Proteomic diversification of spermatostyles among six species of whirligig beetles.

Seminal fluid protein composition is complex and commonly assumed to be rapidly divergent due to functional interactions with both sperm and the female reproductive tract (FRT), both of which evolve rapidly. In addition to sperm, seminal fluid may contain structures, such as mating plugs and spermatophores. Here, we investigate the evolutionary diversification of a lesser-known ejaculate structure: the spermatostyle, which has independently arisen in several families of beetles and true bugs. We characterized the spermatostyle proteome, in addition to spermatostyle and FRT morphology, in six species of whirligig beetles (family Gyrinidae). Spermatostyles were enriched for proteolytic enzymes, and assays confirmed they possess proteolytic activity. Sperm-leucylaminopeptidases (S-LAPs) were particularly abundant, and their localization to spermatostyles was confirmed by immunohistochemistry. Although there was evidence for functional conservation of spermatostyle proteomes across species, phylogenetic regressions suggest evolutionary covariation between protein composition and the morphology of both spermatostyles and FRTs. We postulate that S-LAPs (and other proteases) have evolved a novel structural role in spermatostyles and discuss spermatostyles as adaptations for delivering male-derived materials to females.

Structure and Evolution of Insect Sperm: New Interpretations in the Age of Phylogenomics.

This comprehensive review of the structure of sperm in all orders of insects evaluates phylogenetic implications, with the background of a phylogeny based on transcriptomes. Sperm characters strongly support several major branches of the phylogeny of insects-for instance, Cercophora, Dicondylia, and Psocodea-and also different infraordinal groups. Some closely related taxa, such as Trichoptera and Lepidoptera (Amphiesmenoptera), differ greatly in sperm structure. Sperm characters are very conservative in some groups (Heteroptera, Odonata) but highly variable in others, including Zoraptera, a small and morphologically uniform group with a tremendously accelerated rate of sperm evolution. Unusual patterns such as sperm dimorphism, the formation of bundles, or aflagellate and immotile sperm have evolved independently in several groups.

Ultrastructural Visualization of Vaccine Adjuvant Uptake In Vitro and In Vivo.

Adjuvants are substances that enhance adaptive immune responses when formulated in a vaccine. Alum and MF59 are two vaccine adjuvants licensed for human vaccination. Their mode of action has not been completely elucidated. Here we show the first ultrastructural visualization of Alum and MF59 interaction with immune cells in vitro and in vivo. We observed that Alum is engulfed by cells as inclusions of laminae that are detectable within draining lymph nodes. MF59 is instead engulfed by cells in vitro as low-electron-dense lipid-like inclusions that display a vesicle pattern, as confirmed by confocal microscopy using fluorescently labeled MF59. However, lipid-like inclusions with different high- and low-electron-dense content are detected within cells of draining lymph nodes when injecting MF59. As high-electron-dense lipid-like inclusions are also detected upon injection of Alum, our results suggest that the low-electron-dense inclusions are formed by engulfed MF59, whereas the high-electron-dense inclusions are proper lipid inclusions. Thus, we demonstrated that vaccine adjuvants are engulfed as inclusions by lymph node cells and hypothesize that adjuvant treatment may modify lipid metabolism.

Parasitic castration by Xenos vesparum depends on host gender.

Host castration represents a mechanism used by parasites to exploit energy resources from their hosts by interfering with their reproductive development or to extend host lifespan by removing risks associated with reproductive activity. One of the most intriguing groups of parasitic castrators is represented by the insects belonging to the order Strepsiptera. The macroparasite Xenos vesparum can produce dramatic phenotypic alterations in its host, the paper wasp Polistes dominula. Parasitized female wasps have undeveloped ovaries and desert the colony without performing any social task. However, very little attention has been given to the parasitic impact of X. vesparum on the male phenotype. Here, we investigated the effects of this parasite on the sexual behaviour and the morpho-physiology of P. dominula males. We found that, differently from female wasps, parasitized males are not heavily affected by Xenos: they maintain their sexual behaviour and ability to discriminate between female castes. Furthermore, the structure of their reproductive apparatus is not compromised by the parasite. We think that our results, demonstrating that the definition of X. vesparum as a parasitoid does not apply to infected males of P. dominula, provide a new perspective to discuss and maybe reconsider the traditional view of strepsipteran parasites.

