Enhancement of phycobiliprotein production in nitrogen-fixing cyanobacteria

The influence of several environmental factors on the phycobiliprotein content of two phycoerythrin-rich nitrogen-fixing cyanobacteria has been studied in order to maximize pigment production. Total phycobiliprotein content was enhanced when either temperature within the optimum range for growth or cell density of the culture was increased. The phycobiliprotein level increased also in response to a decrease in irradiance. In all cases the effect was more marked for C-phycoerythrin than for C-phycocyanin and allophycocyanin. The cellular content in C-phycoerythrin was also preferentially enhanced when cultures were irradiated with green light. On the other hand, red light induced an increase in the C-phycocyanin content, but the C-phycoerythrin level decreased considerably.     

Identification and quantification of phytoplankton groups in lakes using new pigment ratios – a comparison between pigment analysis by HPLC and microscopy

1. Pigment analysis by high‐performance liquid chromatography (HPLC) combined with data analysis using the CHEMTAX program has proven to be a fast and precise method for determining the abundance of phytoplankton groups in marine environments. To determine whether CHEMTAX is applicable also to freshwater phytoplankton, 20 different species of freshwater algae were cultured and their pigment/chlorophyll a (Chl a) ratios determined for exponential growth at three different light intensities and for stationary growth at one light intensity. 2. The different treatments had a relatively insignificant impact on the absolute values of the diagnostic pigment/Chl a ratios, with the exception of cyanobacteria and cryptophytes for which the zeaxanthin/Chl a and alloxanthin/Chl a ratios varied considerably. 3. The pigment ratios were tested on samples collected in six different eutrophic Danish lakes during two summer periods using the CHEMTAX program to calculate the biomass of the phytoplankton groups as Chl a. The CHEMTAX‐derived seasonal changes in Chl a biomass corresponded well with the volume of the microscopically determined phytoplankton groups. More phytoplankton groups were detected by the pigment method than by the microscopic method. 4. Applying the pigment ratios developed in this study, the pigment method can be used to determine the abundance of the individual phytoplankton groups, which are useful as biological water quality indicators when determining the ecological status of freshwater lakes.     

Molecular and developmental contributions to divergent pigment patterns in marine and freshwater sticklebacks

Pigment pattern variation across species or populations offers a tractable framework in which to investigate the evolution of development. Juvenile threespine sticklebacks (Gasterosteus aculeatus) from marine and freshwater environments exhibit divergent pigment patterns that are associated with ecological differences. Juvenile marine sticklebacks have a silvery appearance, whereas sticklebacks from freshwater environments exhibit a pattern of vertical bars. We investigated both the developmental and molecular basis of this population‐level variation in pigment pattern. Time course imaging during the transition from larval to juvenile stages revealed differences between marine and freshwater fish in spatial patterns of chromatophore differentiation as well as in pigment amount and dispersal. In freshwater fish, melanophores appear primarily within dark bars whereas iridophores appear within light bars. By contrast, in marine fish, these chromatophores are interspersed across the flank. In addition to spatially segregated chromatophore differentiation, pigment amount and dispersal within melanophores varies spatially across the flank of freshwater, but not marine fish. To gain insight into the molecular pathways that underlie the differences in pigment pattern development, we evaluated differential gene expression in the flanks of developing fish using high‐throughput cDNA sequencing (RNA‐seq) and quantitative PCR. We identified several genes that were differentially expressed across dark and light bars of freshwater fish, and between freshwater and marine fish. Together, these experiments begin to shed light on the process of pigment pattern evolution in sticklebacks.     

