The origin of protein and fatty yolk in Rana pipiens. V. unusual paracrystalline configurations within the yolk precursor complex

Three unusual highly ordered configurations of yolk protein in yolk precursor bodies are described. These differ from the crystalline structure of the main body of mature yolk platelets. One of these is an aggregation of paired membranes with a spacing of about 100 Å between the members of a pair. The paired membranes of such an aggregation may be straight, parallel, and very close together; they may appear as a tight whorl; or they may display an intermediate random arrangement with varying distances between pairs. Another configuration is a tubule with a diameter of about 450 Å, whose wall appears in cross section to consist of particles measuring 50 × 100 Å. A third configuration is a crystalline array of rows of angular-shaped particles with a spacing of about 160 Å. It is suggested that these may represent intermediates in the transition of vitellogenin to lipovitellin and phosvitin.     

Supramolecular organization of lysophosphatidylcholine-packaged gramicidin A′

Heat derived gramicidin A′/l-α-lysophosphatidylcholine complexes were separated on a sucrose gradient to form two fractions: Fraction A which had an approximately constant Gramicidin A′ to phospholipid ratio of 8 to 10 lipid molecules per Gramicidin A′ molecule and Fraction B which had a larger but variable ratio. Fluorescence and circular dichroism studies confirmed Fraction A to be a lipid-incorporated channel state. Electron microscopic studies, using uranyl acetate negative staining, showed fraction A to be a membranous state with the formation of bilayer vesicles, that is, the interaction of peptide and phospholipid micelles causes the lipid to reorganize into a bilayer structure. Freeze-fracture replicas of the channel incorporated state demonstrated the presence of a supramolecular organization of particles exhibiting a tendency to form rows with a 50–60 Å periodicity along the row and with 70–80 Å distance between rows. An idealized working model for the incorporated state is presented.     

Membranous cytochrome c oxidase. A freeze-fracture electron microscopic analysis

Suspensions of membranous cytochrome c oxidase prepared from beef heart mitochondria by Triton extraction were ultra-rapidly cooled (in excess of 10,000 deg.C/s) and analyzed using freeze-fracture and freeze-fracture-etch techniques. The preparations contained non-crystalline and crystalline vesicles as isolated vesicles, vesicles inside other vesicles and stacks of vesicles. In non-crystalline vesicles the particles (about 100 Å diameter) are probably formed by the deviation of hydrophobic fracture planes of the membranes around the large transmembrane enzymes. The intramembrane particles thus formed are compared to particles (about 80 Å diameter) in a vesicle reconstituted from purified enzyme and lipid. Crystalline membranous cytochrome c oxidase vesicles display an unusual fracture pattern in which adjacent crystalline surfaces are separated from each other and from the surrounding ice by fracture steps that are approximately the thickness of a single membrane (100 to 120 Å). In addition adjacent crystalline fracture surfaces have similar low-relief textures, both of which differ significantly from the hydrophobic surfaces normally exposed in membrane fractures. This fracture morphology is interpreted in terms of fractures along hydrophilic surfaces of the membranes. Images of etched crystalline vesicles provide support for this interpretation because etching exposes no new surfaces. It is concluded that the crystalline lattices are derived from the portions of enzymes that protrude from the membrane bilayers and that the interdigitation of the enzymes on the inside surfaces of the vesicles or between vesicles determines the appearance of the crystalline surfaces. The arrangement of the tails of the y-shaped molecules on the cytoplasmic sides of the crystalline membranes can be visualized in micrographs directly and in reconstructions of filtered images. The more complex pattern of arms protruding on the matrix side is obscured by the unidirectional shadowing. Fragmentation of the crystalline membranes during fracturing is indicated by particles sometimes present at the edges of fractured membranes and by deep, irregular pits observed in crystalline surfaces. Particles resting on some crystalline surfaces may be fragments of crystalline membranes removed during fracturing. In other crystalline membranes non-protein is removed during fracturing, leaving globular particles embedded in the lattice, which measure about 118 Å diameter. Comparing these particles to the 3-dimensional arrangement of protein described in the accompanying paper (Frey et al., 1982) suggests that such particles are composed of 2 dimers paired along the a-axis. Intramembrane and fragmentation particles of similar size may also have this protein composition.     

