Ecdysis and the location of the plasma membrane in the dinoflagellateHeterocapsa niei

Summary The outer membrane is the plasma membrane in the pelliculate dinoflagellateHeterocapsa niei (Loeblich) Morrill and Loeblich, except when the cell is preparing to ecdyse and is forming a new amphiesma. At maturity the theca and pellicle are enclosed within a single large amphiesmal vesicle which surrounds the cell; thus, the amphiesmal components are intracellular. The plasma membrane lies outside this vesicle and is continuous with the flagellar membrane. At ecdysis retraction of the flagella and fusion of the innermost or cytoplasmic membrane over the flagellar region facilitates the shedding of all layers external to the cytoplasmic membrane. This membrane eventually becomes the bounding membrane (plasma membrane) of the reformed amphiesma.     

Characteristics of the membranes of the pellicle of the ciliatePseudomicrothorax dubius

Summary The membranes of the pellicle of the ciliatePseudomicrothorax dubius are investigated using thin section electron microscopy and freeze-fracture replicas. The plasma membrane is covered by a surface coat and is connected to the outer alveolar membrane by short, sometimes branched, bridges. The inner alveolar membrane is coated on both sides. The epiplasm lies in intimate contact with the cytoplasmic surface of this membrane, and there is a corresponding deposit on the other surface. This deposit is regularly striated.The epiplasmic layer and the alveoli are interrupted at sites of cytotic activity,e.g., the attachment sites of trichocysts, the cytoproct, and the parasomal sacs. The striated deposit ends where the epiplasm ends, indicating a direct relationship between these two epimembranous layers.There is a deposit along the sides of the first part of the tip of the trichocysts, and in this region the trichocyst membrane is free of intramembranous particles.The membrane of the parasomal sacs has a coat on both surfaces. That on the extraplasmic surface is similar to the surface coat of the plasma membrane. The origin of the cytoplasmic coat is unknown. The cytotic activity of these sacs is indicated by their highly irregular profiles.     

Differentiated pellicle organization and lipopeptide production in standing culture of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains

Pellicle formation and lipopeptide production was analysed in standing cultures of different Bacillus subtilis strains producing two or three families of lipopeptides. Despite its ability to produce surfactin, B. Subtilis ATCC 6633 was unable to form stable pellicle at air–water interface. For the ATTC 21332 and ATCC 9943 strains, it was shown for the first time that the lipopeptides were also produced in standing cultures at productivities similar or lower than those obtained when the culture medium is agitated. A differentiated behaviour was observed between these strains in repetitive batch cultures. B. subtilis 9943 formed a wrinkled, thinner and more resistant pellicle than B. subtilis 21332. The structure of the pellicle determined by electron microscopy observations showed that cells of B. subtilis 9943 formed microcolonies whereas those of B. subtilis 21332 rapidly died. Under these conditions, surfactin production by strain 21332 decreased after 2 days whereas it remained stable for B. subtilis 9943 during the 6 days of the cultures. These data indicate that cells of B. subtilis strains growing in pellicle can produce lipopeptides differently depending on their cellular organisation. M. Chollet-Imbert and F. Gancel have contributed equally to the scientific work.     

The roles of central and peripheral eclosion hormone release in the control of ecdysis behavior in Manduca sexta

1. Ecdysis, a behavior by which insects shed the old cuticle at the culmination of each molt, is triggered by a unique peptide hormone, eclosion hormone (EH). In pupal Manduca sexta, EH is released into the hemolymph just prior to ecdysis, and circulating hormone is sufficient to elicit this behavior. 2. Removal of the proctodeal nerves in prepupal animals eliminated the appearance of blood-borne EH, but ecdysis behavior occurred on schedule. Therefore, circulating EH is not necessary for the triggering of ecdysis. 3. In contrast, a set of dermal glands failed to show their expected bout of secretion after proctodeal nerve removal. Injection of exogenous EH rescued this secretion. Thus, circulating EH appears necessary for action on peripheral but not central targets. 4. A major reduction in EH immunostaining is seen in the proctodeal nerves just preceding ecdysis; this coincides with a greater than 90% reduction in extractable EH from this structure and the appearance of circulating EH. A similar, concomitant reduction was seen in central EH cell processes, suggesting release of peptide within the CNS. 5. Antidromic stimulation of the proctodeal nerve stumps following proctodeal nerve removal triggered precocious ecdysis. This result further supports the conclusion that centrally released EH is sufficient to trigger the motor program.     

