FLAVODOXIN EXPRESSION AS AN INDICATOR OF IRON LIMITATION IN MARINE DIATOMS1

We have prevously shown that a marine chlorophyte expressed flavodoxin under iron limitation but not under other nutrient stress conditions. Here we use polyclonal antiserum raised against the diatom Phaeodactylum tricornutum Bohlin to show that a similar response is observed in this species. Using our antibody, western blotting techniques, and standard colorimetric detection (4-chloro-1-naphthol), we can detect at least a 25–50-fold increase in flavodoxin in iron-depleted compared to iron-replete cells. In iron-limited batch cultures of P. tricornulum, flavodoxin accumulation was inversely proportional to growth rate and was not detectable in cultures containing initially more than 750 nm of iron. We demonstrated that the accumulation of flavodoxin under iron stress is widespread among marine diatoms and that it may be possible to use the presence or absence of flavodoxin in natural marine diatom assemblages to detect iron limitation. However, our polyclonal antisera appears to be specific for diatoms and did not cross-react with Synechococcus sp ., Micromonas pusilla ( Butcher) Manton et Parke , Dunaliella tertiolecta Butcher , Chlorella sp ., Emiliania huxleii ( Lohm.) Hay et Parke, or Isochrysis galbana Parke. A reverse bioassay experiment was conducted with natural phytoplankton assemblages containing mainly diatoms from Long Island Sound and in shelf waters near Cape Hatteras, two areas not suspected to be iron-limited. Although flavodoxin was not detected in situ in these areas, natural populations of diatoms driven into iron limitation expres.sed flavodoxin. Flavodoxin was detected in mats of the diatom Rhizosolenia castracanei Cleve collected from the Equatorial Pacific during a JGOFS cruise in 1992, consistent with the hypothesis that iron may be limiting in this high-nutrient, low-chlorophyll region .     

Kleptoplast distribution,photosynthetic efficiency and sequestration mechanisms in intertidal benthic foraminifera

Foraminifera are ubiquitously distributed in marine habitats, playing a major role in marine sediment carbon sequestration and the nitrogen cycle. They exhibit a wide diversity of feeding and behavioural strategies (heterotrophy, autotrophy and mixotrophy), including species with the ability of sequestering intact functional chloroplasts from their microalgal food source (kleptoplastidy), resulting in a mixotrophic lifestyle. The mechanisms by which kleptoplasts are integrated and kept functional inside foraminiferal cytosol are poorly known. In our study, we investigated relationships between feeding strategies, kleptoplast spatial distribution and photosynthetic functionality in two shallow-water benthic foraminifera (Haynesina germanica and Elphidium williamsoni), both species feeding on benthic diatoms. We used a combination of observations of foraminiferal feeding behaviour, test morphology, cytological TEM-based observations and HPLC pigment analysis, with non-destructive, single-cell level imaging of kleptoplast spatial distribution and PSII quantum efficiency. The two species showed different feeding strategies, with H. germanica removing diatom content at the foraminifer’s apertural region and E. williamsoni on the dorsal site. All E. williamsoni parameters showed that this species has higher autotrophic capacity albeit both feeding on benthic diatoms. This might represent two different stages in the evolutionary process of establishing a permanent symbiotic relationship, or may reflect different trophic strategies.Subject terms: Microbial ecology, Microbial ecology, Microbiology     

