crecimiento bacteriano. El dinoflagelado marino Lingulodinium polyedrum causa proliferaciones algales en diferentes regiones del mundo, incluyendo México, y también se sabe que regula el crecimiento de otras especies en las aguas costeras. En este trabajo, se investiga la taxonomía de este dinoflagelado y se caracteriza la capacidad de sus extractos para inhibir el crecimiento de dos bacterias de importancia médica (Vibrio vulnificus y Staphylococcus aureus) en placas de cultivo de agar. La caracterización taxonómica se realizó por PCR y amplificación del gen de ITS, y se confirmó que la especie aislada en la costa del Pacífico de México fue L. polyedrum. Para demostrar el efecto inhibidor de los extractos de L. polyedrum, los cultivos se cosecharon por centrifugación. Los pellets de tres abundancias celulares se extrajeron con agua, metanol, hexano y cloroformo. Los experimentos en V. vulnificus mostraron una inhibición alta del crecimiento para los cuatro extractos, variando entre 77 y 98 %. Sorprendentemente, la inhibición del crecimiento fue menor cuando los extractos se originaron a partir de una mayor abundancia de células L. polyedrum, varía de 0 a 34 %. Para S. aureus, la inhibición del crecimiento también fue alta, pero no estadísticamente diferente para todos los extractos y abundancias de células, con un rango de 62 hasta 99 %. Esto resultados son prometedores para futuras aplicaciones farmacológicas. La cepa mexicana de L. polyedrum no produjo yesotoxinas detectables.

AbstractThe increased bacterial resistance to antibiotics has caused global concern, prompting the search for new compounds. Because of their abundance and diversity, marine phytoplankton are an important potential source of such compounds. Research on dinoflagellates has led to the discovery of inhibitors of bacterial growth. The marine dinoflagellate Lingulodinium polyedrum blooms in different regions of the world, including Mexico, and is also known to regulate the growth of other species in coastal waters. Here, we investigated the taxonomy of this dinoflagellate and characterized the ability of its extracts to inhibit the growth of two bacteria of medical importance (Vibrio vulnificus and Staphylococcus aureus). Taxonomic characterization was performed by PCR and gene amplification of ITS, and confirmed that the species isolated off the Pacific coast of Mexico was L. polyedrum. To prove the inhibitory effect of L. polyedrum extracts, cultures were harvested by centrifugation. Pellets from three cellular abundances were extracted with water, methanol, hexane and chloroform. The experiments on V. vulnificus showed a high growth inhibition for the four extracts, ranging from 77 to 98 %. Surprisingly, the growth inhibition was lower when the extracts originated from a higher L. polyedrum cell abundance, ranging from 0 to 34 %. For S. aureus, the growth inhibition was also high, but not statistically different for all extracts and cell abundances, ranging from 62 to 99 %. This study obtained promising results for future pharmacological applications. Our Mexican strain of L. polyedrum did not produce any detectable yessotoxins. Rev. Biol. Trop. 64 (2): 805-816. Epub 2016 June 01.

The inhibitory effect of a non-yessotoxin-producing dinoflagellate, Lingulodinium polyedrum (Stein) Dodge, towards Vibrio vulnificus and Staphylococcus aureus.

The increased bacterial resistance to antibiotics has caused global concern, prompting the search for new compounds. Because of their abundance and diversity, marine phytoplankton are an important potential source of such compounds. Research on dinoflagellates has led to the discovery of inhibitors of bacterial growth. The marine dinoflagellate Lingulodinium polyedrum blooms in different regions of the world, including Mexico, and is also known to regulate the growth of other species in coastal waters. Here, we investigated the taxonomy of this dinoflagellate and characterized the ability of its extracts to inhibit the growth of two bacteria of medical importance (Vibrio vulnificus and Staphylococcus aureus). Taxonomic characterization was performed by PCR and gene amplification of ITS, and confirmed that the species isolated off the Pacific coast of Mexico was L. polyedrum. To prove the inhibitory effect of L. polyedrum extracts, cultures were harvested by centrifugation. Pellets from three cellular abundances were extracted with water, methanol, hexane and chloroform. The experiments on V. vulnificus showed a high growth inhibition for the four extracts, ranging from 77 to 98 %. Surprisingly, the growth inhibition was lower when the extracts originated from a higher L. polyedrum cell abundance, ranging from 0 to 34 %. For S. aureus, the growth inhibition was also high, but not statistically different for all extracts and cell abundances, ranging from 62 to 99 %. This study obtained promising results for future pharmacological applications. Our Mexican strain of L. polyedrum did not produce any detectable yessotoxins.

Effects of terrestrial runoff on the coral communities in Santiago Bay, Colima, Mexican Pacific Coast.

