Revision of the Genus Paratylenchus Micoletzky, 1922, and Descriptions of New Species. Part I of 3 Parts

A revision of part of the genus Paratylenchus is reported covering those species with stylets averaging < 22 μm. Thirteen new species are described and further observations are given on the morphology and relationships of nine other species. Paratylenchus gahriciis transferred to the genus Hemicriconemoides. This article is the first of three parts: the second will treat Paratylenchus spp. with stylets averaging 24-40 μm (and include a key to the species described in parts 1 and2), the third will include species with stylets >40μm, respectively.     

Novel Psychropiezophilic Oceanospirillales Species Profundimonas piezophila gen. nov., sp. nov., Isolated from the Deep-Sea Environment of the Puerto Rico Trench

The diversity of deep-sea high-pressure-adapted (piezophilic) microbes in isolated monoculture remains low. In this study, a novel obligately psychropiezophilic bacterium was isolated from seawater collected from the Puerto Rico Trench at a depth of ∼6,000 m. This isolate, designated YC-1, grew best in a nutrient-rich marine medium, with an optimal growth hydrostatic pressure of 50 MPa (range, 20 to 70 MPa) at 8°C. Under these conditions, the maximum growth rate was extremely slow, 0.017 h⁻¹, and the maximum yield was 3.51 × 10⁷ cells ml⁻¹. Cell size and shape changed with pressure, shifting from 4.0 to 5.0 μm in length and 0.5 to 0.8 μm in width at 60 MPa to 0.8- to 1.0-μm diameter coccoid cells under 20 MPa, the minimal pressure required for growth. YC-1 is a Gram-negative, facultatively anaerobic heterotroph. Its predominant cellular fatty acids are the monounsaturated fatty acids (MUFAs) C_16:1 and C_18:1. Unlike many other psychropiezophiles, YC-1 does not synthesize any polyunsaturated fatty acids (PUFAs). Phylogenetic analysis placed YC-1 within the family of Oceanospirillaceae, closely related to the uncultured symbiont of the deep-sea whale bone-eating worms of the genus Osedax. In common with some other members of the Oceanospirillales, including those enriched during the Deepwater Horizon oil spill, YC-1 is capable of hydrocarbon utilization. On the basis of its characteristics, YC-1 appears to represent both a new genus and a new species, which we name Profundimonas piezophila gen. nov., sp. nov.     

Effect of Grain Size on Bacterial Penetration, Reproduction, and Metabolic Activity in Porous Glass Bead Chambers

We determined the effects of grain size and nutritional conditions on the penetration rate and metabolic activity of Escherichia coli strains in anaerobic, nutrient-saturated chambers packed with different sizes of glass beads (diameters, 116 to 767 μm) under static conditions. The chambers had nearly equal porosities (38%) but different calculated pore sizes (range, 10 to 65 μm). Motile strains always penetrated faster than nonmotile strains, and nutrient conditions that resulted in faster growth rates (fermentative conditions versus nitrate-respiring conditions) resulted in faster penetration rates for both motile and nonmotile strains for all of the bead sizes tested. The penetration rate of nonmotile strains increased linearly when bead size was increased, while the penetration rate of motile strains became independent of the bead size when beads having diameters of 398 μm or greater were used. The rate of H₂ production and the final amount of H₂ produced decreased when bead size was decreased. However, the final protein concentrations were similar in chambers packed with 116-, 192-, and 281-μm beads and were only slightly higher in chambers packed with 398- and 767-μm beads. Our data indicated that conditions that favored faster growth rates also resulted in faster penetration times and that the lower penetration rates observed in chambers packed with small beads were due to restriction of bacterial activity in the small pores. The large increases in the final amount of hydrogen produced without corresponding increases in the final amount of protein made indicated that metabolism became uncoupled from cell mass biosynthesis as bead size increased, suggesting that pore size influenced the efficiency of substrate utilization.     