Out of the blue: adaptive visual pigment evolution accompanies Amazon invasion

Incursions of marine water into South America during the Miocene prompted colonization of freshwater habitats by ancestrally marine species and present a unique opportunity to study the molecular evolution of adaptations to varying environments. Freshwater and marine environments are distinct in both spectra and average intensities of available light. Here, we investigate the molecular evolution of rhodopsin, the photosensitive pigment in the eye that activates in response to light, in a clade of South American freshwater anchovies derived from a marine ancestral lineage. Using likelihood-based comparative sequence analyses, we found evidence for positive selection in the rhodopsin of freshwater anchovy lineages at sites known to be important for aspects of rhodopsin function such as spectral tuning. No evidence was found for positive selection in marine lineages, nor in three other genes not involved in vision. Our results suggest that an increased rate of rhodopsin evolution was driven by diversification into freshwater habitats, thereby constituting a rare example of molecular evolution mirroring large-scale palaeogeographic events.     

Preparation of highly purified C-phycoerythrin from marine cyanobacterium Pseudanabaena sp

C-phycoerythrin was isolated and purified from marine Pseudanabaena sp. using two step chromatographic methods. Phycobiliproteins in the marine Pseudanabaena was extracted in 100 mM phosphate buffer (pH 7.2) and precipitated by salting out. The precipitated C-phycoerythrin was purified by gel filtration with Sephadex G-150, and then it was purified by ion exchange chromatography on DEAE cellulose, which was developed by linear ionic strength gradients. Purified phycoerythrin showed absorption maxima at 568 and 541 nm, and displayed a fluorescence maximum at 578 nm. The absorbance ratio A???/A???, a criterion for purity (purity ratio) achieved was 6.86. It showed a single band on examination by polyacrylamide gel electrophoresis (PAGE). The polypeptide analysis of the purified C-phycoerythrin by SDS-PAGE demonstrated that it contained two chromophore-carrying subunits. The yield of purified C-phycoerythrin obtained was 13.6 mg/g of the cell dry weight with 47% of yield. Obtaining highly pure C-phycoerythrin allows one to evaluate its fluorescence properties for future applications in biochemical and biomedical research.     

The use of DGGE analyses to explore eastern Mediterranean and Red Sea marine picophytoplankton assemblages

In vast areas of the oceans, most marine photosynthetic production is performed by cells smaller than 2-3 microm (picoplankton). Here, we report on denaturing gradient gel electrophoresis (DGGE) analyses of naturally occurring marine oxygenic picophytoplankton using the conserved photosynthetic psbA gene. The psbA gene proved to be a good indicator for picophytoplankton presence and was shown to work with DGGE. The DGGE results show the distribution of photosynthetic marine groups belonging to cyanobacteria and the eukaryotic prasinophytes (green algae) in the Red and eastern Mediterranean Seas in the seasons examined. The present study demonstrates the value of DGGE as a tool for rapid analyses of natural marine communities of picophytoplankton.     

Nitrate removal in aquariums by immobilized pseudomonas

Biological denitrification of nitrate to nitrogen gas was examined in a freshwater and a marine aquarium. Nitrate removal in the aquarium water was accomplished with denitrifiers immobilized in a freeze-dried, alginate-starch matrix. Starch served as a bacterial carbon source and cellular matrix-strengthening filler. Freeze-dried beads were placed in canisters through which nitrate-rich aquarium water was recirculated. The freshwater aquarium (100 L) contained goldfish (Carassius auratus) at a total biomass of 390 g, whereas cichlids (Oreochromis mossambicus) were kept at a similar stocking density in the marine aquarium. Denitrification resulted in low ambient nitrate concentrations in both aquariums. The specific nitrate removal rate of the freshwater beads was significantly higher (50 microg of NO(3)-N/bead/day) than that of seawater beads (5 microg of NO(3)-N/bead/day). Differences in ambient nitrate concentrations between both aquariums and diffusion limitation of nitrate to the active denitrification sites within the beads might explain these observed differences.     

