Development of rRNA and rDNA-targeted probes for fluorescence in situ hybridization to detect Heterosigma akashiwo (Raphidophyceae)

Heterosigma akashiwo (Hada) is a fragile, fish-killing alga. Efforts to understand and prevent blooms due to this harmful species to mitigate the impact on aquaculture require the development of methods for rapid and precise identification and quantification, so that adequate warning of a harmful algal bloom may be given. Here, we report the development and application of rRNA and rDNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH) to aid in the detection and enumeration of H. akashiwo. The designed probes were species specific, showing no cross-reactivity with four common HAB causative species: Prorocentrum micans Ehrenberg, P. minimum (Pavillard) Schiller, Alexandrium tarmarense (Lebour) Balech, and Skeletonema costatum (Greville) Cleve, or with four other microalgae, including Gymnodinium sp. Stein, Platy-monas cordiformis (Karter) Korsch, Skeletonema sp.1 Greville and Skeletonema sp.2. The rRNA-targeted probe hybridized to cytoplasmic rRNA, showing strong green fluorescence throughout the whole cell, while cells labeled by rDNA-targeted probe exhibited exclusively fluorescent nucleus. The detection protocols were optimized and could be completed within an hour. For rRNA and rDNA probes, about a corresponding 80% and 70% of targeted cells could be identified and quantified during the whole growth circle, despite the inapparent variability in the average probe reactivity. The established FISH was proved promising for specific, rapid, precise, and quantitative detection of H. akashiwo.     

Mapping the Distribution of Cysts from the Toxic Dinoflagellate Cochlodinium polykrikoides in Bloom-Prone Estuaries by a Novel Fluorescence In Situ Hybridization Assay

Cochlodinium polykrikoides is a cosmopolitan dinoflagellate that is notorious for causing fish-killing harmful algal blooms (HABs) across North America and Asia. While recent laboratory and ecosystem studies have definitively demonstrated that Cochlodinium forms resting cysts that may play a key role in the dynamics of its HABs, uncertainties regarding cyst morphology and detection have prohibited even a rudimentary understanding of the distribution of C. polykrikoides cysts in coastal ecosystems. Here, we report on the development of a fluorescence in situ hybridization (FISH) assay using oligonucleotide probes specific for the large subunit (LSU) ribosomal DNA (rDNA) of C. polykrikoides. The LSU rDNA-targeted FISH assay was used with epifluorescence microscopy and was iteratively refined to maximize the fluorescent reaction with C. polykrikoides and minimize cross-reactivity. The final LSU rDNA-targeted FISH assay was found to quantitatively recover cysts made by North American isolates of C. polykrikoides but not cysts formed by other common cyst-forming dinoflagellates. The method was then applied to identify and map C. polykrikoides cysts across bloom-prone estuaries. Annual cyst and vegetative cell surveys revealed that elevated densities of C. polykrikoides cysts (>100 cm⁻³) during the spring of a given year were spatially consistent with regions of dense blooms the prior summer. The identity of cysts in sediments was confirmed via independent amplification of C. polykrikoides rDNA. This study mapped C. polykrikoides cysts in a natural marine setting and indicates that the excystment of cysts formed by this harmful alga may play a key role in the development of HABs of this species.     

Novel algicidal evidence of a bacterium Bacillus sp. LP-10 killing Phaeocystis globosa,a harmful algal bloom causing species

While searching for effective bio-agents to control harmful algal blooms (HABs), the bacterial strain LP-10, which has strong algicidal activity against Phaeocystis globosa (Prymnesiophyceae), was isolated from surface seawater samples taken from the East China Sea. 16S rDNA sequence analysis and morphological characteristics revealed the strain LP-10 belonged to the genus Bacillus. The lytic effect of Bacillus sp. LP-10 against P. globosa was both concentration- and time-dependent. Algicidal activities of different growth stages of the bacterial culture varied significantly. The lytic effect of different parts of the bacterial cultures indicated that the algal cells were lysed by algicidal active compounds in the cell-free filtrate. Analysis of the properties of the active compounds showed that they had a molecular weight of less than 1000 Da and that the active compounds were stable between −80 and 121 °C. The algicidal range assay indicated that five other algal species were also suppressed by strain LP-10, including: Alexandrium catenella, A. tamarense, A. minutum, Prorocentrum micans and Asterionella japonica. Our results suggested that the algicidal bacterium Bacillus sp. LP-10 could be a potential bio-agent to control the blooms of harmful algal species.     

