A Redescription of Uronema nigricans*

SYNOPSIS. Uronema nigricans, a hymenostome ciliate, is redescribed by modern technics. Anatomic studies were made on 4 strains treated primarily with the Chatton-Lwoff silver impregnation technic. Particular attention was given to the buccal apparatus and its importance to generic assignment in the order Hymenostomatida.     

Observations on the Ultrastructure of Uronema spp., Marine Scuticociliates*

SYNOPSIS. Observations of the ultrastructure of marine scuticociliatids, tentatively assigned to the genus Uronema, were made by light, transmission electron, and scanning electron microscopy. Giant, cortically oriented mitochondria filled the subpellicular, intermeridional areas, and were in close association with the epiplasm immediately under the inner alveolar sac membranes. Reconstructions of serial sections of the posterior poles of ciliates indicated that the intermeridional mitochondria could fuse at that point and the entire chondriome might at times be a single organelle. A system of tubules was observed to be intimately associated with the mitochondria in the posterior region. The tubules anastomosed and were directed posteriorly into the region of the nephridial-contractile vacuole system. The outer surfaces were coated with projections arranged in helical patterns. The system may be regarded as a fluid segregation organelle. The tripartite nature of the polar basal body complex observed by silver impregnation was confirmed by transmission electron microscopy. The 3 structures were the basal body of the caudal cilium and 2 parasomal sacs. A prominent ring around the caudal cilium was observed by scanning electron micrcscopy; it is probably responsible for the silver deposition surrounding the polar basal body complex that can be seen by light microscopy of silver-impregnated specimens. The ultrastructure of the nonmotile caudal cilium and its kinetosome was unremarkable, being like that of the motile, somatic cilia. The micronuclear and macronuclear outer membranes were continuous at several sites. Such interconnections explain the intimate physical relationship between the nuclei during interphase in many ciliates, and could be a structural basis for chemical communication between the 2 nuclear types. Within the cytoplasm surrounding the opening of the cytoproct, numerous clear vesicles were observed. Their position and appearance suggested that the cytoproct may be involved in the elimination of solutions as well as solids. Food vacuoles, cortical microtubules, lamellar vesicles, disc-shaped vesicles, mucocysts, and a contractile vacuole and its pore were also observed.     

Uronema marinum: identification and biochemical characterization of phosphatidylcholine-hydrolyzing phospholipase C

Phosphatidylcholine (PC)-specific phospholipase D (PC-PLD) and phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) activities have been detected in Uronema marinum. Partial purification of PC-PLC revealed that two distinct forms of PC-PLC (named as mPC-PLC and cPC-PLC) were existed in membrane and cytosol fractions. The two PC-PLC enzymes showed the preferential hydrolyzing activity for PC with specific activity of 50.4 for mPC-PLC and 28.3 pmol/min/mg for cPC-PLC, but did not hydrolyze phosphatidylinositol or phosphatidylethanolamine. However, the biochemical characteristics and physiological roles of both enzymes were somewhat different. mPC-PLC had a pH optimum in the acidic region at around, pH 6.0, and required approximately 0.4 mM Ca2+ and 2.5 mM Mg2+ for maximal activity. cPC-PLC had a pH optimum in the neutral region at around, pH 7.0, and required 1.6 mM Ca2+ and 2.5 mM Mg2+ for maximal activity. cPC-PLC, but not mPC-PLC, showed a dose-dependent inhibitory effect on the luminal-enhanced chemiluminescence (CL) responses and the viability of zymosan-stimulated phagocytes of olive flounder, indicating that cPC-PLC may contribute to the parasite evasion against the host immune response. Our results suggest that U. marinum contains PC-PLD as well as two enzymatically distinct PC-PLC enzymes, and that mPC-PLC may play a role in the intercellular multiplication of U. marinum and cPC-PLC acts as a virulence factor, serving to actively disrupt the host defense mechanisms.     

