The eyespot of the flagellateTetraselmis cordiformis stein (Chlorophyceae): Structural spezialization of the outer chloroplast membrane and its possible significance in phototaxis of green algae

Summary The eyespot region of the flagellateTetraselmis cordiformis Stein (Chlorophyceae) was investigated with the freeze-fracture technique. The only fracture faces observed in this region were the two complementary fracture faces (PF and EF) of the outer chloroplast envelope membrane. Intramembranous particle numbers on both fracture faces of this membrane were significantly higher in the eyespot region as compared to regions outside the eye-spot. Higher numbers of particles on the PF face in the eyespot region were mainly caused by an increase in particle numbers of the size class 6–8 mm, while on the EF face particle size distribution was not significantly different between eyespot and other regions. Functional implications are discussed and evidence is presented that the outer chloroplast envelope membrane may be the site of photoreceptor location in green algal phototaxis.     

Development of cellulose synthesizing complexes inBoergesenia andValonia

Summary The development of linear cellulose synthesizing complexes (=TCs) of two selected siphonocladalean algae,Boergesenia forbesii andValonia ventricosa was investigated by following the time course of the regeneration of cell walls with the freeze fracture technique after aplanospore induction. The following structural changes of TC development were examined: (1) TCs initiatede novo; (2) the first nucleation of TC subunits occurs within 2 hr inBoergesenia and 5 hr inValonia after aplanospore induction, immediately followed by the assembly of cellulose microfibrils; (3) TCs increase their length during the assembly of randomly oriented microfibrils; and, (4) TCs stop increasing in length after the assembly of ordered microfibrils begins, with some time lag. The data demonstrate that linear TCs are not artificial products but dynamic entities which are involved in the assembly of cellulose microfibrils.     

Development of a transplantation technique of Cystoseira amentacea var. stricta and Cystoseira compressa

On the North-Western Mediterranean coastline, the genus Cystoseira (Fucales) has a key role on upper infralittoral community structure. Cystoseira amentacea var. stricta and C. compressa are the most common superficial Cystoseira species. In this area, the coastline is subjected to a strong anthropogenic pressure which has caused severe Cystoseira population decreases. The present study consists in the development of a cheap and easy to use transplantation technique of C. amentacea var. stricta and C. compressa in order to either restore former natural populations or help their settlement on artificial substrates. The thalli were fixed with epoxy glue in holes made by a drill. The success of the technique was assessed by counting the number of remaining transplanted thalli after 3 weeks, 3 months and 6 months. Results were found encouraging with 75% of survival for both species after 6 months. Moreover fertile fronds were observed on the transplanted thalli showing the harmlessness of the technique.     

Evaluation of the symbiotic nitrogen-fixing potential of soils by direct microbiological means

A method for estimating the nitrogen-fixing capacity of a population of rhizobia resident in soil is presented. legume test plants, growing under microbiologically-controlled conditions in test tubes packed with a vermiculite substrate moistened with a nitrogen-free plant nutrient solution, are inoculated directly with a suspension of the soil under examination. Rhizobia in the soil nodulate the test plants, and the amount of foliage dry matter produced in the 28 days after inoculation is regarded as an index of their effectiveness. An inoculum of at least 30, and preferably 100, rhizobia is needed to ensure that nitrogen fixation is not masked by delayed nodulation. The new method is tentatively described as the ‘whole-soil inoculation’ technique. Appraisals were made withTrifolium subterraneum L. andRhizobium trifolii and withMedicago sativa L. andR. meliloti. Soil-borne pathogens did not interfere with plant growth. The whole-soil inoculation technique was less tedious and time-consuming than an alternative method which involved extracting representative isolates from the soil and testing their effectiveness individually, and appeared to give more realistic values for the nitrogen-fixing capacity of the soil as a whole. Used in association with a field experiment, the whole-soil inoculation technique confirmed microbiologically that there had been an agronomic response to surface application of inoculant to poorly-nodulatedT. subterraneum pasture. It is submitted that this technique for determining the effectiveness of rhizobia in soil, combined with a plant-infection method for counting rhizobia, can be a reliable guide to the need for inoculation in the field.     