Structural investigations of influenza B virus

Purified preparations of influenza B/Hong Kong/5/72 have been characterized by hydrodynamical measurements, electron microscopy and small-angle neutron scattering. As judged by these techniques the preparations are highly monodisperse, the virus particles being spherical and of molecular weight about 200 × 10⁶. The lipid bilayer is located at a radius of 425 Å and its molecular weight is estimated to be 60 × 10⁶, constituting about 30% of the total virus mass. The external radius is about 580 Å.     

Development of axonal membrane specializations defines nodes of Ranvier and precedes Schwann cell myelin elaboration

The development of the node of Ranvier has been previously described using thin-section electron microscopy. Using freeze-fracture, we have examined the development of glial and axonal membrane specializations before and during myelination. The spinal roots of the newborn rat are composed of bundles of unmyelinated and partially myelinated axons. At this early stage of development, the axons are engulfed by Schwann cells, while certain axons are segregated into a one to one relationship with myelinating cells. Patches of uniformly shaped 150- to 300-Å particles are readily distinguished against a relatively nonparticulate axonal E face. Patches of less uniform particles are found in the axonal P face, however, they are difficult to distinguish from a particulate background. Thin processes are found closely applied to the axonal membrane on the sides of a particle patch. While engulfing the axon with one or two noncompacted windings, the Schwann cell is predominantly restricted to one side of such a particle patch. As the number of windings covering the axon increases, so does the size of the particle patch, until an annulus of particles, similar to that of an adult node, is observed. The paucity of isolated particle patches in axolemma suggests that recognition and segregation of axons by Schwann cells are followed by a rapid initiation of myelination. Throughout the early periods of myelination there is evidence of endocytotic and exocytotic events at the nodal membrane associated with the appearance of 230-Å dimeric particles in the axolemma. Despite the paucity of windings and complete absence of compaction, the fracture faces of the glial and axonal membranes show linear organizations of particles. Scalloped regions in the P face of the nodal axolemma display dimeric-particle rows oriented along the scallop. These rows adopt a more circumferential orientation when the overlying glial process is wound into a paranodal location. While the spacing of dimeric-particle rows is maintained at a constant 360 Å, the number of rows per scallop necessarily decreases with compaction of the paranodal loops until a state similar to that of the adult, in which there are approximately two rows per scallop, is reached. In regions of close apposition between axon and Schwann cell, a linear arrangement of 160- and 75-Å particles in the glial fracture faces occurs prior to the appearance of tight junctions between glial loops and prior to compaction. Though the paranodes on each side of most nodes observed developed symmetrically, some asymmetric half-nodes have been observed.     

Recombination of human erythrocyte apoprotein and lipid. II. Visualization of apoprotein-lipid bilayer complex by freeze-etching

Bilayers of human erythrocyte apoprotein-lipid complexes were made by dipping a mica plate through monolayers of the complex formed at the air-water interface. Stearic acid and erythrocyte lipid alone served as controls. Freeze-fracture images of the complex at high lipid surface pressures (30 dynes/cm) showed particles (average diameter, 109 Å ± 18 Å) similar to those of erythrocyte ghosts (average diameter, 102 Å ± 19 Å). Control surfaces were smooth. We conclude that part or all of the protein molecule penetrated into the lipid bilayer and that erythrocyte apoprotein-lipid complexes yield fracture faces similar to the native erythrocyte membrane.     

Structural analysis of the isolated chloroplast coupling factor and the N,N'-dicyclohexylcarbodiimide binding proteolipid

Negative staining of purified spinach dicyclohexylcarbodiimide (DCCD) sensitive ATPase revealed a population of 110 Å subunits attached by stalks to short string-like aggregates. The interpretation of these data is that 110 Å CF₁ are attached by stalks to an aggregate of CF₀.The CF₁-CF₀ complex was incorporated into phospholipid vesicles; freezefracture analysis of this preparation revealed a homogeneous population of particles spanning the lipid bilayer; these averaged 96 Å in diameter. The DCCD binding proteolipid (apparent molecular weight 7500), an integral component of CF₀, was isolated from membranes by butanol extraction and was incorporated rated into phospholipid vesicles. Freeze-fracture analysis of the DCCD-binding proteolipid/vesicle preparation revealed a population of particles averaging 83 Å in diameter suggesting that the DCCD-binding proteolipid self-associates in lipid to form a stable complex. This complex may be required for proton transport across chloroplast membranes in vivo. The size difference between CF₀ and DCCD-proteolipid freeze-fracture particles may be related to differences in polypeptide composition of the two complexes.     