ACCUMULATION OF FERREDOXIN AND FLAVODOXIN IN A MARINE DIATOM IN RESPONSE TO FE

Despite recognition that Fe availability is significant in regulating oceanic production in some regions, the biogeochemistry of this trace element is poorly understood. To complement contemporary methods of analytical chemistry, we have used an immunological approach to monitor the Fe nutrition of marine phytoplankton. In prokaryotes and numerous microalgae, the redox catalyst ferredoxin is functionally replaced by flavodoxin during periods of Fe deficiency. In this study, antibodies were raised against ferredoxin purified from a marine diatom, and their utility as a diagnostic indicator was assessed. A species survey demonstrated broad reactivity with both pennate and centric diatoms and additionally with several nondiatom taxa. In batch cultures of the diatom Phaeodactylum tricornutum Bohlin, in which Fe levels were varied, accumulation of ferredoxin varied with the physiological state of the culture; in unimpaired cells (F_v/F_m≥ 0.65), ferredoxin levels were high, whereas levels dropped markedly in cells experiencing even slight photochemical impairment. Accumulation of flavodoxin varied inversely with that of ferredoxin. An experiment was performed to demonstrate the temporal pattern of accumulation of ferredoxin upon recovery from Fe limitation. Prior to Fe amendment, cells were physiologically impaired (chlorotic, F_v/F_m < 0.3) and contained flavodoxin but no detectable ferredoxin. Following addition of Fe, constraints on photochemistry were relaxed within hours. Coinciding with this, levels of flavodoxin declined, whereas ferredoxin was accumulated to high levels within 8 h.     

The use of diatoms in monitoring the development of created wetlands at a sandmining site in Western Australia

Former sand-mining pits at Capel, 200 km south of Perth in Western Australia, have been rehabilitated into artificial wetlands since 1975–1979. A chain of fresh water lakes was created as a potential waterbird refuge and an area for passive recreation. Initially, the lakes had low pH, high ammonium, iron and manganese levels and low phosphorus concentration. The lakes were characterised by low diversity of diatoms dominated by acidophilous species. Following an increase in pH in the effluent water discharged into the lake from the mining process plant and landscaping of the lakes since 1988, the diversity of diatoms gradually increased. The system is now dominated by periphytic diatom communities, preferring high conductivity. There has been a marked transition in the diatom community from acidophilous to alkaliphilous species. Planktonic diatom blooms replaced dinoflagellate blooms. Concomitantly, there has been a dramatic increase in the diversity of invertebrates and waterbirds in these lakes. The value of diatoms in assessing the progressive development of created wetlands as self-sustaining ecosystems at sand mines in Australia is discussed.     

Diatoms and Other Epibionts Associated with Olive Ridley (Lepidochelys olivacea) Sea Turtles from the Pacific Coast of Costa Rica

Although the sea turtles have long been familiar and even iconic to marine biologists, many aspects of their ecology remain unaddressed. The present study is the first of the epizoic diatom community covering the olive ridley turtle’s (Lepidochelys olivacea) carapace and the first describing diatoms living on sea turtles in general, with the primary objective of providing detailed information on turtle epibiotic associations. Samples of turtle carapace including the associated diatom biofilm and epizoic macro-fauna were collected from Ostional beach (9° 59´ 23.7´´ N 85° 41´ 52.6´´ W), Costa Rica, during the arribada event in October 2013. A complex diatom community was present in every sample. In total, 11 macro-faunal and 21 diatom taxa were recorded. Amongst diatoms, the most numerous were erect (Achnanthes spp., Tripterion spp.) and motile (Haslea sp., Navicula spp., Nitzschia spp., Proschkinia sp.) forms, followed by adnate Amphora spp., while the most common macro-faunal species was Stomatolepas elegans (Cirripedia). Diatom densities ranged from 8179 ± 750 to 27685 ± 4885 cells mm^-2. Epizoic microalgae were either partly immersed or entirely encapsulated within an exopolymeric coat. The relatively low diatom species number, stable species composition and low inter-sample dissimilarities (14.4% on average) may indicate a mutualistic relationship between the epibiont and the basibiont. Dispersal of sea turtle diatoms is probably highly restricted and similar studies will help to understand both diatom diversity, evolution and biogeography, and sea turtle ecology and foraging strategies.     