Laboratory rearing ofAnaphes sordidatus [Hym.: Mymaridae] on carrot weevil eggs [Col.: Curculionidae]

A technique for rearingAnaphes sordidatus (Girault) on eggs of laboratory-reared carrot weevil,Listronotus oregonensis (Le Conte), is described. Individual rearing was possible by using polyethylene embedding capsules that enabled easy manipulation of parasitized carrot weevil eggs for use in subsequent experimental procedures. The technique described resulted in 65% parasitization of carrot weevil eggs and 90 mn per day were sufficient to obtainca. 200 parasites daily.      

Probing the archaeal diversity of a mixed thermophilic bioleaching culture by TGGE and FISH

The archaeal community present in a sample of Mixed Thermophilic Culture-B (MTC-B) from a laboratory-scale thermophilic bioleaching reactor was investigated by temperature gradient gel electrophoresis (TGGE) and fluorescence in situ hybridisation (FISH). Both techniques were specifically adapted for use on native state bioleaching samples, with a view to establishing convenient means for monitoring culture composition. Using the TGGE protocol developed, the relative species composition of the thermophilic bioleaching sample was analysed, and included four phylotypes belonging to the Sulfolobales, which were related to Stygiolobus azoricus, Metallosphaera sp. J1, Acidianus infernus and Sulfurisphaera ohwakuensis. However, the St. azoricus-like phylotype was difficult to resolve and some micro-heterogeneity was observed within this phylotype. Specific FISH probes were designed to qualitatively assess the presence of the phylotypes in MTC-B. The sample was dominated by Sf. ohwakuensis-like Archaea. In addition, the St. azoricus-like, Metallosphaera species-like and Acidianus species-like cells appeared in similar low abundance in the community. Most strikingly, FISH identified Sulfolobus shibatae-like cells present in low numbers in the sample even though these were not detected by PCR-dependent TGGE. These results highlight the importance of using more than one molecular technique when investigating the archaeal diversity of complex bioleaching reactor samples.     

A cultivation technique for <Emphasis Type="Italic">E. coli</Emphasis> fed-batch cultivations operating close to the maximum oxygen transfer capacity of the reactor

A cultivation strategy combining the advantages of temperature-limited fed-batch and probing feeding control is presented. The technique was evaluated in fed-batch cultivations with E. coli BL21(DE3) producing xylanase in a 3 liter bioreactor. A 20% increase in cell mass was achieved and the usual decrease in specific enzyme activity normally observed during the late production phase was diminished with the new technique. The method was further tested by growing E. coli W3110 in a larger bioreactor (50 l). It is a suitable cultivation technique when the O₂ transfer capacity of the reactor is reached and it is desired to continue to produce the recombinant protein.Revisions requested 13 April 2005; Revisions received 6 May 2005     

Biological studies of the flesh flySarcophaga (Parasarcophaga) misera and its effects as a predator of the snailIndoplanorbis exustus

Attempts were made to identify a natural predator of the freshwater snailIndoplanorbis exustus, vector of schistosomiasis. Ochterlony's double immunodiffusion technique was used. The flySarcophaga (Parasarcophaga) misera was identified as a predator ofI. exustus. Various aspects of the biology of this fly and its effects as a predator of snailI. exustus were studied in the laboratory. The possible use of the larvae of this fly to controlI. exustus is discussed as an additional control measure to chemical control.      

Microstructural Reinforcement in the Canine Enamel of the Hyaenid Crocuta crocuta, the FelidPuma concolorand the Late Miocene Canid Borophagus secundus

In bone-eating carnivores such as the hyena Crocuta crocuta, the tooth enamel contains a secondary vertical prism decussation phyletically derived from the wavelike horizontal decussation of primitive carnivores. The structure resists fracture under vertical, oblique, and horizontal tensile stresses, owing to the following modifications of the primitive structure. Positions of wave crests and of wave troughs are synchronized in the vertically successive layers of decussating prisms. Prisms in each successive layer run in a common direction at the crests and in a common but reversed direction at the troughs. Between the crests and troughs, prisms in obliquely slanting layers often retain their primitively reversed prism directions. Near the enamel–dentine junction (EDJ), irregular horizontal decussation is retained. In the upper canine of C. crocuta, a consumer of large bones, secondary vertical decussation is largely confined to the labial and anterior sides of the crown toward the tip where modeling of the static stresses predicts the tensile stresses to be highest and aligned vertically. In Puma concolor, which does not consume large bones, secondary vertical decussation is absent, indicating stress magnitude to be a critical factor in the selection for secondary vertical decussation. The canine enamel in Borophagus secundus, an extinct canid with derived aspects of skull and dental shape like those in hyenas, has dental structures similar to those in C. crocuta but which differ in several ways. The wavelike shapes of the decussation planes are better developed in transverse sections in B. secundus than in C. crocuta, suggesting either the folds are less modified or they dip at a steeper angle. Secondary vertical decussation in B. secundus is more extensive around the circumference of the canine than in C. crocuta, related to a difference in cross-sectional shape of the tooth. Vertical prism decussation may have been more frequently attained in carnivorous mammals than in ungulates because of the more random orientation of dental stresses which creates a selective advantage for wavy decussation planes—a structural transition to vertical decussation.     

