David Mercati

The ancestral eukaryotes presumably had an MTOC (microtubule organizing center) which late gave origin to the centriole and the flagellar axoneme. The centrosome of insect early spermatids is in general composed of two components: a single centriole and a cloud of electron-dense pericentriolar material (PCM). During spermiogenesis, the centriole changes its structure and gives rise to a flagellar axoneme, while the proteins of PCM, gamma tubulin in particular, are involved in the production of microtubules for the elongation and shaping of spermatid components. At the end of spermiogenesis, in many insects, additional material is deposited beneath the nucleus to form the centriole adjunct (ca). This material can also extend along the flagellum in two accessory bodies (ab) flanking the axoneme. Among Homoptera Sternorrhyncha, a progressive modification of their sperm flagella until complete disappearance has been verified. In the Archaeococcidae Matsucoccus feytaudi, however, a motile sperm flagellum-like structure is formed by an MTOC activity. This finding gives support to the hypothesis that an evolutionary reversal has occurred in the group and that the cell, when a non-functional centriole is present, activates an ancestral structure, an MTOC, to form a polarized motile bundle of microtubules restoring sperm motility. The presence and extension of the centriole adjunct in the different insect orders is also enlisted.

The sperm of the heteropteran bug Raphigaster nebulosa (Poda) are of two types, differing in length and size of their flagella. The thicker sperm are shorter than the thinner ones and have large mitochondrial derivatives. The presence of virus particles associated with the plasma membrane of thinner sperm is described for the first time; thicker sperm are immune to virus infection. The fact that virus particles are present on thinner sperm only initiates considerations on the transmission of virus.

The sperm structure and spermiogenesis of the morphologically peculiar heteropteran Coptosoma scutellatum has been examined with electron microscopy. The sperm has a short monolayer acrosome, a cylindrical nucleus, a 9+9+2 axoneme and two mitochondrial derivatives with crystallized matrix. It shows the synapomorphies typical of the group, consisting of (a) two crystallized areas in the mitochondrial derivatives; (b) the presence of two bridges connecting the intertubular material of the flagellar axoneme to the flattened cisterns adhering to the inner sides of the mitochondrial derivatives; (c) the absence of accessory bodies. The most interesting feature is the presence, in the early spermatid, of an extended centriole adjunct material with the shape of a large ribbon-like structure. This material surrounds the posterior nuclear region and extends anteriorly along the nucleus. It is a microtubule-organizing-center (MTOC) producing the many microtubules surrounding the sperm components At the end of spermiogenesis, the centriole adjunct material modifies into two kidney-like structures accompanying the nuclear posterior end and surrounding the modified centriole.

Previous studies on the spermatogenesis of tenebrionid beetles showed the unusual formation of two antiparallel sperm bundles per cyst. In this work we reported this feature also in Tribolium castaneum using light and transmission electron microscopy. The sperm structure of T. castaneum, similar to other tenebrionids, consists of a three-layered acrosome, an elongated nucleus and a flagellum with a 9+9+2 axoneme, two accessory bodies and two asymmetric mitochondrial derivatives. The presence of two antiparallel sperm bundles per cyst also in Meloidae and Rhipiphoridae suggests that it is a strong trait synapomorphic for Tenebrionoidea. The huge degeneration of whole sperm cells in several cysts of testes during spermiogenesis is also described.

The sperm structure of the jumping bristletail Machilontus sp has been described. The species shares several sperm characteristics with other genera of the same order Archaeognatha. During late spermiogenesis the spermatid bends at half of its length with the two limbs closely apposed within the same plasma membrane. The sperm has a helicoidal bi-layered acrosome with a filamentous perforatorium and a long nucleus. The elongated flagellum consists of an axoneme with 9 accessory microtubules external to the 9+2, two rows of conventional mitochondria and two accessory bodies. The accessory bodies are located lateral to the axoneme and are probably responsible for the shifting of the accessory tubules in two opposite groups of 5 and 4 tubules, respectively. These sperm characteristics represent common traits of all Archaeognatha.