U.V. Irradiation Inhibits The Electrical Block to Polyspermy in Echinoderms*

Oocytes of the sea urchin Sphaerechinus granularis and the startish Marthasterias glacialis have been submitted to U.V. irradiation before fertilization. This treatment significantly increased the incidence and severity of polyspermy in the sea urchin and was also found effective on starfish oocytes. These were found more resistant to damage than sea urchin eggs and U.V. irradiation did not affect either their response to the hormone l-methyladenine or the rate of elevation of the fertilization envelope, which assures the late and definitive block to polyspermy. Electrophysiological measurements performed on M. glacialis oocytes definitively demonstrate that U.V. irradiation completely inactivates voltage-dependent sodium channels, without altering the other main conductances, Cl^?, K⁺ or Ca²⁺. After such a treatment, the relative permeability of the membrane to Na⁺ as compared to K⁺ shifted from 0.019±0.003 to 0.003±0.002 and only the calcium component of the action potentials could be observed. However, a fertilization potential, preceded by small sperm induced steps, is still present in these conditions, although its peak and plateau level are greatly reduced. These new findings are discussed, which confirm the electrical nature of the fast block to polyspermy but question about the specificity of those sperm-gated channels which are supposed to trigger the fertilization potential.     

Redescriptions of Neodiplogaster tropica Cobb and N. pinicola Steiner (Nematoda: Diplogasteridae)

Neodiplogaster tropica is redescribed from syntypes, and N. pinicola from syntypes and newly collected material. Information on the previously undescribed female tail and reproductive system is given for N. tropica, and details of the stoma, female gonad, spicules, gubernaculum, and excretory system are emphasized for N. pinicola. Lectotypes are designated for both species. The predaceous feeding of N. pinicola is described.     

Redescriptions of Phallodrilus parthenopaeus Pierantoni and P. obscurus Cook (Oligochaeta, Tubificidae)

The type species of Phallodrilus Pierantoni, 1902, P. parthenopaeus Pierantoni, 1902; is redescribed on the basis of new material from W Scotland. The species is characterized by its specific spermathecal and penial setae, its very slender atria, and its long spermathecal ducts. Sexually fully mature specimens of P. obscurus Cook, 1969 are described for the first time, on the basis of material from New Jersey (E USA). The new material shows that this species possesses 5 to 9 penial setae in each ventral bundle of segment XI, and that its atria are coated with a relatively thick musculature.     

Environmental factors and chemical agents affecting the growth of the pathogenic marine ciliate Uronema nigricans.

The scuticociliate Uronema nigricans is an opportunistically parasitic marine ciliate known to cause disease in some aquacultural environments with epizootics documented from marine larval rearing systems, marine aquaria and in southern bluefin tuna Thunnus macoyii growout enclosures. This study examined growth responses of laboratory cultures of the ciliate and prey bacteria to variations in temperature and salinity, and the efficacy of potential chemotherapeutants for control of U. nigricans infections. Differences in ciliate growth responses were marginal at temperatures of 10 to 25 degrees C and at salinities between 15 and 35 ppt, though 3.5 ppt or less was lethal. Ciliates were found to be sensitive to fluctuations in bacterial densities, which may be a factor in the seasonal occurrence of the ciliate-related disease in tuna. Commonly used chemotherapeutants such as formalin, malachite green and hydrogen peroxide were all effective against the ciliate during in vitro trials.     

Molecular cloning and characterization of Cathepsin B from a scuticociliate, Uronema marinum

A cDNA encoding cathepsin B was cloned from the scuticociliate, Uronema marinum, which invades the olive flounder, Paralichthys olivaceus, leading to high mortalities in culturing fish. The full-length scuticociliate cathepsin B (ScCtB) gene contains an open reading frame of 1053 base pairs encoding 350 amino acids. A homology search revealed that ScCtB shares sequence identity with several piscine cathepsin Bs (48%-45%). The protein of ScCtB from U. marinum extracts was purified 12.8-fold by a one step purification process using a DEAE-Sephagel high performance liquid chromatography (HPLC) column. It had a molecular mass of 30 kDa, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, which was consistent with predicting molecular mass of mature protein (29.2 kDa) of ScCtB. The protease activity of the ScCtB enzyme was demonstrated by electrophoresis in a gelatin-acrylamide copolymerized gel. Its activity was quantified by cleaving a synthetic fluorogenic peptide substrate, Z-arginyl-arginyl-7-amido-4-methylcoumarin (Z-Arg-Arg-AMC). The optimum pH for the protease activity was 5.5. Typical of cysteine proteases, the enzyme was inhibited by trans-epoxysuccinyl-L-leucyl-amido(4-guanidino)butane (E-64) and leupeptin.     

