IFT cargo and motors associate sequentially with IFT trains to enter cilia of C. elegans.

Intraflagellar transport (IFT) orchestrates entry of proteins into primary cilia. At the ciliary base, assembled IFT trains, driven by kinesin-2 motors, can transport cargo proteins into the cilium, across the crowded transition zone. How trains assemble at the base and how proteins associate with them is far from understood. Here, we use single-molecule imaging in the cilia of C. elegans chemosensory neurons to directly visualize the entry of kinesin-2 motors, kinesin-II and OSM-3, as well as anterograde cargo proteins, IFT dynein and tubulin. Single-particle tracking shows that IFT components associate with trains sequentially, both in time and space. Super-resolution maps of IFT components in wild-type and mutant worms reveal ciliary ultrastructure and show that kinesin-II is essential for axonemal organization. Finally, imaging cilia lacking kinesin-II and/or transition zone function uncovers the interplay of kinesin-II and OSM-3 in driving efficient transport of IFT trains across the transition zone.

MAP9/MAPH-9 supports axonemal microtubule doublets and modulates motor movement.

Microtubule doublets (MTDs) comprise an incomplete microtubule (B-tubule) attached to the side of a complete cylindrical microtubule. These compound microtubules are conserved in cilia across the tree of life; however, the mechanisms by which MTDs form and are maintained in vivo remain poorly understood. Here, we identify microtubule-associated protein 9 (MAP9) as an MTD-associated protein. We demonstrate that C. elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. We find that loss of MAPH-9 causes ultrastructural MTD defects, including shortened and/or squashed B-tubules with reduced numbers of protofilaments, dysregulated axonemal motor velocity, and perturbed cilia function. Because we find that the mammalian ortholog MAP9 localizes to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in regulating ciliary motors and supporting the structure of axonemal MTDs.

Axonemal structures reveal mechanoregulatory and disease mechanisms.

Motile cilia and flagella beat rhythmically on the surface of cells to power the flow of fluid and to enable spermatozoa and unicellular eukaryotes to swim. In humans, defective ciliary motility can lead to male infertility and a congenital disorder called primary ciliary dyskinesia (PCD), in which impaired clearance of mucus by the cilia causes chronic respiratory infections1. Ciliary movement is generated by the axoneme, a molecular machine consisting of microtubules, ATP-powered dynein motors and regulatory complexes2. The size and complexity of the axoneme has so far prevented the development of an atomic model, hindering efforts to understand how it functions. Here we capitalize on recent developments in artificial intelligence-enabled structure prediction and cryo-electron microscopy (cryo-EM) to determine the structure of the 96-nm modular repeats of axonemes from the flagella of the alga Chlamydomonas reinhardtii and human respiratory cilia. Our atomic models provide insights into the conservation and specialization of axonemes, the interconnectivity between dyneins and their regulators, and the mechanisms that maintain axonemal periodicity. Correlated conformational changes in mechanoregulatory complexes with their associated axonemal dynein motors provide a mechanism for the long-hypothesized mechanotransduction pathway to regulate ciliary motility. Structures of respiratory-cilia doublet microtubules from four individuals with PCD reveal how the loss of individual docking factors can selectively eradicate periodically repeating structures.

Expansion microscopy of Plasmodium gametocytes reveals the molecular architecture of a bipartite microtubule organisation centre coordinating mitosis with axoneme assembly.

Transmission of malaria-causing parasites to mosquitoes relies on the production of gametocyte stages and their development into gametes. These stages display various microtubule cytoskeletons and the architecture of the corresponding microtubule organisation centres (MTOC) remains elusive. Combining ultrastructure expansion microscopy (U-ExM) with bulk proteome labelling, we first reconstructed in 3D the subpellicular microtubule network which confers cell rigidity to Plasmodium falciparum gametocytes. Upon activation, as the microgametocyte undergoes three rounds of endomitosis, it also assembles axonemes to form eight flagellated microgametes. U-ExM combined with Pan-ExM further revealed the molecular architecture of the bipartite MTOC coordinating mitosis with axoneme formation. This MTOC spans the nuclear membrane linking cytoplasmic basal bodies to intranuclear bodies by proteinaceous filaments. In P. berghei, the eight basal bodies are concomitantly de novo assembled in a SAS6- and SAS4-dependent manner from a deuterosome-like structure, where centrin, γ-tubulin, SAS4 and SAS6 form distinct subdomains. Basal bodies display a fusion of the proximal and central cores where centrin and SAS6 are surrounded by a SAS4-toroid in the lumen of the microtubule wall. Sequential nucleation of axonemes and mitotic spindles is associated with a dynamic movement of γ-tubulin from the basal bodies to the intranuclear bodies. This dynamic architecture relies on two non-canonical regulators, the calcium-dependent protein kinase 4 and the serine/arginine-protein kinase 1. Altogether, these results provide insights into the molecular organisation of a bipartite MTOC that may reflect a functional transition of a basal body to coordinate axoneme assembly with mitosis.

