The sperm structure of the gall-midges Anaretella and Lestremia (Insecta, Diptera, Cecidomyiidae)

The sperm structure of some dipteran flies belonging to the Lestremiini tribe have been examined. Anaretella cincta was shown to have an axoneme made of 20-21 microtubular doublets, disposed in a circle in a cross section and surrounding a mitochondrion. Other crystal-containing mitochondria flank the axoneme; a second species (Anaretella sp.) was provided with 21-22 axonemal doublets. Lestremia is characterized by a flattened axoneme, consisting of about 150 doublets arranged in 2 antiparallel rows and surrounding a few mitochondria. These mitochondria, in Lestremia sp., have a crystalline core that is missing in Lestremia cinerea. The structure of microtubular doublets is quite similar in the 2 related genera and a derivation of the flattened axoneme found in Lestremia from that circular of Anaretella is suggested. Sperm structure suggests that Lestremia cinerea is not a uniform species.     

Flagellar Axonemes with 10 Microtubular Doublets in Spermatozoa from Gall-midges (Diptera,Cecidomyiidae)

Abstract The spermatozoa of some gall-midges (Cecidomyiidae, Lestremiinae), belonging to the tribe Micromyini, were seen to have an axoneme that consists of 10, instead of nine, microtubular doublets surrounding a central cylinder. In some related species within the same tribe the axoneme was found to contain a similar cylinder but to have nine doublets, as in typical flagella, or to have nine doublets and no central structure. These three types of axonemes can be given the shorthand designations “10+cyl”,“9+cyl”, and “9+0”. The tribe Lestremiini is characterized by a giant axoneme having 150 doublets in two rows reversely oriented. Other characteristics of examined spermatozoa are the electron density of the B-tubules of the axoneme, a feature shared by all members of the subfamily Lestremiinae, and the presence of a prominent cytoplasmic droplet containing numerous, regularly spaced microtubules, which is shared by all Micromyidi. These axonemal models are discussed from a phylogenetic point of view.     

Geometry drives the "deviated-bending" of the bi-tubular structures of the 9 + 2 axoneme in the flagellum

The axoneme "9 + 2" is basically a system constituted of a cylinder of 9 microtubule doublets surrounding a central pair of microtubules. These bi-tubular structures are considered as the support system of the active molecular complexes that generate and regulate the axonemal movement. Schoutens has calculated their moments of inertia [Schoutens, 1994: Journal of Theoretical Biology 171:163-177]. The results obtained allowed us to assume that these bi-tubular systems are endowed with dynamic properties that could be involved in the regulation of the axonemal machinery. For the first time, using the finite elements methods and the resistance of material principles, we have now calculated that the curvature of the axoneme induces the deviated-bending of the bi-tubular structures of the axoneme, because of their geometry only; they behave as beams in a framework. This approach is similar to the one used to measure the deflection of a single microtubule [Kasas et al., 2004: Chem Phys Chem 5:252-257]. These behaviors induce internal movement or constraints of either couples or triplets of doublets within the axonemal cylinder that could be directly involved in a constrained or a spontaneous "convergence/divergence" equilibrium of the cylindrical generatrices that they draw along the axonemal cylinder, which could apparently regulate the activity of the axonemal motors (the dynein arms). These results are discussed here, taking into consideration the dynamic propagation of the wave train along the flagellar axoneme, and the regulated balance between the activities of the two opposite sides of the axoneme during the beat. This study raises a few questions about the architecture-activity duo of the axonemal doublets.     

Heterogeneity of dynein structure implies coordinated suppression of dynein motor activity in the axoneme

Axonemal dyneins provide the driving force for flagellar/ciliary bending. Nucleotide-induced conformational changes of flagellar dynein have been found both in vitro and in situ by electron microscopy, and in situ studies demonstrated the coexistence of at least two conformations in axonemes in the presence of nucleotides (the apo and the nucleotide-bound forms). The distribution of the two forms suggested cooperativity between adjacent dyneins on axonemal microtubule doublets. Although the mechanism of such cooperativity is unknown it might be related to the mechanism of bending. To explore the mechanism by which structural heterogeneity of axonemal dyneins is induced by nucleotides, we used cilia from Tetrahymena thermophila to examine the structure of dyneins in a) the intact axoneme and b) microtubule doublets separated from the axoneme, both with and without additional pure microtubules. We also employed an ATPase assay on these specimens to investigate dynein activity functionally. Dyneins on separated doublets show more activation by nucleotides than those in the intact axoneme, both structurally and in the ATPase assay, and this is especially pronounced when the doublets are coupled with added microtubules, as expected. Paralleling the reduced ATPase activity in the intact axonemes, a lower proportion of these dyneins are in the nucleotide-bound form. This indicates a coordinated suppression of dynein activity in the axoneme, which could be the key for understanding the bending mechanism.     

