Electrophoretic variability within the Protogonyaulax tamarensis/catenella species complex: Pyridine linked dehydrogenases

Extracts from 20 isolates of the Protogonyaulax (= Gonyaulax) tamarensis/catenella species complex from diverse geographical locations, including ten contemporaneous isolates from the same geographical population, were subjected to enzyme electrophoresis. Analysis of isozyme banding patterns produced by eight pyridine-linked dehydrogenases revealed a high degree of genetic polymorphism within and between morphotypes and geographical populations. Since there was little apparent correlation between electromorphs and variants assigned provisionally to the catenelloid or tamarensoid morphotype, and because morphological intermediates exist, the presently used morphological characters used to discriminate unequivocally between P. catenella and P. tamarensis appear to be inadequate. Nevertheless, within a given morphotype, isolates from the same location were more similar than to those from elsewhere. Isozyme electrophoresis offers a means of biochemically discriminating between isolates of this species complex, for which the plate patterns are substantially the same.     

Copepods induce paralytic shellfish toxin production in marine dinoflagellates

Among the thousands of unicellular phytoplankton species described in the sea, some frequently occurring and bloom-forming marine dinoflagellates are known to produce the potent neurotoxins causing paralytic shellfish poisoning. The natural function of these toxins is not clear, although they have been hypothesized to act as a chemical defence towards grazers. Here, we show that waterborne cues from the copepod Acartia tonsa induce paralytic shellfish toxin (PST) production in the harmful algal bloom-forming dinoflagellate Alexandrium minutum. Induced A. minutum contained up to 2.5 times more toxins than controls and was more resistant to further copepod grazing. Ingestion of non-toxic alternative prey was not affected by the presence of induced A. minutum. The ability of A. minutum to sense and respond to the presence of grazers by increased PST production and increased resistance to grazing may facilitate the formation of harmful algal blooms in the sea.     

Reevaluation of production of paralytic shellfish toxin by bacteria associated with dinoflagellates of the Portuguese coast

Paralytic shellfish toxins (PSTs) are potent neurotoxins produced by certain dinoflagellate and cyanobacterial species. The autonomous production of PSTs by bacteria remains controversial. In this study, PST production by two bacterial strains, isolated previously from toxic dinoflagellates, was evaluated using biological and analytical methods. Analyses were performed under conditions determined previously to be optimal for toxin production and detection. Our data are inconsistent with autonomous bacterial PST production under these conditions, thereby challenging previous findings for the same strains.     

Environmental drivers of paralytic shellfish toxin producing Alexandrium catenella blooms in a fjord system of northern Southeast Alaska

Paralytic shellfish poisoning (PSP) is a persistent problem that threatens human health and the availability of shellfish resources in Alaska. Regular outbreaks of marine dinoflagellates in the genus Alexandrium produce paralytic shellfish toxins (PSTs) that make shellfish consumption unsafe, and impose economic hardships on Alaska’s shellfish industry. Phytoplankton and environmental monitoring spanning 2008–2016, and a pilot benthic cyst survey in 2016, were focused in the Juneau region of Southeast Alaska to investigate Alexandrium catenella distributions and conditions favorable to bloom development. Overwintering Alexandrium cysts were found in near-shore sediments throughout the study region. Alexandrium catenella cells were present in the water column across a range of sea surface temperatures (7–15 °C) and surface salinities (S = 4–30); however, an optimal temperature/salinity window (10–13 °C, 18–23) supported highest cell concentrations. Measurable levels of PSTs were associated with lower concentrations (100 cells L⁻¹) of A. catenella, indicating high cell densities may not be required for shellfish toxicity to occur. Several interacting local factors were identified to support A. catenella blooms: 1) sea surface temperatures ≥7 °C; 2) increasing air temperature; 3) low to moderate freshwater discharge; and 4) several consecutive days of dry and calm weather. In combination, these bloom favorable conditions coincide with toxic bloom events during May and June in northern Southeast Alaska. These findings highlight how integrated environmental and phytoplankton monitoring can be used to enhance early warning capacity of toxic bloom events, providing more informed guidance to shellfish harvesters and resource managers in Alaska.     