The Plecoptera community of a small stream in Oregon,U.S.A. *

The Plecoptera community of a small woodland stream in Oregon was studied by examining density and species composition. Four sites were studied using benthos sampling for larvae and emergence trap collections for adults. Sixteen genera and forty-three species were identified with most species occurring in relatively low numbers. A comparison with the species list from an earlier study suggested that the species composition has not changed markedly in 40 years but there were several changes in relative abundance. Species diversity of stoneflies decreased with distance from the headwaters. These changes were correlated with decrease in habitat complexity, decrease in stability of the substrate, and increase in water depth. Examples of longitudinal succession are given as a method enabling the large number of species to partition the habitat. Concurrent benthos and emergence sampling demonstrated differences in emergence success that were correlated with habitat suitability.     

Association of Self-perceived Body Weight Status with Dietary Reporting by U.S. Teens*

Objective: Children's self perception of body weight and the medical definition of obesity show poor correlation. This study examined the independent associations of body mass index (BMI) and self-perceived weight status (considered self over-, under-, or right weight) with food reporting, nutrient intake estimates, and biomarkers of dietary exposure. Research Methods and Procedures: Dietary (one 24-hour recall), anthropometric, and biochemical data were from the third National Health and Nutrition Examination Survey (1269 boys and 1385 girls, ages 12 to 18 years). Sex-specific multiple regression analyses were used to determine the association of BMI and self-perceived body weight status with reported intakes of energy, macronutrients, low-nutrient-dense foods, micronutrients, and serum concentrations of selected vitamins and carotenoids. Results: Reported intakes of energy, macronutrients, number of eating occasions, percentage of energy from low-nutrient-dense foods, likelihood of meeting the standard of intake of micronutrients, and biomarkers were not different among boys and girls who perceived themselves as overweight relative to those who perceived themselves at the right weight (p > 0.05). In boys, BMI was a negative predictor of percentage of energy from low-nutrient-dense foods (p = 0.004) and intake of ascorbic acid (p = 0.04). BMI was inversely related to serum concentrations of most carotenoids (p ≤ 0.002). Discussion: Perceiving oneself as overweight was not associated with reporting of low-nutrient-dense foods, macronutrients, micronutrients, and biomarker status; BMI, however, was a significant predictor of several outcomes.     

7Beta-oxygenated limonoids from Trichilia elegans ssp. elegans

Five 7beta- and 7alpha-oxygenated obacunone-type limonoids were obtained on reinvestigation of the seeds of Trichilia elegans ssp. elegans: 7-deoxo-7beta-acetoxykihadanins A and B, 7-deoxo-7beta-hydroxykihadanins A and B and 7-deoxo-7alpha-hydroxykihadanin A, the last three being isolated after acetylation procedures as their mono- and/or diacetate derivatives. This is the first report of the natural occurrence of C-7 beta-substituted limonoids without any oxygenated function at C-6. The structures of these compounds have been established on the basis of 1D- and 2D-NMR spectral techniques, ESI-mass spectrum and X-ray crystallographic data.     

U2AF binding selects for the high conservation of the C. elegans 3' splice site

Caenorhabditis elegans is unusual among animals in having a highly conserved octamer sequence at the 3' splice site: UUUU CAG/R. This sequence can bind to the essential heterodimeric splicing factor U2AF, with U2AF65 contacting the U tract and U2AF35 contacting the splice site itself (AG/R). Here we demonstrate a strong correspondence between binding to U2AF of RNA oligonucleotides with variant octamer sequences and the frequency with which such variations occur in splice sites. C. elegans U2AF has a strong preference for the octamer sequence and exerts much of the pressure for 3' splice sites to have the precise UUUUCAG/R sequence. At two positions the splice site has a very strong preference for U even though alternative bases can also bind tightly to U2AF, suggesting that evolution can select against sequences that may have a relatively modest reduction in binding. Although pyrimidines are frequently present at the first base in the exon, U2AF has a very strong bias against them, arguing there is a mechanism to compensate for weakened U2AF binding at this position. Finally, the C in the consensus sequence must remain adjacent to the AG/R rather than to the stretch of U's, suggesting this C is recognized by U2AF35.     

