Pigmented variants of Tolypocladium inflatum in relation to cyclosporin A production

Summary We have isolated four distinct colony types of the fungus Tolypocladium inflatum, the producer of the immunosuppresive agent cyclosporin A: morphologically normal white, red, and orange colonies and morphologically diverse tiny brown colonies. In liquid cultures, white normal and brown colonies developed into yellow broths. The broth of the brown colony had a low final pH and low cyclosporin production, whereas orange and red colonies had dark brown and even black broths with higher final pH and high cyclosporin production. The specific production of cyclosporin A by the red colony was three times that of the white normal colonies. Offprint requests to: S. N. Agathos     

CONDITIONAL PRODUCTION OF A FUNCTIONAL FISH GROWTH HORMONE IN THE TRANSGENIC LINE OF NANNOCHLOROPSIS OCULATA (EUSTIGMATOPHYCEAE)1

Plasmid phr‐YPGHc, containing the fish growth hormone (GH) cDNA driven by a heat shock protein 70A promoter and a RUBISCO SSU 2 promoter, was transferred into the protoplast of marine microalga Nannochloropsis oculata (Droop) D. J. Hibberd by electroporation. Four transgenic clones were obtained in which the transferred phr‐YPGHc was integrated into the genome and existed stably at least until the 50th generation. When we treated these transgenic microalgae by heat shock, the heterologous fish GH was produced in the amount of 0.42 to 0.27 μg · mL^?1 from the 50 mL of medium. We incubated artemia with the wildtype and transgenic N. oculata for 6 h and then fed these microalgae‐treated artemia to red‐tilapia larvae. After feeding, the growth of larvae that were fed artemia incubated with transgenic microalgae was greater (i.e., statistically significant: P < 0.05) than that of larvae that were fed artemia incubated with nontransgenic microalgae: 316% versus 104% in weight gain, and 217% versus 146% in body length increase, respectively. Therefore, the N. oculata enables production of functional GH, and we propose that it might be an excellent bioreactor material.     

Freeze-dried microalgae of Nannochloropsis oculata improve soybean oil's oxidative stability

Marine microalga Nannochloropsis oculata possesses nutrients valuable for human health. In this study, we added freeze-dried N. oculata powder to soybean oil and observed a remarkable inhibition in oil oxidation. The amount of microalgae powder added was positively correlated to the increase in oil stability. The addition of 5.0 % (w/w) microalgae powder increased the oil stability index (OSI) values of soybean oil more than twofold at the tested temperatures 120 and 130 °C. N. oculata contains high levels of both phenolic compounds and α-tocopherols that could be the contributors to such an increase of the OSI. Two methods were conducted to assay the active ingredients released from microalgae: one employed three solvent systems to extract the microalgae and the other was the soybean oil added with microalgae. Analyses of free radical scavenging and reducing power suggested that the phenolic compounds dominated the antioxidation activities in soybean oil when it was infused with the microalgae powder. Our results suggest that N. oculata could potentially be used as an additive in cooking oil to increase the shelf life and nutritional value of the oil and to reduce the production of free radicals from lipid oxidation when the oil is used at high-temperature cooking processes.     

Effects of nitrogen supplementation of the culture medium on the growth, total lipid content and fatty acid profiles of three microalgae (Tetraselmis subcordiformis, Nannochloropsis oculata and Pavlova viridis)