Geographic differences in paralytic shellfish poisoning toxin profiles among Japanese populations of Alexandrium tamarense and A. catenella (Dinophyceae)

To reconsider whether toxin profile could be used as a marker for populations from different geographical areas, clonal isolates of the toxic dinoflagellates Alexandrium tamarense (Lebour) Balech and Alexandrium catenella (Whedon et Kofoid) Balech from Ofunato Bay (Iwate Prefecture), Atsumi Bay (Aichi Prefecture), Tanabe Bay (Wakayama Prefecture), Harima‐Nada (Kagawa Prefecture), Uranouchi Bay (Kochi Prefecture), Hiroshima Bay (Hiroshima Prefecture) and Yamakawa Bay (Kagoshima Prefecture), which were identified on the basis of morphotaxonomy, immunological and molecular biological techniques, were subjected to analysis of paralytic shellfish poisoning toxins by high performance liquid chromatography‐fluorometric method. All the isolates except A. tamarense OF152 from Ofunato Bay contained mainly N‐sulfocarbamoyl toxins (C1 +2) with various amounts of derivatives, and a typical north‐to‐south trend of decreasing toxicity was observed. In both A. tamarense and A. catenella, toxin profiles were rather constant within a geographical area and divergent among different geographical areas. The toxin profiles of A. tamarense from Harima‐Nada were well conserved among different bloom years. Toxin profile showed that isolates of A. tamarense from Ofunato Bay, A. tamarense from Harima‐Nada isolated in 1988 and A. catenella from Uranouchi Bay were heterogeneous. However, only two or three groups of isolates with different toxin profiles were observed in a geographical region, suggesting that several representative isolates express the genotype in a given region. These observations confirmed that toxin composition could be used as a marker to discriminate different geographical populations of these species.     

41 Incidence of paralytic shellfish toxin in bivalve mollusc tissue from the oregon coast

Saxitoxin and domoic acid sequestration by bivalve molluscs occurs periodically along the Oregon coast, presumably as a result of harmful algal blooms (HABs). Since 1958 and more continuously since 1979, the Oregon Shellfish Program (OSP) has assayed toxin levels in these molluscs as part of a monitoring program for paralytic (PSP) and amnesic (ASP) shellfish poisoning. We have created a working data base for all PSP sampling by the OSP between 1958 and 2001 and have examined the data for spatial and temporal trends in the appearance of toxin in shellfish, amount of toxin, and apparent duration of toxic events. In this report, we examine the data from the five stations with the longest record of continuous sampling (1979–2000) for evidence of correlation with El Niño events, upwelling, and/or a pattern of increasing frequency or intensity of toxic events. We also compare the pattern of appearance of toxin at open coast stations with the timing of first appearance of toxin in shellfish at adjacent estuarine stations. This is an important analysis because, in Oregon, shellfish closures due to PSP occur frequently in mussel beds on the open coast and the source of toxin‐producing organisms is unknown.     

Variation in species composition and species richness within Phragmites australis dominated riparian zones

In riparian wetlands total standing crop often fails to account for a significant part of the observed variation in species richness and species composition within communities. In this study, we used abundance of the dominant species instead of total standing crop as the biotic predictor variable and investigated its relationships with species composition and species richness in communities dominated by Phragmites australis (Cav.) Trin. ex Steudel. This was done by measuring soil organic matter content, litter cover and elevation, Phragmites abundance (standing crop and stem density) and species composition in 78 relevés. In addition, we tried to identify the environmental boundaries of Phragmites communities by sampling relevés in neighbouring communities.Two gradients were related to a decline in Phragmites abundance: one gradient, perpendicular to the shoreline, was mainly related to increased elevation and the second gradient ran parallel to the shoreline and was related to increased amounts of soil organic matter. Within the relevés dominated by Phragmites, stem density of Phragmites and litter cover were the only factors significantly related to species composition in the RDA solution. Litter cover and standing crop of the dominant accounted for 64% of the variation in species richness within the Phragmites-dominated community. These results show that dead and living biomass of the dominant species may account for a substantial part of the variation in species composition and species richness within a single community.     

