Identification of the pathogenic ciliate Pseudocohnilembus persalinus (Oligohymenophorea: Scuticociliatia) by fluorescence in situ hybridization

Many scuticociliatid ciliates are regarded as devastating pathogens in aquaculture. Among these, Pseudocohnilembus persalinus is a facultative pathogen that often results in refractory diseases of mariculture fish. Although traditional silver staining methods have been successfully used to identify these ciliates, their identification is hampered by their small size and their morphological similarity to closely related species. We designed an alternative method of identification of P. persalinus using an SSU-rDNA targeted oligonucleotide probe labeled with a fluorochrome, and optimized in a fluorescence in situ hybridization (FISH) protocol. The assay results in a clear identification by strong fluorescence signals from the oligonucleotide probe. The method can be used for quick and early detection of P. persalinus infections on host fish, as well as other susceptible organisms in aquiculture water. It may also be used in studies of the geographical distribution of this scuticociliate.     

Grazing rates of the freshwater ciliate Balanion planctonicum determined by flow cytometry

Feeding of Balanion planctonicum on the cryptomonad Rhodomonassp. was recorded in vivo at 2–3 min intervals by flowcytometry. Ingestion rates were 1.6–1.7 algal cells ciliate^–1h^–1. On average, 20–30 min elapsed between ingestionand egestion.     

Inorganic carbon uptake by an Antarctic sea-ice diatom,Nitzschia frigida

There have been no studies to date on the mechanisms of inorganic carbon acquisition by Antarctic microalgae. Consequently, we have examined inorganic carbon (DIC) use inNitzschia frigida, a diatom typical of the Antarctic bottom-ice community. The K_0.5(CO₂) of photosynthesis in this organism was estimated to be 1.09 μM at pH 7.5. The internal concentration of DIC was approximately 4050 μM at an external [DIC] of 45 μM. At air-equilibration levels of inorganic carbon this would be sufficient for a ten-fold accumulation ratio of CO₂. Cells ofN. frigida are capable of carbon-dependent photosynthesis at rates that exceed that expected from uncatalysed CO₂ supply to the cell. About 25% of the total carbonic anhydrase activity appears to be associated with the cell surface inN. frigida. These results support the hypothesis thatN. frigida, like many microalgae from temperate waters, has an active carbon-concentrating mechanism, associated with the ability to utilize external HCO ₃ ⁻ for photosynthesis.     

Biology of Euplotes focardii,an Antarctic ciliate

Euplotes focardii, a ciliate species recently collected from sand sediments of Terra Nova Bay (Ross Sea, Antarctica) reproduced in the laboratory with a duplication time of approximately 72 h, at 4°C. Strains representative of two different mating types were identified and mixed together to produce mating pairs. These showed traits rather unusual for Euplotes species. The two pair members remained united for at least 8–10 days. However, only one carried out fertilization and was able to give rise to a new clone of vegetative cells; the other underwent cell body shrinking after 4–5 days of union, lost the locomotory ciliary apparatus, and eventually died. By analyses of mating pairs formed in mixtures of cell samples cytologically distinct from each other, it was ascertained that the different cell behavior is strain-specific.     

Temperature dependence of inorganic nitrogen uptake and assimilation in Antarctic sea-ice microalgae

Summary The influence of temperature on NO ₃ ^- and NH ₄ ⁺ uptake, and the activity of the assimilatory enzyme NO ₃ ^- reductase (NR) was compared to inorganic C uptake (photosynthesis) in natural assemblages of Antarctic sea-ice microalgae. NO ₃ ^- and NH ₄ ⁺ uptake reached a maximum between 0.5°–2.0°C and 2.0°–3.0°C, respectively, which was close to that for photosynthesis (2.5°–3.0°C). NR showed a distinctly higher temperature maximum (10.0°–12.0°C) and a lower Q₁₀ value than inorganic N and C transport. Our data imply that, owing to differential temperature characteristics between N transport and N assimilation at in situ temperature (-1.9°C), the incorporation of extracellular NO ₃ ^- into cellular macromolecules, may be limited by transport of NO ₃ ^- into the cell rather than the intracellular reduction of NO ₃ ^- to NH ₄ ⁺ . Despite differences in temperature maxima between N transport and N assimilation, the overall low temperature maxima of inorganic N metabolism characterizes Antarctic sea-ice microalgae as psychrophilic. Our study is the first to examine the temperature dependence of inorganic N uptake and assimilation in sea-ice microbial communities.     