Evidence for Production of Sexual Resting Cysts by the Toxic Dinoflagellate Karenia mikimotoi in Clonal Cultures and Marine Sediments

The toxic dinoflagellate Karenia mikimotoi has been well-known for causing large-scale and dense harmful algal blooms (HABs) in coastal waters worldwide and serious economic loss in aquaculture and fisheries and other adverse effects on marine ecosystems. Whether K. mikimotoi forms resting cysts has been a puzzling issue regarding to the mechanisms of bloom initiation and geographic expansion of this species. We provide morphological and molecular confirmation of sexually produced thin-walled resting cysts by K. mikimotoi based on observations of laboratory cultures and their direct detection in marine sediments. Light and scanning electron microscopy evidences for sexual reproduction include attraction and pairing of gametes, gamete fusion, formation of planozygote and thin-walled cyst, and the documentation of the thin-walled cyst germination processes. Evidence for cysts in marine sediments was in three aspects: positive PCR detection of cysts using species-specific primers in the DNA extracted from whole sediments; fluorescence in situ hybridization detection of cysts using FISH probes; and single-cell PCR sequencing for cysts positively labeled with FISH probes. The existence of sexually produced, thin-walled resting cysts by K. mikimotoi provides a possible mechanism accounting for the initiation of annually recurring blooms at certain regions and global expansion of the species during the past decades.     

Prorocentrum minimum (Pavillard) Schiller: A review of a harmful algal bloom species of growing worldwide importance

Prorocentrum minimum (Pavillard) Schiller, a common, neritic, bloom-forming dinoflagellate, is the cause of harmful blooms in many estuarine and coastal environments. Among harmful algal bloom species, P. minimum is important for the following reasons: it is widely distributed geographically in temperate and subtropical waters; it is potentially harmful to humans via shellfish poisoning; it has detrimental effects at both the organismal and environmental levels; blooms appear to be undergoing a geographical expansion over the past several decades; and, a relationship appears to exist between blooms of this species and increasing coastal eutrophication. Although shellfish toxicity with associated human impacts has been attributed to P. minimum blooms from a variety of coastal environments (Japan; France; Norway; Netherlands; New York, USA), only clones isolated from the Mediterranean coast of France, and shellfish exposed to P. minimum blooms in this area, have been shown to contain a water soluble neurotoxic component which killed mice. Detrimental ecosystem effects associated with blooms range from fish and zoobenthic mortalities to shellfish aquaculture mortalities, attributable to both indirect biomass effects (e.g., low dissolved oxygen) and toxic effects. P. minimum blooms generally occur under conditions of high temperatures and incident irradiances and low to moderate salinities in coastal and estuarine environments often characterized as eutrophic, although they have been found under widely varying salinities and temperatures if other factors are conducive for growth. The physiological flexibility of P. minimum in response to changing environmental parameters (e.g., light, temperature, salinity) as well as its ability to utilize both inorganic and organic nitrogen, phosphorus, and carbon nutrient sources, suggest that increasing blooms of this species are a response to increasing coastal eutrophication.     

Fiber-Optic Microarray for Simultaneous Detection of Multiple Harmful Algal Bloom Species

Harmful algal blooms (HABs) are a serious threat to coastal resources, causing a variety of impacts on public health, regional economies, and ecosystems. Plankton analysis is a valuable component of many HAB monitoring and research programs, but the diversity of plankton poses a problem in discriminating toxic from nontoxic species using conventional detection methods. Here we describe a sensitive and specific sandwich hybridization assay that combines fiber-optic microarrays with oligonucleotide probes to detect and enumerate the HAB species Alexandrium fundyense, Alexandrium ostenfeldii, and Pseudo-nitzschia australis. Microarrays were prepared by loading oligonucleotide probe-coupled microspheres (diameter, 3 μm) onto the distal ends of chemically etched imaging fiber bundles. Hybridization of target rRNA from HAB cells to immobilized probes on the microspheres was visualized using Cy3-labeled secondary probes in a sandwich-type assay format. We applied these microarrays to the detection and enumeration of HAB cells in both cultured and field samples. Our study demonstrated a detection limit of approximately 5 cells for all three target organisms within 45 min, without a separate amplification step, in both sample types. We also developed a multiplexed microarray to detect the three HAB species simultaneously, which successfully detected the target organisms, alone and in combination, without cross-reactivity. Our study suggests that fiber-optic microarrays can be used for rapid and sensitive detection and potential enumeration of HAB species in the environment.     

