Sergei Skarlato

Analogs of the Golgi complex in microsporidia: structure and avesicular mechanisms of function

Microsporidia are obligatory intracellular parasites, most species of which live in the host cell cytosol. They synthesize and then transport secretory proteins from the endoplasmic reticulum to the plasma membrane for formation of the spore wall and the polar tube for cell invasion. However, microsporidia do not have a typical Golgi complex. Here, using quick-freezing cryosubstitution and chemical fixation, we demonstrate that the Golgi analogs of the microsporidia Paranosema (Antonospora) grylli and Paranosema locustae appear as 300-nm networks of thin (25- to 40-nm diameter), branching or varicose tubules that display histochemical features of a Golgi, but that do not have vesicles. Vesicles are not formed even if membrane fusion is inhibited. These tubular networks are connected to the endoplasmic reticulum, the plasma membrane and the forming polar tube, and are positive for Sec13, γCOP and analogs of giantin and GM130. The spore-wall and polar-tube proteins are transported from the endoplasmic reticulum to the target membranes through these tubular networks, within which they undergo concentration and glycosylation. We suggest that the intracellular transport of secreted proteins in microsporidia occurs by a progression mechanism that does not involve the participation of vesicles generated by coat proteins I and II.

Emerging Microsporidian Infections in Russian HIV-Infected Patients

ABSTRACT Microsporidia were identified in stool specimens by histochemistry and PCR of 30 (18.9%) of 159 HIV-infected patients presenting to the S. P. Botkin Memorial Clinical Hospital of Infectious Diseases, St. Petersburg, Russia. The higher prevalence of Encephalitozoon intestinalis, in 21 (12.8%) patients, than of Enterocytozoon bieneusi, in 2 patients (1.2%), was unexpected. Encephalitozoon cuniculi was detected in three patients: one with strain I and two with strain II. Encephalitozoon hellem was detected in one patient, and two patients were identified as being infected by Microsporidium species. One patient was infected with both E. intestinalis and E. cuniculi. In two patients, the microsporidian species were not identifiable. No statistically significant differences in gender, age, and stage of AIDS were observed between the microsporidian-positive and -negative HIV-infected patients. HIV-infected patients diagnosed with microsporidian infection, however, were significantly more likely to exhibit ≤100 CD4+ T cells/μl blood (20/30 patients [67%]; odds ratio [OR], 3.150; 95% confidence interval [CI95], 1.280 to 7.750; P = 0.0116) and weight loss of >10% of the baseline (19/30 patients [63%]; odds ratio, 2.995; CI95, 1.100 to 8.158; P = 0.0352) than HIV-infected patients not diagnosed with microsporidian infection. In summary, this is the first report describing the diagnosis of microsporidian infection of HIV-infected patients in Russia and the first detection of E. cuniculi strain II in a human.

Macroalgal diversity along the Baltic Sea salinity gradient challenges Remane's species-minimum concept

Remanes species-minimum concept, which states that the lowest number of taxa occurs at the horohalinicum (5-8psu), was tested by investigating macroalgal diversity on hard substrates along the natural salinity gradient in the Baltic Sea. Field data on species occurrence and abundance were collected by SCUBA diving along 10 transects of the Finnish, Swedish and German coasts, covering a salinity range from 3.9 to 27psu. Macroalgal species numbers declined steadily with salinity, decreasing until 7.2psu was reached, but in the horohalinicum, a marked reduction of species number and a change in diversity were indicated by the Shannon index and evenness values. The non-linear decrease in macroalgal diversity at 5-8psu and the lack of increase in species numbers at salinities below 5psu imply a restricted applicability of Remanes species-minimum concept to macroalgae.

Planktonic Ciliates of the Baltic Sea (a Review)

The data on the ciliates species composition are given for the Baltic Sea, a brackish-water semi-closed water body where a considerable part of the planktonic fauna is presented by freshwater species. During the observation period, 789 species of ciliates were found, 160 of which are typical planktonic forms. The ecological characteristics of ciliates are given, along with an assessment of their role in the productivity of the Baltic Sea pelagic communities.

