The sperm ultrastructure of Spermophagus kuesteri Schilsky, 1905 (Chrysomelidae, Bruchinae) and considerations on the relationships of Cucujiformia superfamilies.

The sperm ultrastructure of the bean-weevil Spermophagus kuesteri (Bruchinae) was studied to verify the congruence of the new position of the subfamily within Chrysomelidae. The results indicated a positive answer to the question supporting a close relationship between Chrysomelidae and Curculionidae, a finding confirmed also by molecular data. Moreover, the sperm morphology of Divales cinctus, a member of Melyridae (Cleroidea) allowed to confirm the different sperm organization between members of this superfamily and Phytophaga (Chrysomeloidea + Curculionoidea). While studying the spermiogenesis of S. kuesteri, some sperm cysts showed aberrant cells provided with two flagella in the same plasma membrane. These aberrant sperm could be the result, during early spermiogenesis, of irregular processes involving the canal rings between spermatids.

Ultrastructure of the female reproductive organs of the diving beetle Deronectes moestus incospectus (Leprieur, 1876) (Dytiscidae, Hydroporinae).

We describe the ultrastructure of the female reproductive organs of Deronectes moestus (Dytiscidae Hydroporinae). The long spermathecal duct has a simple epithelium lined internally by a thin cuticle and externally by a thick layer of muscle cells. The wide duct lumen contains electron-dense material, among which remnants of extracellular material are visible. This material consists of tubular structures assembled around sperm bundles previously described in the male deferent ducts. The so-called gland, disposed along the spermathecal duct, is a structure with epithelial cells lined by an irregular cuticle bearing a rich system of microvilli. Many mitochondria are visible in the apical cytoplasm of the epithelial cells, and a few spheroidal bodies are close to the basal nuclei. Since the epithelial ultrastructure of the gland suggests it is involved in fluid uptake from the lumen rather than secretory activity, the term gland, coined by other authors to describe this organ, is inappropriate. The spermatheca is a large structure with a complex epithelium showing secretory and duct-forming cells. The lumen of this organ contains sperm with the distinctive ultrastructural features of those described in the male deferent ducts, namely having a mitochondrial matrix with a small crystallized area and electron-dense dots. Because to its overall organization, the spermatheca of D. moestus can be considered a more integrated organ than those in previously studied hydroporine species.

Morphology of male and female reproductive systems in the ground beetle Apotomus and the peculiar sperm ultrastructure of A. rufus (P. Rossi, 1790) (Coleoptera, Carabidae).

Relatively few studies have focused on evolutionary losses of sexually selected male traits. We use light and electron microscopy to study the male and female reproductive anatomy of Apotomus ground beetles (Coleoptera, Carabidae), a lineage that we reconstruct as likely having lost sperm conjugation, a putative sexually selected trait. We pay particular attention to the structure of the testes and spermatheca. Both of these organs share a strikingly similar shape-consisting of long blind canals arranged into several concentric overlapping rings measuring approximately 18 mm and 19.5 mm in total length, respectively. The similarity of these structures suggests a positive evolutionary correlation between female and male genital organs. Males are characterized by unifollicular testes with numerous germ cysts, which contain 64 sperm cells each, and we record a novel occurrence of sperm cyst "looping", a spermatogenic innovation previously only known from some fruit fly and Tenebrionid beetle sperm. The sperm are very long (about 2.7 mm) and include an extraordinarily long helicoidal acrosome, a short nucleus, and a long flagellum. These findings confirm the structural peculiarity of sperm, testis, and female reproductive tract (FRT) of Apotomus species relative to other ground beetles, which could possibly be the result of shifts in sexual selection.

New Findings on the Sperm Structure of Tenebrionoidea (Insecta, Coleoptera).

The sperm ultrastructure of a few representative species of Tenebrionoidea was studied. Two species belong to the Mordellidae (Mordellistena brevicauda and Hoshihananomia sp.), one species to Oedemeridae (Oedemera nobilis), and one species to Tenebrionidae (Accanthopus velikensis). It is confirmed that Mordellidae are characterized by the lowest number of spermatozoa per cyst (up to 64), a number shared with Ripiphoridae. In contrast, in the two other families, up to 512 spermatozoa per cyst are observed, the same number present, for example, in Tenebrionidae. Also, as in the other more derived families of tenebrionoids studied so far, during spermatogenesis in O. nobilis and A. velikensis, sperm nuclei are regularly distributed in two sets at opposite poles of the cysts. On the contrary, the Mordellidae species do not exhibit this peculiar process. However, during spermiogenesis, the bundles of sperm bend to form a loop in their median region, quite evident in the Hoshihananomia sp., characterized by long sperm. This process, which also occurs in Ripiphoridae, probably enables individuals to produce long sperm without an increase in testicular volume. The sperm looping could be a consequence of the asynchronous growth between cyst size and sperm length. The sperm ultrastructure of the Mordellidae species reveals that they can be differentiated from other Tenebrionoidea based on the shape and size of some sperm components, such as the accessory bodies and the mitochondrial derivatives. They also show an uncommon stiff and immotile posterior flagellar region provided with only accessory tubules. These results contribute to a better knowledge of the phylogenetic relationship of the basal families of the large group of Tenebrionoidea.