Recent insights about relationships between nutrient availability,forms, and stoichiometry,and the distribution,ecophysiology, and food web effects of pelagic and benthic Prorocentrum species

The genus Prorocentrum includes six planktonic species that form high-biomass blooms, and at least nine predominantly benthic toxigenic species. Four of the plankters, including P. minimum, the only plankter reported to be toxigenic, are among the most commonly recognized harmful algae that are increasing in frequency, duration, and magnitude globally. Culture studies suggest a species group that generally grows maximally at inorganic nutrient N:P ratios just below Redfield proportions. However, field studies indicate that planktonic Prorocentrum species bloom when nutrients are at high N:P ratios relative to Redfield proportions. In the benthic species P. lima complex, toxin production has been shown to be inversely related to nutrient limitation, increasing when nutrient ratios are above Redfield proportions. Mixotrophy and allelopathy can play an important role in the interactions among planktonic Prorocentrum species, diatoms and other dinoflagellates, but little information is available for benthic taxa. The available information suggests that there are allelopathic interactions among benthic species and other algae, and that benthic species also can adversely affect finfish and shellfish health. While high growth rates may allow these plankters to initiate blooms, adaptive physiology is hypothesized to allow blooms to be maintained at less than maximal growth rates and at non-optimal N:P ratios. Given the projection for land-based nutrient export to continue to increase, it is expected that there will be further expansion of planktonic harmful Prorocentrum spp. globally and more intensive or more toxic benthic occurrences in the future.     

Utilization of dissolved organic phosphorus by different groups of phytoplankton taxa

The utilization of nine dissolved organic phosphorus (DOP) compounds by five bloom-causing phytoplankton species was studied under batch culture conditions. The DOP compounds included were adenosine 5-triphosphate (ATP), adenosine 5-monophosphate (AMP), cytidine 5-monophosphate (CMP), guanosine 5-monophosphate (GMP), uridine 5-monophosphate (UMP), glucose-6-phosphate (G6P), sodium glycerophosphate (GYP), 4-nitrophenyl phosphate (NPP), and triethyl phosphate (TEP), and the phytoplankton taxa were Skeletonema costatum, Prorocentrum micans, Alexandrium tamarense, Chattonella marina, and Heterosigma akashiwo. The four flagellate taxa, P. micans, A. tamarense, C. marina, and H. akashiwo, grew well under various DOP regimes. P. micans and C. marina were the most capable of using DOP compounds, sustaining better growth on a majority of nucleotides (ATP, AMP, CMP, GMP, and UMP) and phosphomonoesters (G6P and GYP) than in inorganic phosphorus (P) controls. A. tamarense and H. akashiwo showed equivalent growth in most organic and inorganic P cultures, while the diatom species, S. costatum, could only utilize AMP and GMP. Furthermore, A. tamarense and C. marina could endure N, P-depleted conditions. Among the nine DOP compounds tested, the nucleotide compounds had the highest nutritional value for algal cell growth, while TEP could not sustain growth as the sole source of P. These results suggest that enhanced DOP utilization and the endurance of nutrient-limitation by harmful flagellate taxa offer their competitive advantages, which may account for the frequent occurrence of their blooms in coastal waters.     

Roles of mixotrophy in blooms of different dinoflagellates: Implications from the growth experiment