Are the local adjustments of the relative spatial frequencies of the dynein arms and the beta-tubulin monomers involved in the regulation of the "9+2" axoneme?

The “9+2” axoneme is a highly specific cylindrical machine whose periodic bending is due to the cumulative shear of its 9 outer doublets of microtubules. Because of the discrete architecture of the tubulin monomers and the active appendices that the outer doublets carry (dynein arms, nexin links and radial spokes), this movement corresponds to the relative shear of these topological verniers, whose characteristics depend on the geometry of the wave train. When an axonemal segment bends, this induces the compressed and dilated conformations of the tubulin monomers and, consequently, the modification of the spatial frequencies of the appendages that the outer doublets carry. From a dynamic point of view, the adjustments of the spatial frequencies of the elements of the two facing verniers that must interact create different longitudinal periodic patterns of distribution of the joint probability of the molecular interaction as a function of the location of the doublet pairs around the axonemal cylinder and their spatial orientation within the axonemal cylinder. During the shear, these patterns move along the outer doublet intervals at a speed that ranges from one to more than a thousand times that of sliding, in two opposite directions along the two opposite halves of the axoneme separated by the bending plane, respecting the polarity of the dynein arms within the axoneme. Consequently, these waves might be involved in the regulation of the alternating activity of the dynein arms along the flagellum, because they induce the necessary intermolecular dialog along the axoneme since they could be an element of the local dynamic stability/instability equilibrium of the axoneme. This complements the geometric clutch model [Lindemann, C., 1994. A “geometric clutch” hypothesis to explain oscillations of the axoneme of cilia and flagella. J. Theor. Biol. 168, 175-189].     

Morphogenesis of the giant sperm axoneme in Asphondylia ruebsaameni Kertesz (Diptera, Cecidomyiidae)

The formation of the sperm giant axoneme of the gall-midge fly Asphondylia ruebsaameni is described here. The axoneme consists of a great number of microtubular doublets (up to 2,500) arranged in a double spiral wrapping around an axial cluster of mitochondria. Each microtubular doublet is provided with an outer arm only. In the early spermatid the occurrence of a large system of curved multi-layered filamentous material associated with membranous cisternae has been observed in the perinuclear region. Such a system extends throughout the cytoplasm to contact the plasma membrane. The filamentous material appears to act as a nucleating centre for the assembly of the microtubular doublets, which initially have a submembranous location and later are distributed in the interior of the cell. After their assembly, microtubular doublets are associated pairwise and are arranged in a single microtubular row with a zig-zag configuration. This configuration changes during spermiogenesis as a consequence both of a rotation of the microtubular doublet pairs and a compaction of the axonemal complex due to the elimination of the excess cytoplasm. As a result of this process, a double parallel spiral of microtubular doublets is formed.     

Atypical and distinct microtubule radial symmetries in the centriole and the axoneme of Lecudina tuzetae

The centriole is a minute cylindrical organelle present in a wide range of eukaryotic species. Most centrioles have a signature ninefold radial symmetry of microtubules that is imparted to the axonemes of the cilia and flagella they template, with nine centriolar microtubule doublets growing into nine axonemal microtubule doublets. There are exceptions to the ninefold symmetrical arrangement of axonemal microtubules in some species, with lower or higher fold symmetries. In the few cases where this has been examined, such alterations in axonemal symmetries are grounded in similar alterations in centriolar symmetries. Here, we examine the question of microtubule number continuity between centriole and axoneme in flagellated gametes of the gregarine Lecudina tuzetae, which have been reported to exhibit a sixfold radial symmetry of axonemal microtubules. We used time-lapse differential interference microscopy to identify the stage at which flagellated gametes are present. Thereafter, using electron microscopy and ultrastructure-expansion microscopy coupled to stimulated emission depletion superresolution imaging, we uncover that a six- or fivefold radial symmetry in the axoneme is accompanied by an eightfold radial symmetry in the centriole. We conclude that the transition between centriolar and axonemal microtubules can be characterized by unexpected plasticity.     