The sperm ultrastructure of members of basal Tenebrionoidea (Coleoptera).

The sperm ultrastructure of some beetles of Tenebrionoidea was studied with particular attention to those of the Ripiphoridae, Mordellidae, and Meloidae. These three groups are often thought to form a clade, which is the sister group of the remaining Tenebrionoidea. The testes of the two former families have thinner but longer spermatic cysts containing fewer and longer sperm. Within each cyst all sperm cells have the same orientation, but cross sections showed that the orientation of the axonemes alternate between adjacent cysts, possibly due to the cysts bending on themselves. In both families the sperm has a bilayered acrosome and the flagellum, which shows mitochondrial derivatives starting laterally to the nuclear base, has a typical 9 + 9+2 axoneme with accessory tubules provided with 16 protofilaments in their wall, and well-structured triangular shaped accessory bodies. In Mordellistena sp (Mordellidae) sperm, both mitochondrial derivatives and accessory bodies are somewhat asymmetrical. Moreover, the flagellum shows a very thin and long tail end provided with only accessory tubules. Meloidae species have testes with thicker sperm cysts containing numerous shorter sperm. Within the individual cysts the sperm flagella exhibit an alternating orientation of their axonemes as consequence of a peculiar spermatogenetic process. The flagellar structure is similar to that of the above-mentioned species, but the accessory bodies are not well defined and constituted by fuzzy material. In Mylabris hieracii (Meloidae) sperm, the acrosome is flat with a conspicuous perforatorium and its nucleus has a peculiar quadrangular section. Berberomeloe majalis sperm has a large acrosome with an unusual pentagonal perforatorium. The centriolar structure of Mylabris variabilis shows a complex of dense radial links connecting the microtubular structures to the plasma membrane. These results suggest that Ripiphoridae have a closer relationship with Mordellidae than with Meloidae. These findings are in agreement with results obtained with molecular data.

The sperm ultrastructure of Pytho depressus (Linnaeus, 1767) (Coleoptera, Pythidae).

The sperm ultrastructure of Pytho depressus (Pythidae) is described in this study. The sperm are short cells, about 85-90 µm long, with an acrosome consisting of three layers, a cylindrical nucleus, which at its base has the initial region of two mitochondrial derivatives. The flagellum has two well-developed triangular accessory bodies, and a 9 + 9+2 axonemal pattern with accessory tubules provided with 16 protofilaments in their wall. The structure and shape of the accessory bodies are diagnostic characters within the superfamily. The sperm morphology of P. depressus can be easily distinguished from those of Ripiphoridae, Meloidae and Tenebrionidae. The P. depressus sperm are organized in cysts as in other species of the group but the sperm are not well aligned and show an antiparallel orientation, a feature also observed in other tenebrionids. The phylogenetic implications of the observed sperm features are discussed in the context of comparative sperm ultrastructure of other insect species.

Ultrastructure of spermiogenesis and spermatozoa in Marchalina hellenica (Gennadius) (Hemiptera: Sternorrhyncha, Marchalinidae).

The spermiogenesis, the sperm structure and the sperm motility of Marchalina hellenica (Gennadius) were examined. In the early spermiogenesis a centriolar apparatus was identified, but this structure is not involved in the production of the sperm flagellum. As in other Coccoidea, the flagellar axoneme originates by the activity of the thickened tip of the numerous microtubules surrounding the nuclear anterior region close to the periphery of the cell. This region pushes against a narrow cytoplasmic layer, giving rise to a papilla. In this region a novel structure, consisting of a regular network of thin filaments, arranged orthogonally to the bundle of microtubules, is visible. The sperm flagellum consists of a series of about 260 microtubules, regularly arranged in rings around the axial nucleus. This latter extends in the middle part of the sperm length. As usual in scale insects, sperm form a bundle, which in M. hellenica is composed of 64 sperm cells, surrounded by somatic cyst cells. The sperm bundle has an helicoidal array, with a cap of dense material at its apex, lending the anterior and the posterior region of the sperm bundle with a different structural organization. This difference is responsible of the different speed gradient observed in the helical wave propagating along the sperm bundle.

The sperm ultrastructure and spermiogenesis of Tribolium castaneum (Coleoptera: Tenebrionidae) with evidence of cyst degeneration.

