Distribution and genetic diversity of the parasitic dinoflagellate Amoebophrya in coastal waters of China

Amoebophrya is an obligate endoparasite infecting wide ranges of marine organisms in coastal and oceanic waters. The parasitoid has received growing attention, due to its enormous genetic diversity in seawaters and suppressive effects on the growth of host dinoflagellates. Harmful algal blooms (HABs) caused by planktonic dinoflagellates have significantly impacted the coastal environment and mariculture in China. Series of studies have been conducted to reveal the occurrence mechanism and negative impacts of HABs in past decades, while the factors contributing to the recession of HABs have rarely been studied. Thus, the host range, prevalence and diversity of Amoebophrya along the coastline of China were systemically investigated to facilitate future studies on the ecological roles of the parasitoid. Overall, 10 dinoflagellate taxa were found to be infected by Amoebophrya spp., and the prevalence ranged from 0.03% to 2.50%. Sequencing of environmental genomic DNA revealed substantial diversity and significant regional heterogeneity of Amoebophryidae sequences derived from 12 coastal bays, while no significant correlation was observed among geographical locations. Phylogenetic analyses of 18S rDNA sequences derived from individual Amoebophrya-infected cells indicated the host divergence of the parasitoid and lend credence to the multiple species assumption. The results further revealed the broad host range, wide distribution and substantial diversity of Amoebophrya in the coastal waters of China, that should not be neglected in future studies on the succession of HABs, as well as the ecological significance of this parasitoid in marine microbial food webs.     

Occurrence of Amoebophrya spp. infection in planktonic dinoflagellates in Changjiang (Yangtze River) Estuary,China

The parasitic dinoflagellates in the genus of Amoebophrya can infect broad ranges of planktonic dinoflagellates, and transform algal biomass into organic matter that can be recycled within the planktonic community. The ecological significance of Amoebophrya spp. during harmful algal bloom (HAB) events was gradually recognized along with revelation of its host specificity and diversity in picoplankton communities. The eutrophicated coastal waters of China are frequently affected by HABs, particularly in Changjiang (Yangtze River) estuary and the adjacent East China Sea; while, no research has been conducted to explore the ecological roles of parasitism during HAB events and the related dinoflagellate bloom dynamics. For the first time, we confirmed the presence of Amoebophrya infections in the planktonic community of this region; six species of dinoflagellates were infected, including Ceratium tripos, Scrippsiella trochoidea, Gonyaulax spinifera, Gymnodinium sp., Gonyaulax sp. and an Alexandrium sp. Molecular sequences retrieved from environmental water samples revealed high genetic diversity of Amoebophryidae-like organisms in the water column. Amoebophrya-infected dinoflagellates were only observed in high salinity (>20) stations suggesting that salinity may be a factor limiting the distribution of Amoebophyra infections in natural environment. Whereas, no evidence of Amoebophrya infection was observed in the bloom-forming species Karenia mikimotoi, suggesting that K. mikimotoi in this region was likely free of Amoebophridae infection.     

Dinoflagellate-targeted PCR reveals highly abundant and diverse communities of parasitic dinoflagellates in and near Zhubi Reef,South China Sea