Characteristics of the sperm tail of the velvet worm, Euperipatoides leuckarti (Onychophora)

The onychophoran sperm tail contains several kinds of microtubulcs; probably more than that of any other animal group. There are thus a peripheral manchette consisting of many tightly spaced microtubules, a ring of nine 'peripheral singlets' and a central axoneme of the classical 9 + 2 type (nine doublets and two central singlets). The protofilament organization of these various microtubules was examined and compared to the structure and mode of formation of the peripheral singlets with that of its analogues in other animal groups. The onychophoran peripheral singlets were found to differ in two respects from those in insects: they are formed from the manchette rather than from the axonemal doublets and their transient connection to the axoneme is to the A-subtubules of the doublet rather than to the B-subtubules. The manchette microtubules as well as the peripheral singlets consist of 13 protoh'laments. The manchette may serve a mechanical function (to strengthen the unusually thick sperm tail) hut the role of the peripheral singlets remains unknown.     

Ultrastructural studies on euspermatozoa and paraspermatozoa in Mantispidae (Insecta, Neuroptera)

Previous studies have demonstrated the presence of sperm dimorphism in the Mantispidae Perlamantispa perla. We extended the study on several other mantidflies. In all the examined species the occurrence of euspermatozoa (typical) and paraspermatozoa (atypical) was established. The euspermatozoa are characterized by the presence of a cylindrical nucleus surrounded by an envelope that fans out laterally into two thin wings of different length. The acrosome seems to be missing. The nucleus is surrounded by extracellular material. The flagellum is provided with a 9 + 9 + 2 axonemal pattern; the accessory tubules contain 16 protofilaments and the intertubular material has the distribution typical of the taxon. Two elongated accessory bodies flank partially the axoneme and connect this structure with the mitochondrial derivatives. The flagellar axoneme of paraspermatozoa consists of an axoneme and two giant mitochondrial derivatives filled with large globular units. The axoneme exhibits a 9 + 9 + 2 pattern, in which the central 9 + 2 units have a normal structure, in that the microtubular doublets are provided with both dynein arms and radial links. On the contrary, the nine accessory microtubules have a large diameter and their tubular wall consists of 40 protofilaments. This comparative study provided evidences about the uniformity of sperm ultrastructure in Mantispidae. The function of non-fertilizing giant sperm in mantidflies is discussed.     

Dynein as a microtubule translocator in ciliary motility: current studies of arm structure and activity pattern

The dynein arms of ciliary doublet microtubules cause adjacent axonemal doublets to slide apart with fixed polarity. This suggests that there is a unique mechanochemistry to the dynein arm with unidirectional force generation in all active arms and also that not all arms are active at once during a ciliary beat. Negative stain and thin-section images of arms in axonemes treated with beta, gamma methylene adenosine triphosphate (AMP-PCP) show a consistent subunit construction where the globular head of the arm interacts with subfiber B of doublet N+1. This interpretation differs from that provided by freeze etch and STEM interpretations of in situ arm construction and has implications for the mechanochemical cycle of the arm. A computer model of the arms in relation to other axonemal structures has been constructed to test these interpretations. Attachment of the head of the arm subfiber B is directly demonstrable in splayed axonemes in AMP-PCP. About half of the doublets in an axoneme show such attachments, while half do not. This might imply that about half the doublets in an axoneme are active at any given instant and can be identified as such. This information may be useful in probing questions of how active arms differ biochemically from inactive arms and of how microtubule translocators in general become active.     

The architecture of outer dynein arms in situ

Outer dynein arms, the force generators for axonemal motion, form arrays on microtubule doublets in situ, although they are bouquet-like complexes with separated heads of multiple heavy chains when isolated in vitro. To understand how the three heavy chains are folded in the array, we reconstructed the detailed 3D structure of outer dynein arms of Chlamydomonas flagella in situ by electron cryo-tomography and single-particle averaging. The outer dynein arm binds to the A-microtubule through three interfaces on two adjacent protofilaments, two of which probably represent the docking complex. The three AAA rings of heavy chains, seen as stacked plates, are connected in a striking manner on microtubule doublets. The tail of the alpha-heavy chain, identified by analyzing the oda11 mutant, which lacks alpha-heavy chain, extends from the AAA ring tilted toward the tip of the axoneme and towards the inside of the axoneme at 50 degrees , suggesting a three-dimensional power stroke. The neighboring outer dynein arms are connected through two filamentous structures: one at the exterior of the axoneme and the other through the alpha-tail. Although the beta-tail seems to merge with the alpha-tail at the internal side of the axoneme, the gamma-tail is likely to extend at the exterior of the axoneme and join the AAA ring. This suggests that the fold and function of gamma-heavy chain are different from those of alpha and beta-chains.     

