New culture approaches for yessotoxin production from the dinoflagellate Protoceratium reticulatum

Fed-batch and perfusion cultures were carried out in a traditional glass 2-L bioreactor with the toxic dinoflagellate Protoceratium reticulatum. The maximum cell concentration obtained was 2.3 x 105 cell.mL-1, which is almost 1 order of magnitude higher than the maximum previously referenced for this species. L1 medium was shown to be clearly deficient in nitrate and phosphate for this strain, and addition of highly concentrated aliquots of these nutrients allowed higher cell concentrations to be obtained. This species consumed high amounts of nitrate and phosphate, 2.1 x 10-3 and 2.3 x 10-4 micromol.h-1.cell-1, respectively. However, this consumption produced a very low number of cells compared to other classes of microalgae, indicating that this species is, like other dinoflagellates, a poor competitor in terms of utilization of inorganic nutrients. Higher production of toxins and pigments was strongly associated with cell number in the culture, with maximum values of 700 ng.mL-1 and 1321 microg.mL-1, respectively. Most yessotoxins remained within the cells and not in the cell-free culture medium, and their production was not related to either the age of the culture or the cell growth phase.     

Phytoplankton photosynthetic efficiency and primary production rates estimated from fast repetition rate fluorometry at coastal embayments affected by upwelling (Rias Baixas, NW of Spain)

Previous studies have not always found a significant relationshipbetween fast repetition rate fluorometry (FRRF)-derived and¹⁴C-based primary production rates. This apparent discrepancymight be related to environmental control of the coupling betweenphotosynthetic electron transport through photosystem II (PSII)and carbon uptake in phytoplankton. In this study, we lookedat this relationship under upwelling conditions favourable forphytoplankton growth. The combination of both techniques allowedthe calculation of the quantum efficiency of carbon fixation,which averaged 0.12 mol C (mol quanta)^–1 indicating thatphytoplankton populations were very efficient in convertingabsorbed photons into photosynthetically produced organic carbon.The tight coupling observed between phytoplankton photosyntheticelectron transport through PSII and carbon uptake resulted ina statistically significant linear relationship between FRRF-derivedand ¹⁴C-based carbon incorporation rates, with a slope of 1.43.In conclusion, the results of the this study indicate that FRRFcan be a useful tool to derive high spatial and temporal resolutionprimary production estimates under environmental conditionsfavouring the close coupling between PSII electron transportand carbon uptake, such as those characteristic of this coastalupwelling system.     

Unusual Circulation Patterns of the Rias Baixas Induced by Minho Freshwater Intrusion (NW of the Iberian Peninsula)

The Minho River, situated 30 km south of the Rias Baixas, is the most important freshwater source flowing into the Western Galician coast (NW of the Iberian Peninsula). The buoyancy generated by the Minho estuarine plume can reverse the normal circulation pattern inside the Rias Baixas affecting the exchange between the Rias and the ocean, changing the input of nutrients. Nevertheless, this inversion of the circulation patterns is not a well-monitored phenomenon. The only published results based on in situ data related to the presence of the Minho River plume inside the Rias de Vigo and Pontevedra correspond to an event measured on spring 1998. In this case unexpectedly higher inflow surface current velocities were found at the Ria de Pontevedra, located further away from Minho River. Thus, the main aim of this study is to research the main factors inducing this unusual pattern on the circulation of the Rias de Vigo and Pontevedra. A numerical model implementation of MOHID previously developed, calibrated, and validated for this coastal area was used. Several scenarios were performed in order to explain the individual effect of the Minho River, rivers discharging into each Rias, and estuarine morphology changes. According to the model results, the Minho River discharge is a key factor in the establishment of the negative circulation, while small rivers inside the Rias slightly attenuate this circulation. The negative circulation was stronger in Ria de Pontevedra independently of the distance of this coastal system from the Minho River mouth, showing that morphologic estuarine features are the main factor justifying the different local circulation patterns.     

