Flagellar Length Research Articles

Defects in the ratio of the dynein isoform, DHC11 in the long-flagella mutants of Chlamydomonas reinhardtii

The long-flagella mutants (lf1, lf2, lf3 and lf4) of Chlamydomonas reinhardtii are defective in proteins that are required for the assembly of normal flagella, their phenotype being long flagella. In a previous study, we biophysically characterized these mutants for their waveform patterns, swimming speeds, beat frequencies and correlated these parameters with their flagellar lengths. We found an anomaly in this correlation and set out to explore the underlying molecular significance, if any. The diverse inner dynein isoforms are the flagellar motors that convert the chemical energy of ATP into the mechanical energy of motility; we probed the presence of one of these isoforms (DHC11, which might help in bend initiation) in the lf mutants and compared it with the wild-type. Our studies show that the ratio of DHC11 is defective in the long-flagella mutants of Chlamydomonas reinhardtii.

The relationships of Stichaeus nozawae (Jordan et Snyder, 1902) and Stichaeus grigorievi (Herzenstein, 1890) (Pisces: Stichaeidae) inferred from the data of genetic and karyological analyses and ultrastructural study of spermatozoa

A complex study, including genetic and karyological approaches, as well as an analysis of the ultrastructural organization of spermatozoa of the Nozawa’s prickleback Stichaeus nozawae (Jordan et Snyder, 1902) and Grigorev’s prickleback S. grigorievi (Herzenstein, 1890) from Vostok Bay, Sea of Japan, is considered in the present work. The species exhibit differences in the spermatozoon head width and flagellum length. These characters are species-specific and, along with morphological traits, can be used as a proof of the validity of the species under study. The similarity between S. nozawae and S. grigorievi, as established based on genetic and karyological analyses (2n = 48, NF = 70, localization of nucleolar organizers), does not correspond to inter-species differences in morphological and biological characters. The lack of variations in DNA and the chromosome set that are suitable for species differentiation, between Nozawa’s and Grigorev’s pricklebacks is an evidence for the evolutionary adolescence of these species.

Elastohydrodynamic Synchronization of Adjacent Beating Flagella.

It is now well established that nearby beating pairs of eukaryotic flagella or cilia typically synchronize in phase. A substantial body of evidence supports the hypothesis that hydrodynamic coupling between the active filaments, combined with waveform compliance, provides a robust mechanism for synchrony. This elastohydrodynamic mechanism has been incorporated into 'bead-spring' models in which the beating flagella are represented by microspheres tethered by radial springs as they are driven about orbits by internal forces. While these low-dimensional models reproduce the phenomenon of synchrony, their parameters are not readily relatable to those of the filaments they represent. More realistic models which reflect the underlying elasticity of the axonemes and the active force generation, take the form of fourth-order nonlinear PDEs. While computational studies have shown the occurrence of synchrony, the effects of hydrodynamic coupling between nearby filaments governed by such continuum models have been theoretically examined only in the regime of interflagellar distances d large compared to flagellar length L. Yet, in many biological situations d/L ≪ 1. Here, we first present an asymptotic analysis of the hydrodynamic coupling between two extended filaments in the regime d/L ≪ 1, and find that the form of the coupling is independent of the microscopic details of the internal forces that govern the motion of the individual filaments. The analysis is analogous to that yielding the localized induction approximation for vortex filament motion, extended to the case of mutual induction. In order to understand how the elastohydrodynamic coupling mechanism leads to synchrony of extended objects, we introduce a heuristic model of flagellar beating. The model takes the form of a single fourth-order nonlinear PDE whose form is derived from symmetry considerations, the physics of elasticity, and the overdamped nature of the dynamics. Analytical and numerical studies of this model illustrate how synchrony between a pair of filaments is achieved through the asymptotic coupling.