Cytochemical investigations of the alveolar plates of theEuplotidae (Ciliophora,Hypotrichida)

Summary The ultrastructural appearance of organic plates lying in the alveoli of a freshwater species ofEuplotes is described and seen to be similar to those previously reported from the marine speciesE. vannus. Enzymatic digestion using pepsin and trypsin indicates that the plates in both species are mainly composed of protein with a fine coating of polysaccharides, as revealed using the Thiéry-technique for polysaccharide staining.     

Ultrastructure of the pellicle of Euglena gracilis

Deep-etching technique was used to investigate the organization of the pellicle complex of Euglena gracilis. The interpretation of the images was further supported by SEM and TEM investigations. Our results mainly validate data obtained by previous freeze-fracture studies on the E and P faces of the outer cortical membrane. At the level of the ridges, the outer E fracture face is highly organized in a regular striated pattern, whereas the P inner face shows a particulate structure. However, our images reveal that this particulate organization of the P face is not limited to the ridges, but it is displayed also by the grooves. Moreover, this face shows two distinct layers, a particulate layer facing the cytoplasm and a striated layer facing the E face; these layers represent different true fracture levels of the same P face.     

The optical brightener Calcofluor White disorders thecal architecture and inhibits cell division ofCryothecomonas longipes Schnepf et Kühn, a marine nanoflagellate incertae sedis

Summary Calcofluor White inhibits the division of the marine nanoflagellateCryothecomonas longipes and causes the development of monstrously lobed cells. It disturbs the assembly of a putative noncellulosic fibrillar polysaccharide in the theca. The compact layer of the theca becomes thicker and wider, less densely packed and less rigid. The deposition of thecal components becomes uncoordinated. In consequence the initially unaffected coat of the theca and loose portions of the compact layer protrude from the cell surface. These are then convoluted, with the coat on their convex faces. The spacing of the coat ridges is increased here. The tension between the different layers obviously stabilizes the theca and contributes to its rigidity and elasticity.Abbreviations CW Calcofluor White - DCB 2-chloro-6-dichlo-robenzonitrile     

Redundant ecdysis regulatory functions of three nuclear receptor HR3 isoforms in the direct-developing insect Blattella germanica

In hemimetabolous insects, the molecular basis of the 20-hydroxyecdysone (20E)-triggered genetic hierarchy is practically unknown. In the cockroach Blattella germanica, we had previously characterized one isoform of the ecdysone receptor, BgEcR-A, and two isoforms of its heterodimeric partner, BgRXR-S and BgRXR-L. One of the early-late genes of the 20E-triggered genetic hierarchy, is HR3. In the present paper, we report the discovery of three isoforms of HR3 in B. germanica, that were named BgHR3-A, BgHR3-B(1) and BgHR3-B(2). Expression studies in prothoracic gland, epidermis and fat body indicate that the expression of the three isoforms coincides with the peak of circulating ecdysteroids at each nymphal instar. Experiments in vitro with fat body tissue have shown that 20E induces the expression of BgHR3 isoforms, and that incubation with 20E and the protein inhibitor cycloheximide does not inhibit the induction, which indicates that the effect of 20E on BgHR3 activation is direct. This has been further confirmed by RNAi in vivo of BgEcR-A, which has shown that this nuclear receptor is required to fully activate the expression of BgHR3. RNAi has been also used to demonstrate the functions of BgHR3 in ecdysis. Nymphs with silenced BgHR3 completed the apolysis but were unable to ecdyse (they had duplicated and superimposed the mouth parts, the hypopharinge, the tracheal system and the cuticle layers). This indicates that BgHR3 is directly involved in ecdysis. Finally, RNAi of specific isoforms has showed that they are functionally redundant, at least regarding the ecdysis process.     

An ultrastructural comparison of the mitotic apparatus,feeding apparatus,flagellar apparatus and cytoskeleton in euglenoids and kinetoplastids

Summary The euglenoids and kinetoplastids form a diverse assemblage of organisms which show no obvious phylogenetic relationship with other flagellates. An ultrastructural examination and comparison of the flagellar apparatus, the feeding apparatus, and mitotic nucleus indicate a number of shared morphological features which support a common ancestry for the two groups. Of particular interest is the euglenoid,Petalomonas cantuscygni, which shares many of the ultrastructural features common to both groups. Based on the data presented, we hypothesize that a euglenoid with features similar to those now present inP. cantuscygni was ancestral to both the euglenoid and kinetoplastid lines.Abbrevation MTR complex of reinforcing microtubules     