TomoAlign: A novel approach to correcting sample motion and 3D CTF in CryoET.

TomoAlign is a software package that integrates tools to mitigate two important resolution limiting factors in cryoET, namely the beam-induced sample motion and the contrast transfer function (CTF) of the microscope. The package is especially focused on cryoET of thick specimens where fiducial markers are required for accurate tilt-series alignment and sample motion estimation. TomoAlign models the beam-induced sample motion undergone during the tilt-series acquisition. The motion models are used to produce motion-corrected subtilt-series centered on the particles of interest. In addition, the defocus of each particle at each tilt image is determined and can be corrected, resulting in motion-corrected and CTF-corrected subtilt-series from which the subtomograms can be computed. Alternatively, the CTF information can be passed on so that CTF correction can be carried out entirely within external packages like Relion. TomoAlign serves as a versatile tool that can streamline the cryoET workflow from initial alignment of tilt-series to final subtomogram averaging during in situ structure determination.

Postulated Process of Axoneme Organization in the Male Gametogenesis of Malaria Parasite Plasmodium berghei.

The ultrastructural features of axoneme organization within the cytoplasm and exflagellation were investigated in detail in microgametes of a malaria parasite, Plasmodium berghei, by electron and fluorescence microscopy. The kinetosomes (basal bodies) of the microgamete were characterized by an electron dense mass in which singlet microtubules (MTs) were embedded. Around the kinetosomes, several singlet and doublet MTs were recognized in transverse sections. Incomplete doublets with growing B-tubule were also observed. As precursors of the axoneme, arrays of over three doublets showed a tendency to encircle the central pair MTs. Some of the doublet MTs were already equipped with inner and outer dynein arms. In the microgamete, which lacks an intraflagellar transport (IFT) system, self-assembly of microtubular and associated components appeared to proceed stepwise from singlet MTs through arrays of one to nine doublet MTs, surrounding the central pair, to form the complete axoneme in a quite short time. At exflagellation, some extra doublets were occasionally included between the axoneme and the flagellar membrane. At high magnification, the outer dynein arm of the Plasmodium microgamete had a pistol-like shape representing a three-headed dynein molecule like that of other Alveolata.

The multi-scale architecture of mammalian sperm flagella and implications for ciliary motility.

Motile cilia are molecular machines used by a myriad of eukaryotic cells to swim through fluid environments. However, available molecular structures represent only a handful of cell types, limiting our understanding of how cilia are modified to support motility in diverse media. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image sperm flagella from three mammalian species. We resolve in-cell structures of centrioles, axonemal doublets, central pair apparatus, and endpiece singlets, revealing novel protofilament-bridging microtubule inner proteins throughout the flagellum. We present native structures of the flagellar base, which is crucial for shaping the flagellar beat. We show that outer dense fibers are directly coupled to microtubule doublets in the principal piece but not in the midpiece. Thus, mammalian sperm flagella are ornamented across scales, from protofilament-bracing structures reinforcing microtubules at the nano-scale to accessory structures that impose micron-scale asymmetries on the entire assembly. Our structures provide vital foundations for linking molecular structure to ciliary motility and evolution.

Defects in the cytoplasmic assembly of axonemal dynein arms cause morphological abnormalities and dysmotility in sperm cells leading to male infertility.