To improve the properties of microalgae as sources for biodiesel production, three microalgae (Tetraselmis subcordiformis SHOU-S05, Nannochloropsis oculata SHOU-S14 and Pavlova viridis SHOU-S16) were cultured in media supplemented with different amounts of nitrogen (0, 0.22, 0.44, 0.88 and 1.76 mmol N·L^?1). The growth, total lipid contents, lipid classes and fatty acid profiles of the three microalgae were assayed after 10 days of cultivation. The results indicated that the specific growth rates of T. subcordiformis, N. oculata and P. viridis were lowest (0.014, 0.033 and 0.018, respectively) in the treatments without nitrogen supplementation and increased significantly with increasing nitrogen supplementation. The microalgae treated with 0.22 mmol N·L^?1 had the highest total lipid contents, which were 29.77, 35.85 and 32.10 % in T. subcordiformis, N. oculata and P. viridis, respectively. The total lipid contents as well as the proportions of neutral lipid in the three microalgae decreased significantly with increasing nitrogen supplementation between 0.22 and 1.76 mmol N·L^?1. The fatty acid profiles of the three microalgae were significantly different. The obvious characteristic of the fatty acid profile of T. subcordiformis was a high amount (17.68–22.22 %) of 18:3n3. However, EPA (C20:5n3) and C16 fatty acids were significantly high in N. oculata and P. viridis, respectively. N. oculata and P. viridis accumulated more 16-carbon fatty acids and fewer polyunsaturated fatty acids in nitrogen-limited media. It is therefore suggested that a limited nitrogen treatment is effective for improving the properties of P. viridis and N. oculata as sources for biodiesel.     

A novel fossil-species of Meliolinites Selkirk (fossil Meliolaceae) and its life cycle stages associated with an angiosperm fossil leaf from the Siwalik (Mio-Pliocene) of Bhutan sub-Himalaya

Here, we report the in-situ occurrence of a new fossil-species of Meliolinites (fossil Meliolaceae), Meliolinites bhutanensis sp. nov. on the cuticle fragments of a compressed angiosperm dicot leaf recovered from the middle Siwalik (Formation II: latest Miocene to Pliocene) of Bhutan, eastern Himalaya. This unique foliicolous new fossil fungal species features well-preserved mycelia consisting of superficial, brown to dark brown, septate, thick-walled, branching hyphae with bi-cellular appressoria, unicellular phialides, and a characteristic long, slightly curved hyphal seta. The web-like, brown to dark brown fungal colonies also include globose to sub-globose, dark brown ascomata, and oblong to broadly cylindrical, 5-celled, 4-septate, brown to dark brown, mature ascospores. As almost all features of different stages in the life cycle (ascospores, mature germinating ascospores, superficial lateral hyphae, hyphal seta, hyphopodia, mycelial colony, and ascomata) of this new fossil-species are found, we have proposed the first time a possible life cycle of fossil-species of Meliolaceae. The in-situ evidence of M. bhutanensis on the host leaf cuticle indicates the possible existence of a host-ectoparasite relationship in Bhutan sub-Himalaya's ancient warm and humid tropical evergreen forest during the deposition. So, M. bhutanensis might have thrived generally under warm and humid climate conditions for its growth and development in the Mio-Pliocene time, which is in conformity with our recently published quantitative climatic data by CLAMP (Climate Leaf Analysis Multivariate Program) analysis.     

Modes of colony foundation by females of different morphotypes in the paper wasps (Hymenoptera,Vespidae, <Emphasis Type="Italic">Polistes</Emphasis> Latr.)

Paper wasps of the genus Polistes exhibit wide variability both of the color and size of foundresses, and the mode of colony foundation. The colony foundation and social hierarchy were studied in populations of two Palaearctic species, Polistes dominulus (Christ) and P. nimphus (Christ) (April–May 2006, the Crimean Peninsula, Ukraine), and one Neotropical species, P. lanio (F.) (October 2004–January 2005, Trinidad Island). It was shown that foundresses of various color and size morphs tended to found nests in a peculiar way: singly (haplometrosis), by groups (pleometrosis), or occupying several nests simultaneously (polycaly). The relationship between coloration, size, and mode of colony foundation was species-specific. P. dominulus foundresses with a darker clypeus and a paler mesonotum preferred to found nests alone and had a dominant position in pleometrosis. The haplometrotic P. nimphus foundresses more often displayed darker variants of the clypeus and paler variants of the mesonotum than did foundresses from pleometrotic colonies. P. lanio foundresses from pleometrotic colonies differed from those from polycalic colonies in the coloration variability of the scutum, propodeum, and the 2nd metasomal tergite. The dominant and subordinate P. nimphus and P. lanio foundresses differed in the size of head and wings. The possible significance of the subdivision of foundresses into the “generalists” and “specialists” for the variability structuring in the population is considered.     

