Mindy L. Richlen

BIOGEOGRAPHIC ANALYSIS OF THE GLOBALLY DISTRIBUTED HARMFUL ALGAL BLOOM SPECIES ALEXANDRIUM MINUTUM (DINOPHYCEAE) BASED ON rRNA GENE SEQUENCES AND MICROSATELLITE MARKERS 1

The toxic dinoflagellate Alexandrium minutum Halim is one of three species that comprise the “minutum” species complex. This complex is notable due to its role in the etiology of paralytic shellfish poisoning (PSP). Recent increases in PSP incidence and the geographic expansion of toxin‐producing Alexandrium dinoflagellates have prompted the intensive examination of genetic relationships among globally distributed strains to address questions regarding their present distribution and reasons for their apparent increase. The biogeography of A. minutum was studied using large subunit ribosomal DNA gene (LSU rRNA) and internal transcribed spacer (ITS) sequences and genotypic data from 12 microsatellite loci. rRNA gene and ITS sequencing data distinguished between two clades, herein termed the “Global” and the “Pacific”; however, little to no resolution was seen within each clade. Genotypic data from 12 microsatellite loci provided additional information regarding genetic relationships within the Global clade, but it was not possible to amplify DNA from the Pacific clade using these markers. With the exception of isolates from Italy and Spain, strains generally clustered according to origin, revealing geographic structuring within the Global clade. Additionally, no evidence supported the separation of A. lusitanicum and A. minutum as different species. With the use of microsatellites, it is now possible to initiate studies on the origin, history, and genetic heterogeneity of A. minutum that were not previously possible using only rRNA gene sequence data. This study demonstrates the power of combining a marker with intermediate resolution (rRNA sequences) with finer‐scale markers (microsatellites) to examine intraspecies variability among globally distributed isolates and represents the first effort to employ this technique in A. minutum.

Diversity and Dynamics of a Widespread Bloom of the Toxic Dinoflagellate Alexandrium fundyense

Historically, cosmopolitan phytoplankton species were presumed to represent largely unstructured populations. However, the recent development of molecular tools to examine genetic diversity have revealed differences in phytoplankton taxa across geographic scales and provided insight into the physiology and ecology of blooms. Here we describe the genetic analysis of an extensive bloom of the toxic dinoflagellate Alexandrium fundyense that occurred in the Gulf of Maine in 2005. This bloom was notable for its intensity and duration, covering hundreds of kilometers and persisting for almost two months. Genotypic analyses based on microsatellite marker data indicate that the open waters of the northeastern U.S. harbor a single regional population of A. fundyense comprising two genetically distinct sub-populations. These subpopulations were characteristic of early- and late-bloom samples and were derived from the northern and southern areas of the bloom, respectively. The temporal changes observed during this study provide clear evidence of succession during a continuous bloom and show that selection can act on the timescale of weeks to significantly alter the representation of genotypes within a population. The effects of selection on population composition and turnover would be magnified if sexual reproduction were likewise influenced by environmental conditions. We hypothesize that the combined effects of differential growth and reproduction rates serves to reduce gene flow between the sub-populations, reinforcing population structure while maintaining the diversity of the overall regional population.

Extensive genetic diversity and rapid population differentiation during blooms of Alexandrium fundyense (Dinophyceae) in an isolated salt pond on Cape Cod, MA, USA.

In Massachusetts, paralytic shellfish poisoning (PSP) is annually recurrent along the coastline, including within several small embayments on Cape Cod. One such system, the Nauset Marsh System (NMS), supports extensive marshes and a thriving shellfishing industry. Over the last decade, PSP in the NMS has grown significantly worse; however, the origins and dynamics of the toxic Alexandrium fundyense (Balech) populations that bloom within the NMS are not well known. This study examined a collection of 412 strains isolated from the NMS and the Gulf of Maine (GOM) in 2006–2007 to investigate the genetic characteristics of localized blooms and assess connectivity with coastal populations. Comparisons of genetic differentiation showed that A. fundyense blooms in the NMS exhibited extensive clonal diversity and were genetically distinct from populations in the GOM. In both project years, genetic differentiation was observed among temporal samples collected from the NMS, sometimes occurring on the order of approximately 7 days. The underlying reasons for temporal differentiation are unknown, but may be due, in part, to life-cycle characteristics unique to the populations in shallow embayments, or possibly driven by selection from parasitism and zooplankton grazing; these results highlight the need to investigate the role of selective forces in the genetic dynamics of bloom populations. The small geographic scale and limited connectivity of NMS salt ponds provide a novel system for investigating regulators of blooms, as well as the influence of selective forces on population structure, all of which are otherwise difficult or impossible to study in the adjacent open-coastal waters or within larger estuaries.