Studies over the last two decades suggested that mixotrophy could be an important adaptive strategy for some bloom-forming dinoflagellates. In the coastal waters adjacent to the Changjiang River estuary in the East China Sea, recurrent blooms of dinoflagellates Prorocentrum donghaiense, Karenia mikimotoi and Alexandrium catenella started to appear from the beginning of the 21 century, but roles of mixotrophy in the formation of dinoflagellate blooms were not well understood. In the current study, mixotrophy-based growth of four selected bloom-causative dinoflagellate species, i.e. K. mikimotoi, A. catenella, P. donghaiense and Prorocentrum micans, were studied. Dinoflagellates were co-cultured with different prey organisms, including bacterium Marinobacter sp., microalgae Isochrysis galbana and Hemiselmis virescens, under a variant of nutrient conditions. It was found that growth of dinoflagellate K. mikimotoi was significantly promoted with the presence of prey organisms. Growth of P. donghaiense and P. micans was only slightly improved. For A. catenella, the addition of prey organisms has no effects on the growth, while both of the two prey microalgae I. galbana and H. virescens were killed, probably by allelochemicals released from A. catenella. There was no apparent relationship between nutrient conditions and the mixotrophy-based growth of the tested dinoflagellates. Based on the results of the growth experiment, it is implicated that mixotrophy may play different roles in the growth and bloom of the four dinoflagellate species. It can be an important competitive strategy for K. mikimotoi. For the two Prorocentrum species and A. catenella, however, the role of mixotrophy is much limited. They may depend more on other competitive strategies, such as phototrophy-based growth and allelopathic effect, to prevail in the phytoplankton community and form blooms.     

Detection and Differentiation of Chlamydiae by Fluorescence In Situ Hybridization

Chlamydiae are important pathogens of humans and animals but diagnosis of chlamydial infections is still hampered by inadequate detection methods. Fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes is widely used for the investigation of uncultured bacteria in complex microbial communities and has recently also been shown to be a valuable tool for the rapid detection of various bacterial pathogens in clinical specimens. Here we report on the development and evaluation of a hierarchic probe set for the specific detection and differentiation of chlamydiae, particularly C. pneumoniae, C. trachomatis, C. psittaci, and the recently described chlamydia-like bacteria comprising the novel genera Neochlamydia and Parachlamydia. The specificity of the nine newly developed probes was successfully demonstrated by in situ hybridization of experimentally infected amoebae and HeLa 229 cells, including HeLa 229 cells coinfected with C. pneumoniae and C. trachomatis. FISH reliably stained chlamydial inclusions as early as 12 h postinfection. The sensitivity of FISH was further confirmed by combination with direct fluorescence antibody staining. In contrast to previously established detection methods for chlamydiae, FISH was not susceptible to false-positive results and allows the detection of all recognized chlamydiae in one single step.     

Inhibitory effects of Chinese traditional herbs and herb-modified clays on the growth of harmful algae,Phaeocystis globosa and Prorocentrum donghaiense

In order to develop an improved method of mitigating harmful algal blooms (HABs), we assessed the inhibitory effects of five Chinese traditional herbs, Andrographis paniculata (Burm.f.) Nees, Galla chinensis, Punica granatum L., Cortex phellodendri chinensis and Radix scutellariae, as well as clay modified with herb extract on the growth of two harmful algae, Phaeocystis globosa and Prorocentrum donghaiense. The results showed that the five Chinese herbs had varying effects on the target microalgae and inhibitory rates ranged from −35% to 100% at concentration of 0.3 g dry wt./L during 96 h treatments. Among the five herbs, G. chinensis exhibited the strongest inhibitory effect, almost 100% on P. globosa and P. donghaiense. The growth of P. globosa and P. donghaiense was completely inhibited by G. chinensis extracts (0.3 g dry wt./L) during all growth phases, and lag phase cultures were more sensitive than exponential phase and stationary phase cultures. The highest inhibitory rate (100% inhibition) on P. globosa was observed at lag phase, followed by exponential phase (73.1% inhibition), and stationary phase (57% inhibition). The highest inhibitory rate of 100% on P. donghaiense was also found at lag phase, 40.3% at exponential phase, and 23.4% at stationary phase. Furthermore, modified clay with G. chinensis extract significantly enhanced the inhibitory impact. Modified clay (0.3 g L⁻¹) produced 95% of growth inhibition for both algae species at 24 h, and maintained the inhibition thereafter. Our study demonstrated that G. chinensis and clays modified with its extract significantly inhibited the growth of harmful species, therefore may provide ideas and another option for control of harmful algal blooms.     

