Ultrastructural characterisation of cells specialised for nitrogen fixation in a non-heterocystous cyanobacterium,Trichodesmium spp.

Summary Trichodesmium is the first described example of a filamentous cyanobacterium without heterocysts that contains cells specialised for nitrogen fixation. The ultrastructure of cells with and without nitrogenase were compared using primarilyTrichodesmium tenue Wille, but alsoT. thiebautii Gomont andT. erythraeum Ehrenberg et Gomont. Immunohistochemistry demonstrated that the cytoplasm of certain cells was densely labelled with antibodies against Fe-protein (dinitrogenase reductase). Comparative TEM-image analysis revealed that these cells were also distinguished by a denser thylakoid network, dividing the vacuole-like space into smaller units. The nitrogenase-containing cells also exhibited less extensive gas vacuoles as well as fewer and smaller cyanophycin granules compared to cells which lacked nitrogenase. Carboxysomes were present in both cell types in equal proportion. Longitudinal sections showed that cells with nitrogenase were arranged adjacent to each other, and that groups of cells with and without nitrogenase may coexist in the same trichome. The correlation between modifications in ultrastructure and the presence of nitrogenase suggests a new type of cyanobacterial cell specialisation related to nitrogen fixation. The results obtained also question the systematic affiliation of the genusTrichodesmium.     

Regulation of nitrogen-fixation by different nitrogen sources in the marine non-heterocystous cyanobacterium Trichodesmium sp. NIBB1067

The effect of various nitrogen sources on the synthesis and activity of nitrogenase was studied in the marine, non-heterocystous cyanobacterium Trichodesmium sp. NIBB1067 grown under defined culture conditions. Cells grown with N₂ as the sole inorganic nitrogen source showed light-dependent nitrogenase activity (acetylene reduction). Nitrogenase activity in cells grown on N₂ was not suppressed after 7 h incubation with 2 mM NaNO₃ or 0.02 mM NH₄Cl. However, after 3 h of exposure to 0.5 mM of urea, nitrogenase was inactivated. Cells grown in medium containing 2 mM NaNO₃, 0.5 mM urea or 0.02 mM NH₄Cl completely lacked the ability to reduce acetylene. Western immunoblots tested with polyclonal antisera against the Fe-protein and the Mo–Fe protein, revealed the following: (1) both the Fe-protein and the Mo–Fe protein were synthesized in cells grown with N₂ as well as in cells grown with NaNO₃ or low concentration of NH₄Cl; (2) two bands (apparent molecular mass of 38 000 and 40 000) which cross-reacted with the antiserum to the Fe-protein, were found in nitrogen-fixing cells; (3) only one protein band, corresponding to the high molecular mass form of the Fe-protein, was found in cells grown with NaNO₃ or low concentration of NH₄Cl; (4) neither the Fe-protein nor the Mo–Fe protein was found in cells grown with urea; (5) the apparent molecular mass of the Fe-protein of Trichodesmium sp. NIBB1067 was about 5000 dalton higher than that of the heterocystous cyanobacterium, Anabaena cylindrica IAM-M1.     

Ingestion of the cyanobacterium Trichodesmium spp. by pelagic harpacticoid copepods Macrosetella,Miracia and Oculosetella