SILICON METABOLISM IN DIATOMS: IMPLICATIONS FOR GROWTH

Diatoms are the world's largest contributors to biosilicification and are one of the predominant contributors to global carbon fixation. Silicon is a major limiting nutrient for diatom growth and hence is a controlling factor in primary productivity. Because our understanding of the cellular metabolism of silicon is limited, we are not fully knowledgeable about intracellular factors that may affect diatom productivity in the oceans. The goal of this review is to present an overview of silicon metabolism in diatoms and to identify areas for future research. Numerous studies have characterized parameters of silicic acid uptake by diatoms, and molecular characterization of transport has begun with the isolation of genes encoding the transporter proteins. Multiple types of silicic acid transporter gene have been identified in a single diatom species, and multiple types appear to be present in all diatom species. The controlled expression and perhaps localization of the transporters in the cell may be factors in the overall regulation of silicic acid uptake. Transport can also be regulated by the rate of silica incorporation into the cell wall, suggesting that an intracellular sensing and control mechanism couples transport with incorporation. Sizable intracellular pools of soluble silicon have been identified in diatoms, at levels well above saturation for silica solubility, yet the mechanism for maintenance of supersaturated levels has not been determined. The mechanism of intracellular transport of silicon is also unknown, but this must be an important part of the silicification process because of the close coupling between silica incorporation and uptake. Although detailed ultrastructural analyses of silica deposition have been reported, we know little about the molecular details of this process. However, proteins occluded within silica that promote silicification in vitro have recently been characterized, and the application of molecular techniques holds the promise of great advances in this area. Cellular energy for silicification and transport comes from aerobic respiration without any direct involvement of photosynthetic energy. As such, diatom silicon metabolism differs from that of other major limiting nutrients such as nitrogen and phosphorous, which are closely linked to photosynthetic metabolism. Cell wall silicification and silicic acid transport are tightly coupled to the cell cycle, which results in a dependency in the extent of silicification on growth rate. Silica dissolution is an important part of diatom cellular silicon metabolism, because dissolution must be prevented in the living cell, and because much of the raw material for mineralization in natural assemblages is supplied by dissolution of dead cells. Perhaps part of the reason for the ecological success of diatoms is due to their use of a silicified cell wall, which has been calculated to impart a substantial energy savings to organisms that have them. However, the growth of diatoms and other siliceous organisms has depleted the oceans of silicon, such that silicon availability is now a major factor in the control of primary productivity. Much new progress in understanding silicon metabolism in diatoms is expected because of the application of molecular approaches and sophisticated analytical techniques. Such insight is likely to lead to a greater understanding of the role of silicon in controlling diatom growth, and hence primary productivity, and of the mechanisms involved in the formation of the intricate silicified structures of the diatom cell wall.     

BORON AS A GROWTH REQUIREMENT FOR DIATOMS1

A boron requirement has been shown for 12 species of marine pennate diatoms, 4 species of marine centric diatoms, and S freshwater diatom species. It can be concluded that boron is essential for the growth of most, probably all, diatoms. It is much easier to demonstrate a requirement for the marine species than for the freshwater species. Some species of marine algal flagellates also require boron for growth; others apparently do not.     

Biofilm diatom community structure: influence of temporal and substratum variability

Diatoms, which are early autotrophic colonisers, are an important constituent of the biofouling community in the marine environment. The effects of substratum and temporal variations on the fouling diatom community structure in a monsoon-influenced tropical estuary were studied. Fibreglass and glass coupons were exposed every month for a period of 4 days and the diatom population sampled at 24 h intervals, over a period of 14 months. The planktonic diatom community structure differed from the biofilm community. Pennate diatoms dominated the biofilms whilst centric diatoms were dominant in the water column. Among the biofilm diatoms, species belonging to the genera Navicula, Amphora, Nitzschia, Pleurosigma and Thalassionema were dominant. On certain occasions, the influence of planktonic blooms was also seen on the biofilm community. A comparative study of biofilms formed on the two substrata revealed significant differences in density and diversity. However species composition was almost constant. In addition to substratum variations, the biofilm diatom community structure also showed significant seasonal variations, which were attributed to physico-chemical and biological changes in both the water and substratum. Temporal variations in the tychopelagic diatoms of the water were also observed to exert an influence on the biofilm diatom community. Variations in diatom communities may determine the functional ecosystem of the benthic environment.     