Effect of high ferric ion concentrations on total lipids and lipid characteristics of Tetraselmis subcordiformis,Nannochloropsis oculata and Pavlova viridis

Microalgae as sources for biodiesel production have been widely investigated. Microalgae biomass, lipid content and fatty acid profiles of microalgae are limiting factors for the cost-effective production of biodiesel. In this paper, the effects of high ferric ion concentrations on three species of microalgae (Tetraselmis subcordiformis, Nannochloropsis oculata and Pavlova viridis) were studied. The microalgae were cultured in different concentrations (1.2?×?10^?2, 1.2?×?10^?1, 1.2 and 12 mmol L^?1) of ferric ion. The growth, lipid content and fatty acid profiles of the three microalgae were analysed. When algae were cultured in 1.2 mmol L^?1 ferric ion for 10 days, the final cell density and specific growth rates of T. subcordiformis, N. oculata and P. viridis decreased significantly (p?<?0.05), and the total lipid contents of the microalgae, 33.72, 37.34 and 29.48 % (dry mass) in T. subcordiformis, N. oculata and P. viridis, respectively, were higher than those at other concentrations. The neutral lipid/total lipid ratios of the three microalgae species increased with increasing ferric ion concentration. Neutral lipids accounted for 50.75, 48.37 and 46.59 % of the total lipid in T. subcordiformis, N. oculata and P. viridis, respectively, when cultured in 12 mmol L^?1 ferric ion. The proportions of saturated fatty acids in all three species cultured in 12 mmol L^?1 ferric ion were significantly higher than those cultured in lower ferric ion concentrations. An optimum ferric ion concentration can improve the properties of T. subcordiformis, N. oculata and P. viridis as sources for biodiesel.     

Genetic analysis of genes involved in melanin biosynthesis of Cochliobolus miyabeanus

Color mutants of Cochliobolus miyabeanus defective in melanin biosynthesis were isolated. Although the wild-type strain KU-13 formed dark green colonies, color mutants formed white, brown, and gray colonies or white colonies with red pigment secretion. From the white mutant which secreted red pigment, designated scy, a melanin precursor which restored melanization of albino mutants alm-1 was isolated and identified as scytalone. This indicated that scy mutant was defective in the conversion of scytalone to 1,3,8-trihydroxynaphthalene and that melanin of this fungus is of pentaketide origin formed from oxidation of 1,8-dihydroxynaphthalene. Albino mutants alm-1 were considered to be defective in pentaketide cyclization and brown mutants brm were considered to be defective in the conversion of 1,3,8-trihydroxynaphthalene to vermelone. Albino mutants alm-2 whose coloration was not restored by application of scytalone were also isolated. The alm-2 gene was believed to be a gene transactively regulating the pentaketide cyclization and conversion of scytalone. From crossing experiments among the color mutants, it was indicated that alm-1, alm-2, and brm were linked and that scy segregates independently of these three mutant loci. Crossing of a methionine requiring mutant with alm and scy indicated that the three loci segregate independently of each other.     

A new troglomorphic species of Harmonicon (Araneae,Mygalomorphae, Dipluridae) from Pará, Brazil,with notes on the genus

A new species of Harmonicon F. O. Pickard-Cambridge, 1896 (Araneae, Dipluridae) is described, from a medium-sized lateritic cave in Parauapebas, Pará, Brazil. The male holotype and only specimen known of H. cerberus sp. n. was found near the entrance of Pequiá cave. This taxon is the fourth species described and the southernmost record for the genus. The new species displays some troglomorphic characteristics, such as reduction and merging of the posterior median and both pairs of lateral eyes and pale yellow to light brown coloration. Both characters are diagnostic when compared to the normal separated eyes and reddish to dark brown of other Harmonicon species. Other diagnostic characteristics are isolated, long, rigid setae distal to the lyra and the shape of the copulatory bulb. This is the second troglomorphic mygalomorph species from Brazil and the first from the Amazonian region.     