Trichodesmium is a filamentous, colonial nitrogen fixing cyanobacteria, ubiquitous in tropical and subtropical regions of the world's oceans. Trichodesmium fixes atmospheric nitrogen and can comprise a significant fraction of total primary production in oceanic surface waters. Therefore, the consumption and fate of Trichodesmium has important consequences for understanding carbon and nitrogen cycling in the open ocean. The pelagic harpacticoid copepod Macrosetella gracilis uses Trichodesmium not only as a physical substrate for juvenile development, but also as a food source. Several different types of pelagic copepods (including several species of calanoids, harpacticoids and a poecilostomatoid species) were tested for ingestion of Trichodesmium by labelling the cyanobacteria with ¹⁴C. Only the pelagic harpacticoids ingested Trichodesmium. Here we report the first grazing rates based on ¹⁴C-uptake measurements for Macrosetella gracilis (0.173 µg C copepod^–1 h^–1), and the first quantitative measurements of both Miracia efferata (0.402 µg C copepod^–1 h^–1) and Oculosetella gracilis (0.126 µg C copepod^–1 h^–1) ingesting this cyanobacteria. Ingestion rates of M. gracilis and M. efferata on the two different species of Trichodesmium, T. thiebautii and T. erythraeum, as well as the two different colonial morphologies of T. thiebautii, spherical-shaped (puffs) and fusiform (tufts), were also compared. Both Miracia and Macrosetella had higher ingestion rates on the puff colonies than the tuft colonies of T. thiebautii.. Both also had higher ingestion rates of T. erythraeum than T. thiebautii. Trichodesmium thiebautii contains a previously reported neurotoxin which may be an important factor in determining trophodynamic interactions. Our results suggest that pelagic harpacticoid copepods can be quantitatively important in determining the fate of Trichodesmium carbon and nitrogen.     

Laboratory culture studies of Trichodesmium isolated from the Great Barrier Reef Lagoon, Australia

Cultures of Trichodesmium from the Northern and Southern Great Barrier Reef Lagoon (GBRL) have been established in enriched seawater and artificial seawater media. Some cultures have been maintained with active growth for over 6years. Actively growing cultures in an artificial seawater medium containing organic phosphorus (glycerophosphate) as the principal source of phosphorus have also been established. Key factors that contributed to the successful establishment of cultures were firstly, the seed samples were collected from depth, secondly, samples were thoroughly washed and thirdly, incubations were conducted under relatively low light intensities (PAR 40–50molquantam^–2s^–1). N₂ fixation rates of the cultured Trichodesmium were found to be similar to those measured in the GBRL. Specific growth rates of the cultures during the exponential growth phase in all enriched media were in the range 0.2–0.3day^–1 and growth during this phase was characterised by individual trichomes (filaments) or small aggregations of two to three trichomes. Characteristic bundle formation tended to occur following the exponential growth phase, which suggests that the bundle formation was induced by a lack of a necessary nutrient e.g. Fe. Results from some exploratory studies showed that filament-dominated cultures of Trichodesmium grew over a range of relatively low irradiances (PAR 5–120molquantam^–2s^–1) with the maximum growth occurring at 40–50molquantam^–2s^–1. These results suggest that filaments of the tested strain are well adapted for growth at depth in marine waters. Other studies showed that growth yields were dependent on salinity, with maximum growth occurring between 30 and 37psu. Also the cell yields decreased by an order of magnitude with the reduction of Fe additions from 450 to 45nM. No active growth was observed with the 4.5nM Fe addition.     

Growth,N2 fixation and photosynthesis in a cyanobacterium,Trichodesmium sp., under Fe stress

Trichodesmium sp., isolated from the Great Barrier Reef lagoon, was cultured in artificial seawater media containing a range of Fe concentration. Fe additions stimulated growth, N₂ fixation, cellular chlorophyll a content, light-saturated chlorophyll a-specific gross photosynthetic capacity (P_m ^chla) and the dark respiration rate (R_d ^chla). Cell yields only doubled for 9 nM Fe relative to zero added Fe, whereas N₂ fixation increased 11-fold considerably for 450 nM Fe. The results suggest that N₂ fixation of Trichodesmium is more sensitive to Fe limitation than are the cell yields.     

