Michael L. Brosnahan

Evolution of Saxitoxin Synthesis in Cyanobacteria and Dinoflagellates

Dinoflagellates produce a variety of toxic secondary metabolites that have a significant impact on marine ecosystems and fisheries. Saxitoxin (STX), the cause of paralytic shellfish poisoning, is produced by three marine dinoflagellate genera and is also made by some freshwater cyanobacteria. Genes involved in STX synthesis have been identified in cyanobacteria but are yet to be reported in the massive genomes of dinoflagellates. We have assembled comprehensive transcriptome data sets for several STX-producing dinoflagellates and a related non-toxic species and have identified 265 putative homologs of 13 cyanobacterial STX synthesis genes, including all of the genes directly involved in toxin synthesis. Putative homologs of four proteins group closely in phylogenies with cyanobacteria and are likely the functional homologs of sxtA, sxtG, and sxtB in dinoflagellates. However, the phylogenies do not support the transfer of these genes directly between toxic cyanobacteria and dinoflagellates. SxtA is split into two proteins in the dinoflagellates corresponding to the N-terminal portion containing the methyltransferase and acyl carrier protein domains and a C-terminal portion with the aminotransferase domain. Homologs of sxtB and N-terminal sxtA are present in non-toxic strains, suggesting their functions may not be limited to saxitoxin production. Only homologs of the C-terminus of sxtA and sxtG were found exclusively in toxic strains. A more thorough survey of STX+ dinoflagellates will be needed to determine if these two genes may be specific to SXT production in dinoflagellates. The A. tamarense transcriptome does not contain homologs for the remaining STX genes. Nevertheless, we identified candidate genes with similar predicted biochemical activities that account for the missing functions. These results suggest that the STX synthesis pathway was likely assembled independently in the distantly related cyanobacteria and dinoflagellates, although using some evolutionarily related proteins. The biological role of STX is not well understood in either cyanobacteria or dinoflagellates. However, STX production in these two ecologically distinct groups of organisms suggests that this toxin confers a benefit to producers that we do not yet fully understand.

A Quantitative Assessment of the Role of the Parasite Amoebophrya in the Termination of Alexandrium fundyense Blooms within a Small Coastal Embayment

Parasitic dinoflagellates of the genus Amoebophrya infect free-living dinoflagellates, some of which can cause harmful algal blooms (HABs). High prevalence of Amoebophrya spp. has been linked to the decline of some HABs in marine systems. The objective of this study was to evaluate the impact of Amoebophrya spp. on the dynamics of dinoflagellate blooms in Salt Pond (MA, USA), particularly the harmful species Alexandrium fundyense. The abundance of Amoebophrya life stages was estimated 3–7 days per week through the full duration of an annual A. fundyense bloom using fluorescence in situ hybridization coupled with tyramide signal amplification (FISH- TSA). More than 20 potential hosts were recorded including Dinophysis spp., Protoperidinium spp. and Gonyaulax spp., but the only dinoflagellate cells infected by Amoebophrya spp. during the sampling period were A. fundyense. Maximum A. fundyense concentration co-occurred with an increase of infected hosts, followed by a massive release of Amoebophrya dinospores in the water column. On average, Amoebophrya spp. infected and killed ∼30% of the A. fundyense population per day in the end phase of the bloom. The decline of the host A. fundyense population coincided with a dramatic life-cycle transition from vegetative division to sexual fusion. This transition occurred after maximum infected host concentrations and before peak infection percentages were observed, suggesting that most A. fundyense escaped parasite infection through sexual fusion. The results of this work highlight the importance of high frequency sampling of both parasite and host populations to accurately assess the impact of parasites on natural plankton assemblages.