Microbial Community Response during the Iron Fertilization Experiment LOHAFEX

Iron fertilization experiments in high-nutrient, low-chlorophyll areas are known to induce phytoplankton blooms. However, little is known about the response of the microbial community upon iron fertilization. As part of the LOHAFEX experiment in the southern Atlantic Ocean, Bacteria and Archaea were monitored within and outside an induced bloom, dominated by Phaeocystis-like nanoplankton, during the 38 days of the experiment. The microbial production increased 1.6-fold (thymidine uptake) and 2.1-fold (leucine uptake), while total cell numbers increased only slightly over the course of the experiment. 454 tag pyrosequencing of partial 16S rRNA genes and catalyzed reporter deposition fluorescence in situ hybridization (CARD FISH) showed that the composition and abundance of the bacterial and archaeal community in the iron-fertilized water body were remarkably constant without development of typical bloom-related succession patterns. Members of groups usually found in phytoplankton blooms, such as Roseobacter and Gammaproteobacteria, showed no response or only a minor response to the bloom. However, sequence numbers and total cell numbers of the SAR11 and SAR86 clades increased slightly but significantly toward the end of the experiment. It seems that although microbial productivity was enhanced within the fertilized area, a succession-like response of the microbial community upon the algal bloom was averted by highly effective grazing. Only small-celled members like the SAR11 and SAR86 clades could possibly escape the grazing pressure, explaining a net increase of those clades in numbers.     

Colorimetric DNA-based assay for the specific detection and quantification of Ostreopsis cf. ovata and Ostreopsis cf. siamensis in the marine environment

Ostreopsis is a toxic benthic dinoflagellate largely distributed worldwide in tropical and temperate areas. In the Mediterranean Sea, periodic summer blooms have been reported and have become a serious concern due to their direct impact on human health and the environment. Current microalgae identification is performed via light microscopy, which is time-consuming and is not able to differentiate among Ostreopsis species. Therefore, there is mature need for rapid, specific and easy-to-use detection tools. In this work, a colorimetric assay exploiting a combination of recombinase polymerase amplification (RPA) and a sandwich hybridisation assay was developed for O. cf. ovata and O. cf. siamensis detection and quantification. The specificity of the system was demonstrated by cross-reactivity experiments and calibration curves were successfully constructed using genomic DNA, achieving limits of detection of 10 and 14 pg/μL for O. cf. ovata and O. cf. siamensis, respectively. The assay was applied to the analysis of planktonic and benthic environmental samples from different sites of the Catalan coast. Species-specific DNA quantifications were in agreement with qPCR analysis, demonstrating the reliability of the colorimetric approach. Significant correlations were also obtained between DNA quantifications and light microscopy counts. The approach may be a valuable tool to provide timely warnings, facilitate monitoring activities or study population dynamics, and paves the way towards the development of in situ tools for the monitoring of harmful algal blooms.     

Harmful dinoflagellate blooms caused by Cochlodinium sp.: Global expansion and ecological strategies facilitating bloom formation

The past two decades have witnessed an expansion in the reported occurrences of harmful algal blooms (HABs) caused by the dinoflagellate Cochlodinium. Prior to 1990, blooms had been primarily reported in Southeast Asia, with South Korea alone reporting more than $100M USD in annual fisheries losses during the 1990s. Since then, time blooms have expanded across Asia, Europe, and North America, with recognition of multiple species and ribotypes that exhibit similar ecophysiological and harmful characteristics. Here, we summarize the current state of knowledge regarding taxonomy, phylogeny, detection, distribution, ecophysiology, life history, food web interactions, and mitigation of blooms formed by Cochlodinium. We review this recent expansion of Cochlodinium blooms and characterize the ecological strategies utilized by Cochlodinium populations to form HABs. Although Cochlodinium is comprised of more than 40 species, we focus primarily on the two HAB-forming species, C. polykrikoides and C. fulvescens, specifically describing their flexible nutrient acquisition strategies, inhibition of grazing by inducing rapid mortality in a diverse set of predators, and allelopathic inhibition of a broad range of competing phytoplankton. Finally, we summarize the available information on prevention, control, and mitigation strategies specific to this genus, and discuss pressing questions regarding this increasingly important HAB organism.     