Genetic indicators of iron limitation in wild populations of Thalassiosira oceanica from the northeast Pacific Ocean

Assessing the iron (Fe) nutritional status of natural diatom populations has proven challenging as physiological and molecular responses can differ in diatoms of the same genus. We evaluated expression of genes encoding flavodoxin (FLDA1) and an Fe-starvation induced protein (ISIP3) as indicators of Fe limitation in the marine diatom Thalassiosira oceanica. The specificity of the response to Fe limitation was tested in cultures grown under Fe- and macronutrient-deficient conditions, as well as throughout the diurnal light cycle. Both genes showed a robust and specific response to Fe limitation in laboratory cultures and were detected in small volume samples collected from the northeast Pacific, demonstrating the sensitivity of this method. Overall, FLDA1 and ISIP3 expression was inversely related to Fe concentrations and offered insight into the Fe nutritional health of T. oceanica in the field. As T. oceanica is a species tolerant to low Fe, indications of Fe limitation in T. oceanica populations may serve as a proxy for severe Fe stress in the overall diatom community. At two shallow coastal locations, FLD1A and ISIP3 expression revealed Fe stress in areas where dissolved Fe concentrations were high, demonstrating that this approach may be powerful for identifying regions where Fe supply may not be biologically available.     

Iron acquisition by Streptococcus species: An updated review

Streptococcus is a genus of spherical Gram-positive bacteria responsible for many cases of meningitis, bacterial pneumonia, endocarditis, erysipelas, and necro-tizing fasciitis. To survive in the host environment with limited free iron available, Streptococcus species have developed various mechanisms to uptake iron as an essential nutrient. They can directly extract the metal ions from host iron-containing proteins such as ferritin, transferrin, lactoferrin, and hemoproteins. Other iron-uptake strategies, which are broadly distributed in the strains, include the employment of specialized secreted hemophores to acquire heme and the usage of small molecules called siderophores as high-affinity ferric chelators. This review intends to discuss the most recent discoveries of these iron acquisition systems and their relevant regulators in Streptococcus species.     

广东鉴江水系底栖硅藻多样性与时空分布特征

为了解广东省鉴江水系底栖硅藻多样性和时空分布特征,对全流域进行了底栖硅藻采样调查。结果表明,从19个采样点4次采样中共检出底栖硅藻10科52属242种,其中舟形藻属(Navicula)、菱形藻属(Nitzschia)和异极藻属(Gomphonema)是优势类群,出现频次和相对丰度较高。硅藻多样性指数(丰富度、真香农多样性指数和真辛普森多样性指数)随河流等级呈现一定的空间分布特征,但它们季节变化不明显。底栖硅藻群落相异性在上游和下游河段较高,从一级到三级河流递减,四级河流又增加。底栖硅藻群落结构空间变化明显,季节变化显著。群落丰富度的稀疏曲线表明,热带河流底栖硅藻群落以400个体计数,不能完整反映底栖硅藻多样性。这些为鉴江水系河流健康监测和水生态保护奠定了基础。     

The diversity and distribution of diatoms: from cosmopolitanism to narrow endemism

It has been claimed that microbial taxa will not exhibit endemism because their enormous populations remove dispersal as an effective constraint on geographical range. Here we review evidence that challenges this ubiquity hypothesis for the most speciose group of microbial eukaryotes, the diatoms. Detailed taxonomic inventories using fine-grained morphological characteristics, molecular markers, and crossing experiments have revealed that the geographic distribution of diatoms ranges from global to narrow endemic. Records of human-mediated introductions of exotic species further provide a strong indication that geographic dispersal was limiting in the past. Finally, recent studies have revealed that diatom community structure and diversity are influenced by geographical factors independent of environmental conditions. Diatom communities are thus regulated by the same processes that operate in macro-organisms, although possibly to a different degree, implying that dispersal limitation is significant and the endemism observed in isolated areas is real. These results underscore the pressing need to (1) continue research into diatom biology, ecology and the factors driving diatom species diversity and geographic distributions, and (2) protect relatively isolated areas against further introductions of exotic species. Special Issue: Protist diversity and geographic distribution. Guest editor: W. Foissner.     