While diversity of symbiodiniacean dinoflagellates has been a focus of coral reef ecological research, information on the diversity of planktonic dinoflagellates in reef ecosystems remains limited. We used dinoflagellate-targeted PCR to investigate dinoflagellate diversity for a coral reef ecosystem. In the summer of 2007, plankton samples were collected from a lagoon, atoll, and open sea area of Zhubi Reef in the Nansha Islands, South China Sea. Sequencing of dinoflagellate-specific SSU rDNA clone libraries from samples in each of these habitats revealed high diversity and numerous novel dinoflagellate lineages. Gymnodiniales were most abundantly represented in all three water areas. Lagoon assemblages were co-dominated by Syndiniales and Gonyaulacales, the atoll by Gonyaulacales and Peridiniales, and the open sea by Syndiniales and Prorocentrales taxa. Species in the Syndiniales (group II) genus Amoebophrya were represented by eight new sequences and 13 previously described clades and were dominated by species reported to infect Gymnodiniales, Gonyaulacales, Peridiniales, and Prorocentrales taxa. And Amoebophrya were particularly abundant and diverse in the lagoon. Our results suggest that Amoebophrya probably play an important role in regulating dynamics of dinoflagellate assemblages in the Zhubi Reef coral ecosystem. In contrast, the few symbiodiniacean taxa detected occurred only in the open sea, suggesting planktonic aposymbiotic Symbiodiniaceae rarely occur in the reef ecosystem. We demonstrate the usefulness of a dinoflagellate-specific molecular technique for profiling dinoflagellate communities, and uncover diversity and the potential importance of parasitic lineages in a coral reef ecosystem.

     

Development of a Dinoflagellate-Oriented PCR Primer Set Leads to Detection of Picoplanktonic Dinoflagellates from Long Island Sound

We developed dinoflagellate-specific 18S rRNA gene primers. PCR amplification using these oligonucleotides for a picoplanktonic DNA sample from Long Island Sound yielded 24 clones, and all but one of these clones were dinoflagellates primarily belonging to undescribed and Amoebophrya-like lineages. These results highlight the need for a systematic investigation of picodinoflagellate diversity in both coastal and oceanic ecosystems.     

Molecular cloning reveals co-occurring species behind red tide blooms of the harmful dinoflagellate Cochlodinium polykrikoides

Red tides caused by the marine dinoflagellate Cochlodinium polykrikoides Margalef pose significant environmental problems worldwide. Recently, the existence of severe blooms attributable to a single Cochlodinium Schütt species has been questioned by many researchers. Herein we investigated the dinoflagellate composition of harmful algal blooms (HABs) attributed to C. polykrikoides in Korean coastal waters at nine different stations (St.). The component species of Cochlodinium blooms were examined by using microscopic and gene-cloning methods. In the nine study areas, C. polykrikoides was the predominant species of HABs in St. 2, 4, 7, and St. 9. Based on the morphological identification, the bloom was initially thought to be caused only by C. polykrikoides; however, we detected additional bloom-forming dinoflagellates (Polykrikos schwartzii Bütschli and Polykrikos kofoidii Chatton), and diatoms (Pseudo-nitzschia americana (Hasle) Fryxell) along with C. polykrikoides. The parasitic dinoflagellates Amoebophrya Koeppen and Euduboscquella Coats, Bachvaroff & Delwiche were found to be co-located with Cochlodinium in our study, and for the first time, Cochlodinium fulvescens Iwataki, Kawami & Matsuoka was detected in Korea (west coast). These results suggest co-existence of multiple dinoflagellates in bloom populations of Cochlodinium and describe the composition of other dinoflagellate blooms (e.g., Polykrikos spp.) in Korean coastal regions. This co-occurrence may be considered during efforts to monitor and control HABs.     

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.     

Characterization of the Parasitic Dinoflagellate Amoebophrya sp. Infecting Akashiwo sanguinea in Coastal Waters of China

The endoparasitic dinoflagellate Amoebophrya infects a number of free‐living marine dinoflagellates, including harmful algal bloom species. The parasitoid eventually kills its host and has been proposed to be a significant loss factor for dinoflagellate blooms in restricted coastal waters. For several decades, the difficulties of culturing host‐parasitoid systems have been a great obstacle for further research on the biology of Amoebophrya. Here, we established an Akashiwo sanguinea‐Amoebophrya sp. coculture from Chinese coastal waters and studied the parasitoid's generation time, dinospore survival and infectivity, as well as its host specificity. The lifespan of Amoebophrya sp. ex. A. sanguinea was approximately 58 h. The infective dinospores can survive up to 78 h in ambient waters but gradually lose their infectivity. The parasitoid was unable to infect other dinoflagellate species, its infection rate reached as high as 91% when the ratio of dinospores to host cells was 20:1. The high infectivity of dinospores suggests that the Amoebophrya strain was capable of removing a considerable fraction of host biomass within a short period, but that it is probably unable to maintain high infection levels under nonbloom conditions of its host, due to limited survival and time constraints in encountering host cells.     