Effects of acidity on the growth of two Euglena species

Our study separates the effects of elevated protons (at pH <3) and elevated metals (Al, Cd, Cu, Fe, Ni, Zn) on the growth of E. mutabilis Schmitz, a pioneering phototroph in acid mine drainage (AMD) and E. gracilis Klebs, a closely-related species rarely found in severely AMD-impacted sites. Both species were acid tolerant, growing optimally at pH 2.5–7. At pH values typical of AMD (pH 2.5–4) in the absence of elevated metals, E. gracilis outcompeted E. mutabilis (growth rates of 1.0 and 0.8 div d^–1, respectively). Relative metal toxicities were evaluated based on the Effective Exposure causing 50% growth reduction (= EE50). With total metal additions similar to AMD levels, E. mutabilis demonstrated significantly greater tolerance to all metals, except Cu. E. gracilis showed two-fold higher tolerance to Cu²⁺ than E. mutabilis (EE50 of 91.6 vs. 45.7 pmol cell^–1). The EE50 for Zn²⁺ was similar for both species (368 pmol cell^–1 for E. gracilis and 423 pmol cell^–1 for E. mutabilis). With Cd and Ni, E. mutabilis tolerated an order of magnitude higher exposure than E. gracilis(EE50 of 1.6 vs. 0.2 pmol Cd²⁺ cell^–1; EE50 of 942 vs. 87 pmol Ni²⁺ cell^–1). Al and Fe were tolerated at high total metal concentrations (up to 100 mM) by E. mutabilis, but toxicity was evident with E. gracilisat much lower levels. E. mutabilis grew at double the Al³⁺ exposure tolerated by E. gracilis (EE50 of 398 vs. 188 pmol Al³⁺ cell^–1). There was an 18-fold difference in Fe tolerance levels between E. mutabilis and E. gracilis with EE50s of 8773 and 502 pmol Fe²⁺ cell^–1, respectively. We conclude that differential metal tolerance, particularly to Fe²⁺, accounts for the mutually exclusive distribution of E. gracilis and E. mutabilis in AMD-impacted habitats.     

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.     

Investigation of roles of divalent cations in Shewanella oneidensis pellicle formation reveals unique impacts of insoluble iron

Background

Bacteria adopt a variety of lifestyles in their natural habitats and can alternate among different lifestyles in response to environmental changes. At high cell densities, bacteria can form extracellular matrix encased cell population on submerged tangible surfaces (biofilms), or at the air–liquid interface (pellicles). Compared to biofilm, pellicle lifestyle allows for better oxygen access, but is metabolically more costly to maintain. Further understanding of pellicle formation and environmental cues that influence cellular choices between these lifestyles will definitely improve our appreciation of bacterial interaction with their environments.

Methods

Shewanella oneidensis cells were cultured in 24-well plates with supplementation of varied divalent cations, and pellicles formed under such conditions were evaluated. Mutants defective in respiration of divalent cations were used to further characterize and confirm unique impacts of iron.

Results and conclusions

Small amount of Fe^2 + was essential for pellicle formation, but presence of over-abundant iron (0.3 mM Fe^2 + or Fe^3 +) led to pellicle disassociation without impairing growth. Such impacts were found due to S. oneidensis-mediated formation of insoluble alternative electron acceptors (i.e., Fe₃O₄) under physiologically relevant conditions. Furthermore, we demonstrated that cells preferred a lifestyle of forming biofilm and respiring on such insoluble electron acceptors under tested conditions, even to living in pellicles.

General significance

Our finding suggests that bacterial lifestyle choice involves balanced evaluation of multiple aspects of environmental conditions, and yet-to-be-characterized signaling mechanism is very likely underlying such processes.     

Kryptoperidinium foliaceum blooms in South Carolina: a multi-analytical approach to identification

Observations following the discovery of Kryptoperidinium foliaceum blooms in South Carolina (SC), USA, suggest that a multi-analytical approach, using a standard, minimal set of criteria, should be adopted for determining dinoflagellate species identity and taxonomic placement. A combination of morphological, molecular, and biochemical analyses were used to determine the identity of this “red tide” dinoflagellate, first documented in SC waters in the spring of 1998. Results from thecal plate tabulations (based on scanning electron and epifluorescence microscopy), gene sequence data, species-specific PCR probe assays, and microalgal pigment profiles were analyzed and compared to reference cultures of K. foliaceum. Comparative data showed marked inconsistencies among the K. foliaceum reference culture isolates. In addition, the SC bloom isolate was shown to be mononucleate, contrary to previous reports for K. foliaceum, suggesting a more transient endosymbiotic association than previously considered.     