Cytochemical aspects of the nucleolar organizer in Allium cepa microspores

In Allium cepa mierospores, the nucleolar organizing region appears as an area of low density situated between two nucleolar masses. It consists of a series of zones with a density similar to that of the chromatin surrounded by areas of lower density. The dense zones are sometimes arranged in an orderly pattern of 2–4 rows. The organizing region consists of filaments, about 100 Å in diameter, which are seen to be concentrated in the dense areas, and more scattered in the rest of the region. — The alcoholic PTA staining technique reveals the presence of an appreciable quantity of arginine-Iysine rich histones in the organizing region, as well as a similarity between the dense areas of this region and the rest of the chromatin: a similarity which is also brought out by the thallium alcoholate technique, used for DNA staining. By means of uranyl-BDTA-lead RNP material can be shown to be predominantly located in the low-density areas of the organizing region in the form of fibres of about 80 Å in diameter, presumably representing the newly synthetized r-RNA (45 S RNA). A pattern is suggested for the organizing region, in which areas of functional chromatin (euchromatin) would appear alternating with areas of non-functional chromatin (heterochromatin).     

On the structure of liver ribosomes

The morphology of liver ribosomes and their subparticles, large and small, has been investigated. Analysis of the images has been carried out by successive selection of models and by X-raying them under conditions simulating negative staining. The relation between the main views has been checked by tilting the specimens in an electron microscope through ± 30 °.The small subparticle consists of an elongated body, to one of the ends of which a short “head” is attached. A model has been proposed, whose projections on rotation with respect to the longitudinal axis would satisfy all observable types of images. According to the proposed model, the “head” is tilted with respect to the elongated portion. The length of the subparticle is 230 Å. The dimensions of the elongated portion in the transverse direction are 110 Å × 75 to 80 Å.The large subparticles in lateral view resemble short “rods” 220 to 240 Å long and about 70 to 95 Å wide. At a distance of about 60 Å from the left end of the particles a projection (60 Å in length) is seen, on the inner side of which a depression, or “channel”, filled with the contrasting substance is always observed. Next to this depression a second projection is located, whose height is about 30 Å. The channel is either a cavity in the body of the large subparticle or a part of the RNA without protein. The length of the channel is about 80 Å, the width is about 50 to 60 Å. The left end of the particles is characterized by two sharpened portions; as a result, a cavity that shows an obtuse angle profile makes its appearance. The opposite end of the particles is cut off at an angle of 45 °. In another view, the subparticles appear to be almost rectangular in shape; they are characterized by dimensions of 150 Å × 220 to 240 Å. It is likely that the large projection is displaced sideways with respect to the longitudinal axis of the particles. The asymmetry associated with this displacement gives rise to preferred arrangements of the subparticles on the supporting film. An analysis has been made of the most typical images of monomeric ribosomes, on the basis of which a suggestion is made about mutual orientation of subparticles in a monomer.     

Impact of intercropping aphid-resistant wheat cultivars withoilseed rape on wheat aphid (Sitobion avenae) and its natural enemies

The effects of intercropping of wheat cultivars and oilseed rape on the densities of wheat aphid, Sitobion avenae, and their arthropod natural enemies were evaluated. Three winter wheat cultivars with different resistant levels to S. avenae were used: ‘KOK’ (high resistance), ‘Xiaobaidongmai’ (low resistance) and ‘Hongmanghong’ (susceptible). The results showed that the densities of S. avenae were significantly higher on the monoculture pattern than on either the 8-2 intercropping pattern (eight rows of wheat with two rows of oilseed rape) or the 8-4 intercropping pattern (eight rows of wheat with four rows of oilseed rape). The mean number of predators and the mummy rates of S. avenae were significantly higher in two intercropping patterns than those in the monoculture pattern. The densities of S. avenae, ladybeetles, and mummy rate of S. avenae were significantly different among different wheat cultivars. The highest densities of S. avenae and ladybeetles were found on wheat cultivar Hongmanghong. The lowest densities of S. avenae associated with high mummy rate of S. avenae were found on wheat cultivar Xiaobaidongmai. The results showed that wheat-oilseed rape intercropping conserved more predators and parasitoids than in wheat monoculture fields, and partial resistance of wheat cultivar Xiaobaidongmai had complementary or even synergistic effects on parasitoid of S. avenae.     