Genetic and phenotypic characterization of Phaeodactylum tricornutum (Bacillariophyceae) accessions1

In the last few years, genome‐based studies in diatoms have received a major boost following the genome sequencing of the centric species Thalassiosira pseudonana Hasle et Heimdal and the pleiomorphic raphid pennate diatom Phaeodactylum tricornutum Bohlin. In addition, molecular tools, such as genetic transformation, have been developed for both species. Despite these molecular advances, relatively little is known regarding the genetic diversity of the available strains of these diatoms. In this study, we have compiled a historical summary of the known P. tricornutum species resources and have provided a genetic and phenotypic overview of 10 different axenic strains. Examination of intraspecies genetic diversity based on internal transcribed spacer 2 (ITS2) sequence and amplified fragment length polymorphism (AFLP) analyses indicate four different genotypes. Seven strains are predominantly fusiform, whereas one strain is predominantly oval, and another is predominantly triradiate. Another is defined as a tropical strain because it appears better acclimated to growth at higher temperatures. Observations in the natural environment indicate that P. tricornutum is a coastal marine diatom that is able to adapt to unstable environments, such as estuaries and rock pools. Because it has rarely been noted in nature, we have developed specific primers to amplify ITS2 sequences and have successfully identified it in environmental samples. These resources should become useful tools for the diatom community when combined with the whole genome sequence and will open up a range of new possibilities for experimental investigations that can exploit the genotypic and phenotypic characteristics described.     

Seasonal changes on planktonic diatom communities along an inshore-offshore gradient in the Gulf of Gabes (Tunisia)

We investigated the composition, biomass and cell size dynamics of the marine planktonic diatom species along a coastal-open sea gradient in relation to the hydrological characteristics, during four oceanographic cruises in July 2005, May–June 2006, September 2006 and March 2007 in the Gulf of Gabes. The study of the marine planktonic diatoms throughout the sampling period showed the presence of 40 different species belonging to 22 pennate and 18 centric diatom species. Centric diatoms were more abundant than the pennate ones; 56% and 44% of total diatom abundance, respectively. Diatoms were very abundant, representing about 60% of the total phytoplankton abundance, with an exception in July 2005 (19%) during which Dictyochophyceae were the most dominant group (41% of the total phytoplankton abundance). Diatoms, which were dominant in the coastal samples, mainly proliferate in the semi-mixing conditions (May–June 2006), whereas they declined in the offshore area, most likely due to silicate shortage. In this period, in spite of the high abundance of diatom planktonic cells, only diatom biomass was correlated with silica amount, proving that biomass was a better ecological bio-indicator than abundance. The results suggest that the marine planktonic diatoms taxa were generally adapted to specific hydrological structure. In fact, the dominance index of diatoms showed that the biodiversity of diatoms increased gradually along the coastal-open sea gradient except in May–June 2006 during which a slight decrease along the inshore towards offshore areas was observed. This index increase depended on a coastal-open sea distance during thermal stratification (July 2005 and September 2006) and mixing periods (March 2007).     