A microtubule organizing centre (MTOC) has been described in the spermatid of the hemipteran Matsucoccus feytaudi (Coccoidea). This structure, revealed as a fluorescent ring by treatment with γ-tubulin antibody, gives rise to a bundle of microtubules which surrounds the elongated cylindrical nucleus. This microtubule bundle has been considered an atypical sperm flagellum provided with sperm motility. A comparison of the M. feytaudi MTOC with the material associated with the centriole of Drosophila melanogaster spermatids confirms the great similarity between the two structures, both involved in the nucleation of microtubules. Like the D. melanogaster material associated with the centriole, the M. feytaudi MTOC is a transient structure which disappears or degenerates at the end of spermiogenesis and is no longer visible in the mature sperm.

Mature spermatozoa of the catantopid orthopteran Pezotettix giornai are characterized by an elaborate external glycocalyx. This coating is removed during storage in spermatheca allowing preparation of sperm to their interaction with female gametes. We have studied this membrane coating in mature sperm by both conventional transmission electron microscopy and quick-freeze, deep-etching. The ultrastructural studies allowed visualization of three different domains in glycocalyx of testicular and deferent duct spermatozoa and only two in sperm cell isolated from seminal vesicles. These observations thus demonstrate that a remodeling of the male germ cell starts already during their storage in the seminal vesicle and is completed in the female genital tract. In this paper a three-dimensional (3D) model of sperm external coating is presented and discussed.

The Ephemeroptera sperm axoneme is devoid of outer dynein arms (ODA) and exhibits a pronounced modification of the central pair complex (CPC), which is substituted by the central sheath (CS): a tubular element of unknown molecular composition. We performed a detailed ultrastructural analysis of sperm axonemes in the genera Cloeon and Ecdyonurus using quick-freeze, deep-etch electron microscopy, showing that the loss of the conventional CPC is not only concomitant with the loss of ODA, but also with a substantial modification in the longitudinal distribution of both radial spokes (RS) and inner dynein arms (IDA). Such structures are no longer distributed following the alternation of different repeats as in the 9 + 2 axoneme, but instead share a 32 nm longitudinal repeat: a multiple of the 8 nm repeat observed along the CS wall. Differently from the conventional CPC, the CS and the surrounding RS possess a ninefold symmetry, coherently with the three-dimensional pattern of motility observed in Cloeon free spermatozoa. Biochemical analyses revealed that ultrastructural modifications are concomitant with a reduced complexity of the IDA heavy chain complement. We propose that these structural and molecular modifications might be related to the relief from the evolutionary constraints imposed by the CPC on the basal 9 + 9 + 2 axoneme and could also represent the minimal set compatible with flagellar beating and progressive motility mechanically regulated as suggested by the geometric clutch hypothesis. © 2014 Wiley Periodicals, Inc.