Effect of the endoparasite Amoebophrya sp. on toxin content and composition in the paralytic shellfish poisoning dinoflagellate Alexandrium fundyense (Dinophyceae)

Members of the Amoebophrya ceratii complex are endoparasitic dinoflagellates that parasitize a number of their dinoflagellate relatives, including toxic and/or harmful algal bloom-forming species. Despite many studies on the occurrence, prevalence, biology and molecular phylogeny of Amoebophrya spp., little attention has been given to toxin dynamics of host population following parasitism. Using Amoebophrya sp. infecting the paralytic shellfish toxin (PSP)-producing dinoflagellate Alexandrium fundyense, we addressed the following questions: (1) does parasitism by Amoebophrya sp. alter toxin content and toxin profiles of the dinoflagellate A. fundyense over the infection cycle? and (2) do parasite dinospores produced at the end of the infection cycle retain host toxins and thus potentially act as a vector to convey PSP toxin through the marine microbial food-web? Toxin time-course experiments showed that the PSP toxin contents did not vary significantly over the infection cycle, but mean toxin content for infected cultures was significantly higher than that for uninfected cultures. Host toxins were not detected in the free-living, dinospore stage of the parasite. Therefore, our results indicate that Amoebophrya sp. does not function as a vector for transferring PSP toxins to higher trophic levels. Rather, Amoebophrya infections appear to play an important role in maintaining healthy ecosystems by transforming potent toxins-producing dinoflagellates into non-toxic dinospores, representing “edible food” for consumers of the marine microbial food-web during toxic algal bloom event.     

Paralytic shellfish poisoning toxins in France linked to a human-introduced strain of Alexandrium catenella from the western Pacific: evidence from DNAand toxin analysis

In 1998, the toxins responsible for paralytic shellfish poisoning(PSP) were detected in Thau Lagoon, France. The causative organismwas identified as Alexandrium tamarense, a member of the ‘tamarensis’species complex. This dinoflagellate was first observed in thelagoon in 1995 by a monitoring programme following more thana decade with no observations of this species. The species isthus new to these waters, but its origins were unknown. In thispaper, morphological and molecular data are analysed for twoclonal cultures established from the 1998 bloom. These dataare compared to results from Alexandrium isolates originatingelsewhere in the world to infer an origin. Thecal plate morphology,restriction fragment length polymorphism, DNA sequencing andtoxin analyses demonstrate that the Thau Lagoon strains areA. catenella, and are closely related to populations of A. catenellafound in temperate Asia, specifically the Japanese TemperateAsian ribotype of the tamarense/catenella/fundyense speciescomplex. They show no homology with strains from western Europeanwaters, including the Mediterranean. Until now, the JapaneseTemperate Asian ribotype has not been reported outside the westernPacific. The most likely scenario is that A. catenella was introducedto Thau Lagoon via the ballast water of a ship docked at Sète,France, a shipping port in direct communication with the lagoon.This case provides a clear example of the dispersal of a toxicAlexandrium species, probably via human activities.     

