Photoreception inEuglena gracilis: Fluence-rate and time dependence of photoaccumulation and photodispersal

The fluence-rate and time dependence for photoaccumulation and photodispersal ofEuglena gracilis was measured for the wild-type strain and three white mutants. For wavelengths of 453 or 463 nm the threshold for photoaccumulation was close to 6×10⁻²Wm⁻². Photoaccumulation increased steadily with time and reached a maximum after about 4 hr. Red light elicited substantial photoaccumulation in the wild type and photodispersal in the white, non-photosynthetic mutant 1224-5/9f. The chromophore mediating the red-light response needs to be a non-photosynthetic pigment which remains presently unidentified. A whiteEuglena mutant, FB, which had retained a reduced stigma and a paraflagellar body, showed weak photoaccumulation. Two white mutants, 1224-5/1f and 1224-5/9f, both of which lacked the stigma and positive phototaxis, displayed during the first 90 min of irradiation photodispersal; after longer irradiations they showed instead photoaccumulation. These results contradict a widely held belief that the presence of a stigma represents a stringent requirement for photoaccumulation. Our results imply that phototaxis is not a prerequisite for photoaccumulation. Exogenous flavins and 5,10-methenyl-tetrahydrofolate (MTHF) influenced in a wavelength-dependent manner photoaccumulation and photodispersal. In the wild type FAD and riboflavin (RB) caused at 453 nm an increase of the responsiveness for photoaccumulation. The photoaccumulation of the white mutant FB, was sensitized by FMN and FAD. In the white mutant 1224-5/9f exogenous flavins lowered the threshold for photodispersal. FMN, which absorbs only blue light, altered also the responsiveness to red light: in the wild type FMN reduced photoaccumulation and in the white mutant 1224-5/9f it reduced photodispersal.     

Alterations induced by dimethyl sulfoxide inEuglena gracilis with emphasis on the side effects

Summary In a previous study, we demonstrated that 5% dimethyl sulfoxide (DMSO) alters the contractile system responsible for cell motility (euglenoid movements) and cytokinesis inEuglena gracilis. However, the nucleus continued to divide and most cells were larger than normal and binucleated.The present study reveals that DMSO, besides altering the cell functions requiring microfilaments, also affects other cell parts. More precisely, the materials normally covering the plasma membrane detach from it; the nucleus shows an irregular outline and aberrations in the nucleolus and chromosomes; the chloroplasts decompose the internal structures and, in a number of cells, transform into proplastid-like organelles. Also, the development of the proplastid into chloroplast in etiolated algae exposed to the light in the presence of DMSO is highly disturbed.These results show that DMSO has remarkable side effects like all the cytoskeletal poisons experimented up to now. An interpretation of the nuclear and chloroplast alterations is advanced.The study was supported by grants from Consiglio Nazionale delle Ricerche (CNR) and Ministero della Pubblica Istruzione of Italy.     

The mechanism of growth inhibition by threonine inEuglena gracilis: Regulation of isoleucine-valine biosynthesis by 2-oxobutyrate

InEuglena gracilis the growth inhibition by threonine was accompanied by a rapid accumulation of isoleucine in the cells. Among threonine-catabolizing enzymes only threonine dehydratase was detected in high activity inEuglena, and 2-oxobutyrate, the dehydratase products of threonine, also inhibited as did threonine. Threonine dehydratase was located in the cytosol, and its activity was not affected by isoleucine and related amino acids. 2-Oxobutyrate strongly inhibited the synthesis of valine from pyruvate while augmented the synthesis of isoleucine in mitochondria.     

Cytoskeletal structures inEuglena gracilis after triton X-100 extraction and dry cleaving

Summary A three-dimensional network of structural filaments was visible with common electron microscopes in the cytoplasm ofEuglena gracilis green cells extracted with buffers containing the nonionic detergent Triton X-100. A similar filamentous web was detected at the periphery of critical point dried cells cleaved on grids by means of an adhesive tape. SDS-polyacrylamide gel electrophoresis of the detergent-resistent cytoskeleton showed that actin or actin-like proteins of molecular weight in the range of 43–45 K are not among the components having a structural role inEuglena. The significance of these findings was discussed in relation to the capability of the alga to change the cell shape.The study was supported by grants from Consiglio Nazionale delle Ricerche (CNR) and Ministero della Pubblica Istruzione of Italy.     