Axonemal protein complexes, such as outer (ODA) and inner (IDA) dynein arms, are responsible for the generation and regulation of flagellar and ciliary beating. Studies in various ciliated model organisms have shown that axonemal dynein arms are first assembled in the cell cytoplasm and then delivered into axonemes during ciliogenesis. In humans, mutations in genes encoding for factors involved in this process cause structural and functional defects of motile cilia in various organs such as the airways and result in the hereditary disorder primary ciliary dyskinesia (PCD). Despite extensive knowledge about the cytoplasmic assembly of axonemal dynein arms in respiratory cilia, this process is still poorly understood in sperm flagella. To better define its clinical relevance on sperm structure and function, and thus male fertility, further investigations are required. Here we report the fertility status in different axonemal dynein preassembly mutant males (DNAAF2/ KTU, DNAAF4/ DYX1C1, DNAAF6/ PIH1D3, DNAAF7/ZMYND10, CFAP300/C11orf70 and LRRC6). Besides andrological examinations, we functionally and structurally analyzed sperm flagella of affected individuals by high-speed video- and transmission electron microscopy as well as systematically compared the composition of dynein arms in sperm flagella and respiratory cilia by immunofluorescence microscopy. Furthermore, we analyzed the flagellar length in dynein preassembly mutant sperm. We found that the process of axonemal dynein preassembly is also critical in sperm, by identifying defects of ODAs and IDAs in dysmotile sperm of these individuals. Interestingly, these mutant sperm consistently show a complete loss of ODAs, while some respiratory cilia from the same individual can retain ODAs in the proximal ciliary compartment. This agrees with reports of solely one distinct ODA type in sperm, compared to two different ODA types in proximal and distal respiratory ciliary axonemes. Consistent with observations in model organisms, we also determined a significant reduction of sperm flagellar length in these individuals. These findings are relevant to subsequent studies on the function and composition of sperm flagella in PCD patients and non-syndromic infertile males. Our study contributes to a better understanding of the fertility status in PCD-affected males and should help guide genetic and andrological counselling for affected males and their families.

Motility of efferent duct cilia aids passage of sperm cells through the male reproductive system.

Motile cilia line the efferent ducts of the mammalian male reproductive tract. Several recent mouse studies have demonstrated that a reduced generation of multiple motile cilia in efferent ducts is associated with obstructive oligozoospermia and fertility issues. However, the sole impact of efferent duct cilia dysmotility on male infertility has not been studied so far either in mice or human. Using video microscopy, histological- and ultrastructural analyses, we examined male reproductive tracts of mice deficient for the axonemal motor protein DNAH5: this defect exclusively disrupts the outer dynein arm (ODA) composition of motile cilia but not the ODA composition and motility of sperm flagella. These mice have immotile efferent duct cilia that lack ODAs, which are essential for ciliary beat generation. Furthermore, they show accumulation of sperm in the efferent duct. Notably, the ultrastructure and motility of sperm from these males are unaffected. Likewise, human individuals with loss-of-function DNAH5 mutations present with reduced sperm count in the ejaculate (oligozoospermia) and dilatations of the epididymal head but normal sperm motility, similar to DNAH5 deficient mice. The findings of this translational study demonstrate, in both mice and men, that efferent duct ciliary motility is important for male reproductive fitness and uncovers a novel pathomechanism distinct from primary defects of sperm motility (asthenozoospermia). If future work can identify environmental factors or defects in genes other than DNAH5 that cause efferent duct cilia dysmotility, this will help unravel other causes of oligozoospermia and may influence future practices in genetic and fertility counseling as well as ART.

Cilia and centrosomes: Ultrastructural and mechanical perspectives.

Cilia and centrosomes of eukaryotic cells play important roles in cell movement, fluid transport, extracellular sensing, and chromosome division. The physiological functions of cilia and centrosomes are generated by their dynamics, motions, and forces controlled by the physical, chemical, and biological environments. How an individual cilium achieves its beat pattern and induces fluid flow is governed by its ultrastructure as well as the coordination of associated molecular motors. Thus, a bottom-up understanding of the physiological functions of cilia and centrosomes from the molecular to tissue levels is required. Correlations between the structure and motion can be understood in terms of mechanics. This review first focuses on cilia and centrosomes at the molecular level, introducing their ultrastructure. We then shift to the organelle level and introduce the kinematics and mechanics of cilia and centrosomes. Next, at the tissue level, we introduce nodal ciliary dynamics and nodal flow, which play crucial roles in the organogenetic process of left-right asymmetry. We also introduce respiratory ciliary dynamics and mucous flow, which are critical for protecting the epithelium from drying and exposure to harmful particles and viruses, i.e., respiratory clearance function. Finally, we discuss the future research directions in this field.