Ordered membrane particles in rhabdomeric microvilli of the housefly (Musca domestica L.)

Rhabdomeric microvilli of the housefly were freeze-fractured (FF) and thin sectioned (TS) for ultrastructural examination. Ordered files of closely packed membrane particles (82 Å wide, 250 Å long) were seen (FF) on the microvillar membrane (usually E face). The long axis of each particle was canted about 45° to that of the microvillus. Occasionally particles in this array appeared on the P face. It is hypothesized that ordered particles may represent either a photopigment precursor stock, a second photolabile pigment, or the newly discovered sensitizing, UV-absorbing, photostable visual pigment. In the underlying membrane leaflet (P face) were found spherical (85 Å diameter) unoriented particles in a concentration of about 6,000/μm². The size, shape and density of these structures are compatible with those of rhodopsin particles. These particles also covered the basal area of each microvillus. The findings from TS material were difficult to correlate with those from FF replicas. At high magnification the former showed that the plasma membrane of the transected microvillus is composed of spherical, hollow subunits (averaging 43 Å diameter), sometimes fused to form double, 86 Å units. These substructures were closely packed and continuous around the microvillus. This beaded plasma membrane, in rare cases, was doubled around the microvillus. In other instances the plasma membranes were continuous between neighboring microvilli. The physiological implications of these ultrastructural features are discussed.     

Freeze-fracture study of adenovirus-induced KB cell surface alterations.

A technique of destabilization of KB cell plasma membrane by treatment with hypotonic citrate buffer was employed. The technique did not alter the viability or ability of the cells to synthesize macromolecules or produce virus, but did permit the visualization, by the freeze-fracture technique, of plasma membrane modifications occurring in response to adenovirus adsorption. The modifications consisted of a rearrangement of the membrane particles (MPs) on both protoplasmic and external leaflets of the plasma membrane. The rearrangement delineated bare areas, 139 nm in mean diameter, devoid of MPs and protruding outwards. The membrane changes were transient and were only observed when KB cells were maintained with adenovirus particles at 0 °C. The changes disappeared rapidly upon warming to 37 °C, reforming the ‘random pattern’ of MPs, normally visible on the cell plasma membrane. The same type of study was carried out with purified adenovirus capsid components (hexon, penton, penton base and fiber), with smaller virus particles (poliovirus) and with larger ‘artificial’ adenovirus particles made of latex beads coated with adenovirus pentons. The dimensions of the bare regions devoid of MPs appeared to be related to the size of the particles used, suggesting the existence of a ‘recognition pattern’ specific for virus particles.     

Evolutionary conservation and structural localizations suggest a physical trace of metabolism’s progressive geochronological emergence

Abstract

We studied multiple sequence alignment (MSA) consensus amino acid distributional patterns in 2844 amino acid sequences of the eight enzymes of the Kreb’s oxidative tricarboxylic acid pathway (oTCA) in Archaea, Bacteria and Eukarya and 5545 sequences of 33 bacteria as geochronologically separated enzymes with MSA consensus site modal identities. The 33 bacteria were 20 presumptive examples of early-oldest (Hadean-Archaean) (‘Epoch I’) or 13 late-newest (contemporary) (‘Epoch III’) appearing enzymes on Earth. The enzyme’s MSA consensus sites were identified by their modal identity, % Occupancy in one of nine-graded evolutionary-conservation zones (CZs) and the Euclidean distance (Å) from each of their consensus MSA Cɑs to the same atom (Anchor-atom) in their reported functional center. These MSA consensus sites are tetrad-data points called recovered-amino acids (RAA). Across Domains, the % Occupancies of the eight-dominant RAAs of the Kreb’s cycle and the 33 bacteria were found to be similarly ranked. Compared to Trifonov’s ‘putative ranked temporal order of the appearance of amino acids on Earth’ (TOAE), the greatest statistical concordance with tetrad-RAAs across Domains were those characterized as within the most-evolutionary conserved conservation zone (CZ9), typically nearest (Å) their enzyme’s catalytic/active center. The geochronologically characterized early-oldest Hadean-Archaean Bacteria ‘Epoch I’ enzymes, compared to late-newest Bacteria enzymes, had greater average numbers of amino acid residues/sequence and a statistically significant larger variability in their RAA compositional-ų-volumes. The late-newest ‘Epoch III’ enzymes had statistically significant lower volumetric values, specifically, their native ų-volume, void-volume and volume change on unfolding. Our enzyme data suggest a geochronological trace of ‘metabolism’s progressive emergence’.