Comparison of Fluorescently Labeled Oligonucleotide and Polynucleotide Probes for the Detection of Pelagic Marine Bacteria and Archaea

We compared the detection of bacteria and archaea in the coastal North Sea and at Monterey Bay, Calif., after fluorescence in situ hybridization (FISH) either with rRNA-targeted oligonucleotide probes monolabeled with the cyanin dye Cy3 (oligoFISH) or with fluorescein-labeled polyribonucleotide probes (polyFISH). During an annual cycle in German Bight surface waters, the percentages of bacteria visualized by polyFISH (annual mean, 77% of total counts) were significantly higher than those detected by oligoFISH (53%). The fraction of total bacteria visualized by oligoFISH declined during winter, whereas cell numbers determined by polyFISH remained constant throughout the year. Depth profiles from Monterey Bay showed large differences in the fraction of bacterial cells visualized by polyFISH and oligoFISH in the deeper water layers irrespective of the season. Image-analyzed microscopy indicated that the superior detection of cells by polyFISH with fluorescein-labeled probes in bacterioplankton samples was less a consequence of higher absolute fluorescence intensities but was rather related to quasi-linear bleaching dynamics and to a higher signal-to-background ratio. The relative abundances of archaea in North Sea and Monterey Bay spring samples as determined by oligoFISH were on average higher than those determined by polyFISH. However, simultaneous hybridizations with oligonucleotide probes for bacteria and archaea suggested that the oligoFISH probe ARCH915 unspecifically stained a population of bacteria. Using either FISH technique, blooms of archaea were observed in North Sea surface waters during the spring and summer months. Marine group II archaea (Euryarchaeota) reached >30% of total picoplankton abundances, as determined by polyFISH. We suggest that studies of pelagic microbial community structure using oligoFISH with monolabeled probes should focus on environments that yield detections ≥70% of total cell counts, e.g., coastal surface waters during spring and summer.     

Rapid detection of natural cells of Alexandrium tamarense and A. catenella (Dinophyceae) by fluorescence in situ hybridization

The marine toxic dinoflagellates Alexandrium tamarense (Lebor) Balech and A. catenella (Whedon and Kofoid) Taylor that cause paralytic shellfish poisoning (PSP) are identified on the basis of morphological features in routine monitoring. Rapid and simple identification is, however, often difficult because of the morphological similarity. Fluorescent in situ hybridization (FISH) using ribosomal RNA (rRNA)-targeted probes has been studied as a method of easily identifying and enumerating species responsible for harmful algal blooms (HABs). Its application to monitoring natural populations of HAB species, however, is limited. Here, we applied the FISH method to identify and enumerate cells of A. tamarense and A. catenella in natural plankton assemblages collected from Japanese coastal waters. A. tamarense-specific (Atm1) and A. catenella-specific (Act1) probes were established based on the D2 region of the large-subunit ribosomal RNA gene (28S rDNA). With these two probes, natural cells of A. tamarense or A. catenella in field samples could easily be identified when the following three conditions were met. First, cells should be concentrated by filtration, not centrifugation, in order to avoid the loss of cells. Second, autofluorescence should be minimized; acetone was an effective decolorization reagent. Third, samples should be stored at −20 or −80 °C for long-term preservation. The results indicate that FISH is a useful tool for the rapid identification of toxic Alexandrium spp. and can facilitate the analysis of numerous natural samples.     

北部湾海洋原甲藻的形态特征及其系统进化分析

为探讨广西北部湾海洋原甲藻(Prorocentrum micans)的形态特征及其系统进化意义,利用光学显微镜、分子生物学方法,对海洋原甲藻BBW-01 藻株的形态特征进行了描述,并分析了其分子系统进化关系.结果表明,各地理株系的海洋原甲藻的形态特征相似,仅在细胞大小上存在差异.海洋原甲藻BBW-01 与采自于广东大亚湾的海洋原甲藻形态特征最为接近,其壳板后端的7 个呈“V”字形对称排列的大孔可作为海洋原甲藻鉴定的重要指标.18S rDNA 序列同源检索和系统进化分析表明,海洋原甲藻BBW-01 与源自中国南海的海洋原甲藻的亲缘关系最近,并与其他2 株海洋原甲藻聚成一支,属于浮游、兼性浮游类原甲藻.因此,对赤潮原因种的准确识别有助于预防和减轻赤潮对海洋环境和人类带来的危害.     