Despite their enormous diversity, spermatozoa are only scarcely studied in arachnids. In spiders, harvestmens and mites, it has been shown that spermatozoa are a potential source of characters which can support, contradict or even resolve previous phylogenetic hypotheses. In scorpions, the spermatozoa of most superfamilies (except the taxon-rich Scorpionoidea and the obscure Chaeriloidea) are known in more or less detail revealing considerable differences, especially between buthid and non-buthid scorpions. However, this apparent diversity is based on descriptions of only 12 scorpion species. In the present study, we investigated the spermatozoa of a species of the superfamily Scorpionoidea (Opistophthalmus penrithorum, Scorpionidae) for the first time by means of light and electron microscopy. The scorpionoid spermatozoa are characterized by (1) an asymmetrical, cap-like acrosomal vacuole at the tip of the anterior pole of the nucleus, (2) a short acrosomal filament, (3) a nucleus, which is tapered anteriorly, (4) a midpiece with 5–6 mitochondria and (5) an axoneme with a 9 + 0 pattern with a helical beating movement. Moreover, we provided a detailed description regarding sperm packages, that consist of approximately 250 sperm cells bent in the midpiece region, and lack a secretion sheath. Overall, the organization and general morphology of spermatozoa of the observed species is similar to what is known for non-buthids. To evaluate the evolutionary and phylogenetic implications of our results, we performed a comparative revision of sperm morphology in scorpions for the first time and proposed potential informative characters (six for spermatozoa and six for sperm packages). We concluded so far that sperm structure in scorpions could provide new characters for future comprehensive phylogenetic studies of the order, and that they could even represent synapomorphies not only for the order but for the major scorpion clades as well.A pesar de su enorme diversidad, la morfología de los espermatozoides ha sido escasamente estudiada en arácnidos. En arañas, opiliones y ácaros, se ha mostrado que los espermatozoides son una fuente potencial de caracteres filogenéticos. En escorpiones, los espermatozoides de la mayoría de las superfamilias (excepto la especiosa superfamilia Scorpionoidea y la enigmática Chaeriloidea) han sido estudiados en más o menos el detalle, y revelan diferencias considerables, sobre todo entre buthidos y no-buthidos. Sin embargo, esta diversidad aparente está basada en descripciones de sólo doce especies de escorpiones. En el presente estudio, investigamos por primera vez los espermatozoides de una especie de la superfamilia Scorpionoidea (Opistophthalmus penrithorum, Scorpionidae) por medio de microscopia optica y electrónica. Los espermatozoides de O. penrithorum están caracterizados por (1) una vacuola acrosómica asimétrica, con forma de gorra en el extremo del polo anterior del núcleo, (2) un filamento acrosomal corto, (3) un núcleo ahusado anteriormente, (4) una pieza media con 5–6 mitocondrias y (5) un axonema con un patrón 9 + 0 y con movimiento pulsátil helicoidal. Además, proporcionamos una descripción detallada sobre los paquetes de esperma, que consisten en aproximadamente 250 espermatozoides arqueados en la región de la pieza media, y carecen de una vaina de secreción. En general, la organización y la morfología general del espermatozoide de la especie observada son similares a lo que ha sido reportado para escorpiones no-buthidos. Para evaluar las implicaciones evolutivas y filogenéticas de nuestros resultados, realizamos la primera de la literatura comparada sobre la morfologia del esperma de escorpiones y proponemos caracteres informativos potenciales (seis para el espermatozoide y seis para paquetes de esperma). Concluimos que la estructura del esperma en escorpiones podría proporcionar nuevos caracteres morfológicos para futuros estudios filogenéticos a nivel del orden, y que los mismos podrían representar no sólo sinapomorfías para el orden, sino también para los principales clados de escorpiones.

Thrips spermiogenesis is characterized by unusual features in the differentiating spermatid cells. Three centrioles from which three individual short flagella are initially assembled, make the early spermatid a tri-flagellated cell. Successively, during spermatid maturation, the three basal bodies maintain a position close to the most anterior end of the elongating nucleus, so that the three axonemes are progressively incorporated in the spermatid cytoplasm, where they run in parallel to the main nuclear axis. Finally, the three axonemes amalgamate to form a microtubular bundle. The process starts with the formation of rifts at three specific points in each axonemal circumference, corresponding to sites 1,3,7 and leads to the formation of 9 microtubular rows of different length, i.e. 3 “dyads”, 3 “triads” and 3 “tetrads”. In the spermatozoon, the nucleus, the mitochondrion and the bundle of microtubules are arranged in a helicoidal pattern. The elongation of the spermatozoon is allowed by the deep anchorage of the spermatid to the cyst cell through a dense mass of material which, at the end of spermiogenesis, becomes a long anterior cylindrical structure. This bizarre “axoneme” does not show any trace of progressive movement but it is able to beat. According to the presence of dynein arms, sliding can take place only within each row and not between the rows. The possible molecular basis underlying the peculiar instability of thrips axonemes is discussed in light of the present knowledge on the organization of the axoneme in mutant organisms carrying alterations of the tubulin molecule. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc.

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 α-tubulin subunit, (2) a high level of glutamylation in the β-subunit, (3) an extremely low total extent of glycylation, with regard to both monoglycylated and polyglycylated sites, either in α- or in β-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. Cell Motil. Cytoskeleton 58:160–174, 2004. © 2004 Wiley-Liss, Inc.