Metabolic engineering of Thermobifida fusca for direct aerobic bioconversion of untreated lignocellulosic biomass to 1-propanol

Biofuel production from renewable resources can potentially address lots of social, economic and environmental issues but an efficient production method has yet to be established. Combinations of different starting materials, organisms and target fuels have been explored with the conversion of cellulose to higher alcohols (1-propanol, 1-butanol) being one potential target. In this study we demonstrate the direct conversion of untreated plant biomass to 1-propanol in aerobic growth conditions using an engineered strain of the actinobacterium, Thermobifida fusca. Based upon computational predictions, a bifunctional butyraldehyde/alcohol dehydrogenase was added to T. fusca leading to 1-propanol production during growth on glucose, cellobiose, cellulose, switchgrass and corn stover. The highest 1-propanol titer (0.48 g/L) was achieved for growth on switchgrass. These results represent the first demonstration of direct conversion of untreated lignocellulosic biomass to 1-propanol in an aerobic organism and illustrate the potential utility of T. fusca as an aerobic, cellulolytic bioprocess organism.     

Structural alterations inEuglena gracilis exposed to the herbicide isopropyl-N-phenylcarbamate (IPC)

Summary Ultrastructural abnormalities of various kinds and severity appeared inEuglena gracilis green cells after a 48-hour exposure to 4 × 10^–4M of isoprophyl-N-phenylcarbamate (IPC), a widely used herbicide thought to affect microtubules and/or microtubule organizing centers in susceptible plant and animal species.A high proportion of cells contained nuclei in the G 2 phase of the cycle; in a significant percentage of organisms, however, structural aberrations of the nucleolus and chromosomes were evident. The pellicle outline, chloroplasts, mitochondria, and dictyosomes were also affected. The cytoplasm was rich in dense bodies which sometimes showed granular, fibrillar or tubular materials. Furthermore IPC partially inhibited flagellum regeneration after mechanical amputation.The mechanism by which IPC causes these responses remains unclear. Nevertheless, some indications suggest that the herbicide acts primarily on microtubule organizing centers. However, mitochondria, chloroplasts and nuclear constituents appear as possible additional targets of the compound.The study was supported by a grant from Italian Research Council (CNR), contract No. 80.00420.04.     

Ultrastructural changes inChlorella fusca during iron deficiency and vanadium treatment

Summary The ultrastructure of the unicellular green algaChlorella fusca, grown in the absence and presence of vanadium (4 · 10^–7 M as NH₄VO₃) and under iron deficiency, is investigated by electron microscopy. Iron and vanadium deficiencies are characterized by an accumulation of intracellular starch, the observation being confirmed by chemical analysis of the amount of algal total starch. While iron deficiency causes dishorders in the chloroplast lamellar system and very often disintegration of the whole cell, the presence of vanadate prevents these symptoms. V-grown cells form only little starch, but show an enlarged thylakoid system within the chloroplast. The metabolic significance of these observations is discussed.Taken in part from the forthcoming doctoral thesis of L. J. M. Becker.     

Duration of diapause in the stem borers, Busseola fusca and Chilo partellus

The duration of diapause in the stem borers Busseola fusca (Fuller) and Chilo partellus (Swinhoe) was studied in South Africa by collecting diapausing larvae from the field throughout winter (April–August). B. fusca larvae emerged as moth around the middle of October regardless of the date of collection and the length of time they were kept in the laboratory under constant 21 °C. C. partellus larvae collected in April–June emerged in November, those collected in July emerged in October, and those collected in August emerged in September. Regardless of the collection date C. partellus started to emerge from diapause earlier and moth emergence lasted up to twice as long as in B. fusca. Under laboratory conditions at 60% RH both borer larvae lost about 50% of their body mass during diapause. When provided with water B. fusca larvae lost about 30% of their body mass and adults emerged 20 days earlier than when kept dry. C. partellus, on the other hand, lost only 13% of their body weight and emerged 34 days earlier. The differences between the two species are discussed in light of different types of diapause; i.e., obligatory diapause in B. fusca and facultative diapause in C. partellus.     

Possible role of cyclic AMP in the synthesis of chlorophyll in Chlorella fusca

The intracellular concentration of cAMP in the green alga Chlorella fusca was in the range of 2 · 10^-9 to 10^-8 moles/g dry weight and was strongly dependent on the growth conditions. The cAMP level was high with high light intensity, low nitrate or glucose concentration. Intracellular cAMP increased only by factor of 2 when high amounts (up to 10^-3 M) of cAMP were added to the medium. Most of the given cAMP was converted to 5-AMP.Addition of cAMP had little effect on the chlorophyll content of the cells, only at 10^-6 M some enhancement in photoautotrophic cultures was observed. On the other hand high amounts of cAMP in the medium increased the growth rate. DBcAMP^* showed a positive effect on chlorophyll synthesis and growth rate at much lower concentrations compared to cAMP.Stimulation effects of exogenous cAMP on the synthesis of chlorophyll were also observed in mixotrophic cultures with a high glucose/nitrate ratio, conditions where chlorophyll synthesis is repressed. Similar to autotrophic conditions DBcAMP was more effective than cAMP.These data indicate that cAMP may act in a system controlling the chlorophyll content of the cells in response to nutrients or light.Abbreviation DBcAMP^* N⁶-2-O-dibutyryl-adenosine-35-monophosphate     