Distribution of U3 small nucleolar RNA and fibrillarin during early embryogenesis in Caenorhabditis elegans

U3 small nucleolar RNA (snoRNA) is one of the members of the box C/D class of snoRNA and is essential for ribosomal RNA (rRNA) processing to generate 18S rRNA in the nucleolus. Although U3 snoRNA is abundant, and is well conserved from yeast to mammals, the genes encoding U3 snoRNA in C. elegans have long remained unidentified. A recent RNomics study in C. elegans predicted five distinct U3 snoRNA genes. However, characterization of these candidates for U3 snoRNA has yet to be performed. In this study, we isolated and characterized four candidate RNAs for U3 snoRNA from the immunoprecipitated RNAs of C. elegans using an antibody against the 2,2,7-trimethylguanosine (TMG) cap. The sequences were identical to the predicted U3 sequences in the RNomics study. Here, we show the several lines of evidence that the isolated RNAs are the true U3 snoRNAs of C. elegans. Moreover, we report the novel expression pattern of U3 snoRNA and fibrillarin, which is an essential component of U3 small nucleolar ribonucleoprotein complex, during early embryo development of C. elegans. To our knowledge, this is the first observation of the inconsistent localization U3 snoRNA and fibrillarin during early embryogenesis, providing novel insight into the mechanisms of nucleologenesis and ribosome production during early embryogenesis.     

Systematics of the marine microfilamentous green algae Uronema curvatum and Urospora microscopica (Chlorophyta)

The microfilamentous green alga Uronema curvatum is widely distributed along the western and eastern coasts of the north Atlantic Ocean where it typically grows on crustose red algae and on haptera of kelps in subtidal habitats. The placement of this marine species in a genus of freshwater Chlorophyceae had been questioned. Molecular phylogenetic analysis of nuclear-encoded small and large subunit rDNA sequences reveal that U. curvatum is closely related to the ulvophycean order Cladophorales, with which it shares a number of morphological features, including a siphonocladous level of organization and zoidangial development. The divergent phylogenetic position of U. curvatum, sister to the rest of the Cladophorales, along with a combination of distinctive morphological features, such as the absence of pyrenoids, the diminutive size of the unbranched filaments and the discoid holdfast, warrants the recognition of a separate genus, Okellya, within a new family of Cladophorales, Okellyaceae. The epiphytic Urospora microscopica from Norway, which has been allied with U. curvatum, is revealed as a member of the cladophoralean genus Chaetomorpha and is herein transferred to that genus as C. norvegica nom. nov.     

YACs and the C. elegans genome

During the past decade, it has become apparent that it is within our grasp to understand fully the development and functioning of complex organisms. It is widely accepted that this undertaking must include the elucidation of the genetic blueprint – the genome sequence – of a number of model organisms. As a prelude to the determination of these sequences, clonebased physical maps of the genomes of a number of multicellular animals and plants are being constructed. Yeast artificial chromosome (YAC) vectors, by virtue of their relatively unbiased cloning capabilities and capacity to carry large inserts, have come to play a central role in the construction of these maps. The application of YACs to the physical map of the Caenorhabditis elegans genome has enabled cosmid clone ‘islands’ to be linked together in an efficient manner. The long-range continuity has improved the linkage between the genetic and physical maps, greatly increasing its utility. Since the genome can be represented by a relatively small number of YACs, it has been possible to make replica filters of genomically ordered YACs available to the community at large.     

MyoD and myogenesis in C. elegans

One of the goals in developmental biology is the identification of key regulatory genes that govern the transition of embryonic cells from a pluripotent potential to a specific, committed cell fate. During vertebrate skeletal myogenesis, this transition is regulated by the MyoD family of genes. C. elegans has muscle analogous to vertebrate skeletal muscle and has a gene(hlh-1) related to the MyoD family. The molecular and genetic characterization of hlh-1 shows that it is very similar to the vertebrate MyoD family in many respects, including its expression pattern and DNA binding activity. The hlh-1 product is required for proper myogenesis, but it is not required for myogenic commitment during embryogenesis in the nematode. The role of this MyoD-related gene in nematode myogenesis is discussed and compared to those of the vertebrate MyoD family.     

C. elegans: of neurons and genes

The human brain contains 100 billion neurons and probably one thousand times more synapses. Such a system can be analyzed at different complexity levels, from cognitive functions to molecular structure of ion channels. However, it remains extremely difficult to establish links between these different levels. An alternative strategy relies on the use of much simpler animals that can be easily manipulated. In 1974, S. Brenner introduced the nematode Caenorhabditis elegans as a model system. This worm has a simple nervous system that only contains 302 neurons and about 7,000 synapses. Forward genetic screens are powerful tools to identify genes required for specific neuron functions and behaviors. Moreover, studies of mutant phenotypes can identify the function of a protein in the nervous system. The data that have been obtained in C. elegans demonstrate a fascinating conservation of the molecular and cellular biology of the neuron between worms and mammals through more than 550 million years of evolution.