Male reproductive system and sperm ultrastructure of Furcatopanorpa longihypovalva (Hua and Cai, 2009) (Mecoptera: Panorpidae) and its phylogenetic implication

The male reproductive system and sperm ultrastructure of the scorpionfly Furcatopanorpa longihypovalva (Hua & Cai) were investigated using light and transmission electron microscopy. A peculiar feature of the male reproductive system is the position of the epididymis, separately from the base of testis within the peritoneal sheath, unlike the species of other genera of Panorpidae pressed against the lateral base of testis. The mature spermatozoon is filiform, approximately 1600μm in length. It has largely maintained a mecopteran groundplan condition, with a bilayered acrosome, an elongate nucleus, a neck region, and a long flagellum. As for the sperm features, similarly to other species of Panorpidae, the nucleus has two lateral grooves and a helical appearance. The neck region shows a sheath-shaped centriolar adjunct, beneath the nucleus. The flagellum is helical and consists of a 9+2 axoneme, two mitochondrial derivatives of unequal size, one accessory body and two extra-axonemal accessory structures, as in the genus Panorpa of Panorpidae. The uniqueness of spermatozoa of F. longihypovalva lies in the specifically hammer-shaped mitochondrial derivative along most of the flagellum length. The phylogenetic position of the genus Furcatopanorpa in Panorpidae is briefly discussed based on our findings and literature in this respect.

Chemotaxis in the anisogamous brown alga Mutimo cylindricus (Cutleriaceae, Tilopteridales)

:Locomotion of female and male gametes of Mutimo cylindricus was analysed using a high-speed recording system. Female and male gametes morphologically differed in terms of their cell size and flagellar length. Male gametes swam significantly faster than female gametes; although, the flagellar waveforms were almost the same. Thus, the different swimming velocities of male and female gametes may have been due to differences in their cell size. Chemotactic male gametes treated with the sex pheromone from settled female gametes behaved in a similar manner to the male gametes of the isogamous Ectocarpus siliculosus. The results of the study revealed the following: (1) free-swimming male gametes exhibited thigmotaxis, (2) the beat frequency of the anterior flagellum and the swimming velocity decreased, (3) the deflection angle of the anterior flagellum increased, (4) the unilateral beat of the posterior flagellum was affected by the sex pheromone gradient, and (5) the tracking radius of male gametes around female gametes decreased.

Morphology of gametes, post-fertilization events and the effect of temperature on the embryonic development of Astyanax altiparanae (Teleostei, Characidae).

The aim of this study was to describe the morphology of gametes, post-fertilization events and subsequent temperature effects on the early developmental stages of the neotropical species Astyanax altiparanae. The sperm of this species presents a typical morphology of teleost sperm with a spherical head (diameter = 1.88 µm), midpiece (diameter = 0.75 µm) and a single flagellum (length = 18.67 µm). The extrusion of the second polar body and fusion of male and female pronucleus were reported for the first time in this species. Additionally, we observed the formation of the fertilization cone, which prevents polyspermic fertilization. Developmental stages at 22°C, 26°C and 30°C gave rise to fertilization rates at 91.12, 91.42 and 93.04% respectively. Hatching occurred at 25 hpf at 22°C, 16 hpf at 26°C and 11 hpf at 30°C and the hatching rates were 61.78%, 62.90% and 59.45%, respectively. At 22°C, the second polar body was extruded at ≈6 mpf and the male and female pronucleus fused at ≈10 mpf. This fundamental information is important for the field and opens up new possibilities in fish biotechnology, including micromanipulation and chromosome-set manipulation.

Greater sperm complexity in the Australasian old endemic rodents (Tribe: Hydromyini) is associated with increased levels of inter-male sperm competition.

Spermatozoa exhibit considerable interspecies morphological variation across mammals, especially among murid rodents. In Australasia, most murids in the tribe Hydromyini have a spermatozoon with a highly complex head exhibiting an apical hook, characteristic of most murids, and two projections that extend from its upper concave surface, the ventral processes. In the present study we performed a phylogenetically controlled comparison of sperm morphology across 45 species of hydromyine rodents to test the hypothesis that the length and angle of both the apical hook and ventral processes, as well as the length of the sperm tail, increase with relative testes mass as a proxy for differences in levels of inter-male sperm competition. Although both sperm head protrusions exhibited considerable variation in their length and angle across species, only the angles increased significantly in relation to relative testes mass. Further, the length of the sperm flagellum was positively associated with relative testes mass. These results suggest that, in hydromyine rodents, the angle of the apical hook and ventral processes of the sperm head, as well as the sperm tail length, are likely to be sexually selected traits. The possible functional significance of these findings is briefly discussed.