The peculiar sperm axoneme of the dipteran Asphondylia ruebsaameni is characterized by an extraordinarily high number of microtubule doublets (up to 2,500) arranged in double parallel spirals. Doublets of the inner row of each spiral are tilted, so that their outer arms point towards the B-tubule of the next doublet in the outer row. Doublets are provided with only the outer arm, and no structure related to the central pair/radial spoke complex is present. When analyzed by quick-freeze, deep-etch electron microscopy, the structure of the dynein arms was shown to share the same organization described in other organisms; however, it appears to be somewhat more complex than that previously found in a related dipteran species,Monarthropalpus flavus, since the foot region of the arms displays a globular extra-domain that is intercalated between adjacent arms. Treatment of demembranated sperm with ATP and vanadate induced conformational changes in the dynein arms. SDS-page suggested the presence of a single dynein high molecular weight band or, in the gels with the best electrophoretic resolution, of two very closely spaced bands. This polypeptide positively reacted with a polyclonal antibody raised against a specific amino acid sequence located in the phosphate-binding loop of the dynein catalytic site. Dynein heavy chain-related DNA sequences corresponding to the catalytic phosphate-binding region were amplified by RT-PCR. Two distinct fragments (Asph-ax1 and Asph-ax2) encoding axonemal dynein sequences were identified. Southern blot analysis performed on genomic DNA using these sequences as a probe showed that they are part of different genes. An intron was identified in the Asph-ax1 fragment at a position corresponding to the site of a nucleotide deletion in the putative pseudogene of Monarthropalpus. Asphondylia spermatozoa exhibited in vivo a whirling movement both in the deferent duct and in the spermatheca, but they were unable to undergo processive movement in vitro. They propagated a three-dimensional wave only when constrained in a bent configuration by some mechanical means. The phylogenetic relationships between the two dipteran species, Monarthopalpus and Asphondylia, based on these biochemical and molecular data are also discussed. Cell Motil. Cytoskeleton 50:129–146, 2001. © 2001 Wiley-Liss, Inc.

Various aspects of the reproductive anatomy of the spider crab Inachus phalangium are investigated utilizing light and electron microscopy. Spermatozoal ultrastructure reveals the presence of a glycocalyx in the peripheral region of the periopercular rim, never recorded before in crustacean sperm cells. Sperm cell morphological traits such as semi-lunar acrosome shape, centrally perforate and flat operculum, and absence of a thickened ring, are shared only with Macropodia longirostris, confirming a close phylogenetic relationship of these species and their separation from the other members of the family Majidae. Spermatozoa are transferred to females inside spermatophores of different sizes, but during ejaculate transfer, larger spermatophores might be ruptured by tooth-like structures present on the ejaculatory canal of the male first gonopod, releasing free sperm cells. Such a mechanism could represent the first evidence of a second form of sperm competition in conflict with sperm displacement, the only mechanism of sperm competition known among Brachyura, enabling paternity for both dominant and smaller, non-dominant, males. In addition, we propose several hypotheses concerning the remote and proximal causes of the existence of large seminal receptacles in females of I. phalangium. Among these, genetically diverse progeny, reduction of sexual harassment and phylogenetic retention seem the most plausible, while acquisition of nutrients from seminal fluids, demonstrated in other arthropods, and suggested by previous studies, could be discarded on the basis of the presented data. J. Morphol., 2008. © 2007 Wiley-Liss, Inc.

Centrioles are microtubule-based cylindrical organelles with a 9-fold symmetry. They are essential for axoneme formation in cilia and flagella and for centrosome organization. In the basal hexapods Acerentomon microrhinus, we discovered unusually large centrioles composed of 14 doublet microtubules that serve as templates for cilia and flagella and organize mitotic and meiotic spindles. These observations challenge the long-standing view that centriole symmetry is highly conserved among eukaryotes. Strikingly, daughter centrioles contain a transient cartwheel that is lost after maturation. The length of radial spokes is like that found in 9-fold cartwheels, whereas the diameter of the hub varies according to the dimensions of the centriole cylinder. This suggests that the hub may dictate the master plan for centriole geometry. Finally, the finding that 14-doublet centrioles arise from 9-doublet mothers points to an alternative model for centriole assembly. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc.

