Meta‐analysis reveals enhanced growth of marine harmful algae from temperate regions with warming and elevated CO2 levels

Elevated pCO₂ and warming may promote algal growth and toxin production, and thereby possibly support the proliferation and toxicity of harmful algal blooms (HABs). Here, we tested whether empirical data support this hypothesis using a meta‐analytic approach and investigated the responses of growth rate and toxin content or toxicity of numerous marine and estuarine HAB species to elevated pCO₂ and warming. Most of the available data on HAB responses towards the two tested climate change variables concern dinoflagellates, as many members of this phytoplankton group are known to cause HAB outbreaks. Toxin content and toxicity did not reveal a consistent response towards both tested climate change variables, while growth rate increased consistently with elevated pCO₂. Warming also led to higher growth rates, but only for species isolated at higher latitudes. The observed gradient in temperature growth responses shows the potential for enhanced development of HABs at higher latitudes. Increases in growth rates with more CO₂ may present an additional competitive advantage for HAB species, particularly as CO₂ was not shown to enhance growth rate of other non‐HAB phytoplankton species. However, this may also be related to the difference in representation of dinoflagellate and diatom species in the respective HAB and non‐HAB phytoplankton groups. Since the proliferation of HAB species may strongly depend on their growth rates, our results warn for a greater potential of dinoflagellate HAB development in future coastal waters, particularly in temperate regions.     

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.     

Molecular data and the evolutionary history of dinoflagellates

We have sequenced small-subunit (SSU) ribosomal RNA (rRNA) genes from 16 dinoflagellates, produced phylogenetic trees of the group containing 105 taxa, and combined small- and partial large-subunit (LSU) rRNA data to produce new phylogenetic trees. We compare phylogenetic trees based on dinoflagellate rRNA and protein genes with established hypotheses of dinoflagellate evolution based on morphological data. Protein-gene trees have too few species for meaningful in-group phylogenetic analyses, but provide important insights on the phylogenetic position of dinoflagellates as a whole, on the identity of their close relatives, and on specific questions of evolutionary history. Phylogenetic trees obtained from dinoflagellate SSU rRNA genes are generally poorly resolved, but include by far the most species and some well-supported clades. Combined analyses of SSU and LSU somewhat improve support for several nodes, but are still weakly resolved. All analyses agree on the placement of dinoflagellates with ciliates and apicomplexans (=Sporozoa) in a well-supported clade, the alveolates. The closest relatives to dinokaryotic dinoflagellates appear to be apicomplexans, Perkinsus, Parvilucifera, syndinians and Oxyrrhis. The position of Noctiluca scintillans is unstable, while Blastodiniales as currently circumscribed seems polyphyletic. The same is true for Gymnodiniales: all phylogenetic trees examined (SSU and LSU-based) suggest that thecal plates have been lost repeatedly during dinoflagellate evolution. It is unclear whether any gymnodinialean clades originated before the theca. Peridiniales appear to be a paraphyletic group from which other dinoflagellate orders like Prorocentrales, Dinophysiales, most Gymnodiniales, and possibly also Gonyaulacales originated. Dinophysiales and Suessiales are strongly supported holophyletic groups, as is Gonyaulacales, although with more modest support. Prorocentrales is a monophyletic group only in some LSU-based trees. Within Gonyaulacales, molecular data broadly agree with classificatory schemes based on morphology. Implications of this taxonomic scheme for the evolution of selected dinoflagellate features (the nucleus, mitosis, flagella and photosynthesis) are discussed.     

Development of an oligonucleotide microarray for the detection and monitoring of marine dinoflagellates