Visualization of early golgi compartments at proliferate and sporogenic stages of a microsporidian Nosema grylli.

Vesicular and tubular structures associated with Golgi complex (GC) are observed in sporoblasts during polar filament maturation. Staining with thiamin pyrophosphatase (TPP) proved them to be homologous to trans-Golgi cistemae of other eukaryotes in spite of the lack of the classic flattened organization [8]. At the same time we know very little about localization of the early secretory compartments (intermediate endoplasmic reticulum-GC and cis-GC) in the mirosporidian cells throughout the life cycle and actually nothing about the presence of GC at the proliferate stage of microsporidian development [9 for review 1.

Planktonic ciliates of the Neva Estuary (Baltic Sea): community structure and spatial distribution

Ciliate communities in open waters of the meso-eutrophic Neva Estuary (the Baltic Sea) were studied in summer 2010. Abun- dance and biomass of ciliates were surprisingly low (0.03-1.9 ind ml -1 and 0.04-2.4 × 10 -3 μg C ml -1 ), especially in samples with high detritus content. During this study we detected four ciliate species which are new for the Baltic Sea. Mixotrophic ciliates dominated at the majority of stations (28-67% of overall ciliate numbers). Their contribution was signifi cantly higher in the outfall area and northern part of the Neva Estuary (Resort District), where total density of ciliates was low. Medium-sized ciliates (30-60 μm) were the most diverse and abundant (average contribution 59% of total abundance). The two parts of the estuary, separated from each other by a storm-surge barrier, differed slightly in their community structure (p < 0.05) but did not signifi cantly differ in ciliate numbers and biomass values.

Obtaining Spheroplasts of Armored Dinoflagellates and First Single-Channel Recordings of Their Ion Channels Using Patch-Clamping

Ion channels are tightly involved in various aspects of cell physiology, including cell signaling, proliferation, motility, endo- and exo-cytosis. They may be involved in toxin production and release by marine dinoflagellates, as well as harmful algal bloom proliferation. So far, the patch-clamp technique, which is the most powerful method to study the activity of ion channels, has not been applied to dinoflagellate cells, due to their complex cellulose-containing cell coverings. In this paper, we describe a new approach to overcome this problem, based on the preparation of spheroplasts from armored bloom-forming dinoflagellate Prorocentrum minimum. We treated the cells of P. minimum with a cellulose synthesis inhibitor, 2,6-dichlorobenzonitrile (DCB), and found out that it could also induce ecdysis and arrest cell shape maintenance in these microalgae. Treatment with 100–250 µM DCB led to an acceptable 10% yield of P. minimum spheroplasts and was independent of the incubation time in the range of 1–5 days. We show that such spheroplasts are suitable for patch-clamping in the cell-attached mode and can form 1–10 GOhm patch contact with a glass micropipette, allowing recording of ion channel activity. The first single-channel recordings of dinoflagellate ion channels are presented.

Superposition of Individual Activities: Urea-Mediated Suppression of Nitrate Uptake in the Dinoflagellate Prorocentrum minimum Revealed at the Population and Single-Cell Levels