Cold-adapted signal proteins: NMR structures of pheromones from the Antarctic ciliate Euplotes nobilii

Cell type-specific signal proteins, known as pheromones, are synthesized by ciliated protozoa in association with their self/nonself mating-type systems, and are utilized to control the vegetative growth and mating stages of their life cycle. In species of the most ubiquitous ciliate, Euplotes, these pheromones form families of structurally homologous molecules, which are constitutively secreted into the extracellular environment, from where they can be isolated in sufficient amounts for chemical characterization. This paper describes the NMR structures of En-1 and En-2, which are members of the cold-adapted pheromone family produced by Euplotes nobilii, a species inhabiting the freezing coastal waters of Antarctica. The structures were determined with the proteins from the natural source, using homonuclear (1)H NMR techniques in combination with automated NOESY peak picking and NOE assignment. En-1 and En-2 have highly homologous global folds, which consist of a central three-alpha-helix bundle with an up-down-up topology and a 3(10)-helical turn near the N-terminus. This fold is stabilized by four disulfide bonds and the helices are connected by bulging loops. Apparent structural specificity resides in the variable C-terminal regions of the pheromones. The NMR structures of En-1 and En-2 provide novel insights into the cold-adaptive modifications that distinguish the E. nobilii pheromone family from the closely related E. raikovi pheromone family isolated from temperate waters.     

Heterologous expression and folding analysis of a beta-tubulin isotype from the Antarctic ciliate Euplotes focardii.

Mammalian tubulins and actins attain their native conformation following interactions with CCT (the cytosolic chaperonin containing t-complex polypeptide 1). To study the beta-tubulin folding in lower eukaryotes, an isotype of beta-tubulin (beta-T1) from the Antarctic ciliate Euplotes focardii, was expressed in Escherichia coli. Folding analysis was performed by incubation of the 35S-labeled, denatured beta-T1 in the presence, or absence, of purified rabbit CCT and cofactor A, a polypeptide that stabilizes folded monomeric beta-tubulin. We show for the first time in protozoa that beta-tubulin folding is assisted by CCT and requires cofactor A. In addition, we observed that E. focardiibeta-T1 competes with human beta5 tubulin isotype for binding to CCT. The affinity of CCT to E. focardiibeta-T1 and beta5 tubulin are compared. Finally, the mitochondrial chaperonin mt-cpn60 binds to beta-T1 but is unable to release it in a native or quasi-native state.     

Characterization of the pheromone gene family of an Antarctic and Arctic protozoan ciliate, Euplotes nobilii

Allelic genes encoding water-borne signal proteins (pheromones) were amplified and sequenced from the somatic (macronuclear) sub-chromosomic genome of Antarctic and Arctic strains of the marine ciliate, Euplotes nobilii. Their open reading frames appeared to be specific for polypeptide sequences of 83 to 94 amino acids identifiable with cytoplasmic pheromone precursors (pre-pro-pheromones), requiring two proteolytic steps to remove the pre- and pro-segments and secrete the mature pheromones. Differently from most of the macronuclear genes that have so far been characterized from Euplotes and other hypotrich ciliates, the 5′ and 3′ non-coding regions of all the seven E. nobilii pheromone genes are much longer than the coding regions (621 to 700 versus 214 to 285 nucleotides), and the 5′ regions in particular show nearly identical sequences across the whole set of pheromone genes. These structural peculiarities of the non-coding regions are likely due to the presence of intron sequences and provide presumptive evidence that they are site of basic, conserved activities in the mechanism that regulates the expression of the E. nobilii pheromone genes.     

Interannual variability in the distribution of the phytoplankton standing stock across the seasonal sea-ice zone west of the Antarctic Peninsula

The spatial distribution of phytoplankton cell abundance, carbon(C) biomass and chlorophyll a (Chl a) concentration was analysedduring three summers (1996, 1997 and 1999) in a seasonal sea-icearea, west of the Antarctic Peninsula. The objective of thestudy was to assess interannual variability in phytoplanktonspatial distribution and the mechanisms that regulate phytoplanktonaccumulation in the water column. Phytoplankton C biomass andChl a distributions were consistent from year to year, exhibitinga negative on/offshore gradient. The variations in C concentrationhad a close and non-linear relationship with the upper mixedlayer depth, suggesting that the vertical mixing of the watercolumn is the main factor regulating phytoplankton stock. Themagnitude of C gradients was 5-fold higher during 1996 thanduring 1997 and 1999. This was ascribed to interannual variationsin the concentration of diatom blooms in the region influencedby sea-ice melting. Vertical distribution of the phytoplankton,as estimated from Chl a profiles, also varied along an on/offshoregradient: Chl a was distributed homogeneously in the upper mixedlayer in coastal and mid-shelf stations and concentrated inthe deep layer (40–100 m) occupied by the winter waters(WW, remnants of the Antarctic surface waters during summer)in more offshore stations. The region with a deep Chl a maximumlayer (DCM layer) was dominated by a phytoplankton assemblagecharacterized by a relatively high concentration of diatoms.The extent of this region varied from year to year: it was restrictedto pelagic waters during 1996, extended to the shelf slope during1997 and occupied a major portion of the area during 1999. Itis hypothesized that iron depletion in near surface waters dueto phytoplankton consumption, and a higher concentration inWW, regulated this vertical phytoplankton distribution pattern.Furthermore, we postulate that year-to-year variations in thespatial distribution of the DCM layer were related to interannualvariations in the timing of the sea-ice retreat. The similaritybetween our results and those reported in literature for otherareas of the Southern Ocean allows us to suggest that the mechanismsproposed here as regulating phytoplankton stock in our areamay be applicable elsewhere.     