Quorum Sensing Is a Language of Chemical Signals and Plays an Ecological Role in Algal-Bacterial Interactions

ABSTRACT Algae are ubiquitous in the marine environment, and the ways in which they interact with bacteria are of particular interest in the field of marine ecology. The interactions between primary producers and bacteria impact the physiology of both partners, alter the chemistry of their environment, and shape microbial diversity. Although algal-bacterial interactions are well known and studied, information regarding the chemical-ecological role of this relationship remains limited, particularly with respect to quorum sensing (QS), which is a system of stimuli and response correlated to population density. In the microbial biosphere, QS is pivotal in driving community structure and regulating behavioral ecology, including biofilm formation, virulence, antibiotic resistance, swarming motility, and secondary metabolite production. Many marine habitats, such as the phycosphere, harbor diverse populations of microorganisms and various signal languages (such as QS-based autoinducers). QS-mediated interactions widely influence algal-bacterial symbiotic relationships, which in turn determine community organization, population structure, and ecosystem functioning. Understanding infochemicals-mediated ecological processes may shed light on the symbiotic interactions between algae host and associated microbes. In this review, we summarize current achievements about how QS modulates microbial behavior, affects symbiotic relationships, and regulates phytoplankton chemical-ecological processes. Additionally, we present an overview of QS-modulated co-evolutionary relationships between algae and bacterioplankton, and consider the potential applications and future perspectives of QS.

Characterization and comparison of toxin-producing isolates of Dinophysis acuminata from New England and Canada.

Following the identification of the first toxic isolate of Dinophysis acuminata from the northwestern Atlantic, we conducted detailed investigations into the morphology, phylogeny, physiology, and toxigenicity of three isolates from three sites within the northeastern U.S./Canada region: Eel Pond and Marthas Vineyard, Massachusetts, and the Bay of Fundy. Another isolate, collected from the Gulf of Mexico, was grown under the same light, temperature, and prey conditions for comparison. Despite observed phenotypic heterogeneity, morphometrics and molecular evidence classified the three northwestern Atlantic isolates as D. acuminata Claparède & Lachmann, whereas the isolate from the Gulf of Mexico was morphologically identified as D. cf. ovum. Physiological and toxin analyses supported these classifications, with the three northwestern Atlantic isolates being more similar to each other with respect to growth rate, toxin profile, and diarrhetic shellfish poisoning (DSP) toxin content (okadaic acid + dinophysistoxin 1/cell) than they were to the isolate from the Gulf of Mexico, which had toxin profiles similar to those published for D. cf. ovum F. Schütt. The DSP toxin content, 0.01–1.8 pg okadaic acid (OA) + dinophysistoxin (DTX1) per cell, of the three northwestern Atlantic isolates was low relative to other D. acuminata strains from elsewhere in the world, consistent with the relative scarcity of shellfish harvesting closures due to DSP toxins in the northeastern U.S. and Canada. If this pattern is repeated with the analyses of more geographically and temporally dispersed isolates from the region, it would appear that the risk of significant DSP toxin outbreaks in the northwestern Atlantic is low to moderate. Finally, the morphological, physiological, and toxicological variability within D. acuminata may reflect spatial (and/or temporal) population structure, and suggests that sub‐specific resolution may be helpful in characterizing bloom dynamics and predicting toxicity.