Rapid growth and concerted sexual transitions by a bloom of the harmful dinoflagellate Alexandrium fundyense (Dinophyceae)

Abstract Transitions between life cycle stages by the harmful dinoflagellate Alexandrium fundyense are critical for the initiation and termination of its blooms. To quantify these transitions in a single population, an Imaging FlowCytobot (IFCB), was deployed in Salt Pond (Eastham, Massachusetts), a small, tidally flushed kettle pond that hosts near annual, localized A. fundyense blooms. Machine‐based image classifiers differentiating A. fundyense life cycle stages were developed and results were compared to manually corrected IFCB samples, manual microscopy‐based estimates of A. fundyense abundance, previously published data describing prevalence of the parasite Amoebophrya, and a continuous culture of A. fundyense infected with Amoebophrya. In Salt Pond, a development phase of sustained vegetative division lasted approximately 3 weeks and was followed by a rapid and near complete conversion to small, gamete cells. The gametic period (∼3 d) coincided with a spike in the frequency of fusing gametes (up to 5% of A. fundyense images) and was followed by a zygotic phase (∼4 d) during which cell sizes returned to their normal range but cell division and diel vertical migration ceased. Cell division during bloom development was strongly phased, enabling estimation of daily rates of division, which were more than twice those predicted from batch cultures grown at similar temperatures in replete medium. Data from the Salt Pond deployment provide the first continuous record of an A. fundyense population through its complete bloom cycle and demonstrate growth and sexual induction rates much higher than are typically observed in culture.

2302) Proposal to reject the name Gonyaulax catenella (Alexandrium catenella) (Dinophyceae)