Distribution of Castanea pollen in Galician honeys (NW Spain)

With the aim to know the importance of theCastanea pollen in the Galician honey(N. W. Spain), the interannual oscillations withinthe areas of production and the most activephenological period for bees, we have madethe melissopalynological analysis of: 599samples of honey collected during the period1990–1999 in Galicia, the production of 40beehives during three consecutive years(1990, 1991, 1992), the partial production during1990 of two beehives situated at distant pointsin our geography.     

The life history of the toxic marine dinoflagellate Protoceratium reticulatum (Gonyaulacales) in culture

Asexual and sexual life cycle events were studied in cultures of the toxic marine dinoflagellate Protoceratium reticulatum. Asexual division by desmoschisis was characterized morphologically and changes in DNA content were analyzed by flow cytometry. The results indicated that haploid cells with a C DNA content occurred only during the light period whereas a shift from a C to a 2C DNA content (indicative of S phase) took place only during darkness. The sexual life cycle was documented by examining the mating type as well as the morphology of the sexual stages and nuclei. Gamete fusion resulted in a planozygote with two longitudinal flagella, but longitudinally biflagellated cells arising from planozygote division were also observed, so one of the daughter cells retained two longitudinal flagella while the other daughter cell lacked them. Presumed planozygotes (identified by their longitudinally biflagellated form) followed two life-cycle routes: division and encystment (resting cyst formation). Both the division of longitudinally biflagellated cells and resting cyst formation are morphologically described herein. Resting cyst formation through sexual reproduction was observed in 6.1% of crosses and followed a complex heterothallic pattern. Clonal strains underwent sexuality (homothallism for planozygote formation and division) but without the production of resting cysts. Ornamental processes of resting cysts formed from the cyst wall under an outer balloon-shaped membrane and were fully developed in <1 h. Obligatory dormancy period was of ∼4 months. Excystment resulted in a large, rounded, pigmented, longitudinally biflagellated but motionless, thecate germling that divided by desmoschisis. Like the planozygote, the first division of the germling yielded one longitudinally biflagellated daughter cell and another without longitudinal flagella. The longitudinal biflagellation state of both sexual stages and of the first division products of these cells is discussed.     

The distribution of dinoflagellate cysts along the Galician (NW Spain) coast

We have studied the distribution of dinoflagellate cysts along10 Galician rías and part of their adjacent continentalshelf. Cyst abundance in the area averaged 856 cysts ml^–1,which is of the same order of magnitude as those found in otherareas of the western European coast. It was higher in the ríasthan in the shelf, having a very heterogeneous distribution,especially in the former. Cyst assemblages in these two areaswere different, suggesting that differences are due to cystproduction rather than to accumulation. Principal componentanalysis, cluster analysis, distribution of macroscopic characteristicsof cyst populations and distribution of single species suggestthat local factors control the distribution in the rías.Nevertheless, a general pattern that splits the whole area intotwo-to the north and to the south of the ría de Camariñas-canbe distinguished. This latter trend was also observed in theshelf and, in our opinion, It should be attributed to threeconcurrent causes: the effect of different upwelling intensitiesor frequencies, the effect of the different numbers and sizesof the ría in each area, and the effect of the presenceof different water masses in these areas. The cyst distributionof a number of individual species was examined and showed threegeneral groups: species with very restricted distribution, suchas Alexandrium sp2 or Scrippsiella sp4, species with a widespreaddistribution along the rías, such as several Scrippsiellaspecies, and species mainly distributed along the shelf, suchas Gymnodinium catenatum. The distribution of cysts belongingto red tide organisms fits quite well with that of their correspondingmotile phases dunng the three previous years for most of theorganisms studied and also during the 1992–1993 period(7–8 years later), but the role of this resting stagein initiating such blooms seems to be highly variable with species.     