Extensive variation in sperm morphology in a frog with no sperm competition.

BackgroundRecent comparative studies of several taxa have found that within-species variation in sperm size decreases with increasing levels of sperm competition, suggesting that male-male gamete competition selects for an optimal sperm phenotype. Previous studies of intraspecific sperm length variation have all involved internal fertilizers where some other factors—e.g., sperm storage and sperm movement along the walls of the female’s reproductive tract—probably also influence and reduce sperm size variation. Thus external fertilizers, where those factors are absent, might be expected to exhibit even more variation when there is little or no sperm competition. To test that idea, we studied the sperm morphology of a North American chorus frog, the spring peeper (Pseudacris crucifer), a species in which males encounter little or no sperm competition.ResultsAs expected, sperm size was highly variable in the spring peeper, largely due to variation in flagellum length within and among individual males, among populations and between mitochondrial lineages in southwestern Ontario. In addition, a large proportion of spermatozoa in all males was abnormal in such a way that the ability of abnormal spermatozoa to fertilize was probably compromised. There were no differences in the frequencies of abnormalities among populations or mitochondrial lineages.ConclusionsIn the absence of sperm competition, we suggest that genetic drift has probably played a role in the generation of diversity in sperm morphology in this species, potentially resulting in the observed differences among populations. Such interpopulation difference in sperm morphology might be expected to increase the degree of reproductive isolation between populations even before other isolating mechanisms evolve.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0601-8) contains supplementary material, which is available to authorized users.

A NIMA-related kinase, CNK4, regulates ciliary stability and length.

NIMA-related kinases (Nrks or Neks) have emerged as key regulators of ciliogenesis. In human, mutations in Nek1 and Nek8 cause cilia-related disorders. The ciliary functions of Nrks are mostly revealed by genetic studies; however, the underlying mechanisms are not well understood. Here we show that a Chlamydomonas Nrk, CNK4, regulates ciliary stability and length. CNK4 is localized to the basal body region and the flagella. The cnk4-null mutant exhibited long flagella, with formation of flagellar bulges. The flagella gradually became curled at the bulge formation site, leading to flagellar loss. Electron microscopy shows that the curled flagella involved curling and degeneration of axonemal microtubules. cnk4 mutation resulted in flagellar increases of IFT trains, as well as its accumulation at the flagellar bulges. IFT speeds were not affected, however, IFT trains frequently stalled, leading to reduced IFT frequencies. These data are consistent with a model in which CNK4 regulates microtubule dynamics and IFT to control flagellar stability and length.

Together, the IFT81 and IFT74 N-termini form the main module for intraflagellar transport of tubulin.

The assembly and maintenance of most cilia and flagella rely on intraflagellar transport (IFT). Recent in vitro studies have suggested that, together, the calponin-homology domain within the IFT81 N-terminus and the highly basic N-terminus of IFT74 form a module for IFT of tubulin. By using Chlamydomonas mutants for IFT81 and IFT74, we tested this hypothesis in vivo. Modification of the predicted tubulin-binding residues in IFT81 did not significantly affect basic anterograde IFT and length of steady-state flagella but slowed down flagellar regeneration, a phenotype similar to that seen in a strain that lacks the IFT74 N-terminus. In both mutants, the frequency of tubulin transport by IFT was greatly reduced. A double mutant that combined the modifications to IFT81 and IFT74 was able to form only very short flagella. These results indicate that, together, the IFT81 and IFT74 N-termini are crucial for flagellar assembly, and are likely to function as the main module for IFT of tubulin.

Intraflagellar transport is required for the maintenance of the trypanosome flagellum composition but not its length.