Effects of experimentally induced cyanobacterial blooms on crustacean zooplankton communities

SUMMARY 1. Large in situ enclosures were used to study the effects of experimentally induced cyanobacterial blooms on zooplankton communities. A combination of N and P was added to shallow (2 m) and deep enclosures (5 m) with the goal of reducing the TN : TP ratio to a low level (∼5 : 1) to promote cyanobacterial growth. After nutrient additions, high biomass of cyanobacteria developed rapidly in shallow enclosures reaching levels only observed during bloom events in eutrophic lakes.
2. In the shallow enclosures, particulate phosphorus (PP) was on average 35% higher in comparison with deep enclosures, suggesting that depth plays a key role in P uptake by algae. Phytoplankton communities in both deep and shallow enclosures were dominated by three cyanobacteria species – Aphanizomenon flos-aquae , Anabaena flos-aquae and Microcystis aeruginosa – which accounted for up to 70% of total phytoplankton biomass. However, the absolute biomass of the three species was much higher in shallow enclosures, especially Aphanizomenon flos-aquae . The three cyanobacteria species responded in contrasting ways to nutrient manipulation because of their different physiology.
3. Standardised concentrations of the hepatotoxic microcystin-LR increased as a result of nutrient manipulations by a factor of four in the treated enclosures. Increased biomass of inedible and toxin producing cyanobacteria was associated with a decline in Daphnia pulicaria biomass caused by a reduction in the number of individuals with a body length of >1 mm. Zooplankton biomass did not decline at moderate cyanobacteria biomass, but when cyanobacteria reached high biomass large cladocerans were reduced.
4. Our results demonstrate that zooplankton communities can be negatively affected by cyanobacterial blooms and therefore the potential to use herbivory to reduce algal blooms in such eutrophic lakes appears limited.     

Quorum sensing control of phosphorus acquisition in Trichodesmium consortia

Colonies of the cyanobacterium Trichodesmium are abundant in the oligotrophic ocean, and through their ability to fix both CO₂ and N₂, have pivotal roles in the cycling of carbon and nitrogen in these highly nutrient-depleted environments. Trichodesmium colonies host complex consortia of epibiotic heterotrophic bacteria, and yet, the regulation of nutrient acquisition by these epibionts is poorly understood. We present evidence that epibiotic bacteria in Trichodesmium consortia use quorum sensing (QS) to regulate the activity of alkaline phosphatases (APases), enzymes used by epibionts in the acquisition of phosphate from dissolved-organic phosphorus molecules. A class of QS molecules, acylated homoserine lactones (AHLs), were produced by cultivated epibionts, and adding these AHLs to wild Trichodesmium colonies collected at sea led to a consistent doubling of APase activity. By contrast, amendments of (S)-4,5-dihydroxy-2,3-pentanedione (DPD)—the precursor to the autoinducer-2 (AI-2) family of universal interspecies signaling molecules—led to the attenuation of APase activity. In addition, colonies collected at sea were found by high performance liquid chromatography/mass spectrometry to contain both AHLs and AI-2. Both types of molecules turned over rapidly, an observation we ascribe to quorum quenching. Our results reveal a complex chemical interplay among epibionts using AHLs and AI-2 to control access to phosphate in dissolved-organic phosphorus.     

Programmed cell death in the marine cyanobacterium Trichodesmium mediates carbon and nitrogen export

The extent of carbon (C) and nitrogen (N) export to the deep ocean depends upon the efficacy of the biological pump that transports primary production to depth, thereby preventing its recycling in the upper photic zone. The dinitrogen-fixing (diazotrophic) Trichodesmium spp. contributes significantly to oceanic C and N cycling by forming extensive blooms in nutrient-poor tropical and subtropical regions. These massive blooms generally collapse several days after forming, but the cellular mechanism responsible, along with the magnitude of associated C and N export processes, are as yet unknown. Here, we used a custom-made, 2-m high water column to simulate a natural bloom and to specifically test and quantify whether the programmed cell death (PCD) of Trichodesmium mechanistically regulates increased vertical flux of C and N. Our findings demonstrate that extremely rapid development and abrupt, PCD-induced demise (within 2–3 days) of Trichodesmium blooms lead to greatly elevated excretions of transparent exopolymers and a massive downward pulse of particulate organic matter. Our results mechanistically link autocatalytic PCD and bloom collapse to quantitative C and N export fluxes, suggesting that PCD may have an impact on the biological pump efficiency in the oceans.     