Communicated by Ramaswamy H. Sarma     

The plasmalemmal vesicular system in striated muscle capillaries and in pericytes

By ultrathin serial sectioning of frog mesenteric capillaries it was recently demonstrated that the many apparently free vesicles in electron microscope (EM) sections of endothelial cells may be artefacts due to conventional (500–700 Å thick) sectioning (Frøkjaer-Jensen, 1980). The vesicles were found to be part of two sets of invaginations of the cell surfaces; one set connected to the lumen, the other to the interstitium. The present study extends this view to comprise the vesicle organization in frog striated muscle capillaries. By analysis of the three-dimensional organization of the plasmalemmal vesicles in 21 ultrathin serial sections (120–150 Å) of two muscle capillaries it is demonstrated that less than 1% of the about 70% apparently free vesicles seen in conventional thin sections of the same capillaries in fact represent truly free vesicular units. By analysis of 15 conventional EM cross-sections of capillaries from the frog cutaneous-pectoris muscle containing plasmaproteins in high concentration it is furthermore demonstrated that 48% of the total vesicle population connect to the lumen at the time of fixation. This organization of the vesicular system seems incompatible with the concept that macromolecules are transferred across the capillary wall by vesicular transport or by a series of fusions and fissions between individual cytoplasmic vesicles but is compatible with the notion that macromolecules exchange across capillary walls by means of passive processes such as diffusion and convection through rare ‘large pores’. The study emphasizes that any attempts to classify vesicles in conventional thin sections as ‘luminal’, ‘cytoplasmic’ and ‘abluminal’ is impossible and may lead to erroneous interpretations of vesicle involvement in transcapillary exchange of macromolecules. The rare occurrence of transendothelial channels compared to the number of vesicle invaginations suggests that the main function of the vesicular system relates to functions other than transport.     

Contribution of aqueous interphases to the permeability barrier of lipid bilayers for non-electrolytes

Equilibrium dialysis experiments are used to measure excluded volumes for the non-electrolyte permeant [U-¹⁴C] erythritol in lipid bilayer systems. The data indicate amounts of water associated with the lipid membranes which correspond with amounts calculated from calorimetric measurements.The membrane systems can be described as composite elements consisting of the lipid bilayers and adjacent water layers on both sides. The finding that the permeant is excluded indicates that the water layers contribute to the permeability barrier.The mean thickness of the water layers is about 6 Å for planar bilayers in multilayered liposomes and 10 Å for curved bilayers in sonicated vesicles. Next to the difference in thickness of the water layers differences in interfacial adsorption between the two systems are apparent.     

A jumping spider that lays eggs like an insect

The South East Asian salticid, Asemonea tenuipes, lay its eggs like an insect: as individuated capsules in geometrically precise rows. The occurrence of this behaviour is described in a population from Thailand that lays eggs on the underside of mango tree (Mangifera indica) leaves. The rows were vertically arranged at a 90° angle that was parallel to, and varied in relation to the angle of the central leaf rib, indicating that the rib serves as the primary visual cue for orienting the row. Analysis of within-egg row symmetry found evidence that micro-topography – in particular, vertical ‘stepping’ produced by leaf sub-ribs – of the leaf surface plane was responsible for 97% of variability in the relative symmetry of egg placement. Three strategies were identified: (1) crossing the sub-rib and producing localized asymmetry in the egg row; (2) introducing a gap at the sub-rib to maintain symmetry; and (3) ‘compressing’ the egg row into the gap between sub-ribs. The incidence of asymmetry showed a highly significant linear relationship with egg load. It is hypothesized that the use of rows probably represents a solution to the problem of how to surveil temporally staggered clutches of eggs and newly hatched spiderlings simultaneously.     