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 of Matsucoccus feytaudi (Insecta, Coccoidea): Can the microtubular bundle be considered as a true flagellum?

In the present work the spermiogenesis and sperm structure of Matsucoccus feytaudi, a primary pest of the maritime pine in southern eastern Europe, is studied. In addition to the already known characteristics of coccid sperm, such as the absence of the acrosome and mitochondria, and the presence of a bundle of microtubules responsible for sperm motility, a peculiar structure from which the microtubule bundle takes origin is described. Such a structure--a short cylinder provided with a central hub surrounded by several microtubules with a dense wall--is regarded as a Microtubule Organizing Centre (MTOC). During spermiogenesis, quartets of fused spermatids are formed; from each spermatid, a bundle of microtubules, generated by the MTOC, projects from the cell surface. Each cell has two centrioles, suggesting the lack of a meiotic process and the occurrence of parthenogenesis. At the end of the spermiogenesis, when the cysts containing bundles of sperm are formed, part of the nuclear material together with the MTOC structure is eliminated. Based on the origin of the microtubular bundle from the MTOC, the nature of the bundle as a flagellum is discussed.

The sperm structure of Cryptocercus punctulatus Scudder (Blattodea) and sperm evolution in Dictyoptera.

Sperm of the dictyopteran key taxon Cryptocercus punctulatus was examined. It has largely maintained a blattodean groundplan condition, with a three-layered acrosome, an elongate nucleus, a single centriole, a conspicuous centriole adjunct material, two connecting bands (=accessory bodies), and a long functional flagellum with a 9+9+2 axoneme provided with accessory tubules with 16 protofilaments and intertubular material. These sperm characters are shared with several other polyneopterans. The sperm of C. punctulatus is very similar to what is found in Periplaneta americana and species of other groups of roaches, including the sperm of Loboptera decipiens described here for the first time. The general sperm organization here described can be assumed for the groundplan of Insecta and Pterygota. The following evolutionary path can be suggested: after the split between Cryptocercidae and the common ancestor of Isoptera, the typical pattern of sperm formation was altered very distinctly, resulting in a duplication or multiplication (Mastotermitidae) of the centrioles. Mastotermes has maintained a certain sperm motility, but with a very unusual apparatus of multiple flagella with a 9+0 axoneme pattern. After the split into Mastotermitidae and the remaining Isoptera, sperm motility was completely abandoned, and different modifications of sperm components occurred, and even the loss of the sperm flagellum.

Ultrastructure of the sperm axoneme and molecular analysis of axonemal dynein in Ephemeroptera (Insecta).

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.

p66Shc regulates vesicle-mediated secretion in mast cells by affecting F-actin dynamics.

The extracellular vesicular compartment has emerged as a novel system of intercellular communication; however, the mechanisms involved in membrane vesicle biogenesis and secretion are as yet unclear. Among immune cells releasing membrane vesicles-mast cells that reside near tissues exposed to the environment-are master modulators of immune responses. Here, we have addressed the role of p66Shc, a novel regulator of mast cell activation and homeostasis, in the dynamic reorganization of the actin cytoskeleton that is associated with morphological changes during secretion. We show that p66Shc is recruited as a complex with the lipid phosphatase SHIP1 to the F-actin skeleton and impairs antigen-dependent cortical F-actin disassembly and membrane ruffling through the inhibition of Vav and paxillin phosphorylation. We also show that in addition to acting as a negative regulator of antigen-dependent mast cell degranulation, p66Shc limits the basal release of granule contents by inhibiting microvesicle budding from the plasma membrane and piecemeal degranulation. These findings identify p66Shc as a critical regulator of actin dynamics in mast cells, providing a basis for understanding the molecular mechanisms involved in vesicle-mediated secretion in these cells.

The spermatodesm of Cloeon dipterum (L.): fine structure and sperm movement.

Sperm bundles of the ephemeropteran Cloeon dipterum (L.) were found in the seminal vesicle. They are apically protected by cyst cells and sperm anterior regions are embedded in a secretion produced by these cells. Cyst cells further degenerate making sperm cells free. The sperm bundles are able to perform a forward progression only when sperm are stick together with their tail posterior tips. On the contrary, when sperm have their tail ends free, they beat but are unable to progress. The functional significance of the sperm bundles is discussed.

The spermatogenesis and the sperm structure of Terebrantia (Thysanoptera, Insecta).