Glycosidase activity in the post-ecdysial cuticle of the blue crab, Callinectes sapidus

We have previously demonstrated a marked change in sugar moieties of glycoproteins of the cuticle of the blue crab, Callinectes sapidus, between 0.5 and 3 h post-ecdysis. The present study has identified a glycosidase that appears in the cuticle during the early post-ecdysial hours. The enzyme has affinities for p-nitrophenyl derivatives of both N-acetylglucosamine and N-acetylgalactosamine. Both activities are competitively inhibited by chitobiose, suggesting that the enzyme could be a N-acetylhexosaminidase (EC 3.2.1.52). Atypical of N-acetylhexosaminidases described to date, this enzyme has a pH optimum of 7.0. The enzyme activity is high during the post-ecdysial period coincident with the changes in glycoprotein profiles observed in vivo. Partial purification of the enzyme has been accomplished by Sephacryl size-exclusion chromatography followed by concanavalin A affinity chromatography.     

A consideration of Euglena gracilis W3BUL as a cytoplasmic control for the wild-type phenylalanyl-tRNA synthetase system

The studies described indicate that the UV bleached mutant, Euglena gracilis W₃BUL does not serve as a suitable cytoplasmic control for the phenylalanyl-tRNA synthetase system. Chromatography of wild-type E. gracilis on Sephadex G100 revealed three peaks of activity identified as the chloroplastic, cytoplasmic and mitochondrial enzymes. The chloroplastic activity was greater in log than in stationary phase cells and was the only activity recovered from purified chloroplasts. Cell-free extracts of the achloroplastic mutant, E. gracilis W₃BUL, contained wild-type levels of the cytoplasmic and mitochondrial phenylalanyl-tRNA synthetases. However, no chloroplastic synthetase was detected in the mutant extracts. Anomalies in the aminoacylation behavior of the W₃BUL system were observed which suggest the possibility of a mutation affecting non-chloroplastic tRNAs in this UV-induced mutant. These anomalies significantly reduce the ability of the E. gracilis W₃BUL mutant to serve as a cytoplasmic control in the phenylalanyl-tRNA synthetase system.     

Uptake and incorporation of pyrimidines in Euglena gracilis

In photoorganotrophically grown cells of Euglena gracilis the uptake and incorporation degree of 12 different pyrimidines were tested. The rate of uptake of pyrimidines has distinct maxima in the late log phase and in the stationary phase of cell multiplication. The kinetics of uptake are linear in the first 2 h, do not show saturation at various concentrations and increase with the concentrations. No accumulation of the pyrimidines at various concentrations could be observed in the first 2 h of incubation. Membrane inhibitors as uranyl acetate inhibit the uptake of the reference substance -AIB, which is wellknown transported by an active transport mechanism, but have no effect on uptake rate of uracil and cytosine. It could not be observed an energy requirement tested in temperature dependence and with electron transport inhibitors. Uptake of uridine, uracil, barbituric acid and -AIB is inhibited by cycloheximide in a different manner after 5–10 min.Abbreviations Barb barbituric acid - 5-BrU 5-bromouracil - Cyd cytidine - Cyt cytosine - DHU dihydrouracil - dUrd deoxyuridine - dThd thymidine - 5-FU 5-fluorouracil - Ora orotic acid - Thy thymidine - Ura uracil - Urd uridine - CHI cycloheximide - -AIB -aminoisobutyric acid Dedicated to Prof. Dr. Dr. h.c. mult. K. Mothes on the occasion of his 75th birthday     

Development ofDentalium following removal of D-quadrant blastomeres at successive cleavage stages

Summary Each primary micromere and macromere of the D-quadrant ofDentalium was deleted, through the mesentoblast stage, to investigate the way in which the polar lobe cytoplasm exerts its influence on development.-D and -1D embryos form an apical tuft but no posttrochal structures.-2D embryos form an apical tuft and a reduced posttrochal region without a shell. -3D and -4D are externally similar to control embryos. -1d embryos and -1c embryos have an apical tuft with a reduced number of cilia. Embryos in which both 1c and 1d are deleted lack the apical tuft.-2d embryos lack shell and most other posstrochal structures. -3d and-4d embryos appear externally equivalent to controls.The polar lobe cytoplasm exerts its influence sequentially, and as inIlyanassa the maximal effect is at the third quartet stage.     