Toxic blooms of cyanobacteria in the Patos Lagoon Estuary,southern Brazil

The Patos Lagoon is the largest lagoonal system in South America. Its waters are formed by a huge drainage basin (201,600 km²) situated in the most industrialized areas of the Southern state of Rio Grande do Sul. On its margins more than 3 million inhabitants live in several cities and towns. The lagoon waters are used for leisure, drinking, industry, fisheries, agriculture and navigation. A monitoring and sampling program was developed from February 1994 to January 1996 with emphasis on the estuarine area, aiming to evaluate the occurrence of algal blooms. In the last 15 years, several cyanobacterial (blue-green algal) blooms of theMicrocystis aeruginosa have been registered in the lagoon estuary. HighM. aeruginosa biomass (50 to 9,000 g chla l^–1) was observed in the whole region in late summer and autumn 1994, and early summer 1995. The LD50 of toxic bloom samples tested in mice varied from 22 to 250 mg dry weight kg body weight^–1 while levels of toxicity (LC50) in the brine shrimp varied from 0.47 to 2.44 mg ml^–1. Toxicity varied in different blooms, in the distances along the scum and with time, within the same bloom. The hepatotoxin microcystin-LR was identified in almost all samples.     

Toxicity and isolation of the cyanobacterium Nodularia spumigena from the southern Baltic Sea in 1986

Three water bloom samples were collected in August 1986 from the southern Baltic Sea. Acute toxicity of the samples was determined by mouse bioassay and the toxins were further studied by HPLC. The bloom samples contained equal amounts of cyanobacteria Nodularia spumigena and Aphanizomenon flos-aquae and were hepatotoxic. Two hepatotoxic Nodularia spumigena strains were isolated from the samples. The isolates produce a toxic peak indistinguishable from the bloom material in the HPLC analysis. The toxicity of the fractions was verified by mouse bioassay. Thus the toxicity of the bloom samples was in all likelihood caused by Nodularia spumigena.     

Field and experimental studies on the combined impacts of cyanobacterial blooms and small algae on crustacean zooplankton in a large,eutrophic, subtropical,Chinese lake

Field and experimental studies were conducted to evaluate the combined impacts of cyanobacterial blooms and small algae on seasonal and long-term changes in the abundance and community structure of crustacean zooplankton in a large, eutrophic, Chinese lake, Lake Chaohu. Seasonal changes of the crustacean zooplankton from 22 sampling stations were investigated during September 2002 and August 2003, and 23 species belonging to 20 genera were recorded. Daphnia spp. dominated in spring but disappeared in mid-summer, while Bosmina coregoni and Ceriodaphnia cornuta dominated in summer and autumn. Both maximum cladoceran density (310 ind. l⁻¹) and biomass (5.2 mg l⁻¹) appeared in autumn. Limnoithona sinensis, Sinocalanus dorrii and Schmackeria inopinus were the main species of copepods. Microcystis spp. were the dominant phytoplankton species and formed dense blooms in the warm seasons. In the laboratory, inhibitory effects of small colonial Microcystis on growth and reproduction of Daphnia carinata were more remarkable than those of large ones, and population size of D. carinata was negatively correlated with density of fresh large colonial Microcystis within a density range of 0–100 mg l⁻¹ (r = −0.82, P< 0.05). Both field and experimental results suggested that seasonal and long-term changes in the community structure of crustacean zooplankton in the lake were shaped by cyanobacterial blooms and biomass of the small algae, respectively, i.e., colonial and filamentous cyanobacteria contributed to the summer replacement of dominant crustacean zooplankton from large Daphnia spp. to small B. coregoni and C. cornuta, while increased small algae might be responsible for the increased abundance of crustacean zooplankton during the past decades.     