Microtubule-associated proteins and emerging links to primary cilium structure, assembly, maintenance, and disassembly.

The primary cilium is a microtubule-based structure that protrudes from the cell surface in diverse eukaryotic organisms. It functions as a key signaling center that decodes a variety of mechanical and chemical stimuli and plays fundamental roles in development and homeostasis. Accordingly, structural and functional defects of the primary cilium have profound effects on the physiology of multiple organ systems including kidney, retina, and central nervous system. At the core of the primary cilium is the microtubule-based axoneme, which supports the cilium shape and acts as the scaffold for bidirectional transport of cargoes into and out of cilium. Advances in imaging, proteomics, and structural biology have revealed new insights into the ultrastructural organization and composition of the primary cilium, the mechanisms that underlie its biogenesis and functions, and the pathologies that result from their deregulation termed ciliopathies. In this viewpoint, we first discuss the recent studies that identified the three-dimensional native architecture of the ciliary axoneme and revealed that it is considerably different from the well-known '9 + 0' paradigm. Moving forward, we explore emerging themes in the assembly and maintenance of the axoneme, with a focus on how microtubule-associated proteins regulate its structure, length, and stability. This far more complex picture of the primary cilium structure and composition, as well as the recent technological advances, open up new avenues for future research.

On being the right shape: Roles for motile cilia and cerebrospinal fluid flow in body and spine morphology.

How developing and growing organisms attain their proper shape is a central problem of developmental biology. In this review, we investigate this question with respect to how the body axis and spine form in their characteristic linear head-to-tail fashion in vertebrates. Recent work in the zebrafish has implicated motile cilia and cerebrospinal fluid flow in axial morphogenesis and spinal straightness. We begin by introducing motile cilia, the fluid flows they generate and their roles in zebrafish development and growth. We then describe how cilia control body and spine shape through sensory cells in the spinal canal, a thread-like extracellular structure called the Reissner fiber, and expression of neuropeptide signals. Last, we discuss zebrafish mutants in which spinal straightness breaks down and three-dimensional curves form. These curves resemble the common but little-understood human disease Idiopathic Scoliosis. Zebrafish research is therefore poised to make progress in our understanding of this condition and, more generally, how body and spine shape is acquired and maintained through development and growth.

Rapid estimation of cytosolic ATP concentration from the ciliary beating frequency in the green alga Chlamydomonas reinhardtii.

Determination of cellular ATP levels, a key indicator of metabolic status, is essential for the quantitative analysis of metabolism. The biciliate green alga Chlamydomonas reinhardtii is an excellent experimental organism to study ATP production pathways, including photosynthesis and respiration, particularly because it can be cultured either photoautotrophically or heterotrophically. Additionally, its cellular ATP concentration, [ATP], is reflected in the beating of its cilia. However, the methods currently used for quantifying the cellular ATP levels are time consuming or invasive. In this study, we established a rapid method for estimating cytosolic [ATP] from the ciliary beating frequency in C. reinhardtii. Using an improved method of motility reactivation in demembranated cell models, we obtained calibration curves for [ATP]-ciliary beating frequency over a physiological range of ATP concentrations. These curves allowed rapid estimation of the cytosolic [ATP] in live wild-type cells to be ∼2.0 mM in the light and ∼1.5 mM in the dark: values comparable to those obtained by other methods. Furthermore, we used this method to assess the effects of genetic mutations or inhibitors of photosynthesis or respiration quantitatively and noninvasively. This sensor-free method is a convenient tool for quickly estimating cytosolic [ATP] and studying the mechanism of ATP production in C. reinhardtii or other ciliated organisms.

A comparative study of the ultrastructural characteristics of the mature spermatozoa of two fellodistomids Tergestia clonacantha and T. laticollis and contribution to the phylogenetic knowledge of the Gymnophalloidea.