Coral reef ecosystems are under stress of different origins, from factors including sedimentation, fragmentation, overfishing, and tourism, depending on their geographical location, depth, and proximity to recreation areas. In this study of Juluapan Lagoon, we examined the relationship between various water-quality attributes and the status indicators of the coral community at La Boquita reef. During 2011 (12 months of sampling), six monitoring stations in the Juluapan lagoon were established in order to observe the gradient of the distribution of the physicochemical parameters: three stations on the upper part, or BI, (S4 to S6) and three more in the lower part, or BII, (S1 to S3). A control station (CS) was located in the coral reef close to the lagoon channel, and where dissolved inorganic nutrients and cellular carbon content were determined. Additionally, we considered the monitoring of three of the eight largest coral structures/headlands of this community: the first was the station closest to the channel communicating with Juluapan lagoon (C1), the second was in the intermediate region with respect to that lagoon (C2), and the third was farthest from the channel (C3). Three line intercept transects (LIT) 30 m in length and perpendicular to the coast provenance were established in each station, and the parameters indicative of the status of corals were evaluated in an area of 60 m2 on each transect (180 m2 by the station). Turbidity, evidence of fishing, signs of settling, algal coverage, abundance of fish, rate of sediment, and coral health records (as for CoralWach chart) were determined in situ and from digital photographs and videos. Considering various community status indicators used in the reef area, we could recognize a state of general deterioration, which was reflected in the loss of 17 % of coral coverage. The main anthropogenic disturbances in adjacent areas to La Boquita reef included wastewater discharges into the lagoon, tourist developments in the coastal zone, deforestation and erosion resulting from inappropriate development, and the runoff of nutrients from agricultural lands nearby the lagoon. All these significantly contributed to the nutrient-enriched waters of the lagoon, especially in summer, with negative effects on the coral community. Continued exposure to these factors, coupled with the lack of control over other anthropogenic components, has promoted the maintenance of a chronic stress state in the studied coral community. Our findings highlight the need for the development of appropriate coastal management and conservation policies for the coral reefs of the Mexican Pacific Coast.

A non-toxigenic but morphologically and phylogenetically distinct new species of Pseudo-nitzschia, P. sabit sp. nov. (Bacillariophyceae).

A new species of Pseudo-nitzschia (Bacillariophyceae) is described from plankton samples collected from Port Dickson (Malacca Strait, Malaysia) and Manzanillo Bay (Colima, Mexico). The species possesses a distinctive falcate cell valve, from which they form sickle-like colonies in both environmental samples and cultured strains. Detailed observation of frustules under TEM revealed ultrastructure that closely resembles P. decipiens, yet the new species differs by the valve shape and greater ranges of striae and poroid densities. The species is readily distinguished from the curve-shaped P. subcurvata by the presence of a central interspace. The morphological distinction is further supported by phylogenetic discrimination. We sequenced and analyzed the nuclear ribosomal RNA genes in the LSU and the second internal transcribed spacer, including its secondary structure, to infer the phylogenetic relationship of the new species with its closest relatives. The results revealed a distinct lineage of the new species, forming a sister cluster with its related species, P. decipiens and P. galaxiae, but not with P. subcurvata. We examined the domoic acid (DA) production of five cultured strains from Malaysia by Liquid chromatography-mass spectrometry (LC-MS), but they showed no detectable DA. Here, we present the taxonomic description of the vegetative cells, document the sexual reproduction, and detail the molecular phylogenetics of Pseudo-nitzschia sabit sp. nov.

Bloom of Gymnodinium catenatum in Bahía Santiago and Bahía Manzanillo, Colima, Mexico.

Gymnodinium bloom events are of concern, since they produce toxins, which have unfavorable consequences to marine ecosystems, human health and the economy. This report describes the physico-chemical conditions that were present during the algal bloom event on May 2010 in Bahía Manzanillo and Bahía Santiago, Colima, Mexico. For this, seawater nutrient analysis, phytoplankton counts, identification, and toxicity tests were undertaken. Nutrients in seawater were determined using colorimetric techniques, the higher concentrations (8.88 microM DIN, 0.78 microM PO4 and 24.34 microM SiO2) were related with upwelling waters that promoted the algal bloom that began after registering the year lowest sea-surface temperature, favoring the rapid growth of G. catenatum (up to 1.02 x 10(7) cells/L). Phytoplankton counting was carried out using sedimentation chambers and cells enumerated on appropriated area. The bloom persisted in the bays for approximately two weeks and was associated with toxicity (determined with HPLC) in local oysters (1525.8 microg STXeq/100g), and in phytoplankton (10.9 pg STXeq/cells) samples. Strong variations in cell toxicity (1.4 to 10.9pg STXeq/cells), most likely reflected the availability of inorganic nutrients. The toxin profile of the phytoplankton samples consisted of 11 toxins and resembled those recorded for several strains of G. catenatum isolated from other coastal areas of Mexico.