A comparative study of the food niches of lake-dwelling triclads and leeches

The food niches of four species of triclads and three species of non-parasitic leeches living in the littoral zone of British lakes of different trophic status were investigated, over one year, using a serological technique. Antisera against ten potential prey groups were employed. The basic data were adjusted to compensate for differences in predator size and seasonal changes in field temperatures. Data on two of the leech species have been presented elsewhere, but comparisons of the diet of all seven predator species are made in this paper. Much overlap in diet between the various predator species occurs, but, with one exception, each of the genera has a major food resource. The Polycelis spp. feed extensively on oligochaetes, Dugesia polychroa on molluscs, Dendrocoelum lacteum on Asellus, Erpobdella octoculata on chironomids and Glossiphonia complanata on molluscs. Helobdella stagnalis is a generalist feeder. Problems of coexistence of the leech species, and of the leech and triclad species are discussed.     

Generation of trangenic Xenopus laevis using the Sleeping Beauty transposon system

Using the Sleeping Beauty (SB) transposon system, we have developed a simple method for the generation of Xenopus laevis transgenic lines. The transgenesis protocol is based on the co-injection of the SB transposase mRNA and a GFP-reporter transposon into one-cell stage embryos. Transposase-dependent reporter gene expression was observed in cell clones and in hemi-transgenic animals. We determined an optimal ratio of transposase mRNA versus transposon-carrying plasmid DNA that enhanced the proportion of hemi-transgenic tadpoles. The transgene is integrated into the genome and may be transmitted to the F1 offspring depending on the germline mosaicism. Although the transposase is necessary for efficient generation of transgenic Xenopus, the integration of the transgene occurred by an non-canonical transposition process. This was observed for two transgenic lines analysed. The transposon-based technique leads to a high transgenesis rate and is simple to handle. For these reasons, it could present an attractive alternative to the classical Restriction Enzyme Mediated Integration (REMI) procedure.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Meiotic behaviour of Un,D and R genomes in the amphiploid Aegilops ventricosa -Secale cereale and the parental species

Summary Meiotic pairing frequencies of the Un and D genomes of Ae. ventricosa and the R of S. cereale could be easily established at metaphase I in Aegilops ventricosa — Secale cereale amphiploid plants as well as in its parental species by using the C-banding technique procedure. The results show a high diminution of chromosome pairing for all genomes in the amphiploid with respect to its parental species probably due to C-heterochromatin content and/or genotypic or cryptic interactions between the three genomes.     

DNA Sequence-Specific Ligands: XI.* The Synthesis and Binding to DNA of bis-Netropsins with the C-Ends of Their Netropsin Fragments Tethered by Tetra- or Pentamethylene Linkers

Bis-Netropsins with the C-ends of their netropsin fragments tethered via tetra- or pentamethylene linkers and with Gly or L-Lys-Gly residues on their N-ends were synthesized. The footprinting technique was used to study the specificity of bis-netropsin binding to the specially constructed DNA fragments containing various clusters of A · T pairs. It was found that the linker length affects the binding of bis-netropsins, with the tetramethylene linker providing better protection than the pentamethylene linker. It was shown that the newly synthesized bis-netropsins bind tighter to the 5"-A ₄ T ₄-3" sequence, whereas the bis-netropsin with a linker between the netropsin N-ends binds better to 5"-T ₄ A ₄-3" sequences.     