Variation at the common polysaccharide antigen locus drives lipopolysaccharide diversity within the Pseudomonas syringae species complex

The common polysaccharide antigen (CPA) of the lipopolysaccharide (LPS) from Pseudomonas syringae is highly variable, but the genetic basis for this is poorly understood. We have characterized the CPA locus from P. syringae pv. actinidiae (Psa). This locus has genes for l - and d -rhamnose biosynthesis and an operon coding for ABC transporter subunits, a bifunctional glycosyltransferase and an o -methyltransferase. This operon is predicted to have a role in the transport, elongation and termination of the CPA oligosaccharide and is referred to as the TET operon. Two alleles of the TET operon were present in different biovars (BV) of Psa and lineages of the closely related pathovar P. syringae pv. actinidifoliorum. This allelic variation was reflected in the electrophoretic properties of purified LPS from the different isolates. Gene knockout of the TET operon allele from BV1 and replacement with that from BV3, demonstrated the link between the genetic locus and the biochemical properties of the LPS molecules in Psa. Sequence analysis of the TET operon from a range of P. syringae and P. viridiflava isolates displayed a phylogenetic history incongruent with core gene phylogeny but correlates with previously reported tailocin sensitivity, suggesting a functional relationship between LPS structure and tailocin susceptibility.     

Spatial and seasonal changes in the species composition of armored dinoflagellates in the Southwestern Atlantic Ocean

Summary To compare the spatial and temporal (seasonal) distribution of dinoflagellates, vertical net hauls were taken along similar cruise tracks in the Scotia Sea, Weddell Sea and across the Polar Front Zone in the austral spring and the austral fall. Sixty-three species of armored dinoflagellates were identified and enumerated. Chisquare and hierarchical cluster analyses were performed to define spatial and seasonal patterns in genera and species assemblages. The dominant genera were Protoperidinium, Dinophysis and Ceratium. The Polar Front Zone was an important biogeographical barrier with Blepharocysta, Gonyaulax, Heteroschisma, Oxytoxum and Podolampas occurring mainly north of the Front. Species found primarily in the austral spring were Ceratium fusus, Ceratium lineatum, Dinophysis antarctica, Dinophysis simplex, Gonyaulax digitale, Protoperidinium pyriforme and Protoperidinium variegatum. Austral fall species included Dinophysis tuberculata and Protoperidinium elegantissum. Distribution of armored dinoflagellates in the Southwestern Atlantic Ocean is influenced at the generic level by spatial considerations, particular with relation to the Polar Front Zone, whereas species composition can be effected by both region and season.     

Distribution,occurrence and biotoxin composition of the main shellfish toxin producing microalgae within European waters: A comparison of methods of analysis

Harmful algal blooms (HABs) are a natural global phenomena emerging in severity and extent. Incidents have many economic, ecological and human health impacts. Monitoring and providing early warning of toxic HABs are critical for protecting public health. Current monitoring programmes include measuring the number of toxic phytoplankton cells in the water and biotoxin levels in shellfish tissue. As these efforts are demanding and labour intensive, methods which improve the efficiency are essential. This study compares the utilisation of a multitoxin surface plasmon resonance (multitoxin SPR) biosensor with enzyme-linked immunosorbent assay (ELISA) and analytical methods such as high performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for toxic HAB monitoring efforts in Europe. Seawater samples (n = 256) from European waters, collected 2009–2011, were analysed for biotoxins: saxitoxin and analogues, okadaic acid and dinophysistoxins 1/2 (DTX1/DTX2) and domoic acid responsible for paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. Biotoxins were detected mainly in samples from Spain and Ireland. France and Norway appeared to have the lowest number of toxic samples. Both the multitoxin SPR biosensor and the RNA microarray were more sensitive at detecting toxic HABs than standard light microscopy phytoplankton monitoring. Correlations between each of the detection methods were performed with the overall agreement, based on statistical 2 × 2 comparison tables, between each testing platform ranging between 32% and 74% for all three toxin families illustrating that one individual testing method may not be an ideal solution. An efficient early warning monitoring system for the detection of toxic HABs could therefore be achieved by combining both the multitoxin SPR biosensor and RNA microarray.     