Cephalopod paralarvae and upwelling conditions off Galician waters (NW Spain)

A total of 103 cephalopod paralarvae were sampled during June 1995 in Galician waters (NW Spain). Samples were taken with Bongo nets of 300 and 500 m mesh size at 48 sampling stations along 10 transverse transects ranging from 80 to 600 m water depth. Paralarvae of loliginid squid were most abundant (40%). The Rhynchoteuthion paralarvae of ommastrephid squid accounted for 25%, whereas sepiolids comprised 23% of the total sample. Octopods were scarce, at only 6.6%. Other cephalopod families accounted for 5%. Sizes of paralarvae ranged from 1.0 to 7.1 mm mantle length. Temperature and salinity distribution showed the presence of an intense upwelling during the survey period. The sampling data obtained before and during the presence of upwelled water off Rias of Pontevedra and Vigo (southern zone) showed that paralarval cephalopod abundance and distribution were closely related to the upwelled Eastern North-Atlantic Central Water (ENACW).      

Growth and bioactive secondary metabolites of arctic Protoceratium reticulatum (Dinophyceae)

Harmful algal blooms are mainly caused by marine dinoflagellates and are known to produce potent toxins that may affect the ecosystem, human activities and health. Such events have increased in frequency and intensity worldwide in the past decades. Numerous processes involved in Global Change are amplified in the Arctic, but little is known about species specific responses of arctic dinoflagellates. The aim of this work was to perform an exhaustive morphological, phylogenetical and toxinological characterization of Greenland Protoceratium reticulatum and, in addition, to test the effect of temperature on growth and production of bioactive secondary metabolites. Seven clonal isolates, the first isolates of P. reticulatum available from arctic waters, were phylogenetically characterized by analysis of the LSU rDNA. Six isolates were further characterized morphologically and were shown to produce both yessotoxins (YTX) and lytic compounds, representing the first report of allelochemical activity in P. reticulatum. As shown for one of the isolates, growth was strongly affected by temperature with a maximum growth rate at 15 °C, a significant but slow growth at 1 °C, and cell death at 25 °C, suggesting an adaptation of P. reticulatum to temperate waters. Temperature had no major effect on total YTX cell quota or lytic activity but both were affected by the growth phase with a significant increase at stationary phase. A comparison of six isolates at a fixed temperature of 10 °C showed high intraspecific variability for all three physiological parameters tested. Growth rate varied from 0.06 to 0.19 d⁻¹, and total YTX concentration ranged from 0.3 to 15.0 pg  YTX cell⁻¹ and from 0.5 to 31.0 pg YTX cell⁻¹ at exponential and stationary phase, respectively. All six isolates performed lytic activity; however, for two isolates lytic activity was only detectable at higher cell densities in stationary phase.     

A multimetric diatom index to assess the ecological status of coastal Galician rivers (NW Spain)

There are many rivers in northwest Spain as a consequence of the mountainous landscape and the granitic geology subjected to Atlantic influences. Water and epilithic diatoms samples were collected at 72 sites in Galicia flowing into the Atlantic Ocean and Cantabrian Sea in summer 2002–2003 and spring 2004. These sites included minimally disturbed sites, defined as reference sites, and impacted sites which were influenced by different human pressures. We used the diatom assemblages to calculate diatom indices using the Omnidia software, but we also developed new metrics based on the similarity of species composition in reference sites. The response of the metrics was tested in relation to physicochemical variables. We developed a diatom multimetric index (MDIAT) as a combination of metric values. The sensitivity of the MDIAT to organic and nutrient stressors supports the use of this index to classify the ecological status of Galician rivers. The MDIAT showed higher correlations with some variables and nutrients than the individual metrics. According to the MDIAT, 69% of the sites sampled in Galician coastal rivers achieve good ecological status. The MDIAT has been developed specifically for Galician granitic rivers (NW Spain), and has been intercalibrated at the European level in the Central Baltic Rivers GIG. Our study validates the application of this multimetric index to evaluate the water quality in coastal Galician rivers.     