Intraflagellar transport (IFT) is required for construction of most cilia and flagella. Here, we used electron microscopy, immunofluorescence and live video microscopy to show that IFT is absent or arrested in the mature flagellum of Trypanosoma brucei upon RNA interference (RNAi)-mediated knockdown of IFT88 and IFT140, respectively. Flagella assembled prior to RNAi did not shorten, showing that IFT is not essential for the maintenance of flagella length. Although the ultrastructure of the axoneme was not visibly affected, flagellar beating was strongly reduced and the distribution of several flagellar components was drastically modified. The R subunit of the protein kinase A was no longer concentrated in the flagellum but was largely found in the cell body whereas the kinesin 9B motor was accumulating at the distal tip of the flagellum. In contrast, the distal tip protein FLAM8 was dispersed along the flagellum. This reveals that IFT also functions in maintaining the distribution of some flagellar proteins after construction of the organelle is completed.

The Importance of Ejaculate Volume for the Physical Parameters of Ejaculates and Sperm Morphology of Hypor Boars

The study aimed at analysing the effects of ejaculate volume on the physical parameters of ejaculates and the sperm morphology of Hypor boars. The analyses involved 114 ejaculates collected from 12 Hypor insemination boars. The ejaculates were classified according to the criterion of ejaculate volume. Three groups were specified: ejaculates with a volume of 251 ml or lower (Group I), ejaculates with a volume of 252-310 ml (Group II), and ejaculates with a volume of 311 ml or higher (Group III). The ejaculates were assessed to identify the basic physical traits and determine the incidence of morphological abnormalities in the spermatozoa, specifying major and minor abnormalities. Furthermore, the morphological structure indices for the spermatozoa were also calculated. Rising ejaculate volume accompanied with a rise in the total number and motility of spermatozoa, and a simultaneous slight fall in sperm concentration in the ejaculates. The ejaculates with the highest volumes turned out to contain more morphologically well-formed spermatozoa. We also determined that rising ejaculate volume is accompanied with increasing sperm dimensions, especially those of the head. The increased parameters were the length and the width of sperm heads, as well as their perimeters and areas. Ejaculate volume has an impact on the shape of Hypor boar spermatozoa. As the ejaculate volume increases, the shape of sperm heads becomes increasingly more oval. Additionally, spermatozoa in ejaculates with greater volumes have larger heads in relation to flagellum length.

Basal body multipotency and axonemal remodelling are two pathways to a 9+0 flagellum.

Eukaryotic cilia/flagella exhibit two characteristic ultrastructures reflecting two main functions; a 9+2 axoneme for motility and a 9+0 axoneme for sensation and signalling. Whether, and if so how, they interconvert is unclear. Here we analyse flagellum length, structure and molecular composition changes in the unicellular eukaryotic parasite Leishmania during the transformation of a life cycle stage with a 9+2 axoneme (the promastigote) to one with a 9+0 axoneme (the amastigote). We show 9+0 axonemes can be generated by two pathways: by de novo formation and by restructuring of existing 9+2 axonemes associated with decreased intraflagellar transport. Furthermore, pro-basal bodies formed under conditions conducive for 9+2 axoneme formation can form a 9+0 axoneme de novo. We conclude that pro-centrioles/pro-basal bodies are multipotent and not committed to form either a 9+2 or 9+0 axoneme. In an alternative pathway structures can also be removed from existing 9+2 axonemes to convert them to 9+0.

Sperm traits in farmed and wild Atlantic salmonSalmo salar

Differences in sperm metabolism and morphology between wild and non-local farmed Atlantic salmon Salmo salar were assessed by measuring metabolic enzyme activities and length of sperm flagella. No differences were observed between wild and farmed S. salar sperm with regards to cell counts or any of the biochemical variables assessed. Flagella of sperm cells were significantly longer in wild than farmed S. salar; however, this did not result in higher energy levels or different fertilization rates.