Cryopreservation of some useful microalgae species for biotechnological exploitation

Culture collections of microalgae represent a biological resource for scientific research and biotechnological applications. When compared to the current methods of maintenance and sub-culturing, cryopreservation minimizes labor costs and is an effective method for maintaining a large range of species over long periods with high stability. In order to determine the best cryopreservation method for microalgae species with great biotechnological potential, three freezing protocols were employed using different cryoprotectants (dimethyl sulfoxide—Me₂SO; methanol—MeOH). Three marine microalgae species (Thalassiosira weissflogii; Nannochloropsis oculata, and Skeletonema sp.) were cooled by directly plunging into liquid nitrogen (?196°C) and with two-step controlled cooling protocols (?18°C and ?80°C pre-treatments). After storage periods ranging from 10 to 120 days, viability was determined by the ability of cells to actively grow again. Results obtained for T. weissflogii showed that this species could be preserved at ultra-low temperature (?196°C) for 10 and 30 days with 10?% Me₂SO and 5?% MeOH when employed a controlled cooling protocol (?80°C). N. oculata was successfully cryopreserved either by direct freezing or with controlled cooling protocols. N. oculata samples presented good responses when treated with 5?% Me₂SO, 10?% Me₂SO, 5?% MeOH and even without any cryoprotectant. Skeletonema sp. did not survive cryopreservation in any of the tested conditions. The results indicate the difficulty in establishing common protocols for different microalgae species, being necessary further studies for a better understanding of cell damages during freezing and thawing conditions for each species.     

Genetically engineered termite gut bacteria (Enterobacter cloacae) deliver and spread foreign genes in termite colonies

Indigenous gut bacteria of the Formosan subterranean termite (Coptotermes formosanus Shiraki, Isoptera: Rhinotermitidae) were used as shuttle systems to deliver, express and spread foreign genes in termite colonies. The gut bacterium Enterobacter cloacae was transformed with a recombinant plasmid (pEGFP) containing genes encoding ampicillin resistance and green fluorescent protein (GFP). In laboratory experiments, termite workers and soldiers from three colonies were fed with filter paper inoculated with transformed bacteria. Transformed bacteria were detected in termite guts by growing the entire gut flora under selective conditions and checking the cultures visually for fluorescence. We demonstrated that (1) transformed bacteria were ingested within a few hours and the GFP gene was expressed in the termite gut; (2) transformed bacteria established a persistent population in the termite gut for up to 11 weeks; (3) transformed bacteria were efficiently transferred throughout a laboratory colony, even when the donor (termites initially fed with transformed bacteria) to recipient (not fed) ratio was low; (4) transformed E. cloacae were transferred into soil; however, they did not accumulate over time and the GFP plasmid was not transferred to other soil bacteria. In the future, transgenic bacteria may be used to shuttle detrimental genes into termite colonies for improved pest control.     