Nitrogen fixation by the benthic freshwater cyanobacterium Lyngbya wollei

The ability of the benthic cyanobacterium Lyngbya wollei to fix nitrogen was studied using field samples and axenic cultures. L. wollei was collected and isolated from Lake Okeechobee, Florida, where it forms extensive mats. Rates of acetylene reduction up to 39.1 nmol mg dry wt⁻¹ h⁻¹ were observed for field samples. The maximum observed rate of acetylene reduction in axenic laboratory cultures was 200 nmol mg dry wt⁻¹ h⁻¹. Aerobic conditions limited nitrogen fixation activity, but dark/light cycles promoted the development of activity. Reduced oxygen levels appeared to be required for the development of significant levels of nitrogenase activity. The level of irradiance also had a significant impact on the level of activity. The potential significance of nitrogen fixation to Lyngbya production is discussed.     

Nitrogen-fixing cyanobacterium <Emphasis Type="Italic">Trichormus variabilis</Emphasis> of the Lake Baikal phytoplankton

A new filamentous cyanobacterial strain BAC 9610 was isolated from the lake Baikal pelagial. Data obtained by light, scanning, and transmission electron microscopy, along with 16S rRNA gene sequence analysis, allowed the bacterium identification as Trichormus variabilis, previously known as Anabaena variabilis. Trichormus is a cyanobacterial genus not presented in the list of Baikal plankton algae; A. variabilis also hasn’t been previously detected in Baikal phytoplankton. T. variabilis nitrogen fixation ability was demonstrated. The gene responsible for nitrogen fixation, nifH, was identified by PCR and was partially sequenced. No hepatotoxin synthesis genes were revealed in the strain.     

Low fish predation pressure in London reservoirs: II. Consequences to zooplankton community structure

The 1992 survey of zooplankton structure in fourteen London supply reservoirs showed the overall dominance of large-bodied zooplankton, mainly species of Daphnia. These reservoirs can be considered as ‘anti-fish’ by virtue of their steeply sloping concrete or brick sides. The average biomass of large Daphnia spp (retained on a 710 μm sieve) in the total zooplankton biomass was higher than 20% for twelve out of fourteen reservoirs. The cladoceran-copepod ratio was inversely correlated with both dominance of large-bodied Daphnia magna and cladoceran body-size structure. Parallelly, there were tendency of more efficient utilization of lowered algal crops in reservoirs dominated by large-bodied Daphnia spp. A graphical model is presented which relates daphnid species composition and zooplankton size structure to a presumed gradient of fish biomass in these reservoirs.     

A genetically engineered mosquitocidal cyanobacterium

Larvae of the mosquitoAedes aegypti ingested, and developed into adults, on a diet of 1O of 14 different species of cyanobacteria includingAgmenellum quadruplicatum PR-6 (=Synechococcus PCC7002). Mosquito larvae ingested and grew on cells of PR-6 adapted to growth in the absence of NaCl. ThecryIVD gene ofBacillus thuringiensis var.israelensis was cloned into a PR-6 expression vector to form pAQRM56, which was transformed into PR-6. Expression of the CryIVD protein in PR-6 was demonstrated by immunocytochemistry and larvicidal activity. Immunogold labelling indicated production of an electron-dense material among the thylakoid membranes of PR-6. Cells of PR-6 carrying pAQRM56 were toxic to the larvae ofA. aeqypti whereas control cells were not. Growth of PR-6 cells carrying pAQRM56 was slower than the growth of control cells and these cells were also larger.     