Spermatogenesis and the sperm structure of the terebrantian Aeolothrips intermedius Bagnall are described. Spermatogenesis consists of two mitotic divisions; the second is characterized by the loss of half of the spermatids, which have pyknotic nuclei. Early spermatids have two centrioles, but when spermiogenesis starts, a third centriole is produced. The three basal bodies give rise to three flagella; later these fuse into a single flagellum which contains three 9+0 axonemes. The basal bodies are surrounded by a large amount of centriole adjunct material. During spermiogenesis this material contributes to the shifting of the three axonemes towards the anterior sperm region parallel to the elongating nucleus, and it is transformed into a dense cylinder. In the mature spermatids the three axonemes amalgamate to create a bundle of 27 doublet microtubules. Near the end of spermiogenesis the dense cylinder of the centriole adjunct lies parallel to the nucleus and the axonemes. It ends where the mitochondrion appears at half-sperm length. We confirm that Terebrantia testes have a single sperm cyst; their sperm are characterized by a cylindrical nucleus, three axonemes fused into one, a small mitochondrion and a short cylindrical centriole adjunct which corresponds to the dense body described in a previous work. The acrosome is lacking. At the midpoint of the anterior half of the sperm the outline of the cross-section is bilobed, with the nucleus contained in a pocket evagination of the plasma membrane. These characters are discussed in light of a comparison between Tubulifera and Terebrantia.

The ultrastructure of malpighian tubules and the chemical composition of the cocoon of Aeolothrips intermedius Bagnall (Thysanoptera).

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 beta-N-acetyl-glucosamine, the main component of chitin, which is also present in the cocoons of Neuroptera and some Coleoptera.

Ultrastructural analysis of the aberrant axoneme morphogenesis in thrips (Thysanoptera, Insecta).

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.

Three-dimensional reconstruction of axonemal outer dynein arms in situ by electron tomography.

We present here for the first time a 3D reconstruction of in situ axonemal outer dynein arms. This reconstruction has been obtained by electron tomography applied to a series of tilted images collected from metal replicas of rapidly frozen, cryofractured, and metal-replicated sperm axonemes of the cecidomid dipteran Monarthropalpus flavus. This peculiar axonemal model consists of several microtubular laminae that proved to be particularly suitable for this type of analysis. These laminae are sufficiently planar to allow the visualization of many dynein molecules within the same fracture face, allowing us to recover a significant number of equivalent objects and to improve the signal-to-noise ratio of the reconstruction by applying advanced averaging protocols. The 3D model we obtained showed the following interesting structural features: First, each dynein arm has two head domains that are almost parallel and are obliquely oriented with respect to the longitudinal axis of microtubules. The two heads are therefore positioned at different distances from the surface of the A-tubule. Second, each head domain consists of a series of globular subdomains that are positioned on the same plane. Third, a stalk domain originates as a conical region from the proximal head and ends with a small globular domain that contacts the B-tubule. Fourth, the stem region comprises several globular subdomains and presents two distinct points of anchorage to the surface of the A-tubule. Finally, and most importantly, contrary to what has been observed in isolated dynein molecules adsorbed to flat surfaces, the stalk and the stem domains are not in the same plane as the head.

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.

Helicobacter pylori toxin VacA is transferred to host cells via a novel contact-dependent mechanism.

Helicobacter pylori is the causative agent of peptic ulcer disease. A major virulence factor of H. pylori is VacA, a toxin that causes massive vacuolization of epithelial cell lines in vitro and gastric epithelial erosion in vivo. Although VacA is exported over the outer membrane and is released from the bacteria, a portion of the toxin remains associated with the bacterial surface. We have found surface-associated toxin to be biologically active and spatially organized into distinct toxin-rich domains on the bacterial surface. Upon bacterial contact with host cells, toxin clusters are transferred directly from the bacterial surface to the host cell surface at the bacteria-cell interface, followed by uptake and intoxication. This contact-dependent transfer of VacA represents a cost-efficient route for delivery of VacA and potentially other bacterial effector molecules to target cells.

F-actin dynamics control segregation of the TCR signaling cascade to clustered lipid rafts.

Following ligand binding the TCR segregates to plasma membrane microdomains, termed lipid rafts, characterized by a highly ordered lipid structure favoring partitioning of glycosyl phosphatidyl inositol-linked costimulatory receptors and acylated signaling molecules. Here we show that the inducible association of the TCR and key signaling proteins with lipid rafts is dependent on the actin cytoskeleton through a mechanism involving raft coalescence. Although lipid rafts are required for full activation of the TCR-dependent tyrosine phosphorylation cascade and sustained signaling, triggering of TCR-proximal events, including Fyn activation and a first wave of Vav phosphorylation, is independent of lipid rafts, while a second wave of raft-dependent Vav phosphorylation occurs after raft coalescence, as also supported by the finding that Vav is phosphorylated in response to lipid raft clustering by GM1 aggregation. The constitutive association found between Vav and the CD3zeta chain suggests a model whereby the TCR-associated signaling machinery initiates raft aggregation by promoting F-actin reorganization, which permits full activation of the tyrosine phosphorylation cascade, further reorganization of the actin cytoskeleton and sustained signaling, leading to cell activation.