Toxin and molecular analysis of Gymnodinium catenatum (Dinophyceae) strains from Galicia (NW Spain) and Andalucia (S Spain)

Nineteen strains of Gymnodinium catenatum were isolated fromone bloom in Andalucía (S Spain) and from different bloomsin Galicia (NW Spain). The PSP toxin profiles of 16 of the strainswere analyzed, and although the saxitoxin was exclusive to theGalician strains, the corresponding dendogram showed no clusteringof the isolates from this location. However, nine out of elevenAndalusian strains were included in the same cluster. In orderto compare toxin with molecular analysis, a fragment of thelarge subunit ribosomal (LSU) RNA gene was partially sequencedfor all of the strains and fully sequenced for the five strainsthat had shown most different growth curves. Since all the strainswere identical in the LSU sequenced region, another fragmentcomprising the internal transcribed spacer 1 (ITS 1), 5.8S rRNAgene and ITS 2, was sequenced and compared among all the strains.Although this region has been used before for the detectionof intraspecific variability, it was similar in all our strains.Finally, to detect molecular differences in the strains, a randomamplified polymorphic DNA (RAPD) analysis was performed. Thecorresponding cluster analysis grouped the strains in threeclusters: one of them comprised all the Galician strains plusthree from Andalucía, another one included eight Andalusianstrains, and the last one, more separated from the two previous,was constituted by two Andalucía isolates. Although theresults of the toxin and RAPD analysis were different, sevenAndalusian strains were clustered together in both dendograms.Since neither the toxin nor the RAPD analysis brought out aclear geographic signal, we can conclude that differences intoxin content and RAPD profile between the isolates of G. catenatumare probably not linked to the location in which the strainswere collected.     

Cryptic speciation in Protoceratium reticulatum (Dinophyceae): Evidence from morphological,molecular and ecophysiological data

The cosmopolitan, potentially toxic dinoflagellate Protoceratium reticulatum possesses a fossilizable cyst stage which is an important paleoenvironmental indicator. Slight differences in the internal transcribed spacer ribosomal DNA (ITS rDNA) sequences of P. reticulatum have been reported, and both the motile stage and cyst morphology of P. reticulatum display phenotypic plasticity, but how these morpho-molecular variations are related with ecophysiological preferences is unknown. Here, 55 single cysts or cells were isolated from localities in the Northern (Arctic to subtropics) and Southern Hemispheres (Chile and New Zealand), and in total 34 strains were established. Cysts and/or cells were examined with light microscopy and/or scanning electron microscopy. Large subunit ribosomal DNA (LSU rDNA) and/or ITS rDNA sequences were obtained for all strains/isolates. All strains/isolates of P. reticulatum shared identical LSU sequences except for one strain from the Mediterranean Sea that differs in one position, however ITS rDNA sequences displayed differences at eight positions. Molecular phylogeny was inferred using maximum likelihood and Bayesian inference based on ITS rDNA sequences. The results showed that P. reticulatum comprises at least three ribotypes (designated as A, B, and C). Ribotype A included strains from the Arctic and temperate areas, ribotype B included strains from temperate regions only, and ribotype C included strains from the subtropical and temperate areas. The average ratios of process length to cyst diameter of P. reticulatum ranged from 15% in ribotype A, 22% in ribotype B and 17% in ribotype C but cyst size could overlap. Theca morphology was indistinguishable among ribotypes. The ITS-2 secondary structures of ribotype A displayed one CBC (compensatory change on two sides of a helix pairing) compared to ribotypes B and C. Growth response of one strain from each ribotype to various temperatures was examined. The strains of ribotypes A, B and C exhibited optimum growth at 15 °C, 20 °C and 20–25 °C, respectively, thus corresponding to cold, moderate and warm ecotypes. The profiles of yessotoxins (YTXs) were examined for 25 strains using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The parent compound yessotoxin (YTX) was produced by strains of ribotypes A and B, but not by ribotype C strains, which only produced the structural variant homoyessotoxin (homoYTX). Our results support the notion that there is significant intra-specific variability in Protoceratium reticulatum and the biogeography of the different ribotypes is consistent with specific ecological preferences.     