Amoebophrya spp. from the Bloom‐forming Dinoflagellate Cochlodinium polykrikoides: Parasites not Nested in the “Amoebophrya ceratii Complex”

Members of Amoebophrya ceratii complex are known to infect a number of free‐living dinoflagellates including harmful algal bloom species. In August and October 2012, Amoebophrya infections during two bloom events of the dinoflagellate Cochlodinium polykrikoides were observed along southern coastal waters of Korea. Microscopic observations and molecular data revealed that two different Amoebophrya parasites infected the same host species. In addition, while one developed in the host's nucleus, the other in the host's cytoplasm. Phylogenetic analyses showed that the two parasites were not nested in the previously recognized “Amoebophrya ceratii complex clade”, which contained sequences of parasites infecting numerous dinoflagellate species. Instead, they branched as sister taxa to the isolate (possibly Amoebophrya) from radiolarians Hexacontium gigantheum. Our result indicates that the two Amoebophrya parasites infecting C. polykrikoides may be different species from those inside the “complex.”     

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.     

Dynamics of Amoebophrya parasites during recurrent blooms of the ichthyotoxic dinoflagellate Cochlodinium polykrikoides in Korean coastal waters

During the bloom events of the harmful dinoflagellate Cochlodinium polykrikoides in August and October, 2012, infections by two different Amoebophrya species were observed in Korean coastal waters. To investigate the dynamics of the two parasites and their relative impact on the host populations, a quantitative real-time PCR (qPCR) method was applied to detect and quantify the parasites in the free-living and parasitic stages. Each specific primer set of the target species, Amoebophrya sp. 1 and sp. 2 was designed on the large subunit (LSU) and the first internal transcribed spacer (ITS1) of ribosomal RNA (rRNA) gene, respectively. Dynamics of the two Amoebophrya species via qPCR assay showed distinct patterns during the C. polykrikoides bloom events. Amoebophrya sp. 1 showed peaks during both bloom events in August and October with relatively low copies (10⁶ to 10⁷ copies L⁻¹), while Amoebophrya sp. 2 appeared only during the bloom event in October with very high copies (10⁹ to 10¹⁰ copies L⁻¹). Overall, the qPCR measurements for the dynamics of two Amoebophrya species in the parasitic stage (> 5 μm fractions) were consistent with parasite prevalence through microscopic observations. Amoebophrya sp. 1 infections were observed during both bloom events in August and October with relatively low parasite prevalence (0.1–1.5%), while Amoebophrya sp. 2 infections were detected only during the bloom event in October with high prevalence (up to 45%). Taken together, Amoebophrya sp. 1 may be a generalist and C. polykrikoides may not be its primary host, while Amoebophrya sp. 2 may be a specialist which can substantially impact host population dynamics.     