Heliobacterium chlorum: cell organization and structure

The basic cellular organization of Heliobacterium chlorum is described using the freeze-etching technique. Internal cell membranes have not been observed in most cells, leading to the conclusion that the photosynthetic apparatus of these organisms must be localized in the cell membrane of the bacterium. The two fracture faces of the cell membrane are markedly different. The cytoplasmic (PF) face is covered with densely packed particles averaging 8 nm in diameter, while the exoplasmic (EF) face contains far fewer particles, averaging approximately 10 nm in diameter. Although a few differentiated regions were noted within these fracture faces, the overall appearance of the cell membrane was remarkably uniform. The Heliobacterium chlorum cell wall is a strikingly regular structure, composed of repeating subunits arranged in a rectangular pattern at a spacing of 11 nm in either direction. We have isolated cell wall fragments by brief sonication in distilled water, and visualized the cell wall structure by negative staining as well as deep-etching.Abbreviations PF protoplasmic fracture face - EF exoplasmic fracture face     

The Selection of an Isolate of the Hyphomycete Fungus,Metarhizium anisopliae,for Control of Termites in Australia

Ninety-three isolates ofMetarhizium anisopliae,mostly derived from a survey of termite material, were screened for activity againstNasutitermes exitiosusandCoptotermes frenchiorC. acinaciformisusing a grooming assay technique. Twenty-six of the most promising isolates were further evaluated by bioassay againstN. exitiosusandC. acinaciformis.All isolates were pathogenic withCoptotermesspp. being more susceptible thanN. exitiosus.A group of nine isolates, chosen for their level of pathogenicity for one or other genus of termites and to represent a genetically diverse group, was finally compared in a minicolony test using termite colonies in 1 liter jars. The isolate, code-named FI-610 (derived from nest-mound material ofC. lacteusin SE New South Wales), was one of the most effective isolates against termites from both of the two colonies tested. This isolate also grew relatively well on agar plates at 36°C. FI-610 was thus selected for field trials and was found to be effective in killing colonies ofC. acinaciformiswhen 10 g (=3 × 10¹¹conidia) or more of conidial powder was blown into the center of the large mound colonies.     

Ultrastructure of chytridiomycete and oomycete zoospores using spray-freeze fixation

Summary The ultrastructure of zoospores of several zoosporic fungi was examined using a modified cryofixation technique. An atomizer was used to spray a zoospore suspension into the cold propane reservoir of a conventional plunge freeze-substitution apparatus. Spray-freeze fixation and freeze-substitution of zoospores porvided better fixation of vacuolar structures, membranes and the extracellular coat than that obtained with chemical fixation. The overall shape of cryofixed spores was closer to that seen in living zoospores. Two types of vacuoles were seen in cryofixed zoospores ofMonoblepharella andChytridium. One type of vacuole contained electron-opaque material within the lumen while the other type had no visible internal material in the lumen and appeared to be part of the water expulsion vacuole complex. Coated pits and coated vesicles were observed associated with both the water expulsion vacuoles and the plasma membrane inMonoblepharella andPhytophthora, suggesting that endocytosis of the plasma membrane and expulsion vacuoles is part of membrane recycling during osmoregulatory events. An extracellular coat was seen on the outer surface of cryofixed zoospores ofMonoblepharella sp.,Chytridium confervae andPhytophthora palmivora without the use of carbohydrate-specific stains. The spray-freeze method gave good and reproducible fixation of the wall-less spores in quantities greater than those obtained in previously described zoospore cryofixation studies. The technique is potentially useful for cell suspensions in that freeze damage from excess water is limited.Abbreviations ddH₂O deionized distilled water - PME Pipes/MgCl2/EGTA buffer - WEV water expulsion vacuole     

Ultrastructure of sperm cells and the male germ unit in pollen tubes ofNicotiana tabacum

Summary The structure of sperm cells and their association with the vegetative nucleus in pollen tubes ofNicotiana tabacum grown in styles were observed with the electron microscope, demonstrating the existence of a male germ unit. The two sperm cells are arranged in tandem and are closely associated with the vegetative nucleus, which always takes the lead. The leading sperm cell (SC 1) has a long and narrow cytoplasmic projection which lies within the enclaves of the much lobed vegetative nucleus, thus forming a physical association. The trailing sperm cell (SC 2) and the SC 1 are not only joined by a common transverse cell wall but also are surrounded by a periplasm bounded by the plasma membrane of the sperm cells and that of the vegetative cell, thus forming a structural connection. The sperm cells are elongated, with cytoplasmic projections at the anterior end of the SC 1 and at both ends of the SC 2. The cytoplasm of both sperm cells includes mitochondria, endoplasmic reticulum, dictyosomes, ribosomes, small vacuoles and axially oriented microtubules. No plastids were observed.Abbreviations DAPI 4,6-diamino-2-phenylindole - MGU male germ unit - MT microtubule - SC 1 the leading sperm cell physically associated with the vegetative nucleus - SC 2 the trailing sperm cell     