Comparative analysis of gamete ultrastructure in big-scaled redfin Tribolodon hakonensis (Cyprinidae) from Southern Primorye and Sakhalin

Considerable differences in the structure of gametes of big-scaled redfin Tribolodon hakonensis from Southern Primorye (southern form) and rivers of the Sea of Okhotsk basin (northern form) are found. In spermatozoa of the southern form of T. hakonensis, the mitochondrial apparatus includes up to five mitochondria, and flagellum length is, on average, 36.2 μm. In spermatozoa of the northern form, up to three mitochondria are observed, and flagellum length is, on average, 42.3 μm. In oocytes of the southern form, the zona radiata has well-expressed striation, and radial canals are dense and large. Oocytes of the northern form are characterized by a poor expressed striation, and radial canals are rarely located and small. Observed differences confirm the specificity of gametes in the southern and northern forms of T. hakonensis, and they can be used as additional phylogenetic traits.

Effect of aliphatic, monocarboxylic, dicarboxylic, heterocyclic and sulphur-containing amino acids on Leishmania spp. chemotaxis.

In the sand-fly mid gut, Leishmania promastigotes are exposed to acute changes in nutrients, e.g. amino acids (AAs). These metabolites are the main energy sources for the parasite, crucial for its differentiation and motility. We analysed the migratory behaviour and morphological changes produced by aliphatic, monocarboxylic, dicarboxylic, heterocyclic and sulphur-containing AAs in Leishmania amazonensis and Leishmania braziliensis and demonstrated that L-methionine (10-12 m), L-tryptophan (10-11 m), L-glutamine and L-glutamic acid (10-6 m), induced positive chemotactic responses, while L-alanine (10-7 m), L-methionine (10-11 and 10-7 m), L-tryptophan (10-11 m), L-glutamine (10-12 m) and L-glutamic acid (10-9 m) induced negative chemotactic responses. L-proline and L-cysteine did not change the migratory potential of Leishmania. The flagellum length of L. braziliensis, but not of L. amazonensis, decreased when incubated in hyperosmotic conditions. However, chemo-repellent concentrations of L-alanine (Hypo-/hyper-osmotic conditions) and L-glutamic acid (hypo-osmotic conditions) decreased L. braziliensis flagellum length and L-methionine (10-11 m, hypo-/hyper-osmotic conditions) decreased L. amazonensis flagellum length. This chemotactic responsiveness suggests that Leishmania discriminate between slight concentration differences of small and structurally closely related molecules and indicates that besides their metabolic effects, AAs play key roles linked to sensory mechanisms that might determine the parasite's behaviour.

A NIMA-Related Kinase Suppresses the Flagellar Instability Associated with the Loss of Multiple Axonemal Structures.

CCDC39 and CCDC40 were first identified as causative mutations in primary ciliary dyskinesia patients; cilia from patients show disorganized microtubules, and they are missing both N-DRC and inner dynein arms proteins. In Chlamydomonas, we used immunoblots and microtubule sliding assays to show that mutants in CCDC40 (PF7) and CCDC39 (PF8) fail to assemble N-DRC, several inner dynein arms, tektin, and CCDC39. Enrichment screens for suppression of pf7; pf8 cells led to the isolation of five independent extragenic suppressors defined by four different mutations in a NIMA-related kinase, CNK11. These alleles partially rescue the flagellar length defect, but not the motility defect. The suppressor does not restore the missing N-DRC and inner dynein arm proteins. In addition, the cnk11 mutations partially suppress the short flagella phenotype of N-DRC and axonemal dynein mutants, but do not suppress the motility defects. The tpg1 mutation in TTLL9, a tubulin polyglutamylase, partially suppresses the length phenotype in the same axonemal dynein mutants. In contrast to cnk11, tpg1 does not suppress the short flagella phenotype of pf7. The polyglutamylated tubulin in the proximal region that remains in the tpg1 mutant is reduced further in the pf7; tpg1 double mutant by immunofluorescence. CCDC40, which is needed for docking multiple other axonemal complexes, is needed for tubulin polyglutamylation in the proximal end of the flagella. The CCDC39 and CCDC40 proteins are likely to be involved in recruiting another tubulin glutamylase(s) to the flagella. Another difference between cnk11-1 and tpg1 mutants is that cnk11-1 cells show a faster turnover rate of tubulin at the flagellar tip than in wild-type flagella and tpg1 flagella show a slower rate. The double mutant shows a turnover rate similar to tpg1, which suggests the faster turnover rate in cnk11-1 flagella requires polyglutamylation. Thus, we hypothesize that many short flagella mutants in Chlamydomonas have increased instability of axonemal microtubules. Both CNK11 and tubulin polyglutamylation play roles in regulating the stability of axonemal microtubules.