Margollus bokanicus n. sp. from Iran,the Fourth Species of a Rare Nematode Genus (Dorylaimida,Tylencholaimellidae)

Margollus bokanicus n. sp., collected from natural habitats in Khorasaneh district, Bokan, West Azarbaijan province, Iran, is described. Morphological and morphometric data are provided as well as drawings and light microscopy illustrations. The new species is characterized by a medium size body length (0.60 to 0.73 mm), labial and postlabial sclerotizations, lip region 7-μm wide, offset by constriction and long neck (167 to 207 μm), long pharyngeal basal bulb (27 to 36 μm) or 16% to 17% of total neck length, female genital system monodelphic–opisthodelphic, anterior branch reduced to a uterine sac (26–29 μm) or 1.1 to 1.3 times the body diameter, long posterior uterus (25–28 μm) or 1.1 to 1.3 times the body diameter, V = 40 to 47, cylindroid female tail (17 to 24 μm, c = 31 to 38, c’ = 1.1 to 1.4), and males unknown. This taxon is easily distinguishable from other Margollus species by its smaller general size and more posterior vulva. A compendium of Margollus species is also presented.     

Paurodontella parapitica n. sp. (Nematoda: Hexatylina,Sphaerularioidea) from Kermanshah Province,Western Iran

Paurodontella parapitica n. sp., collected from the rhizosphere of an apple tree in Kermanshah province, western Iran, is described. The new species is characterized by a body length of 505 to 723 µm (females) and 480 to 600 µm (males), lip region continuous by depression; 7 to 8 μm broad, 3 to 4 µm high, stylet length 7 to 9 µm or 1 to 1.3 times the lip region diameter, short postuterine sac of 4 to 6 μm long, lateral fields with five to six incisures; outer incisures crenated and inner incisures weakly crenated, excretory pore situated 90 to 100 µm from anterior end; functional males common in the population, with spicules 24 to 26 μm long. Tail of both sexes similar, almost straight and elongate-conoid. The new species resembles in morphology and morphometrics to four known species of the genus, namely P. apitica, P. minuta, P. myceliophaga, and P. sohailai. The results of phylogenetic analyses based on sequences of D2/D3 expansion region of 28S rRNA gene revealed this genus is polyphyletic in four different clades in Tylenchid.     

Microcystin Production by Microcystis aeruginosa in a Phosphorus-Limited Chemostat

The production of microcystins (MC) from Microcystis aeruginosa UTEX 2388 was investigated in a P-limited continuous culture. MC (MC-LR, MC-RR, and MC-YR) from lyophilized M. aeruginosa were extracted with 5% acetic acid, purified by a Sep-Pak C₁₈ cartridge, and then analyzed by high-performance liquid chromatography with a UV detector and Nucleosil C₁₈ reverse-phase column. The specific growth rate (μ) of M. aeruginosa was within the range of 0.1 to 0.8/day and was a function of the cellular P content under a P limitation. The N/P atomic ratio of steady-state cells in a P-limited medium varied from 24 to 15 with an increasing μ. The MC-LR and MC-RR contents on a dry weight basis were highest at μ of 0.1/day at 339 and 774 μg g⁻¹, respectively, while MC-YR was not detected. The MC content of M. aeruginosa was higher at a lower μ, whereas the MC-producing rate was linearly proportional to μ. The C fixation rate at an ambient irradiance (160 microeinsteins m⁻² s⁻¹) increased with μ. The ratios of the MC-producing rate to the C fixation rate were higher at a lower μ. Accordingly, the growth of M. aeruginosa was reduced under a P limitation due to a low C fixation rate, whereas the MC content was higher. Consequently, increases in the MC content per dry weight along with the production of the more toxic form, MC-LR, were observed under more P-limited conditions.     