Dinoflagellates readily use diverse inorganic and organic compounds as nitrogen sources, which is advantageous in eutrophied coastal areas exposed to high loads of anthropogenic nutrients, e.g., urea, one of the most abundant organic nitrogen substrates in seawater. Cell-to-cell variability in nutritional physiology can further enhance the diversity of metabolic strategies among dinoflagellates of the same species, but it has not been studied in free-living microalgae. We applied stable isotope tracers, isotope ratio mass spectrometry and nanoscale secondary ion mass spectrometry (NanoSIMS) to investigate the response of cultured nitrate-acclimated dinoflagellates Prorocentrum minimum to a sudden input of urea and the effect of urea on the concurrent nitrate uptake at the population and single-cell levels. We demonstrate that inputs of urea lead to suppression of nitrate uptake by P. minimum, and urea uptake exceeds the concurrent uptake of nitrate. Individual dinoflagellate cells within a population display significant heterogeneity in the rates of nutrient uptake and extent of the urea-mediated inhibition of the nitrate uptake, thus forming several groups characterized by different modes of nutrition. We conclude that urea originating from sporadic sources is rapidly utilized by dinoflagellates and can be used in biosynthesis or stored intracellularly depending on the nutrient status; therefore, sudden urea inputs can represent one of the factors triggering or supporting harmful algal blooms. Significant physiological heterogeneity revealed at the single-cell level is likely to play a role in alleviation of intra-population competition for resources and can affect the dynamics of phytoplankton populations and their maintenance in natural environments.

Fine Structural Morphology of the Nucleus of Trypanosoma danilewskyi (Kinetoplastida, Trypanosomatina) during Mitosis

Summary The mitosis in cultured epimastigotes of Trypanosoma danilewskyi has been investigated by electron microscopy, with special reference to early stages. The form of nuclear division in T. danilewskyi can be classified as intranuclear closed orthomitosis without discernible condensed chromosomes. At first signs of cell fission, the nucleus is always situated close to the kinetoplast. The basal bodies replicate and the kinetoplast divides before the onset of mitosis. At the beginning of mitosis, the chromatin components of the interphase nucleus decondense, the nucleolus undergoes fragmentation and its material partly disappears. At the stage presumably corresponding to the metaphase of classical mitosis, at least four complexes of paired sister kinetochores can be observed; these seem to be connected with chromatin fibers (uncondensed chromosomes?). At the same time, two main bundles of microtubules are usually formed in the nucleus. The complexes of sister kinetochores reach 180–190 nm in width and display distinct laminated structures. Thereupon, these complexes divide each into two single kinetochores, about 80 nm in width. At the early stages of mitosis, the microtubules converge on the spindle poles and seem to attach to the nuclear envelope. Thus, at these stages the spindle of microtubules in T. danilewskyi is symmetrical, bipolar and monaxial. However, with the course of nuclear elongation, the two main bundles of microtubules begin to show a strong tendency to splay out in the pole regions and to fuse in the isthmus. No microtubule organizing centres are visible at the nuclear poles. Thereupon, the nucleus becomes dumbbell-shaped and then pinches in two. Just after separation, the daugther nuclei contain very little, if any, condensed chromatin and rather loose nucleoli. The nuclear envelope is maintained intact throughout the division stages.

Mitosis in the flagellate Trypanoplasma borreli (Kinetoplastidea: Bodonida)

Summary The mitosis in both uninucleate and giant multinucleate T. borreli culture forms was studied using transmission electron microscopy. The interphase nucleus is of the vesicular type with one prominent nucleolus and dense peripheral chromatin. Nucleosome-like fibres, occasionally with scattered nodules on them of 21–23 nm in diameter, are found in the karyoplasm. The onset of mitosis is signalled by the decondensation of the peripheral chromatin. No individual chromosomes are visible during the whole division. In the course of mitosis the nucleus elongates, the nucleolus becomes fragmented, the nuclear envelope remains intact, and the kinetochore-like plaques and microtubules appear. At metaphase, the spindle microtubules are connected with fibrillar discs that adhere to the inner envelope membrane near nuclear poles. At anaphase, three main bundles of about 40 microtubules each can be found in the spindle. In giant cells the mitotic figures often lie inside a single outer membrane of the envelope, but at different angles to each other. We infer that mitosis in T. borreli involves a combination of a primitive membraneous mechanism of chromosome separation with a more advanced mode of chromosome transmission including spindle microtubules and associated structures. This type of nuclear division can be designated as intranuclear closed orthomitosis without discernible individual chromosomes.