The sperm tail of bug insects has 9 + 9 + 2 flagellar axonemes and two mitochondrial derivatives showing two to three crystalline inclusions in their matrix. During spermiogenesis, the axoneme is surrounded by a membrane cistern which, at sperm maturity, reduces to two short cisterns on the opposite sides of the axoneme adhering to the mitochondrial derivatives. Filamentous bridges connect the intertubular material of the axoneme to these cisterns. Such bridges, which represent a peculiar feature of bug insects, are resistant to detergent treatment, whereas part of the intertubular material and the inner content of microtubular doublets are affected by the treatment. After freeze-fracture replicas, at the insertion of the bridges to the cisternal membrane, the P-face of this membrane shows a characteristic ribbon consisting of four rows of 11 ± 1 nm staggered intramembrane particles, 13 ± 2 nm apart along each row. The bridges could be able to maintain the axoneme in the proper position during flagellar beating avoiding distortion affecting sperm motility. J. Morphol. 2009. © 2009 Wiley-Liss, Inc.

The secretory activity of the two branched malpighian tubules (MTs) of the second-instar larva in Aeolothrips intermedius is described. MTs of adult thrips have the typical ultrastructure of excretory epithelium with apical microvilli containing long mitochondria and a rich system of basal membrane infoldings. In the second-instar larva just before pupation, the ultrastructure of MT epithelial cells is dramatically different, and there are numerous huge Golgi systems in the cytoplasm. These cells are involved in an intense secretory activity to produce an electron-dense product which is released into the MTs lumen. This secretion is extruded from the hindgut and used by the second-instar larva to build an elaborate protective cocoon for pupation. Electron-spray-ionization mass spectrometry analysis of the cocoon revealed the presence of a β-N-acetyl-glucosamine, the main component of chitin, which is also present in the cocoons of Neuroptera and some Coleoptera. J. Morphol., 2010. © 2009 Wiley-Liss, Inc.

The peculiar sperm axoneme of the dipteran Asphondylia ruebsaameni is characterized by an extraordinarily high number of microtubule doublets (up to 2,500) arranged in double parallel spirals. Doublets of the inner row of each spiral are tilted, so that their outer arms point towards the B-tubule of the next doublet in the outer row. Doublets are provided with only the outer arm, and no structure related to the central pair/radial spoke complex is present. When analyzed by quick-freeze, deep-etch electron microscopy, the structure of the dynein arms was shown to share the same organization described in other organisms; however, it appears to be somewhat more complex than that previously found in a related dipteran species,Monarthropalpus flavus, since the foot region of the arms displays a globular extra-domain that is intercalated between adjacent arms. Treatment of demembranated sperm with ATP and vanadate induced conformational changes in the dynein arms. SDS-page suggested the presence of a single dynein high molecular weight band or, in the gels with the best electrophoretic resolution, of two very closely spaced bands. This polypeptide positively reacted with a polyclonal antibody raised against a specific amino acid sequence located in the phosphate-binding loop of the dynein catalytic site. Dynein heavy chain-related DNA sequences corresponding to the catalytic phosphate-binding region were amplified by RT-PCR. Two distinct fragments (Asph-ax1 and Asph-ax2) encoding axonemal dynein sequences were identified. Southern blot analysis performed on genomic DNA using these sequences as a probe showed that they are part of different genes. An intron was identified in the Asph-ax1 fragment at a position corresponding to the site of a nucleotide deletion in the putative pseudogene of Monarthropalpus. Asphondylia spermatozoa exhibited in vivo a whirling movement both in the deferent duct and in the spermatheca, but they were unable to undergo processive movement in vitro. They propagated a three-dimensional wave only when constrained in a bent configuration by some mechanical means. The phylogenetic relationships between the two dipteran species, Monarthopalpus and Asphondylia, based on these biochemical and molecular data are also discussed. Cell Motil. Cytoskeleton 50:129–146, 2001. © 2001 Wiley-Liss, Inc.

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.