Deficient SUMO Attachment to Flp Recombinase Leads to Homologous Recombination-dependent Hyperamplification of the Yeast 2 μm Circle Plasmid

Many Saccharomyces cerevisiae mutants defective in the SUMO pathway accumulate elevated levels of the native 2 μm circle plasmid (2 μm). Here we show that accumulation of 2 μm in the SUMO pathway mutants siz1Δ siz2Δ, slx5Δ, and slx8Δ is associated with formation of an aberrant high-molecular-weight (HMW) form of 2 μm. Characterization of this species from siz1Δ siz2Δ showed that it contains tandem copies of the 2 μm sequence as well as single-stranded DNA. Accumulation of this species requires both the 2 μm–encoded Flp recombinase and the cellular homologous recombination repair (HRR) pathway. Importantly, reduced SUMO attachment to Flp is sufficient to induce formation of this species. Our data suggest a model in which Flp that cannot be sumoylated causes DNA damage, whose repair via HRR produces an intermediate that generates tandem copies of the 2 μm sequence. This intermediate may be a rolling circle formed via break-induced replication (BIR), because mutants defective in BIR contain reduced levels of the HMW form. This work also illustrates the importance of using cir° strains when studying mutants that affect the yeast SUMO pathway, to avoid confusing direct functions of the SUMO pathway with secondary effects of 2 μm amplification.     

Cryptocotyle lata (Trematoda: Heterophyidae) Adult from a Korean Raccoon Dog,Nyctereutes procyonoides koreensis

Total 513 heterophyid flukes were collected from a carcass of wild Korean raccoon dog, Nyctereutes procyonoides koreensis, in Korea. With morphological and molecular characteristics, the flukes were identified to Cryptocotyle lata. The adult C. lata were minute, transparent, pentagonal, 522 μm long by 425 μm wide. Ceca extended into post-testicular region. Ventrogenital sac elliptical, 79 μm by 87 μm with genital pore and ventral sucker. Two testes semielliptical and slightly lobed, located in the posterior region, right testis 173 μm by 155 μm, left testis 130 μm by 134 μm. In a phylogenetic tree, the fluke specimen of this study was grouped with C. lata divergent from Cryptocotyle lingua. We report here N. procyonoides koreensis first as a natural definitive host of C. lata.     

Absorption and Fluorescence of Water-Soluble Pigments Produced by Four Species of Pseudomonas

Pigments produced by four species of Pseudomonas during growth in three media were examined for visible and ultraviolet absorption. Ultraviolet fluorescence excitation and emission spectra were obtained. In all pigments, an absorption maximum occurs at 405 mμ. Ultraviolet excitation of fluorescence occurs primarily at 400 to 410 mμ, with smaller maxima at 460 mμ, or 525 mμ, depending on pH, bacterial species, or medium. Emission maxima, after excitation at 410 mμ, occur at 390 mμ, and 455 to 475 mμ. The differences in fluorescence spectra may be used for taxonomic classification of the Pseudomonas.     

Brine Assemblages of Ultrasmall Microbial Cells within the Ice Cover of Lake Vida,Antarctica

The anoxic and freezing brine that permeates Lake Vida''s perennial ice below 16 m contains an abundance of very small (≤0.2-μm) particles mixed with a less abundant population of microbial cells ranging from >0.2 to 1.5 μm in length. Fluorescent DNA staining, electron microscopy (EM) observations, elemental analysis, and extraction of high-molecular-weight genomic DNA indicated that a significant portion of these ultrasmall particles are cells. A continuous electron-dense layer surrounding a less electron-dense region was observed by EM, indicating the presence of a biological membrane surrounding a cytoplasm. The ultrasmall cells are 0.192 ± 0.065 μm, with morphology characteristic of coccoid and diplococcic bacterial cells, often surrounded by iron-rich capsular structures. EM observations also detected the presence of smaller unidentified nanoparticles of 0.020 to 0.140 μm among the brine cells. A 16S rRNA gene clone library from the brine 0.1- to 0.2-μm-size fraction revealed a relatively low-diversity assemblage of Bacteria sequences distinct from the previously reported >0.2-μm-cell-size Lake Vida brine assemblage. The brine 0.1- to 0.2-μm-size fraction was dominated by the Proteobacteria-affiliated genera Herbaspirillum, Pseudoalteromonas, and Marinobacter. Cultivation efforts of the 0.1- to 0.2-μm-size fraction led to the isolation of Actinobacteria-affiliated genera Microbacterium and Kocuria. Based on phylogenetic relatedness and microscopic observations, we hypothesize that the ultrasmall cells in Lake Vida brine are ultramicrocells that are likely in a reduced size state as a result of environmental stress or life cycle-related conditions.     