The ultrastructure of the mature spermatozoa of Tergestia clonacantha and T. laticollis collected from the digestive tracts of fishes from New Caledonia is described using transmission electron microscopy and compared to that of related species. The spermatozoa of the two species exhibit the general pattern described in most digeneans, namely two axonemes with the 9 + "1" pattern of the Trepaxonemata, nucleus, mitochondrion, cortical microtubules, an external ornamentation of the plasma membrane, spine-like bodies and granules of glycogen. The spermatozoa of T. clonacantha and T. laticollis show the same ultrastructural model with some specificities in each case, particularly in the disposition of the structures in the posterior extremities of the spermatozoon. This study confirms that ultrastructural characters of the mature spermatozoon are useful tools for the phylogenetic analysis of the Digenea.

TITLE: Étude comparative des caractéristiques ultrastructurales des spermatozoïdes mûrs de deux Fellodistomidae, Tergestia clonacantha et T. laticollis, et contribution à la connaissance phylogénétique des Gymnophalloidea. ABSTRACT: L'ultrastructure des spermatozoïdes mûrs de Tergestia clonacantha et T. laticollis, prélevés dans le tube digestif de poissons de Nouvelle-Calédonie, est décrite par microscopie électronique à transmission et comparée à celle d'espèces apparentées. Les spermatozoïdes des deux espèces présentent la structure générale décrite chez la plupart des digènes, à savoir deux axonèmes du type 9 + « 1 ¼ des Trepaxonemata, un noyau, une mitochondrie, des microtubules corticaux, des ornementations externes de la membrane plasmique, des corps épineux et des granules de glycogène. Les spermatozoïdes de T. clonacantha et T. laticollis présentent le même modèle ultrastructural avec quelques spécificités dans chaque cas, notamment dans la disposition des structures aux extrémités postérieures du spermatozoïde. Cette étude confirme que les caractères ultrastructuraux du spermatozoïde mûrs sont des outils utiles pour l'analyse phylogénétique des Digenea.

CFAP45 deficiency causes situs abnormalities and asthenospermia by disrupting an axonemal adenine nucleotide homeostasis module.

Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45-/- mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.

Deficiency of the Tbc1d21 gene causes male infertility with morphological abnormalities of the sperm mitochondria and flagellum in mice.

Approximately 2-15% of couples experience infertility, and around half of these cases are attributed to male infertility. We previously identified TBC1D21 as a sterility-related RabGAP gene derived from infertile men. However, the in vivo function of TBC1D21 in male fertility remains unclear. Here, we show that loss of Tbc1d21 in mice resulted in male infertility, characterized by defects in sperm tail structure and diminished sperm motility. The mitochondria of the sperm-tail had an abnormal irregular arrangement, abnormal diameter, and structural defects. Moreover, the axoneme structure of sperm tails was severely disturbed. Several TBC1D21 interactors were selected via proteomic analysis and functional grouping. Two of the candidate interactors, a subunit protein of translocase in the outer membrane of mitochondria (TOMM20) and an inner arm component of the sperm tail axoneme (Dynein Heavy chain 7, DNAH7), confirmed in vivo physical co-localization with TBC1D21. In addition, TOMM20 and DNAH7 detached and dispersed outside the axoneme in Tbc1d21-deficient sperm, instead of aligning with the axoneme. From a clinical perspective, the transcript levels of TBC1D21 in sperm from teratozoospermia cases were significantly reduced when compared with those in normozoospermia. We concluded that TBC1D21 is critical for mitochondrial and axoneme development of mammalian sperm.

Loss of Rsph9 causes neonatal hydrocephalus with abnormal development of motile cilia in mice.