Uptake and elimination of arsenobetaine by the mussel Mytilus edulis is related to salinity

The high concentrations of the naturally occurring arsenic compound arsenobetaine in marine animals, in comparison with freshwater animals, has led to the suggestion that salinity is a factor in its accumulation. In separate experiments, we investigated the uptake and elimination of arsenobetaine by the mussel Mytilus edulis when maintained under three salinity regimes (32, 24, and 16 practical salinity units). Both uptake and elimination of arsenobetaine depended on the salinity of the water in a manner leading to higher concentrations at the higher salinity. The data are consistent with a proposed role of arsenobetaine as an adventitiously acquired osmolyte, and readily explain field data for freshwater and marine animals.     

Characterization of Hemiselmis amylosa sp. nov. and phylogenetic placement of the blue-green cryptomonads H. amylosa and Falcomonas daucoides

The morphology and ultrastructure of a new freshwater blue-green cryptomonad, Hemiselmis amylosa sp. nov., is described. In addition, a marine blue-green cryptomonad isolate was confirmed as Falcomonas daucoides by electron microscopy and phycobilin analysis so that it could be included in molecular sequence studies, since the original isolate is no longer available. Complete ssu rRNA gene sequences for H. amylosa and F. daucoides were obtained. Our freshwater isolate of Hemiselmis possesses the same general features described for blue-green marine species, but it differs in having an eyespot, and multiple, single thylakoids penetrating the pyrenoid; therefore, a new blue-green, freshwater species is described. Phylogenetic analyses of H. amylosa and F. daucoides, as well as 24 other cryptophyte algae, indicate a monophyletic origin for all blue-green cryptomonads. Falcomonas forms a sister clade to blue-green cryptomonads, indicating that it is the most primitive extant blue-green cryptomonad and probably diverged early from other blue-green genera. Furthermore, we suggest that the eocyte blue-green cryptomonad may have originated from a Proteomonas-like progenitor that underwent a pigment change, resulting in a Falcomonas-like cell. Based on comparative morphology, the Proteomonas haplomorph may be a likely candidate in the evolutionary transformation from red to blue-green in cryptomonads; however, phylogenetic analyses neither support nor refute this hypothesis. Finally, the current status of cryptomonad classification is addressed.     

Relationships between picophytoplankton and environmental variables in lakes along a gradient of water colour and nutrient content

SUMMARY 1. Biomass and production of picophytoplankton, phytoplankton and heterotrophic bacterioplankton were measured in seven lakes, exhibiting a broad range in water colour because of humic substances. The aim of the study was to identify environmental variables explaining the absolute and relative importance of picophytoplankton. In addition, two dystrophic lakes were fertilised with inorganic phosphorus and nitrogen, to test eventual nutrient limitation of picophytoplankton in these systems.
2. Picophytoplankton biomass and production were highest in lakes with low concentrations of dissolved organic carbon (DOC), and DOC proved the factor explaining most variation in picophytoplankton biomass and production. The relationship between picophytoplankton and lake trophy was negative, most likely because much P was bound in humic complexes. Picophytoplankton biomass decreased after the additions of P and N.
3. Compared with heterotrophic bacterioplankton, picophytoplankton were most successful at the clearwater end of the lake water colour gradient. Phytoplankton dominated over heterotrophic bacteria in the clearwater systems possibly because heterotrophic bacteria in such lakes are dependent on organic carbon produced by phytoplankton.
4. Compared with other phytoplankton, picophytoplankton did best at intermediate DOC concentrations; flagellates dominated in the humic lakes and large autotrophic phytoplankton in the clearwater lakes.
5. Picophytoplankton were not better competitors than large phytoplankton in situations when heterotrophic bacteria had access to a non-algal carbon source. Neither did their small size lead to picophytoplankton dominance over large phytoplankton in the clearwater lakes. Possible reasons include the ability of larger phytoplankton to float or swim to reduce sedimentation losses and to acquire nutrients by phagotrophy.     