Behavioural versus physiological photoprotection in epipelic and epipsammic benthic diatoms

Benthic diatoms are dominant primary producers in intertidal marine sediments, which are characterized by widely fluctuating and often extreme light conditions. To cope with sudden increases in light intensity, benthic diatoms display both behavioural and physiological photoprotection mechanisms. Behavioural photoprotection is restricted to raphid pennate diatoms, which possess a raphe system that enables motility and hence positioning in sediment light gradients (e.g. via vertical migration into the sediment). The main physiological photoprotection mechanism is to dissipate excess light energy as heat, measured as Non-Photochemical Quenching (NPQ) of chlorophyll fluorescence. A trade-off between vertical migration and physiological photoprotection (NPQ) in benthic diatoms has been hypothesized before, but this has never been formally tested. We exposed five epipelic diatom species (which move in between sediment particles) and four epipsammic diatom species (which live in close association with individual sand grains) to high light conditions, and characterized both NPQ and the relative magnitude of the migratory response to high light. Our results reveal the absence of a significant downward migratory response in an araphid diatom, but also in several raphid epipsammic diatoms, while all epipelic species showed a significant migratory response upon high light exposure. In all epipsammic species the upregulation of NPQ was rapid and pronounced; NPQ relaxation in low light conditions, however, occurred faster in the araphid diatom, compared with the raphid epipsammic species. In contrast, all epipelic species lacked a strong and flexible NPQ response and showed higher susceptibility to photodamage when not able to migrate. While overall our results support the vertical migration-NPQ trade-off, the lack of strong relationships between the capacity for vertical migration and NPQ within the epipsammic and epipelic groups suggests that other factors as well, such as cell size, substrate type and photoacclimation, may influence photoprotective strategies.     

Biofilm diatom community structure: Influence of temporal and substratum variability

Abstract

Diatoms, which are early autotrophic colonisers, are an important constituent of the biofouling community in the marine environment. The effects of substratum and temporal variations on the fouling diatom community structure in a monsoon-influenced tropical estuary were studied. Fibreglass and glass coupons were exposed every month for a period of 4 days and the diatom population sampled at 24 h intervals, over a period of 14 months. The planktonic diatom community structure differed from the biofilm community. Pennate diatoms dominated the biofilms whilst centric diatoms were dominant in the water column. Among the biofilm diatoms, species belonging to the genera Navicula, Amphora, Nitzschia, Pleurosigma and Thalassionema were dominant. On certain occasions, the influence of planktonic blooms was also seen on the biofilm community. A comparative study of biofilms formed on the two substrata revealed significant differences in density and diversity. However species composition was almost constant. In addition to substratum variations, the biofilm diatom community structure also showed significant seasonal variations, which were attributed to physico-chemical and biological changes in both the water and substratum. Temporal variations in the tychopelagic diatoms of the water were also observed to exert an influence on the biofilm diatom community. Variations in diatom communities may determine the functional ecosystem of the benthic environment.     

Quantification of diatom and dinoflagellate biomasses in coastal marine seawater samples by real-time PCR

Two real-time PCR assays targeting the small-subunit (SSU) ribosomal DNA (rDNA) were designed to assess the proportional biomass of diatoms and dinoflagellates in marine coastal water. The reverse primer for the diatom assay was designed to be class specific, and the dinoflagellate-specific reverse primer was obtained from the literature. For both targets, we used universal eukaryotic SSU rDNA forward primers. Specificity was confirmed by using a BLAST search and by amplification of cultures of various phytoplankton taxa. Reaction conditions were optimized for each primer set with linearized plasmids from cloned SSU rDNA fragments. The number of SSU rDNA copies per cell was estimated for six species of diatoms and nine species of dinoflagellates; these were significantly correlated to the biovolumes of the cells. Nineteen field samples were collected along the Swedish west coast and subjected to the two real-time PCR assays. The linear regression of the proportion of SSU rDNA copies of dinoflagellate and diatom origin versus the proportion of dinoflagellate and diatom biovolumes or biomass per liter was significant. For diatoms, linear regression of the number of SSU rDNA copies versus biovolume or biomass per liter was significant, but no such significant correlation was detected in the field samples for dinoflagellates. The method described will be useful for estimating the proportion of dinoflagellate versus diatom biovolume or biomass and the absolute diatom biovolume or biomass in various aquatic disciplines.