Gastrointestinal helminths in brown trout (Salmo trutta Linnaeus, 1758) captured in Galician rivers (NW Spain)

Specimens of Salmo trutta (n = 613) captured by local anglers in different rivers in Galicia (NW Spain) during the 2015 fishing season (15 March–15 August) were examined. In total 1479 adult helminths were recovered from the gastrointestinal tracts of 221 fish. Moreover, the microscopic observation of the sediments obtained, previous diphasic concentration, revealed the presence of helminth eggs in 485 trout specimens. The following species were identified by morphological and molecular analysis: Crepidostomum metoecus (8.97%) (Trematoda); Salmonema ephemeridarum (16.97%), Raphidascaris acus (9.46%) and Pseudocapillaria sp. (2.12%) (Nematoda); and Echinorhynchus truttae (8.48%) (Acanthocephala). The prevalence, mean intensity and mean abundance of each helminth species were determined in relation to size/age of the fish. The helminth infracommunity comprised a maximum of four species and the species richness was S = 5. The biological cycles of most of the helminth species recovered are dependent on benthic macroinvertebrate fauna, which, in turn, is influenced by the water quality. Therefore, any changes that take place in the aquatic ecosystem (due to anthropogenic activities or climate change) may be reflected in the helminth composition.     

Toxic cyanobacteria strains isolated from blooms in the Guadiana river (southwestern Spain)

This paper describes the occurrence of toxic cyanobacteria along the Guadiana River over its course between Mérida and Badajoz (Extremadura, Spain). Water sampling for phytoplankton quantification and toxin analysis was carried out regularly between 1999 and 2001 in six different locations, including two shallow, slow-flowing river sites, two streamed river sites and two drinking water reservoirs. The cyanobacterial community differed significantly between these locations, especially during the summer. The predominant genera were Microcystis, Oscillatoria, Aphanizomenon and Anabaena. Using an ELISA assay the total microcystin contents of natural water samples from the most eutrophic locations ranged from 0.10 - 21.86 microg mcyst-LR equivalent x L(-1) in Valdelacalzada and 0.10-11.3 microg mcyst-LR equivalent x L(-1) in Vitonogales, and a seasonal variation of toxin content was observed. The amount of microcystins produced by each strain was determined by ELISA assay and the detection and identification of microcystin variants of three toxic strains of Microcystis aeruginosa was performed by high performance liquid chromatography (HPLC). The analysis of microcystins of the cultured strains revealed that toxin production was variable among different strains of M. aeruginosa isolated either from different blooms or from the same bloom.     

Genetic diversity of wine Saccharomyces cerevisiae strains in an experimental winery from Galicia (NW Spain)

Genetic diversity of wine Saccharomyces cerevisiae strains involved in spontaneous fermentations was studied by analysis of mitochondrial DNA restriction patterns. Yeasts were isolated at different stages of fermentations with must from three different white grapevine varieties, Albariño, Godello and Treixadura, which are autochthonous from Galicia. Nineteen different patterns out of a total of 446 strains analysed were identified, but only a few of them appeared at high frequency and therefore were able to lead the fermentation process. Some strains were common to all fermentations; however, most of them were a minority being only found at low frequency for one or two specific grape varieties. The dominant strain was different for each variety except in one case, suggesting that some strains are better adapted to certain must conditions.     

Qualitative and quantitative analysis of carrageenan content in gametophytes of Mastocarpus stellatus (Stackhouse) Guiry along Galician coast (NW Spain)

Qualitative and quantitative differences in carrageenan composition of gametophytes of the rhodophyte Mastocarpus stellatus (Gigartinales) were observed in this study. Carrageenans in gametophytes belong to the kappa family (κ-, ι-, ν-, μ-carrageenan). The dominant fractions were κ- and ι-carrageenan (more than 80 % of the total carrageenans). Mean total carrageenan content in gametophytes was of 37.32?±?1.21 % DW. Spatial and seasonal variations were observed, mainly related to changes on environmental and oceanographic factors and the role of carrageenans in adapting the fronds to these changes. Maximum values in carrageenan content were observed for San Román (Biscay Bay) in May and for Laxe and Mougás (Atlantic coast) in June. The results of this study indicated that spatial differences in carrageenan content were due to interactions of different factors, rather than the effect of a single factor. Fronds from San Román had higher carrageenan content (43.23?±?1.87 % DW) than those collected at two sites of the Atlantic coast, Mougás and Laxe (32.20?±?1.14 % DW). San Román is exposed to the open sea, windy and oriented to the north, and the water temperature is higher in summer than in the Atlantic coast. However, seasonal variations in carrageenan content resulted to be more related to other factors directly correlated with the input of energy in the ecosystems (irradiance, sunshine hours and insolation). Thus, carrageenan content began to increase in early spring when the number of sunlight hours increased. Maximum values were reached in late spring or early summer, just before maximum values of irradiance and air temperature were achieved.     