Rugosity in Grimontia hollisae

Grimontia hollisae, formerly Vibrio hollisae, produces both smooth and rugose colonial variants. The rugose colony phenotype is characterized by wrinkled colonies producing copious amounts of exopolysaccharide. Cells from a rugose colony grown at 30°C form rugose colonies, while the same cells grown at 37°C form smooth colonies, which are characterized by a nonwrinkled, uncrannied appearance. Stress response studies revealed that after exposure to bleach for 30 min, rugose survivors outnumbered smooth survivors. Light scatter information obtained by flow cytometry indicated that rugose cells clumped into clusters of three or more cells (average, five cells) and formed two major clusters, while smooth cells formed only one cluster of single cells or doublets. Fluorescent lectin-binding flow cytometry studies revealed that the percentages of rugose cells that bound either wheat germ agglutinin (WGA) or Galanthus nivalis lectin (GNL) were greater than the percentages of smooth cells that bound the same lectins (WGA, 35% versus 3.5%; GNL, 67% versus 0.21%). These results indicate that the rugose exopolysaccharide consists partially of N-acetylglucosamine and mannose. Rugose colonies produced significantly more biofilm material than did smooth colonies, and rugose colonies grown at 30°C produced more biofilm material than rugose colonies grown at 37°C. Ultrastructurally, rugose colonies show regional cellular differentiation, with apical and lateral colonial regions containing cells embedded in a matrix stained by Alcian Blue. The cells touching the agar surface are packed tightly together in a palisade-like manner. The central region of the colony contains irregularly arranged, fluid-filled spaces and loosely packed chains or arrays of coccoid and vibrioid cells. Smooth colonies, in contrast, are flattened, composed of vibrioid cells, and lack distinct regional cellular differences. Results from suckling mouse studies showed that both orally fed rugose and smooth variants elicited significant, but similar, amounts of fluid accumulated in the stomach and intestines. These observations comprise the first report of expression and characterization of rugosity by G. hollisae and raise the possibility that expression of rugose exopolysaccharide in this organism is regulated at least in part by growth temperature.     

A new species of Hisonotus (Siluriformes,Loricariidae) from rio S?o Francisco basin,Brazil

A new species of Hisonotus is described from the rio São Francisco basin. The new species can be distinguished from congeners by having (1) a unique coloration pattern of caudal fin with one black spot extending from its origin to the ventral lobe and two dark spots at the end of the lobe’s rays; (2) odontodes forming longitudinally aligned rows on head and trunk; (3) a functional V-shaped spinelet; (4) a single rostral plate at the tip of the snout; (5) by lacking contrasting dark geometric spots on the anterodorsal region of the body; (6) a lower caudal-peduncle depth; and (7) lower counts of the lateral median plates and (8) higher premaxillary and dentary teeth. The new species is the second described species of the genus Hisonotus in the rio São Francisco basin. It was found inhabiting the marginal vegetation of the rio São Francisco and three of its tributary, rio das Velhas, rio Paraopeba and rio Formoso.     

Influence of microalgae cell wall characteristics on protein extractability and determination of nitrogen-to-protein conversion factors

Additional evidence about the influence of the cell wall physical and chemical characteristics on protein extractability was determined by calculating the conversion factors of five different microalgae known to have different cell wall composition, and their protein extracts. The conversion factors obtained for crude rigid cell walled Chlorella vulgaris, Nannochloropsis oculata and Haematococcus pluvialis were 6.35, 6.28 and 6.25, respectively, but for their protein extracts the values were lower with 5.96, 5.86 and 5.63. On the other hand, conversion factor obtained for fragile cell walled microalgae Porphyridium cruentum and Athrospira platensis was 6.35 for the former and 6.27 for the latter, with no significant difference for their protein extract with 6.34 for the former and 6.21 for the latter. In addition, the highest hydro-soluble protein percentage recovered from total protein was for P. cruentum 80.3 % and A. platensis 69.5 % but lower for C. vulgaris with 43.3 %, N. oculata with 33.3 % and H. pluvialis with 27.5 %. The study spotted the light on the influence of the cell wall on evaluating the conversion factor and protein extractability. In addition, it showed the necessity of finding the conversion factor every time accurate protein quantification is required, and proved that there is not a universal conversion factor that can be recommended.     