The ultrastructure of the spermatheca of Mordellistena brevicauda (Coleoptera, Tenebrionoidea) and the associated bacterial cells.

The ultrastructural study on the female reproductive system of the beetle M. brevicauda (Mordellidae) confirmed the positive correlation between the length of the sperm and the size of the female seminal receptacle (Spermatheca). The spermatheca of the species is characterized by an apical bulb-like structure where the spermathecal duct forms numerous folds filled with sperm. At this level many bacterial cells are present intermingled with the duct folds. Some are organized in large structures, such as bacteriomes, while other are single bacteriocytes. The latter are often found near the basal lamina of duct epithelium. In addition, some bacteria are visible in the cytoplasm of the duct epithelial cells. Interestingly, bacterial cells have never been observed in the duct lumen. The possible function of the bacterial cells is discussed.

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.

The sperm structure of Clinidium canaliculatum (Costa): A contribution to the systematic position of Rhysodidae (Coleoptera: Carabidae).

The systematic position and the phylogenetic relationship of Rhysodidae members is still debated, with some authors considering the group as a separate family of Adephaga, while for others they could be a subfamily of Carabidae. The group have morphological traits quite different from Carabidae and an aberrant behaviour compared to ground beetles being not predaceous. The sperm ultrastructure of C. canaliculatum was studied comparatively with other species of beetles, Carabidae in particular. The results indicate that the sperm structure of this species is similar to that of the Carabinae species. As in these species, C. canaliculatum has sperm conjugates with an apical conical cap protecting the heads and the initial region of flagella. This sperm appearance is also shared by another species of Rhysodidae, Omoglymmius hamatus. The material of the apical cap consists of an electron-dense material with a peculiar outer net configuration. Many species of Carabidae, however, can present a different type of sperm conjugation, the spermatostyle: a long rod-like structure where the individual sperms have only the most apical part inserted in the cortical area and the flagella are completely free. C. canaliculatum sperm are endowed with a mono-layered acrosome, a nucleus of variable shape along its length, a flagellum consisting of a typical axoneme 9 + 9+2, provided with 16 protofilaments in the tubular wall of accessory tubules, two asymmetric mitochondrial derivatives with the left one larger than the opposite one, and the right accessory body elongated and larger than the opposite one. These sperm characteristics, which are shared also by another member of the group, suggest the demotion of the family Rhysodidae to the subfamily Rhysodinae within Carabidae, a result also supported by recent molecular data.

Sperm morphology of Tingidae Laporte, 1833 (Miroidea: Cimicomorpha).

Here, we describe for the first time the sperm morphology of Tingidae (Heteroptera). They are small insects presenting lacy patterns on their pronotum and hemielytra and are exclusively phytophagous, with many economically important species. We studied five species of the tribe Tingini (Tinginae): Teleonemia scrupulosa, Vatiga illudens, Gargaphia lunulata, Leptopharsa sp., and Corythucha arcuata. Their spermiogenesis process is similar to other Heteroptera, with some differences in the formation of the centriole adjunct. This structure extends in the anteroposterior spermatid axis, flanking the nucleus, possibly contributing to nucleus remodeling and sperm elongation. The mature sperm of Tingidae is also similar to that of other Heteroptera, with features that corroborate the group's monophyly. Our data support previous results for their sister family, Miridae, which exhibits some characteristics exclusive to this taxon, not present in Tingidae or other Heteroptera. They also support the sister relationship of the genera Gargaphia and Leptopharsa and suggest closer relationship between Vatiga and Corythucha. Overall, this study sheds light on the sperm ultrastructure of Tingidae and provides information for understanding the evolution and diversity of Heteroptera. RESEARCH HIGHLIGHTS: The spermiogenesis process and mature sperm are similar to other Heteroptera The centriole adjunct is derived from a strip of a pericentriolar material extending from the centriole Tingidae and Miridae are distinguishable using sperm morphology.