BIODIVERSITY OF PLANKTONIC DINOFLAGELLATE SPECIES IN MANGROVE PONDS,PELICAN CAYS,BELIZE

Information on the population structure of planktonic dinoflagellates is reported in the coral reef‐mangrove ecosystem at Pelican Cays, Belize. Six sites examined included: Cat Cay, Douglas Cay, Elbow Cay, Fisherman's Cay, Lagoon Cay and Manatee Cay. A spectacular and rich dinoflagellate taxa including oceanic, coastal and offshore species are illustrated. The presence of oceanic species in the studied cays is an unexpected observation since dinolfagellate assemblages are virtually enclosed within ponds bordered by coral ridges that limits water exchange with the open ocean except during storm events. I am also reporting significant differences in the dinoflagellate associations among the studied cays. Dominant taxa included 16 Proroperidinium species, 11 Gonyaulax species, and ten Ceratium species. Only six planktonic species were harmful. Bloom forming species included Ceratium furca and Gonyaulax polygramma. A much more diverse authotrophic and heterotrophic dinoflagellate population characterizes the Pelican Cays than previously suspected. Some species are reported the first time: Protoperidinium belizeanum sp. nov., P. pyrum Balech, P. steidingerae Balech, P. depressum (Bailey) Balech, and P. divergens (Ehrenberg) Balech. These results demonstrate that the Belizean coral reef‐mangrove ecosystem is a delicate and species‐rich environment, and as such, should be protected and preserved.     

ALGAE AS WATER QUALITY INDICATORS FOR A DYSTROPHIC POND

Information on the population structure of planktonic dinoflagellates is reported in the coral reef-mangrove ecosystem at Pelican Cays, Belize. Six sites examined included: Cat Cay, Douglas Cay, Elbow Cay, Fisherman's Cay, Lagoon Cay and Manatee Cay. A spectacular and rich dinoflagellate taxa including oceanic, coastal and offshore species are illustrated. The presence of oceanic species in the studied cays is an unexpected observation since dinolfagellate assemblages are virtually enclosed within ponds bordered by coral ridges that limits water exchange with the open ocean except during storm events. I am also reporting significant differences in the dinoflagellate associations among the studied cays. Dominant taxa included 16 Proroperidinium species, 11 Gonyaulax species, and ten Ceratium species. Only six planktonic species were harmful. Bloom forming species included Ceratium furca and Gonyaulax polygramma. A much more diverse authotrophic and heterotrophic dinoflagellate population characterizes the Pelican Cays than previously suspected. Some species are reported the first time: Protoperidinium belizeanum sp. nov., P. pyrum Balech, P. steidingerae Balech, P. depressum (Bailey) Balech, and P. divergens (Ehrenberg) Balech. These results demonstrate that the Belizean coral reef-mangrove ecosystem is a delicate and species-rich environment, and as such, should be protected and preserved.     

Ecological impacts of parasitic chytrids,syndiniales and perkinsids on populations of marine photosynthetic dinoflagellates

Parasitism is a widespread interaction that plays significant roles in ecosystem balance and evolution. Historically the biology of zoosporic parasites is often a neglected field when studying aquatic ecological dynamics, especially in marine ecosystems. In the marine environment, dinoflagellates represent a significantly large component of primary production, and may be infected by a variety of fungal and fungus-like parasites including chytrids, syndiniales, and perkinsids as well as other microorganisms. The relationship between these organisms and their dinoflagellate hosts constitutes a highly significant pathosystem given the increasing importance of aquaculture. Because of their small size and lack of morphological characteristics these organisms are difficult to identify. This review compares the taxonomy, life cycle, host range, infection strategies, and ecological roles of species of Parvilucifera, Amoebophrya and Dinomyces which are parasites of dinoflagellates. Most of these species have only been described recently. Implications for environmental management are discussed.     

Parasitism as a biological control agent of dinoflagellate blooms in the California Current System

Amoebophrya is a marine parasite recently found to infect and kill bloom-forming dinoflagellates in the California Current System (CCS). However, it is unknown whether parasitism by Amoebophrya can control dinoflagellate blooms in major eastern boundary upwelling systems, such as the CCS. We quantified the abundance of a common bloom-forming species Akashiwo sanguinea and prevalence of its parasite (i.e., % infected cells) in surface water samples collected weekly from August 2005 to December 2008 at the Santa Cruz Wharf (SCW), Monterey Bay, CA. Additionally, we measured physical and chemical properties at the SCW and examined regional patterns of wind forcing and sea surface temperature. Relative abundance of the net phytoplankton species was also analyzed to discern whether or not parasitism influences net phytoplankton community composition. Epidemic infection outbreaks (>20% parasite prevalence in the host species) may have contributed to the end or prevented the occurrence of A. sanguinea blooms, whereas low parasite prevalence was associated with short-term (≤2 weeks) A. sanguinea blooms. The complete absence of parasitism in 2007 was associated with an extreme A. sanguinea bloom. Anomalously strong upwelling conditions were detected in 2007, suggesting that A. sanguinea was able to outgrow Amoebophrya and ‘escape’ parasitism. We conclude that parasitism can strongly influence dinoflagellate bloom dynamics in upwelling systems. Moreover, Amoebophrya may indirectly influence net phytoplankton species composition, as species that dominated the net phytoplankton and developed algal blooms never appeared to be infected.     