Hydrocephalus is a brain disorder triggered by cerebrospinal fluid accumulation in brain cavities. Even though cerebrospinal fluid flow is known to be driven by the orchestrated beating of the bundled motile cilia of ependymal cells, little is known about the mechanism of ciliary motility. RSPH9 is increasingly becoming recognized as a vital component of radial spokes in ciliary "9 + 2" ultrastructure organization. Here, we show that deletion of the Rsph9 gene leads to the development of hydrocephalus in the early postnatal period. However, the neurodevelopment and astrocyte development are normal in embryonic Rsph9-/- mice. The tubular structure of the central aqueduct was comparable in Rsph9-/- mice. Using high-speed video microscopy, we visualized lower beating amplitude and irregular rotation beating pattern of cilia bundles in Rsph9-/- mice compared with that of wild-type mice. And the centriolar patch size was significantly increased in Rsph9-/- cells. TEM results showed that deletion of Rsph9 causes little impact in ciliary axonemal organization but the Rsph9-/- cilia frequently had abnormal ectopic ciliary membrane inclusions. In addition, hydrocephalus in Rsph9-/- mice results in the development of astrogliosis, microgliosis and cerebrovascular abnormalities. Eventually, the ependymal cells sloughed off of the lateral wall. Our results collectively suggested that RSPH9 is essential for ciliary structure and motility of mouse ependymal cilia, and its deletion causes the pathogenesis of hydrocephalus.

Tissue specific requirement of Drosophila Rcd4 for centriole duplication and ciliogenesis.

Rcd4 is a poorly characterized Drosophila centriole component whose mammalian counterpart, PPP1R35, is suggested to function in centriole elongation and conversion to centrosomes. Here, we show that rcd4 mutants exhibit fewer centrioles, aberrant mitoses, and reduced basal bodies in sensory organs. Rcd4 interacts with the C-terminal part of Ana3, which loads onto the procentriole during interphase, ahead of Rcd4 and before mitosis. Accordingly, depletion of Ana3 prevents Rcd4 recruitment but not vice versa. We find that neither Ana3 nor Rcd4 participates directly in the mitotic conversion of centrioles to centrosomes, but both are required to load Ana1, which is essential for such conversion. Whereas ana3 mutants are male sterile, reflecting a requirement for Ana3 for centriole development in the male germ line, rcd4 mutants are fertile and have male germ line centrioles of normal length. Thus, Rcd4 is essential in somatic cells but is not absolutely required in spermatogenesis, indicating tissue-specific roles in centriole and basal body formation.

Analysis of the sperm flagellar axoneme using gene-modified mice.

Infertility is a global health issue that affects 1 in 6 couples, with male factors contributing to 50% of cases. The flagellar axoneme is a motility apparatus of spermatozoa, and disruption of its structure or function could lead to male infertility. The axoneme consists of a "9+2" structure that contains a central pair of two singlet microtubules surrounded by nine doublet microtubules, in addition to several macromolecular complexes such as dynein arms, radial spokes, and nexin-dynein regulatory complexes. Molecular components of the flagellar axoneme are evolutionally conserved from unicellular flagellates to mammals, including mice. Although knockout (KO) mice have been generated to understand their function in the formation and motility regulation of sperm flagella, the majority of KO mice die before sexual maturation due to impaired ciliary motility, which makes it challenging to analyze mature spermatozoa. In this review, we introduce methods that have been used to overcome premature lethality, focusing on KO mouse lines of central pair components.

Spermatological characteristics of Sclerodistomoides pacificus (Digenea, Sclerodistomoididae) a parasite of the flying fish Cheilopogon pinnatibarbatus (Teleostei, Exocoetidae).

Sclerodistomoides pacificus is the only species described now in Sclerodistomoididae. We present in this paper the first ultrastructural data of the mature spermatozoon of a species from the genus Sclerodistomoides. Adult specimens of S. pacificus (Digenea: Hemiuroidea: Sclerodistomoididae), were parasites of the gall-bladder of the teleost fish Cheilopogon pinnatibarbatus captured in the Atlantic Ocean, near Dakar (Senegal). The male gamete is a filiform cell which exhibits a similar ultrastructural organization to that reported in most species belonging to the Hemiuroidea with two axonemes of the 9 + '1' pattern of trepaxonematans, a nucleus, a mitochondrion, external ornamentation of the plasma membrane not associated with cortical microtubules and located in the anterior region of the spermatozoon, and parallel cortical microtubules disposed in one side of the spermatozoon. However, the present study allowed describing for the first time a moniliform mitochondrion in the Hemiuroidea. The presence of a moniliform mitochondrion and the absence of filamentous external ornamentation described in other Hemiuridae: Lecithochirium microstomum, L. musculus and Hemiurus appendiculatus are a good tool for phylogenetic purposes in the Hemiuroidea. Moreover, spermatological organisation and model are discussed in context with those of previous studies in the Hemiuroidea.