The dinoflagellate species Alexandrium catenella (Whedon & Kof.) Balech (in Anderson & al., Toxic Dinoflagellates: 37. 1985), first published as Gonyaulax catenella Whedon & Kof., was described from marine waters off San Francisco, California, U.S.A. The protologue included the species diagnosis, a detailed description and seven drawings in which the thecal plate pattern in apical, antapical, dorsal and ventral view was provided, as well as a sketch of four cells joined in a chain and two drawings showing the shape and position of the nucleus. The species name was published according to the International Code of Zoological Nomenclature and did not include a Latin diagnosis). The type material for the name, however, was not designated. Gonyaulax catenella was subsequently transferred to the genus Alexandrium by Balech (l.c. 1985), but no lectotype was designated. Alexandrium catenella, together with A. tamarense (M. Lebour) Balech and A. fundyense Balech, comprise the A. tamarense species complex, one of the most studied marine dinoflagellate groups due to their ecological, toxicological and economic importance. Several members of this complex produce saxitoxins, potent neurotoxins that cause paralytic shellfish poisoning. Identification of thecate dinoflagellates such as Alexandrium is largely based on the number, shape and arrangement of the thecal plates that surround vegetative cells. The three morpho-species grouped in the A. tamarense species complex are morphologically very similar, share the same plate pattern and have been distinguished based on the combination of two main characters: the ability to form chains and the presence/absence of a ventral pore between plates 1 and 4 (Balech, l.c. 1985; Genus Alexandrium. 1995). John & al. (in Protist, in review) critically reviewed the taxonomic status of the species grouped into the Alexandrium tamarense species complex. This analysis included a broad range of information on cell morphology, sequences of multiple regions in the rDNA operon, mating compatibility, ITS/5.8S genetic distances, ITS2 compensatory base changes, toxicity and presence of the gene sxtA published over the last several decades. As already shown by various independent studies (for a complete list of references, see John & al., l.c. in review) morphological characters used to identify the three species are not consistent and/or distinctive. Moreover, phylogenies based on multiple rDNA regions (SSU, LSU, ITS) indicate that the sequences from morphologically indistinguishable isolates consistently partition into five clades, designated Groups I–V (John & al. in Molec. Biol. Evol. 20: 1015–1027. 2003; Lilly & al. in J. Phycol. 43: 1329–1338. 2007). The preponderance of evidence supports each of these groups as distinct species (John & al., l.c. in review). A majority of the Group I sequences currently come from isolates in regions adjacent to the type locality for A. fundyense (Bay of Fundy, Canada). Similarly the Group III sequences come primarily from isolates obtained in regions adjacent to the type locality for A. tamarense (Tamar River Estuary, England). Given that these two genetically distinct species are morphologically indistinguishable, it was logical to designate Group I as A. fundyense and Group III as A. tamarense (John & al., l.c. in review). Since most of the published studies on A. fundyense and A. tamarense encompass Group I and Group III sequences, respectively, these revised species designations cause a minimum of confusion with regard to the current literature. The same, however, is not true for A. catenella. Alexandrium catenella cells were originally described as being slightly broader than long and to form chains. Based on these morphological criteria, a majority of the strains isolated in various sites in the Pacific Ocean were reported as A. catenella (see the recent reviews Anderson & al. in Harmful Algae 14: 10–35. 2012; in Annual Rev. Mar. Sci. 4: 143–176. 2012). However, the molecular analysis of these “morphologically” identified strains primarily fell into either Clade I (primarily eastern Pacific along coasts of North, Central and South America) or Clade IV (primarily western Pacific). The isolates sequenced to date from the A. catenella type locality (California) belong to Group I (Ruiz Sebastian & al. in Phycologia 44: 49–60. 2005; Jester & al. in Mar. Biol. 156: 493–504. 2010; Garneau & al. in Appl. Environm. Microbiol. 77: 7669–7680. 2011). These observations indicate that the Group I morphology is more variable than originally described and that the A. catenella species description was incorrectlybased on a population of A. fundyense cells exhibiting chain formation and the shape slightly broader than long (i.e., A. catenella simply represents one of the distinct morphological variants of A. fundyense). Therefore based on Art. 56.1 of the ICN (McNeill & al. in Regnum Veg. 154. 2012) we propose rejection of the basionym of Alexandrium catenella (Whedon & Kof.) Balech for the following reasons: (1) The identity of the type material on which this species was based remains unclear. No type was designated by the author and strains isolated from the region from which the material most likely originated that was the basis of the species description belong to a different species (A. fundyense). (2) Alexandrium catenella could in principle supplant the name A. fundyense and be applied to all Group I strains, because its original description (Whedon & Kofoid, l.c.) predates that of A. fundyense (Balech, l.c. 1985). However, a large number of studies on Group I strains have been published using the name A. fundyense and making this nomenclatural change would cause considerable confusion in the research community. As an alternative, John & al. (l.c. in review) proposed in their revision of the A. tamarense species complex that A. fundyense be retained as an accepted species name. This required formally designating a lectotype and epitype for A. fundyense. (3) Retention of A. catenella would foster continued confusion in the literature concerning whether the data in a given study pertains to Group I or Group IV species. To rectify the existing taxonomic confusion in this group, John & al. (l.c. in review) formally proposed Group I isolates as A. fundyense, Group III isolates as A. tamarense and Group IV isolates as a new species, Alexandrium pacificum Litaker (in John & al., l.c. in review). The designation of Alexandrium pacificum as distinct from A. fundyense will allow the confusion caused by the A. catenella species designation having been simultaneously applied to Group I and IV to be more easily addressed.

Horizontal Gene Transfer is a Significant Driver of Gene Innovation in Dinoflagellates

The dinoflagellates are an evolutionarily and ecologically important group of microbial eukaryotes. Previous work suggests that horizontal gene transfer (HGT) is an important source of gene innovation in these organisms. However, dinoflagellate genomes are notoriously large and complex, making genomic investigation of this phenomenon impractical with currently available sequencing technology. Fortunately, de novo transcriptome sequencing and assembly provides an alternative approach for investigating HGT. We sequenced the transcriptome of the dinoflagellate Alexandrium tamarense Group IV to investigate how HGT has contributed to gene innovation in this group. Our comprehensive A. tamarense Group IV gene set was compared with those of 16 other eukaryotic genomes. Ancestral gene content reconstruction of ortholog groups shows that A. tamarense Group IV has the largest number of gene families gained (314–1,563 depending on inference method) relative to all other organisms in the analysis (0–782). Phylogenomic analysis indicates that genes horizontally acquired from bacteria are a significant proportion of this gene influx, as are genes transferred from other eukaryotes either through HGT or endosymbiosis. The dinoflagellates also display curious cases of gene loss associated with mitochondrial metabolism including the entire Complex I of oxidative phosphorylation. Some of these missing genes have been functionally replaced by bacterial and eukaryotic xenologs. The transcriptome of A. tamarense Group IV lends strong support to a growing body of evidence that dinoflagellate genomes are extraordinarily impacted by HGT.