The spermatogenesis and sperm structure of Acerentomon microrhinus (Protura, Hexapoda) with considerations on the phylogenetic position of the taxon

The spermatogenesis of the proturan Acerentomon microrhinus Berlese, (Redia 6:1–182, 1909) is described for the first time with the aim of comparing the ultrastructure of the flagellated sperm of members of this taxon with that of the supposedly related group, Collembola. The apical region of testes consists of a series of large cells with giant polymorphic nuclei and several centrosomes with 14 microtubule doublets, whose origin is likely a template of a conventional 9-doublet centriole. Beneath this region, there are spermatogonial cells, whose centrosome has two centrioles, both with 14 microtubule doublets; the daughter centriole of the pair has an axial cylinder. Slender parietal cells in the testes have centrioles with nine doublet microtubules. Spermatocytes produce short primary cilia with 14 microtubule doublets. Spermatids have a single basal body with 14 microtubule doublets. Anteriorly, a conical dense material is present, surrounded by a microtubular basket, which can be seen by using an α-anti-tubulin antibody. Behind this region, the basal body expresses a long axoneme of 14 microtubule doublets with only inner arms. An acrosome is lacking. The nucleus is twisted around the apical conical dense structure and the axoneme; this coiling seems to be due to the rotation of the axoneme on its longitudinal axis. The posterior part of the axoneme forms three turns within the spermatid cytoplasm. Few unchanged mitochondria are scattered in the cytoplasm. Sperm consist of encysted, globular cells that descend along the deferent duct lumen. Some of them are engulfed by the epithelial cells, which thus have a spermiophagic activity. Sperm placed in a proper medium extend their flagellar axonemes and start beating. Protura sperm structure is quite different from that of Collembola sperm; and on the basis of sperm characters, a close relationship between the two taxa is not supported.     

Axonemal activity relative to the 2D/3D-waveform conversion of the flagellum

The waveform of the flagellum of the sea urchin spermatozoon is mainly planar, but its 3D-properties were evoked for dynamic reasons and described as helical. In 1975, the apparent twisting pattern of the sea urchin axoneme was described [Gibbons I. 1975. The molecular basis of flagellar motility in sea urchin spermatozoa. In: Inoué S, Stephens R, editors. Molecular and cellular movement. New York: Raven Press, p. 207-232.] and was considered to be one of the main elements involved in axonemal behaviour. Recently, planar, quasi-planar, and helical waveforms were observed when the flagellum of sea urchin sperm cells was submitted to an increase in viscosity. The quasi-planar conformation seemed to be due to the alternating torsion of the inter-bend segments [Woolley D, Vernon G. 2001. A study of helical and planar waves on sea urchin sperm flagella, with a theory of how they are generated. J. Exp. Biol. 204:1333-1345]. These three waveforms, which are due to a change in axonemal activity, are possibly used by the sperm cells to adapt their movement to variations in the physico-chemical characteristics of the medium (seawater) in which the cells normally swim. We constructed a simple model to describe qualitatively the central shear (between the axonemal doublets and the central pair) and the tangential shear (between the doublets themselves). In this model, the 3D-bending is resolved into components in two perpendicular planes and each of the nine planes of inter-doublet interaction defines a potential bending plane that is independently regulated. These shears were calculated for the three waveforms and their inter-conversion. This allowed us to propose that axoneme is resolved in successive modules delineated by abscissas where the sliding is always nil. We discuss these data concerning the axonemal machinery, and especially the alternating activity of opposite sides of (two) neutral surface(s) that seem(s) to be responsible for this inter-conversion, and for the possible twist of the axoneme during the beating.     