Phylogenetic relations of the dinoflagellate Gymnodinium baicalense from Lake Baikal

Freshwater dinoflagellates still remain poorly studied by modern biological methods. This lack of knowledge prevents us from understanding the evolution and colonization patterns of these ecologically important protists. Gymnodinium baicalense is the most abundant, and possibly endemic, planktonic dinoflagellate from the ancient Lake Baikal. This dinoflagellate species blooms in the spring under the ice. This study analyzed the origin of this Baikalian dinoflagellate using three markers (two ribosomal and one mitochondrial DNA). It was found that this species is a true member of the order Gymnodiniales and has close relatives in the glacial melt waters of the Arctic Ocean. It seems that G. baicalense has diversified relatively recently from the arctic marine gymnodinioids. These results shed light on dinoflagellate biogeography and their colonizations in Lake Baikala biodiversity hotspot.     

PLANKTONIC SARCODINES: MICROHABITAT FOR OCEANIC DINOFLAGELLATES1

Two morphologically distinct species of free-swimming dinoflagellates belonging to the genus Gyrodinium utilize the spine and rhizopodial environments of planktonic foraminifera and colonial radiolaria as microhabitats. Up to 84% of the sarcodines examined in a given population were associated with these dinoflagellates at densities up to 20,000 cells per sarcodine in some radiolarian colonies. Both dinoflagellate species possess chloroplasts, indicating they are capable of autotrophy. ¹⁴C-labelling experiments with the radiolarian-associated dinoflagellate demonstrate that it can take up inorganic carbon under both light and dark conditions. Ultrastructural evidence suggests the foraminiferal dinoflagellate may be capable of phagotrophy. Hence, these algae should be considered mixotrophs. An unusual cytoplasmic extension used for attachment and possibly feeding occurs in the foraminiferal-associated Gyrodinium and is documented with electron microscopy. Ultrastructural examination suggests this organelle may be hydrostatically controlled and may be an extension of the sac pusule.     

Use of eriochrome cyanine R for routine histology and histopathology: an improved dichromatic staining procedure

Abstract

A modified dichromatic iron-eriocyanine R (Fe-ECR) staining method is described. Staining obtained with this new technique generally was similar to that of hematoxylin and eosin (H & E). Cell nuclei were stained blue. Cardiac, smooth and skeletal muscle, and red blood cells, were stained different shades of red. Collagen fibers were stained different shades of orange, usually faintly. Decalcified bony tissue was stained pinkish violet. Epithelial cells were strongly stained deep shades of red, magenta and violet. Cartilage matrix, and goblet and mast cells were unstained. Although Fe-ECR staining differed too much from standard H & E staining to be a substitute for diagnostic purposes, the dichromatic method described might usefully replace van Gieson or trichrome stains, especially if muscle is of interest. A pH 0.95 staining solution was used to differentiate initially over-stained sections followed by washing in distilled water. This dichromatic technique is easier to perform and more precisely controllable than other ECR dichromatic methods. The entire procedure can be completed in less than 5 min. The technique has the advantages of greater technical simplicity and speed, a larger range of polychromasia, and a longer shelf-life than H & E. ECR also is more reliably available than hematoxylin and usually is less expensive.