Culture studies on the mycobiont isolated from Parmotrema reticulatum (Taylor) Choisy: metabolite production under different conditions

A strain of the lichen mycobiont isolated from a thallus of Parmotrema reticulatum was cultured axenically on different media. The morphology, anatomy, growth of the colonies, and metabolite production were studied. The isolated fungal colonies developed well and showed a remarkable morphogenetic capacity on most of the assayed solid media, e.g., malt extract 2%-yeast extract 0.2% (MEYE), malt extract 1%-yeast extract 0.4%-sucrose 10% (MY10), and the original Lilly & Barnett medium (LB). The identity of the isolated fungus was confirmed by its ITS rDNA-sequence. Atranorin, the major cortical lichen depside, was produced when the colonies were grown over 5 and 10 months on solid LB medium, combined with a dessication treatment. Atranorin was identified by matching of UV spectra obtained from HPLC running and a reference substance in a spectrum library. Colonies grown on MEYE and MY10 with a dessication treatment did not produce any lichen secondary metabolite. Mycobionts grown for 5 months on solid MEYE without a dessication treatment produced triacylglycerides as the major metabolites, and the fatty acids were characterized as their methyl esters. Analysis by TLC and HPLC-DAD of extracts of colonies grown on LB and MY10 without dessication revealed that the typical secondary compounds of the natural lichen were not produced. The major metabolites of the natural lichen thallus were identified by chromatographic and spectroscopic methods.     

Toxin profiles of natural populations and cultures ofAlexandrium minutum Halim from Galician (Spain) coastal waters

The toxin profiles of three isolates and natural populations of the PSP agentAlexandrium minutum from several Galician rías (NW Spain) was obtained by HPLC. The toxin content of cultures ofA. minutum is dominated by GTX4 (80–90%) and GTX4 (10–15%) with small amounts of GTX3 and GTX2 (less than 3% of each); similar results were obtained for natural populations ofAlexandrium from three different Galician rías, where a mixture ofA. lusitanicum Balech andA. minutum can occur. Important quantitative differences were found between the three isolates, one being highly and two weakly toxic. The results obtained from these isolates and natural populations ofAlexandrium were very similar to those obtained from HPLC analyses of mussels intoxicated during a PSP outbreak in Ría de Ares (Rías Altas) in 1984, confirming thatA. minutum (previously identified asGonyaulax tamarensis Lebour andAlexandrium lusitanicum) was the PSP agent during the toxic outbreak in May 1984. Toxin profiles obtained from natural populations during different PSP outbreaks in different rías and from cultures are fairly consistent and suggest that at least from the toxin point of view,A. lusitanicum andA. minutum are identical, and that the toxin profile ofA. minutum from Galicia can be used as a biochemical marker.     

我国沿海四爿藻的室内培养

在水产养殖中,四爿藻是一种重要的饵料资源,从我国沿海(包括广东、浙江、山东等省)采集到的海洋单细胞绿藻四爿藻,用毛细管法挑取进行单种培养,然后在固体培养基中稀释划线分离得到无菌纯培养物,利用改良的Guillard &; Ryther(Guillard F)培养基、海洋Ⅲ号培养基和改良的海洋Ⅲ号培养基对四爿藻进行了实验室培养。结果发现,改良的海洋Ⅲ号培养基中四爿藻的生长速率最快,最适pH范围为7．0～8．0,加入维生素B12(10μg·L^-1),也可以适当增加四爿藻的生长速率。这种改良的海洋Ⅲ号培养基配制简单,适合于水产养殖中四爿藻的规模生产。