Diet of the brown howler monkeyAlouatta fusca in a semi-deciduous forest fragment of southeastern Brazil

The diet of a group of six brown howlers was studied weekly during 12 months in a reserve of 250 ha of secondary, mesophytic, semi-deciduous forest. The phenology of 186 trees of 72 species and 29 families was monitored simultaneously. Scan sampling was used to record the diet from dawn until dusk on a total of 60 days of observation, yielding 718 hr of animal-observer contact and 2,943 feeding scans. The diet was composed of leaves (73%), flowers (12%), and fruits (5%), from 68 identified plant species.Celtis iguanae, Cassia ferruginea, andInga spp. were the main food sources, accounting for approximately 50% of the diet. Young leaves (59%) were preferred to mature leaves (31%), trees contributing 56% and lianas 41% of the leaf diet. The ingestion of young leaves was correlated to the availability of these items, however, the correlations were not significant for flowers and fruits. The diet was poorer in fruits and richer in young leaves of lianas in comparison to other howler monkey species studies, probably as a consequence of the liana abundance in this forest fragment.     

Enhanced cutinase production of Thermobifida fusca by a two-stage batch and fed-batch cultivation strategy

In this study, cutinase production by Thermobifida fusca WSH03-11 was investigated with mixed short-chain organic acids as co-carbon sources to demonstrate the possibility of producing high value-added products from organic wastes. T. fusca WSH03-11 was cultured with different combinations of butyrate, acetate, and lactate with a purpose of increasing cutinase activity. The optimum proportion of butyrate, acetate, and lactate was 4:1:3. In batch cultivation, acetate and lactate were consumed quickly, while the consumption of butyrate was depressed in the presence of acetate with a concentration higher than 0.5 g/L. Based on these results, a two-stage batch and fed-batch cultivation strategy was proposed: a batch culture with acetate and lactate as the co-carbon sources in the first 10 h, and then a fed-batch culture with a constant butyrate feeding rate of 12 mL/h during 11∼20 h. By this two-stage cultivation strategy, cutinase activity, dry cell weight, and consumption rate of butyrate were increased by 70%, 103.4%, and 4.3-fold, respectively, compared to those of the batch cultivation. These results provided a novel and efficient way to produce high value-added products from organic wastes.     

Development of chloroplast membranes during light induced greening ofChlorella fusca g-2

Summary The development of thylakoid protein complexes during light induced greening of a mutant ofChlorella fusca was studied. Separation of chlorophyll-protein complexes and thylakoid polypeptides by LDS-polyacrylamide gel electrophoresis show that cells grown in the dark contain proteins belonging to coupling factor and cytochrome f/b₆ complex. Parts of both reaction centers are present also. The antennae complexes are specifically lost in yellow cells. The changes in polypeptide pattern at different stages of development in the light are related to ultrastructural changes. The beginning of membrane appression can be correlated with the appearance of the light-harvesting complex II. While the average diameter of EF-particles increases throughout the greening process, their densitiesapart from the rearrangement due to membrane stacking-remain fairly constant. The kinetics of EF_u-particle enlargement are different from those of EF_s-particles.PF-faces in thylakoids grown in the dark contain particles of uniform diameter but some of them protrude more from the fracture plane than do their neighbors. During the first hours of greening, their density increases and two classes develop. From the beginning of membrane stacking, the composition of PF_u-faces remains constant and PF_s-particles increase in number for some time.Results are discussed on the basis of present knowledge of structurefunction relations in thylakoids.Abbreviations CF _o intrinsic membrane complex of the coupling factor - EF, EF _s ,EF _u exoplasmic fracture face, stacked and unstacked region, respectively - LDS lithium dodecyl sulfate - LHCII light-harvesting complex of Photosystem II - PF, PF _s ,PF _u protoplasmic fracture face, stacked and unstacked region, respectively - PS I andPS II Photosystem I and Photosystem II     

Photosynthesis-dependent removal of 2,4-dichlorophenol by Chlorella fusca var. vacuolata

Of 7 green algae, Chlorella fusca var. vacuolata removed about 23% of 2,4-dichlorophenol (DCP) at 10–80 M after 4 d when grown photoautotrophically. Removal of DCP was growth-dependent and was suppressed dose-dependently by the photosynthesis inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea.     