Formation of rough and smooth strains ofSphaerotilus discophorus

Sphaerotilus discophorus forms characteristic filamentous dark brown colonies on dilute organic media containing managanous salts. Spontaneously or by induction the organism may produce an entirely different kind of colony, the S type which is smooth, glistening, hemispherical with a regular edge, and colorless. The latter resembles, therefore, colonies of common bacteria. The S colony may be a temporary modification imposed by nutritional conditions or a mutation.Temporary S-colony modifications occurred when a 0.5% peptone, mineral salts agar was enriched with 0.2 to 1.0% glucose, galactose, mannitol, sucrose or salicin but not with raffinose, glycerol or Na lactate. Two per cent peptone and 0.5% tryptose also stimulated temporary S-colony formation. However, the dark brown pigmentation, i.e. MnO₂ deposition, persisted in contrast to its marked repression by sugars. Phytone and tryptone are partially effective in S-colony formation and proteose-peptone is ineffective.Mutations of R to S strains were obtained by ageing broth cultures of R strains. The mutant S strains were identical to the parent R strains in nutritional and physiological properties but differed apparently in that they did not form sheaths or oxidize manganous salts.     

The morphology of larvae and systematics of the leaf-beetles Chrysolina tundralis and Chrysolina roddi (Coleoptera, Chrysomelidae, Chrysomelinae)

The 1st- and 4th-instar larvae of Chrysolina tundralis and Ch. roddi and the egg of the latter species are described for the first time; the instar-related changes of the larval morphology are discussed. The 4th-instar larva of Ch. tundralis is very similar to that of Ch. septentrionalis, but differs in the smaller sclerite-like plates of the abdominal segments and wider spaces between them (4–7 times as wide as a sclerite-like plate). The 4th-instar larva of Ch. roddi is very similar to that of Ch. pedestris, but differs in the dark brown coloration of the body and a fewer number of setae (9–12) in the dorsolateral areas of the meso- and metathorax. Data on the habitats and host plants of the larvae are given.     

Treatment with Algae Extracts Promotes Flocculation, and Enhances Growth and Neutral Lipid Content in Nannochloropsis oculata—a Candidate for Biofuel Production

Marine microalgae represent a potentially valuable feedstock for biofuel production; however, large-scale production is not yet economically viable. Optimisation of productivity and lipid yields is required and the cost of biomass harvesting and dewatering must be significantly reduced. Microalgae produce a wide variety of biologically active metabolites, many of which are involved in inter- and intra-specific interactions (the so-called infochemicals). The majority of infochemicals remain unidentified or uncharacterised. Here, we apply known and candidate (undefined extracts) infochemicals as a potential means to manipulate the growth and lipid content of Nannochloropsis oculata—a prospective species for biofuel production. Five known infochemicals (β-cyclocitral, trans,trans-2,4-decadienal, hydrogen peroxide, norharman and tryptamine) and crude extracts prepared from Skeletonema marinoi and Dunaliella salina cultures at different growth stages were assayed for impacts on N. oculata over 24?h. The neutral lipid content of N. oculata increased significantly with exposure to three infochemicals (β-cyclocitral, decadienal and norharman); however the effective concentrations affected a significant decrease in growth. Exposure to particular crude extracts significantly increased both growth and neutral lipid levels. In addition, water-soluble extracts of senescent S. marinoi cultures induced a degree of flocculation in the N. oculata. These preliminary results indicate that artificial manipulation of N. oculata cultures by application of algae infochemicals could provide a valuable tool towards achieving economically viable large-scale algae biofuel production.     

Population growth dynamics of the rotifer Brachionus plicatilis cultured in non-limiting food condition

Population growth parameters in batch culture of Brachionus plicatilis under a continuous supply of freeze-dried microalgae powder have been determined. Two B. plicatilis strains (L- and S-types) and four microalgae species (Nannochloropsis oculata, Nannochloropsis gaditana, Nannochloris oculata and Tetraselmis suecica) have been tested, establishing the dynamics of growth at different daily food rations. Cultures showed a short lag phase, an exponential growth phase, a long post-exponential growth phase and long decline with episodic increases. In both rotifer strains, the best growth was obtained with Nannochloropsis oculata and the poorest with Nannochloris oculata.