Divalent cations cooperatively stabilize close membrane contacts in myelin

Intact nerve myelin compacts to a dehydrated structure of closely apposed membranes when exposed to isotonic solutions at least 10 mM in calcium or tetracaine. The repeat period of the membrane pair in the compacted structure measured by X-ray diffraction is about 126 Å in both central and peripheral mammalian nerve myelins whereas the normal periods are about 158 and 178 Å, respectively. The electron density profile of compacted myelin shows an asymmetric membrane unit with thickness similar to that of the symmetric bilayer of flocculated myelin lipids. The centrosymmetrically averaged myelin membrane profile is similar to that of the lipid bilayer except at the surface where residual protein is concentrated. Dispersions of extracted total myelin lipids flocculate under similar conditions to those causing myelin compaction, indicating that similar forces act in both processes. Compaction is always accompanied by lateral segregation of intramembrane particles out of the close-packed domains. Lateral displacement of intramembrane proteins from compacted domains can be driven by the attraction of the lipid surfaces for each other. Rates of compaction vary with compacting reagent, concentration, tissue, and temperature, and probably reflect the permeability of the tissue. Extensive compaction by calcium or tetracaine leads to disruption and vesiculation of the spirally wrapped myelin membranes.     

Simulation of grana stacking in a model membrane system. Mediation by a purified light-harvesting pigment-protein complex from chloroplasts

An isolated light-harvesting pigment-protein complex contains polypeptides which bind chlorophyll a and b. The individual complexes can be purified from detergent-solubilized membranes. The isolated light-harvesting complex, when dialyzed to remove detergents, was examined by freeze-fracture electron microscopy. The material consisted of planar sheets of 80-Å subunits which interacted via an edge-to-edge contact. Addition of cations caused the planar light-harvesting complex sheets to become tightly appressed in multilamellar stacks, with distinct subunits still visible within each lamellar sheet. A transition of particle organization from random to crystalline occurred in parallel with the cation-induced lamellar association. Treatment of the dialyzed light-harvesting complex subunits with low levels of the proteolytic enzyme trypsin removed a 2000 molecular weight segment of the major polypeptide of the light-harvesting complex and blocked all subsequent cation-induced changes in structural organization of the isolated light-harvesting complex lamellar sheets.To gain further evidence for mechanisms of cation effects upon the organization of the light-harvesting complex in native membranes, the light-harvesting complex was incorporated into uncharged (phosphatidylcholine) lipid vesicles. The protein complexes spanned the lipid bilayer and were arranged in either a random pattern or in hexagonal crystalline lattices. Addition of either monovalent or divalent cations to ‘low-salt’ (20 mM monovalent cation) vesicles containing light-harvesting complex caused extensive regions of membrane appression to appear. It is concluded that this cation-induced membrane appression is mediated by surface-exposed segments of the light-harvesting complex since (a) phosphatidylcholine vesicles themselves did not undergo cation-induced aggregation, and (b) mild trypsin digestion of the surface-exposed regions of the light-harvesting complex blocked cation-induced lamellar appression. The particles in the appressed vesicle membranes tended to form long, linear arrays of particles, with occasional mixed quasi-crystalline arrays with an angular displacement near 72°. Surface-mediated interactions among light-harvesting complex subunits of different membranes are, therefore, related to changes in structural organization and interaction of the particles within the lipid phase of the membrane.Numerous previous studies have implicated the involvement of the light-harvesting complex in mediating grana stocking in intact chloroplast membranes. The data presented herein provide a simulation of the membrane appression phenomena using a single class of chloroplast-derived membrane subunits. The data demonstrate that specific surface-localized regions of the light-harvesting complex are involved in membrane-membrane interactions.     

Ultrastructural Observations on the Spermatozoa of Two Temnocephalids (Platyhelminthes)

The spermatozoa of two Temnocephalidae collected in Uruguay, Temnocephala iheringi Haswell, 1893 (Host: Pomacea canaliculata) and Temnocephala axenos Monticelli, 1899 (Host: Parastacus varicosus), were studied with a transmission electron microscope. In both species the spermatozoon is made up of a long sperm body which bears at one extremity two free flagella of the 9+‘1’ flatworm pattern. The sperm body contains the nucleus, mitochondria, dense bodies and parallel, cortical, longitudinal singlet microtubules. Along a part of the sperm body the palissade of the microtubules displays a spiral pattern in transverse sections. A part of the perimeter of the cell is thus lined by two overlapping rows of microtubules. This spiral pattern of the singlets is considered as a synapomorphy of the family Temnocephalidae. The singlet microtubules are interconnected by two kinds of links: tangential links between neighbouring singlets in the same row and radial links between singlets belonging to two rows. The presence of these links suggests that this structure could be a motile system of singlets.