A Portable Anaerobic Microbioreactor Reveals Optimum Growth Conditions for the Methanogen Methanosaeta concilii

Conventional studies of the optimum growth conditions for methanogens (methane-producing, obligate anaerobic archaea) are typically conducted with serum bottles or bioreactors. The use of microfluidics to culture methanogens allows direct microscopic observations of the time-integrated response of growth. Here, we developed a microbioreactor (μBR) with ~1-μl microchannels to study some optimum growth conditions for the methanogen Methanosaeta concilii. The μBR is contained in an anaerobic chamber specifically designed to place it directly onto an inverted light microscope stage while maintaining a N₂-CO₂ environment. The methanogen was cultured for months inside microchannels of different widths. Channel width was manipulated to create various fluid velocities, allowing the direct study of the behavior and responses of M. concilii to various shear stresses and revealing an optimum shear level of ~20 to 35 μPa. Gradients in a single microchannel were then used to find an optimum pH level of 7.6 and an optimum total NH₄-N concentration of less than 1,100 mg/liter (<47 mg/liter as free NH₃-N) for M. concilii under conditions of the previously determined ideal shear stress and pH and at a temperature of 35°C.     

Influence of Environmental Variables on Gambierdiscus spp. (Dinophyceae) Growth and Distribution

Benthic dinoflagellates in the genus Gambierdiscus produce the ciguatoxin precursors responsible for the occurrence of ciguatera toxicity. The prevalence of ciguatera toxins in fish has been linked to the presence and distribution of toxin-producing species in coral reef ecosystems, which is largely determined by the presence of suitable benthic habitat and environmental conditions favorable for growth. Here using single factor experiments, we examined the effects of salinity, irradiance, and temperature on growth of 17 strains of Gambierdiscus representing eight species/phylotypes (G. belizeanus, G. caribaeus, G. carolinianus, G. carpenteri, G. pacificus, G. silvae, Gambierdiscus sp. type 4–5), most of which were established from either Marakei Island, Republic of Kiribati, or St. Thomas, United States Virgin Island (USVI). Comparable to prior studies, growth rates fell within the range of 0–0.48 divisions day^-1. In the salinity and temperature studies, Gambierdiscus responded in a near Gaussian, non-linear manner typical for such studies, with optimal and suboptimal growth occurring in the range of salinities of 25 and 45 and 21.0 and 32.5°C. In the irradiance experiment, no mortality was observed; however, growth rates at 55μmol photons · m^-2 · s^-1 were lower than those at 110–400μmol photons · m^-2 · s^-1. At the extremes of the environmental conditions tested, growth rates were highly variable, evidenced by large coefficients of variability. However, significant differences in intraspecific growth rates were typically found only at optimal or near-optimal growth conditions. Polynomial regression analyses showed that maximum growth occurred at salinity and temperature levels of 30.1–38.5 and 23.8–29.2°C, respectively. Gambierdiscus growth patterns varied among species, and within individual species: G. belizeanus, G. caribaeus, G. carpenteri, and G. pacificus generally exhibited a wider range of tolerance to environmental conditions, which may explain their broad geographic distribution. In contrast, G. silvae and Gambierdiscus sp. types 4–5 all displayed a comparatively narrow range of tolerance to temperature, salinity, and irradiance.     

Dolichodorus aestuarius n. sp. (Nematode: Dolichodoridae)

Dolichodorus aestuarius n. sp. from an estuarine habitat near Cedar Key, Florida is described. This nematode has a stylet range of 62-76 μm in females and 60-72 μm in males. The stylet is shorter than those of all described species except D. brevistilus. The probable host plant is Juncus roemerianus.     