Tubulin folding: the special case of a beta-tubulin isotype from the Antarctic psychrophilic ciliate Euplotes focardii

Folding assistance is a fundamental requirement of certain proteins, and it may be subjected to physicochemical constraints in case of organisms adapted to polar temperatures. Limited information is available about protein folding in the polar environment. Folding of tubulin provides one of the few studied cases. Here, we report a pilot folding analysis of a divergent beta-tubulin isotype, named EFBT3, from the Antarctic psychrophilic ciliate Euplotes focardii. To attain its native monomeric structure, beta-tubulin needs the assistance of the eukaryotic class II chaperonin CCT and cofactor A (CofA). The in vitro folding reaction of EFBT3 with CCT and CofA purified from rabbit did not generate any folded product. In contrast, the reaction performed with the rabbit reticulocyte lysate, that contains all the chaperones required for efficient tubulin folding, was productive, suggesting that additional factors besides purified CCT and CofA are required for EFBT3 to attain its monomeric structure. We also demonstrated that the rare Cys281 of EFBT3 is critical for the folding reaction. Model predictions indicate that EFBT3 binds to CofA differently from yeast beta-tubulin, suggesting a diverse folding mechanism that may be correlated with microtubule cold adaptation.     

Structural characterization of En-1, a cold-adapted protein pheromone isolated from the Antarctic ciliate Euplotes nobilii

The second of two diffusible cell signal proteins (pheromones) purified from a wild-type strain of the Antarctic ciliate, Euplotes nobilii, has been determined by automated Edman degradation of the whole molecule and peptides generated by its chymotryptic digestion. The proposed sequence of 52 amino acids of this new pheromone, designated En-1, is: NPEDWFTPDT(10)CAYGDSNTAW(20)TTCTTPGQTC(30)YTCCSSCFDV(40)VGEQACQMSA(50)QC. In common with the previously determined 60-amino-acid sequence of the other pheromone, En-2, it bears eight cysteines in conserved positions (presumably linked into four conserved intrachain disulfide bonds), and physicochemical features of potential significance for cold adaptation, such as a reduced hydrophobicity, an increased solvent accessibility, and an improved local backbone flexibility. However, En-1 diverges from En-2 for having evolved a threonine cluster in the place of a glycine cluster to apparently make more flexible a region that is likely functionally important.     

Grazing of ciliates on blue–green algae: Effects of ciliate encystment and related phenomena

Grazing of the ciliate Colpoda steinii on the blue–green alga Anacystis nidulans has been studied in batch and chemostat type laboratory cultures. Growth of the populations, grazing of the ciliates on the algae, hydrodynamic washout of the populations (in chemostat cultures), encystment of Colpoda, and transfer of cysts from the liquid culture to the vessel wall and their attachment thereto were all found to have significant effects on the dynamics of this system. In addition, reinoculation of the liquid with ciliates excysted from the wall and with algae detached from the wall may be important. The interaction of all of these processes produces quite complex dynamical phenomena which at present cannot be predicted by a model. The results obtained differ from those found earlier for feeding of ciliates or slime mold amoebas on bacteria in that steady states of coexistence, rather than sustained oscillations, were exhibited by the present system.     

Contrasting effects of the extent of sea-ice on the breeding performance of an Antarctic top predator, the Snow Petrel Pagodroma nivea

Recent studies have shown that the Antarctic Circumpolar Wave and the related sea-ice cover around the Antarctic continent may have a profound effect on the lower trophic levels of the marine environment. In particular, extensive sea-ice cover enhances the survival of krill. However, the effects of sea-ice cover on top predators remain poorly understood. Using time series from 1973 to 1999, we examine the influence of regional sea-ice extent on a number of indices of breeding performance of an avian predator, the Snow Petrel, in Antarctica. The percentage of breeding pairs was highly variable and there were fewer birds breeding when sea-ice cover was extensive during July. By contrast, overall breeding success and fledgling body condition were improved during years with extensive sea-ice cover during the preceding November and July–September. These results indicate that the same sea-ice conditions may have different effects on the breeding performance of a species. The overall increase in winter sea-ice extent during the last decade appears to have resulted in an overall improvement of the quality of fledglings produced, and thus probably of future recruitment.     