Bloom of Gymnodinium catenatum in Bahía Santiago and Bahía Manzanillo, Colima, Mexico

Gymnodinium bloom events are of concern, since they produce toxins, which have unfavorable consequences to marine ecosystems, human health and the economy. This report describes the physico-chemical conditions that were present during the algal bloom event on May 2010 in Bahía Manzanillo and Bahía Santiago, Colima, Mexico. For this, seawater nutrient analysis, phytoplankton counts, identification, and toxicity tests were undertaken. Nutrients in seawater were determined using colorimetric techniques, the higher concentrations (8.88μM DIN, 0.78μM PO4 and 24.34μM SiO2) were related with upwelling waters that promoted the algal bloom that began after registering the year lowest sea-surface temperature, favoring the rapid growth of G. catenatum (up to 1.02 x10(7)cells/L). Phytoplankton counting was carried out using sedimentation chambers and cells enumerated on appropriated area. The bloom persisted in the bays for approximately two weeks and was associated with toxicity (determined with HPLC) in local oysters (1525.8μg STXeq/100g), and in phytoplankton (10.9pg STXeq/cells) samples. Strong variations in cell toxicity (1.4 to 10.9pg STXeq/cells), most likely reflected the availability of inorganic nutrients. The toxin profile of the phytoplankton samples consisted of 11 toxins and resembled those recorded for several strains of G. catenatum isolated from other coastal areas of Mexico.

La proliferación de Gymnodinium son motivo de preocupación, debido a que en algunas circunstancias producen toxinas, que tienen consecuencias desfavorables para los ecosistemas marinos, la salud humana y la economía. Este trabajo describe las condiciones fisicoquímicas presentes durante una proliferación algal detectado en mayo de 2010 en la Bahía de Santiago y Bahía Manzanillo (Colima, México). La proliferación algal inició poco tiempo después de registrarse las temperaturas oceánicas superficiales más bajas del año, las cuales permitieron un aumento de las concentraciones de nutrientes (8.88μM DIN, 0.78μM PO4 and 24.34μM SiO2) que favorecieron el desarrollo de G. catenatum (hasta 1.02 x10(7)cel/L). Esta proliferación se detectó en las bahías durante dos semanas y fue relacionada con toxicidad en ostiones de la localidad (1525.8μg STXeq/100g) y en muestras de fitoplancton (10.9pg STXeq/cel). Fuertes variaciones en la toxicidad de G. catenatum (1.4 a 10.9pg STXeq/cel) pudieron reflejar la disponibilidad de nutrientes inorgánicos. El perfil de toxinas de las muestras del fitoplancton consistieron en 11 toxinas semejantes a las de varias cepas de G. catenatum aisladas de otras áreas de las costas de México.

Quantitative PCR coupled with melt curve analysis for detection of selected pseudo-nitzschia spp. (Bacillariophyceae) from the Northwestern Mediterranean sea.

The frequency and intensity of Pseudo-nitzschia spp. blooms along the coast of Catalonia have been increasing over the past 20 years. As species from this genus that are documented as toxigenic have been found in local waters, with both toxic and nontoxic species cooccurring in the same bloom, there is a need to develop management tools for discriminating the difference. Currently, differentiation of toxic and nontoxic species requires time-consuming electron microscopy to distinguish taxonomic features that would allow identification as to species, and cryptic species can still remain misidentified. In this study, cells of Pseudo-nitzschia from clonal cultures isolated from seawater were characterized to their species identity using scanning electron microscopy, and subsamples of each culture were used to create an internal transcribed spacer 1 (ITS-1), 5.8S, and ITS-2 ribosomal DNA database for development of species-specific quantitative PCR (qPCR) assays. Once developed, these qPCR assays were applied to field samples collected over a 2-year period in Alfaques Bay in the northwestern Mediterranean Sea to evaluate the possibility of a comprehensive surveillance for all Pseudo-nitzschia spp. using molecular methods to supplement optical microscopy, which can discern taxonomy only to the genus level within this taxon. Total Pseudo-nitzschia cell density was determined by optical microscopy from water samples collected weekly and compared to results obtained from the sum of eight Pseudo-nitzschia species-specific qPCR assays using duplicate samples. Species-specific qPCR followed by melt curve analysis allowed differentiation of amplicons and identification of false positives, and results correlated well with the total Pseudo-nitzschia cell counts from optical microscopy.

HOMOTHALLIC AUXOSPORULATION IN PSEUDO-NITZSCHIA BRASILIANA (BACILLARIOPHYTA)(1).

Most pennate diatoms are allogamous, and various types of mating systems have been described. In Pseudo-nitzschia, reproductive stages have been identified in some species, and it is generally accepted that the genus is mainly heterothallic. Here we report homothallic auxosporulation of Pseudo-nitzschia brasiliana Lundholm, Hasle et G. A. Fryxell. To our knowledge, this is the first verified description of homothallic sexual reproduction in the genus. Auxospore formation was observed in all 16 subclones derived from three initial clonal cultures of P. brasiliana. Pairing was followed by production of two gametes per gametangium, which fused to give two zygotes. Each zygote (early auxospore) was initially spherical and adhered to one girdle band of the parental frustule. The two auxospores tended to expand parallel to each other and perpendicular to the parental frustule. Elongation was synchronous, slightly asynchronous, or totally asynchronous. The entire process of sexual reproduction, from gamete formation to the appearance of the initial vegetative cells, took 2-4 d. The occurrence of sex in a homothallic species seems an advantageous life strategy for this species in that any encounter between cells of the right size class is potentially sexual.