Detection of intracellular neutral lipid content in the marine microalgae Prorocentrum micans and Phaeodactylum tricornutum using Nile red and BODIPY 505/515

Diatoms and dinoflagellates not only have extensive distribution and a huge biomass in marine ecosystems, but also have high lipid accumulation in nature or after physiological and genetic modification, which indicates that these organisms may be optimal candidate algal strains for biodiesel production. In this study, we determined the content of intracellular neutral lipids (triacylglycerol [TAG]) in the dinoflagellate Prorocentrum micans and in the diatom Phaeodactylum tricornutum using NR and BODIPY 505/515 staining. The freshwater green alga Scenedesmus obliquus was used as a control. Optimum concentrations of 1.000 and 1.500 μg mL^?1 were determined for neutral lipid Nile red (NR) staining in P. micans and P. tricornutum. Unlike NR staining, the optimal concentrations of BODIPY 505/515 staining in P. micans and P. tricornutum were lower, at 0.100 and 0.075 μg mL^?1, respectively. High correlation coefficients of R ²?=?0.990 and R ²?=?0.989 were obtained for P. micans and P. tricornutum intracellular neutral lipid content and the relative fluorescence intensity with NR staining, while the reference alga, S. obliquus, had a relatively low correlation coefficient of R ²?=?0.908 when stained with NR. The neutral lipid content determined by thin-layer chromatography-flame ionization detector matched the analytical data from fluorescence measurements. These results indicated that NR and BODIPY 505/515 staining can be used as an excellent high-throughput approach to screen marine diatoms and dinoflagellates.     

rRNA Sequence-Based Scanning Electron Microscopic Detection of Bacteria

A new scanning electron microscopic method was developed for gaining both phylogenetic and morphological information about target microbes using in situ hybridization with rRNA-targeted oligonucleotide probes (SEM-ISH). Target cells were hybridized with oligonucleotide probes after gold labeling. Gold enhancement was used for amplification of probe signals from hybridized cells. The hybridized cells released a strong backscatter electron signal due to accumulation of gold atoms inside cells. SEM-ISH was applied to analyze bacterial community composition in freshwater samples, and bacterial cell counts determined by SEM-ISH with rRNA-targeted probes for major phyla within the domain Bacteria were highly correlated to those by fluorescent in situ hybridization (FISH). The bacterial composition on surface of river sediment particles before and after cell dispersion treatment by sonication was successfully revealed by SEM-ISH. Direct enumeration of bacterial cells on the surface of sonicated sediment particles by SEM-ISH demonstrated that members of Cytophaga-Flavobacterium existed tightly on the surface of particles. SEM-ISH allows defining the number and distribution of phylogenetically defined cells adherent to material surfaces, which is difficult in FISH, and it gives new insight into electron microscopic studies of microorganisms in their natural environment.     

Candidatus Prosiliicoccus vernus,a spring phytoplankton bloom associated member of the Flavobacteriaceae

Microbial degradation of algal biomass following spring phytoplankton blooms has been characterised as a concerted effort among multiple clades of heterotrophic bacteria. Despite their significance to overall carbon turnover, many of these clades have resisted cultivation. One clade known from 16S rRNA gene sequencing surveys at Helgoland in the North Sea, was formerly identified as belonging to the genus Ulvibacter. This clade rapidly responds to algal blooms, transiently making up as much as 20% of the free-living bacterioplankton. Sequence similarity below 95% between the 16S rRNA genes of described Ulvibacter species and those from Helgoland suggest this is a novel genus. Analysis of 40 metagenome assembled genomes (MAGs) derived from samples collected during spring blooms at Helgoland support this conclusion. These MAGs represent three species, only one of which appears to bloom in response to phytoplankton. MAGs with estimated completeness greater than 90% could only be recovered for this abundant species. Additional, less complete, MAGs belonging to all three species were recovered from a mini-metagenome of cells sorted via flow cytometry using the genus specific ULV995 fluorescent rRNA probe. Metabolic reconstruction indicates this highly abundant species most likely degrades proteins and the polysaccharide laminarin. Fluorescence in situ hybridisation showed coccoid cells, with a mean diameter of 0.78 mm, with standard deviation of 0.12 μm. Based on the phylogenetic and genomic characteristics of this clade, we propose the novel candidate genus Candidatus Prosiliicoccus, and for the most abundant and well characterised of the three species the name Candidatus Prosiliicoccus vernus.