Stochastic Assembly of Bacteria in Microwell Arrays Reveals the Importance of Confinement in Community Development

The structure and function of microbial communities is deeply influenced by the physical and chemical architecture of the local microenvironment and the abundance of its community members. The complexity of this natural parameter space has made characterization of the key drivers of community development difficult. In order to facilitate these characterizations, we have developed a microwell platform designed to screen microbial growth and interactions across a wide variety of physical and initial conditions. Assembly of microbial communities into microwells was achieved using a novel biofabrication method that exploits well feature sizes for control of innoculum levels. Wells with incrementally smaller size features created populations with increasingly larger variations in inoculum levels. This allowed for reproducible growth measurement in large (20 μm diameter) wells, and screening for favorable growth conditions in small (5, 10 μm diameter) wells. We demonstrate the utility of this approach for screening and discovery using 5 μm wells to assemble P. aeruginosa colonies across a broad distribution of innoculum levels, and identify those conditions that promote the highest probability of survivial and growth under spatial confinement. Multi-member community assembly was also characterized to demonstrate the broad potential of this platform for studying the role of member abundance on microbial competition, mutualism and community succession.     

Revision of the Genus Paratylenchus Micoletzky, 1922 and Descriptions of New Species. Part III of Three parts-Gracilacus

Part III covers species with female stylet length >41 μm which are considered by this author to comprise the genus Gracilacus Raski, 1962. Seven new species of Gracilacus are described and further observations given on 14 other species. Paratylenchus strenzkei (Volz, 1951) Oostenbrink, 1960 is transferred to species inquirendae. A key to the species of Gracilacus is included.     

Production of Hydrocinnamic Acid by Clostridia

Hydrocinnamic acid was found in acid extracts of spent growth medium from cultures of Clostridium sporogenes. The acid was identified by mass spectrometry and its identity was confirmed by gas chromatography. The acid was produced in relatively large amounts (2 to 3 μmoles/ml of medium) by C. sporogenes, toxigenic types A, B, D, and F of C. botulinum, and some strains of C. bifermentans. Other strains of C. bifermentans and strains of C. sordellii and C. caproicum produced only small amounts (0.1 to 0.4 μmoles/ml) of the acid. The acid was not detected in spent medium from toxigenic types C and E of C. botulinum or from 25 other strains representing eight Clostridium species. Resting cell suspensions exposed to l-phenylalanine produced hydrocinnamic and cinnamic acid; the latter compound probably functions as an intermediate in the metabolism of l-phenylalanine.     

Calcaridorylaimus castaneae sp. n. (Nematoda,Dorylaimidae) from Bulgaria with an identification key to the species of the genus

An unknown species belonging to the genusCalcaridorylaimus Andrássy, 1986 was collected from the litter of broadleaf forests dominated by Castanea sativa Mill. and mixed with Quercus daleshampii Ten. and Fagus sylvatica L. on Belasitsa Mountain, south-western Bulgaria. Calcaridorylaimus castaneae sp. n. is characterised by its long body (1.4–2.1 mm), lip region practically not offset, vulva transverse, short odontostyle (14.5–16 μm) and tail (75.5–110.5 μm, c=14.7–23.6; c’=2.9–4.4) in females and 38–46 μm long spicules with small spur before their distant end in males. It is most similar to C. andrassyi Ahmad & Shaheen, 2004, but differs in having transverse vs pore-like vulva and shorter spicules (38–46 μm vs 52–57 μm). An identification key to the species of the genus Calcaridorylaimus is proposed. Phylogenetic analyses were performed on 18S and D2-D3 expansion domains of 28S rRNA genes by Neighbor-Joining, Maximum Likelihood and Bayesian Inference methods. The phylograms inferred from 18S sequences showed closest relationships of the new species with some species belonging to the genus Mesodorylaimus. However, insufficient molecular data for members of both genera do not allow the phylogenetic relationships of Calcaridorylaimus and the new species described herein to be elucidated.     