Molecular cloning, expression, purification and characterization of thioredoxin from Antarctic sea-ice bacteria Pseudoalteromonas sp. AN178

Thioredoxin (Trx) is a highly conserved and multi-functional protein that plays a pivotal role in maintaining the redox state of the cell and in protecting the cell against oxidative stress. Trx gene from Antarctic sea-ice bacteria Pseudoalteromonas sp. AN178 was cloned and expressed as soluble protein in Escherichia coli (designated as PsTrx). Trx gene consisted of an open reading frame of 324-bp nucleotides encoding a protein of 108 amino acids with a calculated molecular mass of 11.88 kDa. The deduced protein included the conserved Cys–Gly–Pro–Cys active-site sequence. After purification by a single step Ni–NTA affinity chromatography, recombinant PsTrx with a high specific activity of 96.67 U/mg was obtained. The purified PsTrx had an optimal temperature and pH of 25 °C and 7.0, respectively, and showed about 55 % of the residual catalytic activity even at 0–10 °C. It had high tolerance to a wide range of NaCl concentrations (0–2 M NaCl) and was stable in the presence of H₂O₂. This research suggested that PsTrx displayed unique catalytic properties.     

Structural characterization of a protein pheromone from a cold-adapted (Antarctic) single-cell eukaryote, the ciliate Euplotes nobilii

Free-living species of ciliated Protozoa control their vegetative (mitotic) proliferation and mating (sexual) processes by diffusible, cell type-specific protein signals (pheromones). One of these molecules, designated En-2, was isolated from a species, Euplotes nobilii, living in the stably cold marine waters of Antarctica, and its complete amino acid sequence of 60 residues was determined by automated Edman degradation of the whole protein and peptides generated by trypsin digestion. The proposed sequence is: DIEDFYTSETCPYKNDSQLA(20)WDTCSGGTGNCGTVCCGQCF(40)SFPVSQSCAGMADSNDCPNA(60). The En-2 structure appears to be characterized by an adaptive insertion of a glycine-rich motif potentially capable to confer more flexibility to a functionally critical region of the molecule.     

Grazing rates of a calanoid copepod (Paralabidocera antarctica) in a continental Antarctic lake

The presence of crustaceans in some Antarctic lakes adds a level of complexity to the food webs of these environments. The grazing pressure of the crustaceans on phytoplankton and other protists, and the role they play in controlling food web structure, has not yet been examined in any detail. This paper presents the first data on grazing pressure of crustacean zooplankton in continental Antarctic lakes. In this study the grazing rates of Paralabidocera antarctica in saline Ace Lake, Vestfold Hills, were assessed using a radio-tracer method. Clearance rates ranged from approximately 1 to 8 ml ind.⁻¹ day⁻¹, depending on developmental stage. Due to their high abundance, nauplii exhibited the highest daily carbon ingestion rates. The population cleared about 3% of the water column per day, accounting for a significant fraction of primary production. P. antarctica has a considerable impact on the carbon cycle in Ace Lake and, by inference, in the other lakes in which it occurs. Accepted: 30 October 1999     

Cold-adaptation in sea-water-borne signal proteins: sequence and NMR structure of the pheromone En-6 from the Antarctic ciliate Euplotes nobilii

Ciliates of Euplotes species constitutively secrete pleiotropic protein pheromones, which are capable to function as prototypic autocrine growth factors as well as paracrine inducers of mating processes. This paper reports the amino acid sequence and the NMR structure of the pheromone En-6 isolated from the antarctic species Euplotes nobilii. The 63-residue En-6 polypeptide chain forms three alpha-helices in positions 18-25, 36-40 and 46-56, which are arranged in an up-down-up three-helix bundle forming the edges of a distorted trigonal pyramid. The base of the pyramid is covered by the N-terminal heptadecapeptide segment, which includes a 3(10)-turn of residues 3-6. This topology is covalently anchored by four long-range disulfide bonds. Comparison with the smaller pheromones of E. raikovi, a closely related species living in temperate waters, shows that the two-pheromone families have the same three-helix bundle architecture. It then appears that cold-adaptation of the En proteins is primarily related to increased lengths of the chain-terminal peptide segments and the surface-exposed loops connecting the regular secondary structures, and to the presence of solvent-exposed clusters of negatively charged side-chains.