LSU rDNA based RFLP assays for the routine identification of Gambierdiscus species

The Gambierdiscus genus is a group of benthic dinoflagellates commonly associated with ciguatera fish poisoning (CFP), which is generally found in tropical or sub-tropical regions around the world. Morphologically similar species within the genus can vary in toxicity; however, species identifications are difficult or sometimes impossible using light microscopy. DNA sequencing of ribosomal RNA genes (rDNA) is thus often used to identify and describe Gambierdiscus species and ribotypes, but the expense and time can be prohibitive for routine culture screening and/or large-scale monitoring programs. This study describes a restriction fragment length polymorphism (RFLP) typing method based on analysis of the large subunit rDNA that can successfully identify at least nine of the described Gambierdiscus species and two Fukuyoa species. The software programs DNAMAN 6.0 and Restriction Enzyme Picker were used to identify a set of restriction enzymes (SpeI, HpyCH4IV, and TaqαI) capable of distinguishing most of the known Gambierdiscus species for which DNA sequences were available. This assay was tested using in silico analysis and cultured isolates, and species identifications of isolates assigned by RFLP typing were confirmed by DNA sequencing. To verify the assay and assess intra-specific heterogeneity in RFLP patterns, identifications of 63 Gambierdiscus isolates comprising ten Gambierdiscus species, one ribotype, and two Fukuyoa species were confirmed using RFLP typing, and this method was subsequently employed in the routine identification of isolates collected from the Caribbean Sea. The RFLP assay presented here reduces the time and cost associated with morphological identification via scanning electron microscopy and/or DNA sequencing, and provides a phylogenetically sensitive method for routine Gambierdiscus species assignment.

Molecular characterization and morphology of Cochlodinium strangulatum, the type species of Cochlodinium, and Margalefidinium gen. nov. for C. polykrikoides and allied species (Gymnodiniales, Dinophyceae)

Photosynthetic species of the dinoflagellate genus Cochlodinium such as C. polykrikoides, one of the most harmful bloom-forming dinoflagellates, have been extensively investigated. Little is known about the heterotrophic forms of Cochlodinium, such as its type species, Cochlodinium strangulatum. This is an uncommon, large (∼200μm long), solitary, and phagotrophic species, with numerous refractile bodies, a central nucleus enclosed in a distinct perinuclear capsule, and a cell surface with fine longitudinal striae and a circular apical groove. The morphology of C. polykrikoides and allied species is different from the generic type. It is a bloom-forming species with single, two or four-celled chains, small cell size (25-40μm long) with elongated chloroplasts arranged longitudinally and in parallel, anterior nucleus, eye-spot in the anterior dorsal side, and a cell surface smooth with U-shaped apical groove. Phylogenetic analysis based on LSU rDNA sequences revealed that C. strangulatum and C. polykrikoides/C. fulvescens formed two distally related, independent lineages. Based on morphological and phylogenetic analyses, the diagnosis of Cochlodinium is emended and C. miniatum is proposed as synonym of C. strangulatum. The new genus Margalefidinium gen. nov., and new combinations for C. catenatum, C. citron, C. flavum, C. fulvescens and C. polykrikoides are proposed.

The prevalence of benthic dinoflagellates associated with ciguatera fish poisoning in the central Red Sea

This study confirms the presence of the toxigenic benthic dinoflagellates Gambierdiscus belizeanus and Ostreopsis spp. in the central Red Sea. To our knowledge, this is also the first report of these taxa in coastal waters of Saudi Arabia, indicating the potential occurrence of ciguatera fish poisoning (CFP) in that region. During field investigations carried out in 2012 and 2013, a total of 100 Turbinaria and Halimeda macroalgae samples were collected from coral reefs off the Saudi Arabian coast and examined for the presence of Gambierdiscus and Ostreopsis, two toxigenic dinoflagellate genera commonly observed in coral reef communities around the world. Both Gambierdiscus and Ostreopsis spp. were observed at low densities (<200 cells g-1 wet weight algae). Cell densities of Ostreopsis spp. were significantly higher than Gambierdiscus spp. at most of the sampling sites, and abundances of both genera were negatively correlated with seawater salinity. To assess the potential for ciguatoxicity in this region, several Gambierdiscus isolates were established in culture and examined for species identity and toxicity. All isolates were morphologically and molecularly identified as Gambierdiscus belizeanus. Toxicity analysis of two isolates using the mouse neuroblastoma cell-based assay for ciguatoxins (CTX) confirmed G. belizeanus as a CTX producer, with a maximum toxin content of 6.50±1.14×10-5pg P-CTX-1 eq. cell-1. Compared to Gambierdiscus isolates from other locations, these were low toxicity strains. The low Gambierdiscus densities observed along with their comparatively low toxin contents may explain why CFP is unidentified and unreported in this region. Nevertheless, the presence of these potentially toxigenic dinoflagellate species at multiple sites in the central Red Sea warrants future study on their possible effects on marine food webs and human health in this region.