Uptake of HCO3? and CO2 in Cells and Chloroplasts from the Microalgae Chlamydomonas reinhardtii and Dunaliella tertiolecta

Mass-spectrometric disequilibrium analysis was applied to investigate CO₂ uptake and HCO₃⁻ transport in cells and chloroplasts of the microalgae Dunaliella tertiolecta and Chlamydomonas reinhardtii, which were grown in air enriched with 5% (v/v) CO₂ (high-Ci cells) or in ambient air (low-Ci cells). High- and low-Ci cells of both species had the capacity to transport CO₂ and HCO₃⁻, with maximum rates being largely unaffected by the growth conditions. In high- and low-Ci cells of D. tertiolecta, HCO₃⁻ was the dominant inorganic C species taken up, whereas HCO₃⁻ and CO₂ were used at similar rates by C. reinhardtii. The apparent affinities of HCO₃⁻ transport and CO₂ uptake increased 3- to 9-fold in both species upon acclimation to air. Photosynthetically active chloroplasts isolated from both species were able to transport CO₂ and HCO₃⁻. For chloroplasts from C. reinhardtii, the concentrations of HCO₃⁻ and CO₂ required for half-maximal activity declined from 446 to 33 μm and 6.8 to 0.6 μm, respectively, after acclimation of the parent cells to air; the corresponding values for chloroplasts from D. tertiolecta decreased from 203 to 58 μm and 5.8 to 0.5 μm, respectively. These results indicate the presence of inducible high-affinity HCO₃⁻ and CO₂ transporters at the chloroplast envelope membrane.     

New and Known Species of Pratylenchus Filipjev, 1936 (Nematoda: Pratylenchidae) from Haryana,India, with Remarks on Intraspecific Variations

Two new monosexual and one bisexual species Pratylenchus Filipjev, 1936 collected from Haryana state of India are described and illustrated. The primary distinguishing features of these species are Pratylenchus microstylus n. sp.: L = 331-458 μm, spear = 11 or 12 μm; Pratylenchus cruciferus n. sp.: L = 648-793 μm, central core of lateral fields with oblique lines, hemizonid 2-8 annules anterior to excretory pore; Pratylenchus ekrami n. sp.: spear = 11-13 μm, spermatheca oblong, post vulval uterine sac with differentiated cells, tail with 26-40 annules, males abundant. Studies on intraspecific variations of P. cruciferus, P. ekrami, and P. coffeae (Zimmermann, 1898) Goodey, 1951 revealed that spear length and value of ''V'' are the least variable characters. Body length and size of post vulval uterine sac varies to varying degrees in different species. Shape of median bulb in P. ekrami, number of incisures in P. coffeae, and tail shape in P. ekrami and P. coffeae exhibit the greatest amount of intraspecific variations. P. zeae Graham, 1936 and P. thornei Sher & Allen, 1953 are the other species collected during the present studies.     

Meloidogyne trifoliophila n. sp. (Nemata: Meloidogynidae), a Parasite of Clover from Tennessee

Meloidogyne trifoliophila n. sp. is described from white clover collected at Ames Plantation, Fayette County, Tennessee. The perineal pattern is rounded, with long, smooth striae and rounded arch, and without distinct lateral lines or perivulval striae. The female stylet is 12.6-15.5 μm long, the excretory pore is level with or up to one stylet length posterior to the stylet knobs, and the vulva is subterminal. The posterior terminus is weakly protuberant. The male lateral field is composed of approximately eight repeatedly broken or forked incisures. The male stylet is 17.0-18.9 μm long, the stylet knobs are rounded and sloping, gradually merging with the shaft, and the head region consists of one large annule. Second-stage juveniles are 357-400 μm long, with a stylet length of 11.9-13.6 μm and one head annule. The tail tapers to a slender tip. This new species is similar to M. graminicola and M. triticoryzae but differs from them in perineal pattern and lateral field morphology, and numerous morphometric characters.