Development of the polyembryonic parasitoid Copidosoma floridanum in Trichoplusia ni

Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) parasitized by the polyembryonic egg-larval parasitoid Copidosoma floridanum (Ashmead) (Hymenoptera: Encyrtidae) attained significantly larger final weights and head capsule widths than unparasitized controls. The difference in weight between parasitized and unparasitized hosts was not entirely accounted for by the weight of the C. floridanum brood. The head capsule widths of all parasitized and unparasitized fifth instars used in the study exceeded the critical threshold of 1.66 mm previously established for T. ni metamorphosis. The critical ratios associated with each T. ni instar of: 1) maximum weight within the instar:head capsule width and 2) maximum weight within the instar:weight at the beginning of the instar differed between parasitized and unparasitized larvae. Development of C. floridanum was synchronized with that of its host. Germ band formation and gastrulation of morulae destined to produce reproductive larvae invariably coincided with the host molt to the ultimate, fifth instar. Reproductive larvae had two instars. Eclosion from the egg to the first instar occurred during day 2 of the host's fifth instar, and ecdysis from the first to the second instar was synchronized with host cocoon spinning. Conversely, embryogenesis of morulae destined to produce precocious larvae began during the host first instar, continued through the second and third instar and ceased during the penultimate, fourth instar. Precocious larvae never molted and died when the host was consumed by the reproductive larvae.

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Résumé T. ni Hübner parasité par le parasitoïde ovo-larvaire C. floridanum Ashmead à développement polyembryonnaire atteint un poids final signficativement plus élevé avec une capsule céphalique plus grosse que les témoins non parasités, sans subir de mues surnuméraires. La différence de poids entre noctuelles parasitées ou non ne correspondait pas entièrement au poids des C. floridanum. Les largeurs des capsules céphaliques de tous les T. ni du cinquième stade dépassaient toutes le seuil critique de 1,66 mm lié à la métamorphose, mais les seuils critiques de taille du corps:largeur de la capsule céphalique et/ou taille et corps, taille initiale du corps au début du stade associé à la mue, différaient chez T. ni parasités ou non. Les développements de T. ni et de C. floridanum étaient synchrones. La formation de la bande germinative et la gastrulation de la morula produisant la multiplication des larves ont coïncidé invariablement avec la mue de l'hôte donnant le dernier stade. Les larves polyembryonnaires ont présenté deux stades. L'éclosion des oeufs s'est produite le deuxième jour du cinquième stade de T. ni, et le passage du premier au second était synchrone de la formation du cocon de l'hôte. Réciproquement, l'embryogenèse de la morula qui donnait des larves précoces commençait pendant le premier stade de l'hôte et se poursuivait à travers les second et troisième, pour cesser pendant le quatrième et pénultième stade.

     

Effects of organic acids on lipid synthesis and ecdysis in Triatoma infestans eggs

Scarce bibliographical data exists on the enzymes in Lepidosiren paradoxa and analysis of several enzymes was considered worthy of investigation. Distribution of ADH, ALP, FBALD, GAPDH, G3PDH, G6PDH, GPI, LDH, MDH, and PGM was identified in ten tissues (retina, heart, muscle, liver, kidney, lung, gut, gills, brain, and ovary) of the South American lungfish and compared with patterns previously described in other vertebrates. Compared with earlier results differences in the number of loci expressed were observed for ADH, G3PDH, GPI, and MDH. The number of loci expressed and/or in tissue specificity of several enzymes (ADH, FBALD, GAPDH, G3PDH, G6PDH and PGM) were found to be similar to those of other vertebrates. Differences were detected in ALP due to the absence of an intestinal-specific form typical of fish, amphibians, reptiles and birds; further differences were observed in GPI and MDH due to their tissue expression. The differences in LDH involve the LDH-A₄ isozyme which was most common in tissues. Overall, comparison with other vertebrates reveals that in L. paradoxa the tissue-restricted expressions of some enzymes are similar, while others have retained an ancestral pattern and exhibit a more widespread tissue expression of genes.     

Antimutagenic effect of heteroxylans,arabinogalactans, pectins and mannans in the Euglena assay

A series of plant cell wall polysaccharides – heteroxylans, arabinogalactans, pectins and mannans exerted antimutagenic (antibleaching) activity against acridine orange- and ofloxacin-induced mutagenicity in the Euglena assay. All polysaccharides tested exhibited a significant dose-dependent antibleaching activity and the percentage inhibition of mutagenicity ranged from 52 to 96%. It can be assumed that the antimutagenicity of the polysaccharides depends on their structural and compositional properties as well as on the different mode of action of both the mutagens tested.