Harmful Algal Blooms (HABs), mainly caused by dinoflagellates and diatoms, have great economic and sanitary implications. An important contribution for the comprehension of HAB phenomena and for the identification of risks related to toxic algal species is given by the monitoring programs. In the microscopy-based monitoring methods, harmful species are distinguished through their morphological characteristics. This can be time consuming and requires great taxonomic expertise due to the existence of morphologically close-related species. The high throughput, automation possibility and specificity of microarray-based detection assay, makes this technology very promising for qualitative detection of HAB species. In this study, an oligonucleotide microarray targeted to the ITS1-5.8S-ITS2 rDNA region of nine toxic dinoflagellate species/clades was designed and evaluated. Two probes (45-47 nucleotides in length) were designed for each species/clade to reduce the potential for false positives. The specificity and sensitivity of the probes were evaluated with ITS1-5.8S-ITS2 PCR amplicons obtained from 20 dinoflagellates cultured strains. Cross hybridization experiments confirmed the probe specificity; moreover, the assay showed a good sensitivity, allowing the detection of up to 2 ng of labeled PCR product. The applicability of the assay with field samples was demonstrated using net concentrated seawater samples, un-spiked or spiked with known amounts of cultured cells. Despite the general application of microarray technology for harmful algae detection is not new, a peculiar group of target species/clades has been included in this new-format assay. Moreover, novelties regarding mainly the probes and the target rDNA region have allowed sensitivity improvements in comparison to previously published studies.     

RNA干扰文库及其在功能基因组学研究中的应用

随着人类基因组大规模测序的完成,下一步的挑战是了解每一个基因的功能 . RNA 干扰文库为大规模基因功能筛选提供了可能 . 虽然用于线虫等模式生物的 RNAi 文库,已经证明是大规模基因功能筛选的有效方法,但这些文库不能用于高等动物的细胞 . 自 2003 年以来,用于人的细胞和哺乳动物细胞的 RNAi 文库取得了突破,相继出现构建已知基因 RNAi 文库和构建随机 RNAi 文库的报道,并成功地应用于大规模基因功能的筛选 . RNAi 文库作为一种简单、高效、大规模、高通量的功能基因组学研究的工具,将在基因功能研究、发现新的药物靶基因、发现疾病相关基因等方面有广阔的应用前景 .     

Rapid identification of dinoflagellates using protein profiling with matrix-assisted laser desorption/ionization mass spectrometry

The occurrence of harmful algal blooms (HABs) or red tides is an important and expanding threat to human health, fishery resources, and the tourism industries. Toxic species post an additional treat of intoxication when consumed either in seafood or directly swallowed. Rapid and accurate identification of the HAB species is critical for minimizing or controlling the damage. We report the use of protein/peptide mass fingerprint profiles obtained with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) for the identification of dinoflagellates, common causative agents of HABs. The method is simple, fast and reproducible. The peptide mass fingerprint spectral patterns are unique for different dinoflagellate species and are easily distinguishable by visual inspection. In addition to the whole mass spectra, several specific biomarkers were identified from the mass spectra of different species. These biomarker ions and the mass spectral patterns form an unambiguous basis for species discrimination.     

RNA干扰技术在几项研究领域的应用

作为一项新的反向遗传学技术 ,RNA干扰技术正在越来越多地应用于包括鉴定基因功能、疾病治疗、植物病毒抗性研究在内的多项研究领域。在鉴定基因功能研究中 ,由于该技术的操作简便性 ,使得在基因组水平进行大范围的基因功能鉴定成为可能 ;而针对致病相关基因、致病病毒基因组进行RNA干扰 ,可以有效抑制病情恶化 ,有可能成为未来疾病治疗的重要手段 ;同时 ,将RNA干扰技术应用于植物抗病毒研究 ,为工程化植物抗病毒遗传育种提供了一个高效、特异的抗性获得手段。对RNA干扰技术在上述三个研究领域的应用作简要综述 ,并对应用过程中需注意的问题进行了探讨 。     

Genetic diversity, morphological uniformity and polyketide production in dinoflagellates (Amphidinium, Dinoflagellata)

Dinoflagellates are an intriguing group of eukaryotes, showing many unusual morphological and genetic features. Some groups of dinoflagellates are morphologically highly uniform, despite indications of genetic diversity. The species Amphidinium carterae is abundant and cosmopolitan in marine environments, grows easily in culture, and has therefore been used as a 'model' dinoflagellate in research into dinoflagellate genetics, polyketide production and photosynthesis. We have investigated the diversity of 'cryptic' species of Amphidinium that are morphologically similar to A. carterae, including the very similar species Amphidinium massartii, based on light and electron microscopy, two nuclear gene regions (LSU rDNA and ITS rDNA) and one mitochondrial gene region (cytochrome b). We found that six genetically distinct cryptic species (clades) exist within the species A. massartii and four within A. carterae, and that these clades differ from one another in molecular sequences at levels comparable to other dinoflagellate species, genera or even families. Using primers based on an alignment of alveolate ketosynthase sequences, we isolated partial ketosynthase genes from several Amphidinium species. We compared these genes to known dinoflagellate ketosynthase genes and investigated the evolution and diversity of the strains of Amphidinium that produce them.     