Recruitment of IFT proteins during flagellum construction in Trypanosoma brucei

Trypanosoma brucei is a protozoan parasite responsible for sleeping sickness in Central Africa. It is transmitted by the bite of the tsetse fly. During its complex life cycle, it undergoes significant morphological changes including extensive variations in flagellum length (3 to 30 µm). The flagellum is an essential organelle for parasite survival as it is involved in morphogenesis, movement, division and adhesion of the trypanosome. Intraflagellar transport (IFT) refers to the movement of protein complexes between the membrane and the microtubules. Like in other eukaryotes, IFT is essential for the construction of the trypanosome flagellum. Studies in the green alga Chlamydomonas suggested that the total amount of IFT proteins injected in the flagellum defines its final length: the higher the amount, the longer the flagellum. Moreover, the total amount of IFT proteins would be injected at once.

Mutations in the Borrelia burgdorferi Flagellar Type III Secretion System Genes fliH and fliI Profoundly Affect Spirochete Flagellar Assembly, Morphology, Motility, Structure, and Cell Division.

ABSTRACTThe Lyme disease spirochete Borrelia burgdorferi migrates to distant sites in the tick vectors and mammalian hosts through robust motility and chemotaxis activities. FliH and FliI are two cytoplasmic proteins that play important roles in the type III secretion system (T3SS)-mediated export and assembly of flagellar structural proteins. However, detailed analyses of the roles of FliH and FliI in B. burgdorferi have not been reported. In this study, fliH and fliI transposon mutants were utilized to dissect the mechanism of the Borrelia type III secretion system. The fliH and fliI mutants exhibited rod-shaped or string-like morphology, greatly reduced motility, division defects (resulting in elongated organisms with incomplete division points), and noninfectivity in mice by needle inoculation. Mutants in fliH and fliI were incapable of translational motion in 1% methylcellulose or soft agar. Inactivation of either fliH or fliI resulted in the loss of the FliH-FliI complex from otherwise intact flagellar motors, as determined by cryo-electron tomography (cryo-ET). Flagellar assemblies were still present in the mutant cells, albeit in lower numbers than in wild-type cells and with truncated flagella. Genetic complementation of fliH and fliI mutants in trans restored their wild-type morphology, motility, and flagellar motor structure; however, full-length flagella and infectivity were not recovered in these complemented mutants. Based on these results, disruption of either fliH or fliI in B. burgdorferi results in a severe defect in flagellar structure and function and cell division but does not completely block the export and assembly of flagellar hook and filament proteins.

Morphometry of boar sperm head and flagellum in semen backflow after insemination

Once deposited in the female reproductive system, sperm begin their competition and undergo a selection to reach the site of fertilization. Little is known about the special characteristics of sperm that reach the oviduct and are able to fertilize, with even less information on the role of sperm dimension and shape in transport and fertilization. Here, we examine whether sperm morphometry could be involved in their journey within the uterus. For this purpose, sperm head dimension (length, width, area, and perimeter) and shape (shape factor, ellipticity, elongation, and regularity), and flagellum length were analyzed in the backflow at different times after insemination (0–15, 16–30, and 31–60 minutes). Sperm morphometry in the backflow was also analyzed taking into account the site of semen deposition (cervical vs. intrauterine). Finally, flagellum length was measured at the uterotubal junction. Sperm analyzed in the backflow were small (head and flagellum) with different head shapes compared with sperm observed in the dose before insemination. The site of deposition influenced head morphometry and tail size both being smaller in the backflow after cervical insemination compared with intrauterine insemination. Mean tail length of sperm collected in the backflow was smaller than that in the insemination dose and at the uterotubal junction. Overall, our results suggest that sperm size may be involved in sperm transport either because of environment or through sperm selection and competence on their way to encounter the female gamete.