Teleost fish osmoregulation: what have we learned since August Krogh, Homer Smith, and Ancel Keys

In the 1930s, August Krogh, Homer Smith, and Ancel Keys knew that teleost fishes were hyperosmotic to fresh water and hyposmotic to seawater, and, therefore, they were potentially salt depleted and dehydrated, respectively. Their seminal studies demonstrated that freshwater teleosts extract NaCl from the environment, while marine teleosts ingest seawater, absorb intestinal water by absorbing NaCl, and excrete the excess salt via gill transport mechanisms. During the past 70 years, their research descendents have used chemical, radioisotopic, pharmacological, cellular, and molecular techniques to further characterize the gill transport mechanisms and begin to study the signaling molecules that modulate these processes. The cellular site for these transport pathways was first described by Keys and is now known as the mitochondrion-rich cell (MRC). The model for NaCl secretion by the marine MRC is well supported, but the model for NaCl uptake by freshwater MRC is more unsettled. Importantly, these ionic uptake mechanisms also appear to be expressed in the marine gill MRC, for acid-base regulation. A large suite of potential endocrine control mechanisms have been identified, and recent evidence suggests that paracrines such as endothelin, nitric oxide, and prostaglandins might also control MRC function.     

Nutrient dynamics and pelagic food web interactions in oligotrophic and eutrophic environments: an overview

The concept of limiting nutrients is a cornerstone of theories concerning the control of production, structure and dynamics of freshwater and marine plankton. The current dogma is that nitrogen is limiting in most marine environments while freshwater ecosystems are mostly phosphorus-limited, although evidence of phytoplankton limitation by either N or P has been found in both environments.However, the same considerations apply to the availability of phosphorus in freshwater as to nitrogen in oceans. In resource-limited environments the plankton dynamics depend mostly on the internal mechanisms which act to recycle the limiting nutrient many times over within the surface waters. As the overall productivity increases, this dependence on nutrient regeneration decreases.The relationship between the stock of limiting nutrient, rates of supply and plankton dynamics must therefore be seen in the light of the processes operating within the entire food chain over quite different time scales. There is strong evidence that process-rates are mostly size-dependent and that food web interactions at the microbial level (picophytoplankton, bacteria, microheterotrophs) strongly effect the production of carbon and the regeneration of nutrients in the pelagic zone.     

Pigment-Based Chemotaxonomy - A Quick Alternative to Determine Algal Assemblages in Large Shallow Eutrophic Lake?

Pigment-based chemotaxonomy and CHEMTAX software have proven to be a valuable phytoplankton monitoring tool in marine environments, but are yet underdeveloped to determine algal assemblages in freshwater ecosystems. The main objectives of this study were (1) to compare the results of direct microscopy and CHEMTAX in describing phytoplankton community composition dynamics in a large, shallow and eutrophic lake; (2) to analyze the efficiency of the pigment-based method to detect changes in phytoplankton seasonal dynamics and during rapid bloom periods; (3) to assess the suitability of specific marker pigments and available marker pigment:chlorophyll a ratios to follow seasonal changes in eutrophic freshwater environment. A 5-year (2009-2013) parallel phytoplankton assessment by direct microscopy and by CHEMTAX was conducted using published marker pigment:chlorophyll a ratios. Despite displaying some differences from microscopy results, the pigment-based method successfully described the overall pattern of phytoplankton community dynamics during seasonal cycle in a eutrophic lake. Good agreement between the methods was achieved for most phytoplankton groups - cyanobacteria, chlorophytes, diatoms and cryptophytes. The agreement was poor in case of chrysophytes and dinoflagellates. Our study shows clearly that published marker pigment:chlorophyll a ratios can be used to describe algal class abundances, but they need to be calibrated for specific freshwater environment. Broader use of this method would enable to expand monitoring networks and increase measurement frequencies of freshwater ecosystems to meet the goals of the Water Framework Directive.     