Occurrence ofDinophysis spp. and toxic shellfish in the Northern Adriatic

The dynamics of the toxicity of the musselMytilus galloprovincialis was compared between two different shellfish farms, 5 km apart, but using the same cultivation technique. The main differences concerned the freshwater influx and the open aspect to the Gulf of Trieste. It is suggested that a deep closed bay and abundant fresh water inflow are the two main conditions for the low toxicity levels in mussels and for shorter periods of danger. A detailed study of the phytoplankton samples revealed the presence of eight species ofDinophysis in the area of both shellfish farms. During the period of the DSP outbreak in Slovenia (autumn and winter 1989).D. fortii andD. acuminata were the most frequentDinophysis species. There was a high positive correlation between the onset of mussel toxicity and the appearance ofDinophysis spp.     

Transport of rosettes from the golgi apparatus to the plasma membrane in isolated mesophyll cells ofZinnia elegans during differentiation to tracheary elements in suspension culture

Summary Rosettes of six particles have been visualized by freeze-fracture in the protoplasmic fracture (PF) faces of: a) the plasma membrane, b) Golgi cisternae, and c) Golgi-derived vesicles in mesophyll cells ofZinnia elegans that had been induced to differentiate synchronously into tracheary elements in suspension culture. These rosettes have been observed previously in the PF face of the plasma membranes of a variety of cellulose-synthesizing cells and are thought to be important in cellulose synthesis. InZinnia tracheary elements, the rosettes are localized in the membrane over regions of secondary wall thickening and are absent between thickenings. The observation of rosettes in the Golgi cisternae and vesicles suggests that the Golgi apparatus is responsible for the selective transport and exocytosis of rosettes in higher plants, as has been previously indicated in the algaMicrasterias (Giddings et al. 1980). The data presented indicate that the Golgi apparatus has a critical role in the control of cell wall deposition because it is involved not only in the synthesis and export of matrix components but also in the export of an important component of the cellulose synthesizing apparatus. The rosettes are present in the plasma membrane and Golgi vesicles throughout the enlargement of the secondary thickening, suggesting that new rosettes must be continually inserted into the membrane to achieve complete cell wall thickening.Abbreviations EF Golgi vesicles, exoplasmic fracture; the plasma membrane, extracellular fracture - PF protoplasmic fracture     

The mechanical significance of the occlusal geometry of great ape molars in food breakdown

Analyses of dental function are an essential component of the study of human evolution. However, with few exceptions, they have utilized the traditional analogizing method of comparative anatomy, and have assumed rather than demonstrated that proposed adaptive characters confer a performance benefit. Since food reduction is a mechanical process, it is appropriate to measure performance using mechanical parameters, specifically the ability of a given morphology to induce failure in food particle by either of the two major regimes: crush and shear, corresponding to simple stresses (tensile and compressive) and shear stress, respectively. We apply finite elements stress analysis to model the relationship between the angulation of the intercuspal occlusal surfaces in a “puncture crushing” mode of mastication. On the basis of morphological data acquired from sectioned great ape molars, we have predicted the nature, magnitude and distribution of stress in a standard food particle by models representing each morphotype. Results indicate that the blunt-cusped molars ofHomo, the gradually-sloping supporting (buccal) cusps but high-angled guiding (lingual) cusps of the lower molars ofPan, and the high angled occlusal surfaces ofGorillaare all more likely to fracture small food particles by shear, while the gradually sloping occlusal surfaces ofPongomolars are more likely to break them down by “crush”. Mechanisms of food failure induced by molars ofPanandHomowill vary according to the orientation of the tooth–food contacting surfaces, which in turn will vary according to the size of the food particle. These genera may be able to break food down either by shear or by “crush”.