Predicting siRNA activity based on back-propagation neural network

RNA interference (RNAi) is a phenomenon of gene silence induced by a double-stranded RNA (dsRNA) homologous to a target gene.RNAi can be used to identify the function of genes or to knock down the targeted genes.In RNAi technology,19 bp double-stranded short interfering RNAs (siRNA) with characteristic 3' overhangs are usually used.The effects of siRNAs are quite varied due to the different choices in the sites of target mRNA.Moreover,there are many factors influencing siRNA activity and these factors are usually nonlinear.To find the motif features and the effect on siRNA activity,we carried out a feature extraction on some published experimental data and used these features to train a backpropagation neural network (BP NN).Then,we used the trained BP NN to predict siRNA activity.     

Predicting siRNA activity based on back-propagation neural network

RNA interference (RNAi) is a phenomenon of gene silence induced by a double-stranded RNA (dsRNA) homologous to a target gene. RNAi can be used to identify the function of genes or to knock down the targeted genes. In RNAi technology, 19 bp double-stranded short interfering RNAs (siRNA) with characteristic 39 overhangs are usually used. The effects of siRNAs are quite varied due to the different choices in the sites of target mRNA. Moreover, there are many factors influencing siRNA activity and these factors are usually nonlinear. To find the motif features and the effect on siRNA activity, we carried out a feature extraction on some published experimental data and used these features to train a back-propagation neural network (BP NN). Then, we used the trained BP NN to predict siRNA activity. __________ Translated from Acta Biophysica Sinica, 2006, 22(6): 429–434 [译自: 生物物理学报]     

RNA interference-mediated simultaneous silencing of four genes using cross-shaped RNA

The structural flexibility of RNA interference (RNAi)-triggering nucleic acids suggests that the design of unconventional RNAi trigger structures with novel features is possible. Here, we report a cross-shaped RNA duplex structure, termed quadruple interfering RNA (qiRNA), with multiple target gene silencing activity. qiRNA triggers the simultaneous down-regulation of four cellular target genes via an RNAi mechanism. In addition, qiRNA shows enhanced intracellular delivery and target gene silencing over conventional siRNA when complexed with jetPEI, a linear polyethyleneimine (PEI). We also show that the long antisense strand of qiRNA is incorporated intact into an RNA-induced silencing complex (RISC). This novel RNA scaffold further expands the repertoire of RNAi-triggering molecular structures and could be used in the development of therapeutics for various diseases including viral infections and cancer.     

OCEAN CLIMATE CHANGE,PHYTOPLANKTON COMMUNITY RESPONSES,AND HARMFUL ALGAL BLOOMS: A FORMIDABLE PREDICTIVE CHALLENGE1

Prediction of the impact of global climate change on marine HABs is fraught with difficulties. However, we can learn important lessons from the fossil record of dinoflagellate cysts; long‐term monitoring programs, such as the Continuous Plankton Recorder surveys; and short‐term phytoplankton community responses to El Niño Southern Oscillation (ENSO) and North Atlantic Oscillation (NAO) episodes. Increasing temperature, enhanced surface stratification, alteration of ocean currents, intensification or weakening of local nutrient upwelling, stimulation of photosynthesis by elevated CO₂, reduced calcification through ocean acidification (“the other CO₂ problem”), and heavy precipitation and storm events causing changes in land runoff and micronutrient availability may all produce contradictory species‐ or even strain‐specific responses. Complex factor interactions exist, and simulated ecophysiological laboratory experiments rarely allow for sufficient acclimation and rarely take into account physiological plasticity and genetic strain diversity. We can expect: (i) range expansion of warm‐water species at the expense of cold‐water species, which are driven poleward; (ii) species‐specific changes in the abundance and seasonal window of growth of HAB taxa; (iii) earlier timing of peak production of some phytoplankton; and (iv) secondary effects for marine food webs, notably when individual zooplankton and fish grazers are differentially impacted (“match‐mismatch”) by climate change. Some species of harmful algae (e.g., toxic dinoflagellates benefitting from land runoff and/or water column stratification, tropical benthic dinoflagellates responding to increased water temperatures and coral reef disturbance) may become more successful, while others may diminish in areas currently impacted. Our limited understanding of marine ecosystem responses to multifactorial physicochemical climate drivers as well as our poor knowledge of the potential of marine microalgae to adapt genetically and phenotypically to the unprecedented pace of current climate change are emphasized. The greatest problems for human society will be caused by being unprepared for significant range expansions or the increase of algal biotoxin problems in currently poorly monitored areas, thus calling for increased vigilance in seafood‐biotoxin and HAB monitoring programs. Changes in phytoplankton communities provide a sensitive early warning for climate‐driven perturbations to marine ecosystems.     