Eukaryotic picophytoplankton community response to copper enrichment in a metal‐perturbed coastal environment

Copper is an essential micronutrient, especially for photosynthetic organisms, but can be toxic at high concentrations. In the past years, coastal waters have been exposed to an increase in copper concentration due to anthropogenic inputs. One well known case is the Chañaral area (Easter South Pacific coast), where a long term coastal copper enrichment event has occurred. That event strongly affected benthic marine diversity, including microbial communities. In this work, microcosm experiments were carried out to address the changes on picophytoplankton community composition of the disturbed area, when challenged to copper additions. Eukaryotic picophytoplankton communities from two areas were analyzed: one in the most copper‐perturbed area and another at the north edge of the perturbed area. Flow cytometry data showed that 25 μg L^?1 of copper addition exerted a positive effect in the growth kinetics on part of the eukaryotic picophytoplankton communities, independently of the site. 16S‐plastid terminal restriction fragment length polymorphisms analysis suggested that eukaryotic picophytoplankton display a short and directional response to high copper levels. Members of the Prasinophyceae class, a Coscinodiscophyceae diatom, as well as Phaeocystis, respond in a short time to the environmental disturbance, making them excellent candidates for further studies to evaluate phytoplanktonic species as sentinels for copper disturbances in coastal marine ecosystems.     

Pattern and process in the biogeography of subterranean amphipods

The comparative data from studies of two ‘superfamily’ groups with large numbers of subterranean taxa, the exclusively freshwater Crangonyctoidea and the predominately marine Hadzioidea, support the hypothesis that distributional patterns and evolutionary processes of stygobiont amphipods are closely linked and that the former can be a useful indicator of the latter. Three major biogeographic patterns are indicated by the distribution of subterranean species in these groups, each apparently reflecting a particular mode of origin: (1) freshwater stygobionts (limnostygobionts) derived from epigean freshwater ancestors through colonizations probably influenced by adaptive shifts, or assisted by stream capture and spring failure; (2) freshwater stygobionts derived from marine/brackish water ancestors by stranding during regression of marine embayments; and (3) marine/brackish water stygobionts (thalassostygobionts) derived from epigean marine/brackish water ancestors through adaptive shifts possibly in concert with fluctuating sea levels.     

Pigment composition and photoacclimation as keys to the ecological success of Gonyostomum semen (Raphidophyceae,Stramenopiles)

Aquatic habitats are usually structured by light attenuation with depth resulting in different microalgal communities, each one adapted to a certain light regime by their specific pigment composition. Several taxa contain pigments restricted to one phylogenetic group, making them useful as marker pigments in phytoplankton community studies. The nuisance and invasive freshwater microalga Gonyostomum semen (Raphidophyceae) is mainly found in brown water lakes with sharp vertical gradients in light intensity and color. However, its pigment composition and potential photoadaptations have not been comprehensively studied. We analyzed the photopigment composition of 12 genetically different strains of G. semen by high performance liquid chromatography after acclimation to different light conditions. We confirmed the pigments chl a, chl c1c2, diadinoxanthin, trans‐neoxanthin, cis‐neoxanthin, α and β carotene, which have already been reported for G. semen. In addition, we identified, for the first time, the pigments violaxan‐thin, zeaxanthin, and alloxanthin in this species. Alloxanthin has never been observed in raphidophytes before, suggesting differences in evolutionary plastid acquisition between freshwater lineages and the well‐described marine species. The amount of total chl a per cell generally decreased with increasing light intensity. In contrast, the increasing ratios of the prominent pigments diadinoxanthin and alloxanthin per chl a with light intensity suggest photoprotective functions. In addition, we found significant variation in cell‐specific pigment concentration among strains, grouped by lake of origin, which might correspond to genetic differences between strains and populations.     