Detection of species within the Xerocomus subtomentosus complex in Europe using rDNA–ITS sequences

Identification of species within the boletoid genus Xerocomus has relied heavily upon the macromorphological features of the basidiomes. However, the phenotypic plasticity of these features has resulted in considerable confusion over the delimitation of taxa. In this study, we examined collections attributed to the X. subtomentosus complex in Europe using morphological and rDNA–ITS sequence data. In total, 45 European collections from a wide range of geographical areas and ecological conditions were included in the study. In spite of detecting considerable genetic variation, even within individual basidiomes of X. subtomentosus, molecular data, spore size, flesh colour, and the colour of the basal mycelium allow for the recognition of four distinct taxa: two correspond to X. subtomentosus (13 collections) and X. ferrugineus (20); one X. chrysonemus sp. nov. (10), to date only found in the UK, is described as new; and the existence of another taxon (two; Italy and UK) is noted but left undescribed owing to lack of material. Eight collections from North America were also included in the study, from which two taxa with a close affinity to X. ferrugineus were recognised.     

In vitro transformation of paralytic shellfish toxins in the clams Mya arenaria and Protothaca staminea

Dissected tissues of two clam species, the Pacific littleneck, Protothaca staminea, and soft-shell, Mya arenaria, were evaluated for in vitro conversion of paralytic shellfish poisoning (PSP) toxins. Tissue homogenates were incubated with purified PSP toxins to determine the time-course of toxin conversion. The effects of boiling and addition of a natural reductant (glutathione) on toxin conversion were also assessed. For P. staminea, the digestive gland showed the greatest capacity for biotransformation, followed by gill, but mantle, adductor muscle, and siphon tissues exhibited very low conversion. In this species, the production of decarbamoyl derivatives was much greater from low potency N-sulfocarbamoyl toxins than from carbamate analogues. Decarbamolyation exhibited apparent specificity for α-epimers of all toxin substrates and this reaction was inhibited by boiling. Glutathione-mediated desulfation was tissue specific and had apparent specificity for β-epimers. These observations on P. staminea suggest that the above reactions are enzyme-mediated. In contrast, there was little toxin conversion in M. arenaria homogenates, but even this low activity was heat-labile and thus likely enzyme-mediated.     

Reactive oxygen species as causative agents in the ichthyotoxicity of the red tide dinoflagellate Cochlodinium polykrikoides

The underlying toxic mechanisms of the red tide dinoflagellate,Cochlodinium polykrikoides, were studied with respect to thereactive oxygen species-mediated toxic effect. Cochlodiniumpolykrikoides generates superoxide anion (O₂^–) and hydrogenperoxide (H₂O₂), as measured by the cytochrome c reduction methodand scopoletin–peroxidase method, respectively. The capabilityof C.polykrikoides to generate these oxygen radicals was relatedto the growth phase: the highest rate in the exponential phaseand a gradual decrease in the stationary phase. Other phytoplankton,such as Eutreptiella gymnastica, Heterosigma akashiwo, Prorocentrummicans, Gymnodinium sanguineum and Alexandrium tamarense, alsoproduce H₂O₂; the rate of H₂O₂ generation by these species waslower than that of C.polykrikoides. The exposure of liposomalsamples to intact or ruptured individuals of C.polykrikoidesresulted in severe membrane damage, such as liposomal lipidperoxidation. Cochlodinium polykrikoides-induced lipid peroxidationwas significantly reduced by oxygen radical scavengers, superoxidedismutase, benzoquinone, catalase and mannitol. In addition,lipid peroxidation of gill tissue of flatfish exposed to C.polykrikoidesincreased with increasing algal cell density. These resultssuggest that reactive oxygen species generated from C.polykrikoidesare responsible for oxidative damage leading to fish kills.     