CHARACTERIZATION OF 17 NEW MICROSATELLITE MARKERS FOR THE DINOFLAGELLATE ALEXANDRIUM FUNDYENSE (DINOPHYCEAE), A HARMFUL ALGAL BLOOM SPECIES.

Alexandrium fundyense is a toxic marine dinoflagellate responsible for “red tide” events in temperate and sub-arctic waters worldwide. In the Gulf of Maine (GOM) and Bay of Fundy in the Northwest Atlantic, blooms of A. fundyense recur annually and are associated with major health and ecosystem impacts. In this region, microsatellite markers have been used to investigate genetic structure and gene flow; however, the loci currently available for this species were isolated from populations from Japan and the North Sea, and only a subset is suitable for the analysis of A. fundyense populations in the Northwest Atlantic. To facilitate future studies of A. fundyense blooms, both in this region and globally, we isolated and characterized 17 polymorphic microsatellite loci from 31 isolates collected from the GOM and from the Nauset Marsh System, an estuary on Cape Cod, Massachusetts, USA. These loci yielded between two and 15 alleles per locus, with an average of 7.1. Gene diversities ranged from 0.297 to 0.952. We then analyzed these same 31 isolates using previously published markers for comparison. We determined the new markers are sufficiently variable and better suited for the investigation of genetic structure, bloom dynamics, and diversity in the Northwest Atlantic.

Microbial Community Structure and Associations During a Marine Dinoflagellate Bloom

Interactions between microorganisms and algae during bloom events significantly impacts their physiology, alters ambient chemistry, and shapes ecosystem diversity. The potential role these interactions have in bloom development and decline are also of particular interest given the ecosystem impacts of algal blooms. We hypothesized that microbial community structure and succession is linked to specific bloom stages, and reflects complex interactions among taxa comprising the phycosphere environment. This investigation used pyrosequencing and correlation approaches to assess patterns and associations among bacteria, archaea, and microeukaryotes during a spring bloom of the dinoflagellate Alexandrium catenella. Within the bacterial community, Gammaproteobacteria and Bacteroidetes were predominant during the initial bloom stage, while Alphaproteobacteria, Cyanobacteria, and Actinobacteria were the most abundant taxa present during bloom onset and termination. In the archaea biosphere, methanogenic members were present during the early bloom period while the majority of species identified in the late bloom stage were ammonia-oxidizing archaea and Halobacteriales. Dinoflagellates were the major eukaryotic group present during most stages of the bloom, whereas a mixed assemblage comprising diatoms, green-algae, rotifera, and other microzooplankton were present during bloom termination. Temperature and salinity were key environmental factors associated with changes in bacterial and archaeal community structure, respectively, whereas inorganic nitrogen and inorganic phosphate were associated with eukaryotic variation. The relative contribution of environmental parameters measured during the bloom to variability among samples was 35.3%. Interaction analysis showed that Maxillopoda, Spirotrichea, Dinoflagellata, and Halobacteria were keystone taxa within the positive-correlation network, while Halobacteria, Dictyochophyceae, Mamiellophyceae, and Gammaproteobacteria were the main contributors to the negative-correlation network. The positive and negative relationships were the primary drivers of mutualist and competitive interactions that impacted algal bloom fate, respectively. Functional predictions showed that blooms enhance microbial carbohydrate and energy metabolism, and alter the sulfur cycle. Our results suggest that microbial community structure is strongly linked to bloom progression, although specific drivers of community interactions and responses are not well understood. The importance of considering biotic interactions (e.g., competition, symbiosis, and predation) when investigating the link between microbial ecological behavior and an algal bloom’s trajectory is also highlighted.