Nitrogen-Fixing Cyanobacterium with a High Phycoerythrin Content

The elemental and molecular composition, pigment content, and productivity of a phycoerythrin-rich nitrogen-fixing cyanobacterium—an Anabaena strain isolated from the coastal lagoon Albufera de Valencia, Spain—has been investigated. When compared with other heterocystous species, this strain exhibits similar chlorophyll a, carotene, and total phycobiliprotein contents but differs remarkably in the relative proportion of specific phycobiliproteins; the content of C-phycoerythrin amounts to 8.3% (versus about 1% in the other species) of cell dry weight. Absorption and fluorescence spectra of intact phycobilisomes isolated from this Anabaena sp. corroborate the marked contribution of phycoerythrin as an antenna pigment, a circumstance that is unusual for cyanobacteria capable of fixing N₂. The pigment content of cells is affected by variations in irradiance and cell density, these adaptive changes being more patent for C-phycoerythrin than for phycocyanins. The Anabaena strain is clumpy and capable of rapid flocculation. It exhibits outdoor productivities higher than 20 g (dry weight) m⁻² day⁻¹ during summer.     

Hydrophysical correlation and water mass indication of optical physiological parameters of picophytoplankton in Prydz Bay during autumn 2008

Flow cytometry (FCM) is efficient in detecting both abundance and optical physiological parameters including cell size and cellular carbon content—side scatter (SSC), carotenoids—green and orange fluorescence (FL1 and FL2), and red fluorescence—chlorophylls (FL3) can be obtained by FCM. The utilization of these physiological parameters in indicating water masses in Prydz Bay was investigated for the first time. Picophytoplankton were very sensitive to hydrophysical changes and present distinct characteristics of water masses: Picophytoplankton in water closer to the Amery Ice Shelf were more affected by salinity than by temperature, while temperature became more important than salinity the nearer the picophytoplankton were to the deep sea. The picophytoplankton dealt with declines in light by increasing the size of cells, which increase the fixation of carbon. This can also be increased by high temperature and salinity. Pure water masses can increase the content of chlorophylls and cellular carbon. Generally, the distributions of all the five parameters at upper water depths were less affected by temperature and salinity than by water masses; and these parameters can be as indicators to Summer Surface Water (SSW), Winter Water (WW) and Continental Shelf Water (CSW).     

Evidence for a Novel Marine Harmful Algal Bloom: Cyanotoxin (Microcystin) Transfer from Land to Sea Otters

“Super-blooms” of cyanobacteria that produce potent and environmentally persistent biotoxins (microcystins) are an emerging global health issue in freshwater habitats. Monitoring of the marine environment for secondary impacts has been minimal, although microcystin-contaminated freshwater is known to be entering marine ecosystems. Here we confirm deaths of marine mammals from microcystin intoxication and provide evidence implicating land-sea flow with trophic transfer through marine invertebrates as the most likely route of exposure. This hypothesis was evaluated through environmental detection of potential freshwater and marine microcystin sources, sea otter necropsy with biochemical analysis of tissues and evaluation of bioaccumulation of freshwater microcystins by marine invertebrates. Ocean discharge of freshwater microcystins was confirmed for three nutrient-impaired rivers flowing into the Monterey Bay National Marine Sanctuary, and microcystin concentrations up to 2,900 ppm (2.9 million ppb) were detected in a freshwater lake and downstream tributaries to within 1 km of the ocean. Deaths of 21 southern sea otters, a federally listed threatened species, were linked to microcystin intoxication. Finally, farmed and free-living marine clams, mussels and oysters of species that are often consumed by sea otters and humans exhibited significant biomagnification (to 107 times ambient water levels) and slow depuration of freshwater cyanotoxins, suggesting a potentially serious environmental and public health threat that extends from the lowest trophic levels of nutrient-impaired freshwater habitat to apex marine predators. Microcystin-poisoned sea otters were commonly recovered near river mouths and harbors and contaminated marine bivalves were implicated as the most likely source of this potent hepatotoxin for wild otters. This is the first report of deaths of marine mammals due to cyanotoxins and confirms the existence of a novel class of marine “harmful algal bloom” in the Pacific coastal environment; that of hepatotoxic shellfish poisoning (HSP), suggesting that animals and humans are at risk from microcystin poisoning when consuming shellfish harvested at the land-sea interface.