A multi-phylum study of grazer-induced paralytic shellfish toxin production in the dinoflagellate Alexandrium fundyense: A new perspective on control of algal toxicity

The present study surveyed grazer-induced stimulation of paralytic shellfish toxin (PST) production by the marine dinoflagellate Alexandrium fundyense. The survey included species, known to graze upon A. fundyense, from five phyla: the protists, Polykrikos kofoidii (Dinoflagellata) and Tiarina fusus (Ciliophora), the bivalve molluscs Mytilus edulis and Mya arenaria (Mollusca), the ascidians, Molgula manhattensis and Botrylloides violaceus (Chordata), and the copepod, Eurytemora herdmani (Arthropoda). Direct (grazers in contact with cells of A. fundyense) and indirect (grazers not in contact with cells of A. fundyense) induction assays were carried out with protists and copepods. Only indirect assays were carried out with molluscs and ascidians. Indirect assays also tested whether induction of PST production occurred via kairomones or feeding-related cues. All metazoan grazers induced PST production. By contrast, neither of the two species of protistan grazer induced PST production. Direct and indirect inductions of PST production were evident for the copepod, with direct induction being significantly higher than indirect induction. Effects upon PST production by phylum, species (nested within phylum), and interactions of phylum by diet, and species by diet, were evident. When induction of PST production occurred, a kairomone effect was apparent, except for M. edulis. Similarly, feeding-related cues were evident, except for E. herdmani. An asymptotic relationship between the magnitude of indirect induction of PST production and total cell ingestion by the grazers suggests a saturation response of grazer-induced PST production.     

Variation in cross‐sectional horn shape within and among rhinoceros beetle species

Sexual selection has equipped male rhinoceros beetles with large horns on their head and prothorax to aid in battle over access to females. Horns are used to pry and dislodge opponents from resource sites that attract females, so an optimal horn should be able both to withstand the high stresses imposed during fights, and to resist deflection in response to these loads. We examined the cross‐sectional morphology of horns using micro‐computed tomography scanning to determine how horn structure changes with horn length to withstand the different fighting loads. Specifically, we measured the second moment of area of horns within and among rhinoceros beetle species to assess whether changes in cross‐sectional morphology accompany changes in body size in order to maintain high strength and stiffness during fights. We find that the second moment of area of horns increases with body size both intra‐specifically and inter‐specifically, and that these relationships closely fit those predicted if horns have been selected to be strong and stiff fighting structures. Our results therefore support the hypothesis that rhinoceros beetle horns are structurally adapted for combat.     

Development of compound microsatellite markers in the harmful red tide species Chattonella ovata (Raphidophyceae)

We isolated 12 polymorphic microsatellites from the noxious red‐tide‐causing alga Chattonella ovata. These loci provide a class of highly variable genetic markers, as the number of alleles ranged from four to 12, and the observed and expected heterozygosities ranged from 0.238 to 0.850 and from 0.310 to 0.889, respectively. These loci are useful for revealing the genetic structure of and gene flow among C. ovata populations.     

Variation in the guild composition of herbivorous insect assemblages among co‐occurring plant species

Variation in plant traits among plant species may promote the development of a characteristic functional assemblage of insect herbivores associated with each plant species. However, only a small number of studies have detailed the representation of several herbivore guilds among co‐occurring plant species to determine whether the functional structure of herbivorous insect assemblages varies widely and consistently among plant species. The present study provides one of the few published data sets reporting on the density of several guilds of insect herbivores among numerous plant species. Variation in guild associations with plant phenology and season are also described. Insect herbivores were divided into 10 guilds, and the representation of these guilds was examined for 18 co‐occurring plant species. Guild densities and assemblage composition varied significantly among plant species, even when variation over time was taken into account. Variation in guild densities and assemblage composition were not strongly related to the taxonomic relationships of the plants. The highest densities of several guilds occurred in spring and summer, although other guilds were not strongly seasonal. Certain guilds were strongly associated with the presence of new leaves, whereas other guilds appeared to prefer mature leaves. This resulted in assemblage differences between samples containing new and mature leaves and samples containing mature leaves only. Even though the timing and duration of leaf and flower production varied among plant species, this did not explain all variation in guild densities among plant species. It is suggested that additional factors, including plant traits, are contributing to the wide and consistent variation in herbivore assemblage composition among plant species.