Hibernation by the lepidopteran stalk borers, Busseola fusca and Chilo partellus on grain sorghum

The lepidopteran stalk borers, the indigenous Busseola fusca (Fuller) (Noctuidae), and the exotic Chilo partellus (Swinhoe) (Pyralidae), were studied in grain sorghum fields, Sorghum bicolor (L.) Moench (cultivar SSK-52), in the dry season of 1986 (April–October) at Delmas and Brits, Transvaal, South Africa. More than 90% of plants at both sites were infested, but as winter progressed, the proportion of plants infested and the level of infestation dropped gradually. The borer larvae hibernated inside the dry stalks, but the location differed: 65% of B. fusca were in the lower third and 30% in the middle third, whereas for C. partellus it was 45 and 50% respectively. Both borers overwintered as larvae in either of the last three instars, but the proportions of larval instars were different. About 82% of B. fusca hibernated as 6th and 16% as 5th larval instars whereas with C. partellus it was 40 and 45% respectively. C. partellus started to emerge from diapause in the second part of August and it lasted until the first week of November, a period of 12 weeks, but with B. fusca on the other hand pupation lasted only 3 weeks during October–November. Parasitism was low on both species, but higher on B. fusca. Three parasites, Chelonus curvinaculatus Cameron, Chelonus sp. and Pristomerus sp. hibernated in the borers, one parasite Iphiaulax sp., hibernated in its cocoon inside the stalks, and two parasites Apanteles sesamiae Cameron and Bracon sp. were active in winter. Two ants, Pheidole megacephala Febricius, and Dorylus helvolus (L.), preyed on the borer larvae in winter.

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Hibernation des chenilles de deux espèces de mineuses, Busseola fusca et Chilo partellu dans les tiges de Sorghum bicolor

Résumé Deux chenilles mineuses de tiges, B. fusca, Noctuidae indigène, et C. partellus, Pyralidae exotique, ont été étudiées dans des champs de S. bicolor (cultivar SSK-52), pendant la saison sèche de 1986 (Avril–Octobre) à Delmas et Brits, au Transvaal en Afrique du Sud. Plus de 90% des pieds des deux stations étaient infestés, mais plus l'hiver avançait, plus la proportion de plantes in festées et le taux d'infestation diminuaient. Les chenilles ont hiverné dans les tiges sèches, mais leurs positions étaient différentes: 65% des B. fusca étaient dans le tiers inférieur et 30% dans le tiers médian, tandis que pour C. partellus, il s'agissait respectivement de 45 et 50%. Les duex chenilles hivernaient à l'un des 3 derniers stades larvaires, mais les proportions étaient différentes. Pour B. fusca environ 82% hivernaient au 6ème stade et 16% au 5ème stade, tandis que pour C. partellus c'étaient 40 et 45% respectivement. C. partellus a commencé à sortir de diapause pendant la seconde moitié d'Août et a continué jusqu'à la première semaine de Novembre, soit durant une période de 12 semaines, mais avec B. fusca la nymphose a duré seulement 3 semaines en Octobre–Novembre. Le parasitisme des deux espèces a été faible, mais plus important chez B. fusca. Trois parasites, Chelonus curvimaculatus Cameron, Chelonus sp. et Pristomerus sp. ont hiverné dans leurs cocons à l'intérieur des tiges, et deux parasites, Apanteles sesamiae Cameron et Bracon sp. étaient actifs en hiver. Deux fourmis, Pheidole megacephala Fabricius et Dorylus helvolus L. capturaient des chenilles de ces mineuses pendant l'hiver.

     

High-salt solutions prevent reactivation of euglenoid movement in detergent-treated cell models ofEuglena gracilis

Summary Detergent-treated cell models ofEuglena gracilis showed rounded-up movement of the cell body upon addition of ATP and Ca²⁺. Reactivation of the cell models was inhibited when the cell models had been treated with solutions containing >150 mM NaCl or >300 mM KC1. When the supernatant of salt-extracted cell models was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two distinctive bands of 120 and 160 kDa were found to be enriched. The cell membrane and associated cytoskeleton (pellicular complex) were isolated after treatment with salt solutions, and examined by electron microscopy to identify essential cortical structures required for reactivating rounding-up cell movement. Among three regularly arranged microtubules, only one and its associated structures were selectively dissolved from the pellicular strips, while the other pellicular elements remained intact. These structures were located in the groove region where sliding between strips is believed to occur during cell shape change. These results suggest a possible involvement of microtubule 2 and its associated bridges in active sliding between adjacent pellicular strips during euglenoid movement.     