The restructuring of the flagellar base and the flagellar necklace during spermatogenesis of Ephestia kuehniella Z. (Pyralidae,Lepidoptera)

Summary Transmission electron microscopy was used to study the development of the flagellar base and the flagellar necklace during spermatogenesis in a moth (Ephestia kuehniella Z.). Until mid-pachytene, two basal body pairs without flagella occur per cell. The basal bodies, which contain a cartwheel complex, give rise to four flagella in late prophase I. The cartwheel complex appears to be involved in the nucleation of the central pair of axonemal microtubules. In spermatids, there is one basal body; this is attached to a flagellum. At this stage, the nine microtubular triplets of the basal body do not terminate at the same proximal level. The juxtanuclear triplets are shifted distally relative to the triplets distant from the nuclear envelope. Transition fibrils and a flagellar necklace are formed at the onset of axoneme elongation. The flagellar necklace includes Y-shaped elements that connect the flagellar membrane and the axonemal doublets. In spindle-containing spermatocytes, the flagellar necklace is no longer detectable. During spermatid differentiation, the transition fibrils move distally along the axoneme and a prominent middle piece appears. Our observations and those in the literature indicate certain trends in sperm structure. In sperms with a short middle piece, we expect the presence of a flagellar necklace. The distal movement of the transition fibrils or equivalent structures is prevented by the presence of radial linkers between the flagellar membrane and the axonemal doublets. On the other hand, the absence of a flagellar necklace at the initiation of spermiogenesis enables the formation of a long middle piece. Thus, in spermatozoa possessing an extended middle piece, a flagellar necklace may be missing.     

Flagellate spermatozoa of Protura (Insecta,Apterygota) are motile

A new model of sperm axoneme with 16 + 0 doublets is described. The spermatozoon of Acerentulus confinis (Apterygota : Protura) has a short conical acrosome, a long helicoidal nucleus, well-developed centriolar adjunct material, and a long flagellum. Using fixation with a glutaraldehyde-tannic acid mixture, without osmium post-fixation, doublet protofilaments, inner dynein arms, radial spokes, nexin bridges, and Y-links of the sperm axoneme of A. confinis and Acerentomon italicum were clearly observed. Optical observation shows that the proturan flagellate spermatozoa are motile cells. The process involving the transformation of the spermatozoa from a coiled to an elongated swimming form was studied by scanning electron microscope. The findings confirmed that flagellar motility is due to the presence of a single dynein arm on doublets in spite of the unusual axonemal pattern.     

Glutamylated and glycylated tubulin isoforms in the aberrant sperm axoneme of the gall-midge fly, Asphondylia ruebsaameni

The axonemal organization expressed in the sperm flagella of the cecidomyiid dipteran Asphondylia ruebsaameni is unconventional, being characterized by the presence of an exceedingly high number of microtubular doublets and by the absence of both the inner dynein arms and the central pair/radial spoke complex. Consequently, its motility, both in vivo and in vitro, is also peculiar. Using monoclonal antibodies directed against posttranslational modifications, we have analyzed the presence and distribution of glutamylated and glycylated tubulin isoforms in this aberrant axonemal structure, and compared them with those of a reference insect species (Apis mellifera), endowed with a conventional axoneme. Our results have shown that the unorthodox structure and motility of the Asphondylia axoneme are concomitant with: (1). a very low glutamylation extent in the alpha-tubulin subunit, (2). a high level of glutamylation in the beta-subunit, (3). an extremely low total extent of glycylation, with regard to both monoglycylated and polyglycylated sites, either in alpha- or in beta-tubulin, (4). the presence of a strong labeling of glutamylated tubulin isoforms at the proximal end of the axoneme, and (5). a uniform distribution of glutamylated as well as glycylated isoforms along the rest of the axoneme. Thus, our data indicate that tubulin molecular heterogeneity is much lower in the Asphondylia axoneme than in the conventional 9+2 axoneme with regard to both isoform content and isoform distribution along the axoneme.     

The role of axonemal components in ciliary motility

1. The axoneme is the detergent-insoluble cytoskeleton of the cilium. 2. All axonemes generate movement by the same fundamental mechanism: microtubule sliding utilizing ATP hydrolysis during a mechanochemical cycling of dynein arms on the axonemal doublets. 3. Structure, fundamental biochemistry and physiology of the axoneme are conserved evolutionarily, but the phenotypes of beating movements and the responses to specific cytoplasmic signals differ greatly from organism to organism. 4. A model of asynchronous dynein arm activity--the switch point hypothesis--has been proposed to account for cyclic beating in the face of unidirectional sliding. The model suggests that the diversity of beat phenotype may be explicable by changes in the timing of switching between active and inactive states of doublet arm activity. Evidence of axonemal splitting in arrested axonemes provides new support for the hypothesis.     