A novel approach for the construction of multiple shRNA expression vectors

The application of RNA interference (RNAi) as a research and therapeutic tool depends on its ability to silence genes in a sequence-specific manner. Recent studies have reported that the effective knockdown of genes can be achieved by multiple short hairpin RNAs (shRNAs) in a single vector. Moreover, this approach can depress several genes simultaneously. However, current methods for the construction of multiple shRNA vectors often suffer from vector instability and are time-consuming. Here, we describe a simple, quick and low-cost approach to construct a single vector expressing four shRNA sequences driven by four different promoters. Using this vector, we were able to improve the gene silencing efficiency and make it possible to silence four different genes simultaneously, further expanding the application spectrum of RNAi, both in functional studies and therapeutic strategies.     

pSAT RNA interference vectors: a modular series for multiple gene down-regulation in plants

RNA interference (RNAi) is a powerful tool for functional gene analysis, which has been successfully used to down-regulate the levels of specific target genes, enabling loss-of-function studies in living cells. Hairpin (hp) RNA expression cassettes are typically constructed on binary plasmids and delivered into plant cells by Agrobacterium-mediated genetic transformation. Realizing the importance of RNAi for basic plant research, various vectors have been developed for RNAi-mediated gene silencing, allowing the silencing of single target genes in plant cells. To further expand the collection of available tools for functional genomics in plant species, we constructed a set of modular vectors suitable for hpRNA expression under various constitutive promoters. Our system allows simple cloning of the target gene sequences into two distinct multicloning sites and its modular design provides a straightforward route for replacement of the expression cassette's regulatory elements. More importantly, our system was designed to facilitate the assembly of several hpRNA expression cassettes on a single plasmid, thereby enabling the simultaneous suppression of several target genes from a single vector. We tested the functionality of our new vector system by silencing overexpressed marker genes (green fluorescent protein, DsRed2, and nptII) in transgenic plants. Various combinations of hpRNA expression cassettes were assembled in binary plasmids; all showed strong down-regulation of the reporter genes in transgenic plants. Furthermore, assembly of all three hpRNA expression cassettes, combined with a fourth cassette for the expression of a selectable marker, resulted in down-regulation of all three different marker genes in transgenic plants. This vector system provides an important addition to the plant molecular biologist's toolbox, which will significantly facilitate the use of RNAi technology for analyses of multiple gene function in plant cells.     

A bacterium that inhibits the growth of Pfiesteria piscicida and other dinoflagellates

Toxic dinoflagellate blooms have increased in estuaries of the east coast of the United States in recent years, and the discovery of Pfiesteria piscicida has brought renewed attention to the problem of harmful algal blooms (HAB) in general. Many bacteria and viruses have been isolated that have algicidal or algistatic effects on phytoplankton, including HAB species. Twenty-two bacterial isolates from the Delaware Inland Bays were screened for algicidal activity. One isolate (Shewanella IRI-160) had a growth-inhibiting effect on all three dinoflagellate species tested, including P. piscicida (potentially toxic zoospores), Prorocentrum minimum, and Gyrodinium uncatenum. This bacterium did not have a negative effect on the growth of any of the other four common estuarine non-dinoflagellate species tested, and in fact had a slight stimulatory effect on a diatom, a prasinophyte, a cryptophyte, and a raphidophyte. Shewanella IRI-160 is the first non-microzooplankton example of a microbe with the ability to control and inhibit the growth of P. piscicida, suggesting that bacteria in the natural environment could play a role in controlling the growth and abundance of P. piscicida and other dinoflagellates. Such bacteria could also potentially be used as management tools to prevent the proliferation of potentially harmful dinoflagellates in estuaries and coastal waters.