Temperature and Residence Time Controls on an Estuarine Harmful Algal Bloom: Modeling Hydrodynamics and Alexandrium fundyense in Nauset Estuary

A highly resolved, 3D model of hydrodynamics and Alexandrium fundyense in an estuarine embayment has been developed to investigate the physical and biological controls on a recurrent harmful algal bloom. Nauset estuary on Cape Cod (MA, USA) consists of three salt ponds connected to the ocean through a shallow marsh and network of tidal channels. The model is evaluated using quantitative skill metrics against observations of physical and biological conditions during three spring blooms. The A. fundyense model is based on prior model applications for the nearby Gulf of Maine, but notable modifications were made to be consistent with the Nauset observations. The dominant factors controlling the A. fundyense bloom in Nauset were the water temperature, which regulates organism growth rates, and the efficient retention of cells due to bathymetric constraints, stratification, and cell behavior (diel vertical migration). Spring-neap variability in exchange altered residence times, but for cell retention to be substantially longer than the cell doubling time, it required both active vertical migration and stratification that inhibited mixing of cells into the surface layer by wind and tidal currents. Unlike in the Gulf of Maine, the model results were relatively insensitive to cyst distributions or germination rates. Instead, in Nauset, high apparent rates of vegetative cell division by retained populations dictated bloom development. Cyst germination occurred earlier in the year than in the Gulf of Maine, suggesting that Nauset cysts have different controls on germination timing. The model results were relatively insensitive to nutrient concentrations, due to eutrophic conditions in the highly impacted estuary or due to limitations in the spatial and temporal resolution of nutrient sampling. Cell loss rates were inferred to be extremely low during the growth phase of the bloom but increased rapidly during the final phase due to processes that remain uncertain. The validated model allows a quantitative assessment of the factors that contribute to the development of a recurrent harmful algal bloom and provides a framework for assessing similarly impacted coastal systems.

Insights into the loss factors of phytoplankton blooms: The role of cell mortality in the decline of two inshore Alexandrium blooms

While considerable effort has been devoted to understanding the factors regulating the development of phytoplankton blooms, the mechanisms leading to bloom decline and termination have received less attention. Grazing and sedimentation have been invoked as the main routes for the loss of phytoplankton biomass, and more recently, viral lysis, parasitism and programmed cell death (PCD) have been recognized as additional removal factors. Despite the importance of bloom declines to phytoplankton dynamics, the incidence and significance of various loss factors in regulating phytoplankton populations have not been widely characterized in natural blooms. To understand mechanisms controlling bloom decline, we studied two independent, inshore blooms of Alexandrium fundyense, paying special attention to cell mortality as a loss pathway. We observed increases in the number of dead cells with PCD features after the peak of both blooms, demonstrating a role for cell mortality in their terminations. In both blooms, sexual cyst formation appears to have been the dominant process leading to bloom termination, as both blooms were dominated by small-sized gamete cells near their peaks. Cell death and parasitism became more significant as sources of cell loss several days after the onset of bloom decline. Our findings show two distinct phases of bloom decline, characterized by sexual fusion as the initial dominant cell removal processes followed by elimination of remaining cells by cell death and parasitism.

Bloom termination of the toxic dinoflagellate Alexandrium catenella: Vertical migration behavior, sediment infiltration, and benthic cyst yield