Indications about the mechanism of action and toxicity of netilmicin on the basis of the antiplastidial activity inEuglena gracilis

Summary The effects of the new antibiotic netilmicin (NT) were studied onEuglena gracilis green cells. It was found that, in the presence of the drug, chlorophyll synthesis was strongly inhibited and plastid structure dramatically altered as revealed by fluorescence and electron microscopic observations. Importantly, NT at low concentrations (10–20 g/ml) and for short periods of time (36–72 hours) induced a marked bleaching effect that was permanent and accompanied by the persistence in the colorless cells of poorly differentiated plastids. Other cell components were not influenced as shown by their appearance and also by the kinetics of growth and the cell viability. On the basis of the results and of the literature on the bleaching agents it is suggested that NT is an untoxic antibiotic which specifically inhibits protein synthesis at prokaryotic level.This work was supported by a grant from Italian Research Council (CNR), Contract N. 82.02016.04.     

In vitro propagation of a Saccharum officinarum (L.) and Sclerostachya fusca (Roxb.) A. Camus hybrid

Summary Callus induction and plant differentiation were obtained in an intergeneric hybrid of Saccharum officinarum and Sclerostachya fusca. The sub clones showed morphological variation. Chromosome numerical variation was not observed but structural aberrations were noticed in some sub clones. The study indicates the use of tissue culture technique for inducing intergeneric gene transfer in Saccharum hybrids.     

Activity pattern of the brown howler monkeyAlouatta fusca,Geoffroy 1812, in a forest fragment of southeastern Brazil

A group of six brown howlers was studied during a year in the Santa Genebra Reserve, a 250ha-forest fragment near the city of Campinas, State of São Paulo, southeastern Brasil. On average, the group rested for 64% of the daylight hours and feeding and moving occupied 18% and 13% of this period, respectively. A greater proportion of time was spent feeding during dry season (24%) than the wet season (15%), and the reverse observed for time spent resting (59% and 69% of the group's daylight hours during dry and wet seasons, respectively). The increase of resting and decrease of feeding during wet season were related to the percentage of leaves in the group's diet, high even during the fruiting season (wet season). However, additional data of other groups are necessary to draw conclusions about the effects of habitat disturbance on this population.     

CO2-Fixation and ATP synthesis in continuous cultures of Chlorella fusca

In continuous cultures of Chlorella fusca under steady state conditions, the CO₂-fixation rate, the ATP-level, the apparent rate of photophosphorylation as calculated from the changes in the ATP-level during light to dark or dark to light transients and the energy charge were measured at various environmental conditions. During growth the energy charge was around 0.64. CO₂-assimilation and the apparent ATP-synthesis were strongly dependant on light intensity, however the ATP-level was independant on it. Since the rates of apparent ATP-synthesis and of the CO₂-fixation do not seem to be strictly correlated in a logic way when environmental factors are changed and furthermore the stoichiometry of 3 ATP necessary per CO₂ fixed was never achieved, the described method frequently used for procaryotes to determine the in vivo rate of phosphorylation does not give valid results in highly compartimented eukaryotic cells.     

Distribution of polyphosphates in cell-compartments of Chlorella fusca as measured by 31P-NMR-spectroscopy

In suspensions of the green alga Chlorella fusca the influence of high pH and high ethylene-diamine-tetraacetic acid concentrations in the external medium, of French-press and perchloric acid extraction of the cells and of alkalization of the intracellular pH on the polyphosphate signal in ³¹P-nuclear magnetic resonance (³¹P NMR) spectra was investigated.The results show that part of the polyphosphates of asynchronous Chlorella cells are located outside the cytoplasmic membrane and complexed with divalent metal-ions. These polyphosphates are tightly bound to the cell wall and/or the cytoplasmic membrane and are not susceptible to hydrolyzation by strong acid at room temperature, in contrast to the intracytoplasmic polyphosphates.Upon alkalization of the internal pH of Chlorella cells, polyphosphates, previously not visible in the spectra become detectable by ³¹P-NMR-spectroscopy. ³¹P-NMR spectroscopic monitoring of polyphosphates during gradual alkalization of the extra-and intracellular space is proposed as a quick method for the estimation of the cellular polyphosphate content and distribution.Abbreviations CCCP Carbonylcyanide-m-chlorophenyl-hydrazone - NTP/NDP Nucleotide triphosphate/-diphosphate - PCA Perchloric acid - ³¹P-NMR ³¹P-nuclear magnetic resonance - PolyP polyphosphates - PP₁, PP₂, PP₃ terminal, second and third phosphate residue of polyphosphates, respectively - PP₄ core phosphate residues of polyphosphates