Pathology of the cytoskeleton of the human sperm flagellum: axonemal and peri-axonemal anomalies

A quantitative ultrastructural study was performed on 56 ejaculates showing anomalies of the sperm axonemal complex. The anomalies comprised either the absence of one, or more often several, axonemal structures, or defective elongation of the doublets. Several characteristics relating to the extent and superimposition of the various anomalies could be described and enabled the definition of 6 groups of anomalies. In decreasing order of frequency these were: absence of the doublets and peripheral junctions, absence of the central complex, of the outer dynein arms, of the central junctions, of both dynein arms, and absence of the inner dynein arms and peripheral junctions. Some anomalies caused total immobility, whereas others caused abnormal movement patterns. Abnormalities of the peri-axonemal structures were found in each group. The various light microscopic characteristics of each of the 6 groups represented 6 seminal profiles which should permit their detection during a routine semen analysis. Several specific associations of axonemal and/or peri-axonemal anomalies would suggest some morphogenetic links between them. Relationships between the absence of doublets or the absence of the central complex and disturbances of microtubular polymerization are discussed. Finally, the study has provided new data on the composition of the axoneme.     

Composition and organization of tubulin isoforms reveals a variety of axonemal models

In the flagellum of mammalian spermatozoa, glutamylated and glycylated tubulin isoforms are detected according to longitudinal gradients and preferentially in axonemal doublets 1-5-6 and 3-8, respectively. This suggested a role for these tubulin isoforms in the regulation of flagellar beating. In the present work, using antibodies directed against various tubulin isoforms and quantitative immunogold analysis, we aimed at investigating whether the particular accessibility of tubulin isoforms in the mammalian sperm flagellum is restricted to this model of axoneme surrounded with periaxonemal structures or is also displayed in naked axonemes. In rodent lung ciliated cells, all studied tubulin isoforms are uniformly distributed in all axonemal microtubules with a unique deficiency of glutamylated tubulin in the transitional region. A similar distribution of tubulin isoforms is observed in cilia of Paramecium, except for a decreasing gradient of glutamylated tubulin labeling in the proximal part of axonemal microtubules. In the sea urchin sperm flagellum, predominant labeling of tyrosinated and detyrosinated tubulin in 1-5-6 and 3-8 doublets, respectively, were observed together with decreasing proximo-distal gradients of glutamylated and polyglycylated tubulin labeling and an increasing gradient of monoglycylated tubulin labeling. In flagella of Chlamydomonas, the glutamylated and glycylated tubulin isoforms are detected at low levels. Our results show a specific composition and organization of tubulin isoforms in different models of cilia and flagella, suggesting various models of functional organization and beating regulation of the axoneme.     

Dynein arm attachment probed with a non-hydrolyzable ATP analog. Structural evidence for patterns of activity

The dynein arms that power ciliary motility are normally permanently attached by one end exclusively to subfiber A of each axonemal doublet (N) while the other (head) end transiently attaches to the subfiber B of the adjacent doublet (N + 1) to produce sliding of the doublets. In Tetrahymena axonemes, sliding of contiguous groups of doublets is induced by ATP suggesting that, in the absence of exogenous protease, there may be sets of potentially active and potentially inactive or refractory arms in a single axoneme. In the presence of a non-hydrolyzable analog of ATP, beta,gamma-methylene adenosine 5'-triphosphate (AMP-PCP), about half the doublets in an axonemal preparation retain all arms bound to subfiber A, but half the doublets show long regions where some arms are pulled away from subfiber A of doublet N and attached to subfiber B of doublet N + 1 by their head ends. In AMP-PCP-induced splaying, positional information regarding arm state is retained. Analysis reveals that throughout regions where B subfiber attachment is found, small groups of about four subfiber B attached arms alternate with groups of about four arms that remain attached to subfiber A. This unique pattern of attachment suggests that arms function co-operatively in groups of four. Further, the repetition of the pattern is reminiscent of metachronal activity seen at higher levels of biological organization. This suggests that in these regions we have instantaneously preserved groups of arms capable of attaching to and detaching from doublet N + 1 in rapid succession. This appearance could be used to delineate the potentially active sets of arm, primed for mechanochemical activity, within an axoneme.