Abstract New resting cyst production is crucial for the survival of many microbial eukaryotes including phytoplankton that cause harmful algal blooms. Production in situ has previously been estimated through sediment trap deployments, but here was instead assessed through estimation of the total number of planktonic cells and new resting cysts produced by a localized, inshore bloom of Alexandrium catenella, a dinoflagellate that is a globally important cause of paralytic shellfish poisoning. Our approach utilizes high frequency, automated water monitoring, weekly observation of new cyst production, and pre‐ and post‐bloom spatial surveys of total resting cyst abundance. Through this approach, new cyst recruitment within the study area was shown to account for at least 10.9% ± 2.6% (SE) of the blooms decline, ∼ 5× greater than reported from comparable, sediment trap based studies. The observed distribution and timing of new cyst recruitment indicate that: (1) planozygotes, the immediate precursor to cysts in the life cycle, migrate nearer to the water surface than other planktonic stages and (2) encystment occurs after planozygote settlement on bottom sediments. Near surface localization by planozygotes explains the ephemerality of red surface water discoloration by A. catenella blooms, and also enhances the dispersal of new cysts. Following settlement, bioturbation and perhaps active swimming promote sediment infiltration by planozygotes, reducing the extent of cyst redistribution between blooms. The concerted nature of bloom sexual induction, especially in the context of an observed upper limit to A. catenella bloom intensities and heightened susceptibility of planozygotes to the parasite Amoebophrya, is also discussed.

The Role of Life Cycle Characteristics in Harmful Algal Bloom Dynamics

Life cycle-based adaptations are integral to the ecology of most organisms. For the toxic microalgal species Pyrodinium bahamense, Alexandrium fundyense, Pseudo-nitzschia spp., and Nodularia spumigena, the properties and behaviours of their life cycle stages enable them to thrive in diverse marine environments. Planktonic blooms of these species are associated with a range of negative impacts including fisheries closures and animal die-offs. As a result, their bloom dynamics have been studied extensively, illustrating the ways that each organism’s life cycle is adaptive to recurring biotic and abiotic stressors. Both P. bahamense and A. fundyense form thick-walled resting cysts that play a major role in the dynamics of episodic blooms and can lie dormant for extended intervals in bottom sediments. These cysts function effectively in both tropical and temperate habitats. Nodularia spumigena uses a related but different strategy by producing akinetes that help in its recurrence. Pseudo-nitzschia species do not form resting or benthic stages, but undergo sexuality to counteract the progressive decrease in cell size due to cell division with a rigid siliceous frustule. These life cycles are clearly adaptable to a broad range of environments as shown by their widespread distribution and abundance. Continued investigation of these life cycles, especially stage-specific interactions with biotic and abiotic conditions, is likely to provide further insights into algal species ecology broadly, including responses to global climate change, ocean acidification, and coastal nutrient enrichment.

Behavioral and mechanistic characteristics of the predator-prey interaction between the dinoflagellate Dinophysis acuminata and the ciliate Mesodinium rubrum.

Predator-prey interactions of planktonic protists are fundamental to plankton dynamics and include prey selection, detection, and capture as well as predator detection and avoidance. Propulsive, morphology-specific behaviors modulate these interactions and therefore bloom dynamics. Here, interactions between the mixotrophic, harmful algal bloom (HAB) dinoflagellate Dinophysis acuminata and its ciliate prey Mesodinium rubrum were investigated through quantitative microvideography using a high-speed microscale imaging system (HSMIS). The dinoflagellate D. acuminata is shown to detect its M. rubrum prey via chemoreception while M. rubrum is alerted to D. acuminata via mechanoreception at much shorter distances (89 ± 39 μm versus 41 ± 32 μm). On detection, D. acuminata approaches M. rubrum with reduced speed. The ciliate M. rubrum responds through escape jumps that are long enough to detach its chemical trail from its surface, thereby disorienting the predator. To prevail, D. acuminata uses capture filaments and/or releases mucus to slow and eventually immobilize M. rubrum cells for easier capture. Mechanistically, results support the notion that the desmokont flagellar arrangement of D. acuminata lends itself to phagotrophy. In particular, the longitudinal flagellum plays a dominant role in generating thrust for the cell to swim forward, while at other times, it beats to supply a tethering or anchoring force to aid the generation of a posteriorly-directed, cone-shaped scanning current by the transverse flagellum. The latter is strategically positioned to generate flow for enhanced chemoreception and hydrodynamic camouflage, such that D. acuminata can detect and stealthily approach resting M. rubrum cells in the water column.