Measuringin situ predation byMysis relicta and observations on underdispersed microdistributions of zooplankton

Described are a method and apparatus that allowin situ measurement of predation on zooplankton byMysis relicta. The method, which can be generalized to other predators, involves lowering paired large-volume (30-1) plankton traps to the depth of interest, with subsequent trapping of the ambient zooplankton assemblage in each trap and release of predators into one of the traps. The statistical adequacy of the method was shown by error propagation theory to depend on the percentage of available prey consumed, on the number of prey captured by the traps, and on the distribution of zooplankton within the volume of water captured by the traps. Repeated casts of the apparatus showed that, in contrast to other studies of zooplankton distribution, various zooplankton categories were statistically underdispersed (evenly dispersed in space) or at least not more statistically dispersed (clumped) than was a random distribution at a space scale of 1 m. An error analysis of many replicated feeding experiments showed that the errors obtained were reasonably small and that they conformed with or were less than those predicted by error propagation theory that assumed random distribution of zooplankton. Thus, these results supported the practical application of the method and corroborated the conclusion of random dispersion or underdispersion drawn from the experiment of repeated casts of the apparatus.     

Relationship of cellular phosphorus in the cyanobacterium Nodularia to phosphorus availability in the Peel-Harvey Estuarine System

A laboratory-determined critical concentration of phosphorus in the cyanobacterium Nodularia spumigena was used to assess the phosphorus sufficiency of field populations in the Peel-Harvey Estuarine System, Western Australia. Nutrient addition bioassays indicated that phosphorus was the limiting nutrient. Since river flow, and thus phosphorus input, to the estuary ceases 1–2 months before the onset of Nodularia blooms, the phosphorus contained in these blooms was derived from sediment release and nutrient recycling. A progressive increase in the mean cellular phosphorus concentration of Nodularia from 1981 to 1983 indicated that sediments were releasing a greater amount of phophorus each year. Although evidence suggests that the biomass of recent Nodularia blooms is now limited by physical factors, the additional phosphorus contained in each bloom will increase the supply of phosphorus to other organisms. Cellular phosphorus concentrations in Nodularia appeared to be correlated with river phosphorus loadings in the Peel Inlet but not in the Harvey Estuary.     

Phylogeography of the invasive cyanobacterium Cylindrospermopsis raciborskii

Cylindrospermopsis raciborskii is a planktonic freshwater cyanobacterium that has become increasingly prevalent in tropical and temperate water bodies world-wide. This species is of concern from a water-quality perspective because of its known ability to produce toxins that can affect the health of humans and other animals. This study investigates genetic variation between strains of C. raciborskii isolated from freshwater rivers and reservoirs in Australia, Brazil, Germany, Hungary, Portugal and the USA. Strains were first characterized by analysis of their 16S rRNA gene nucleotide sequences and were found to have a sequence divergence of 99.1%. A phylogenetic tree, constructed using the 16S rRNA gene sequences showed that strains grouped into Australian, European and North/South American phylotypes. To investigate further the observed separation of strains into geographically distinct groups, we applied a cyanobacterium-specific short tandem repeat sequence technique, HIP1. An electrophoretic comparison of the HIP1 polymerase chain reaction products showed clear distinctions between the C. raciborskii strains. A phylogenetic tree, based on the repeat element banding patterns, also revealed three distinct groups of C. raciborskii strains. The first group consisted of strains from the USA and Brazil; the second comprised European strains from Germany, Hungary and Portugal; and the third were strains from Australia. In general, between-country variation was greater than within-country variation, indicating that this fingerprinting technique can successfully distinguish C. raciborskii strains taken from different global locations. The relationship between toxicity and the observed HIP1 polymerase chain reaction fingerprint profiles was less clear, although it is interesting to note that of the strains analysed in this study, only Australian strains are known to produce cylindrospermopsin and only Brazilian strains have been reported to produce paralytic shellfish poisoning toxins.     

Observations on the zooplankton in the reedbelt area of the Neusiedlersee

Weekly sampling and simultaneous measurements of temperature, pH, and conductivity were made at three stations in the reedbelt, between 1980–82. 20 cladoceran and 14 copepod species were encountered, of whichCyclops vicinus vicinus was found in the lake for the first time. Of the measured environmental features, temperature showed the greatest seasonal changes; pH and conductivity were less variable. There were, both quantitatively and qualitatively, constant differences between the three stations, although they lay close to each other.