Zorotypus in Peninsular Malaysia (Zoraptera: Zorotypidae), with the description of three new species.

Three new species of the uncommonly encountered insect order Zoraptera are described and figured from Peninsular Malaysia--Zorotypus magnicaudelli sp. n., Zorotypus cervicornis sp. n., and Zorotypus impolitus sp. n. Another species from the region, identified as Zorotypus caudelli Karny, 1927, was also collected and is reevaluated herein based on new material. A brief discussion of characters used in zorapteran systematics is provided, and a key to the species of Peninsular Malaysia provided. This is the first report for the order Zoraptera from Peninsular Malaysia.

The sperm structure of the diving beetles Stictonectes optatus (Seidlitz, 1887) and Scarodytes halensis (Fabricius, 1787) (Dytiscidae, Hydroporinae) with evidence of a spermatostyle in the sperm conjugation.

The structure of the male genital organs and spermiogenesis of two diving beetles, Stictonectes optatus and Scarodytes halensis were studied for the first time. S. optatus shows unifollicular testes consisting of a long tubule apically forming a globular structure. The deferent duct epithelia show a secretory activity involved in the spermatostyle organization. They are connected with two very large accessory glands. Sc. halensis has a more common structure of the male genital apparatus with unifollicular cylindrical testes and very long deferent ducts. Sc. halensis accessory glands are smaller than those of S. optatus. The sperm structure in both species is characterized by a small acrosome, a flattened nucleus with a lateral extension containing a centriole from which a long flagellum originates. Both species exhibit sperm conjugation with long sperm bundles showing nuclei orderly arranged in sperm-heads stacks and free flagella. In addition, S. optatus has a thick layer of secretion surrounds these sperm-head stacks. Such a secretion is considered a spermatostyle. This finding represents the first record about the presence of this structure among Dytiscidae. In the flagellum, a typical axoneme with a 9 + 9 + 2 microtubular complex, and two mitochondrial derivatives are present in both species. Those of S. optatus have a peculiar shape with the apical side, in cross-section, displaying pointed corners. Two small accessory bodies are located between the axoneme and the two mitochondrial derivatives.

Sperm structure of the diving beetle Deronectes moestus incospectus (Leprieur, 1876) (Hydroporinae, Dytiscidae) and considerations on extracellular material surrounding sperm bundles.

The sperm cells of the diving beetle Deronectes moestus incospectus are characterized by sperm conjugation leading to the formation of sperm bundles of 64 units each. These bundles are formed at the end of spermatocyte cell divisions occurring in the testes and can be detected in the anterior region of the deferent ducts (first type of sperm conjugation). Fusions of some sperm bundles can occur at the end of the deferent ducts. The sperm bundles show sperm-head stacks (sperm rouleaux) and are surrounded by a cup of extracellular material secreted by the epithelial cells of the deferent ducts. This material extends posteriorly around the sperm bundle to cover the nuclei and the initial region of the sperm flagella. The cup extracellular material consists of fine tubules, and is no longer visible in sperm bundles at the posterior end of the deferent ducts. The sperm cells of D. moestus incospectus have an axoneme with a 9 + 9 + 2 pattern and unusual mitochondrial derivatives having a matrix showing dense dots and a small crystallized domain. Two thin elongated accessory bodies are located between the mitochondrial derivatives and the axoneme. The extracellular material can have different morphologies in the various families of Adephaga, but all are produced by the epithelium of the deferent ducts. Thus it is reasonable to assume that it has the same function in the different groups.

Fine structure of the female genital system of diving beetle Stictonectes optatus (Seidlitz, 1887) (Dytiscidae-Hydroporinae) and evidence of mating plug formation.

The general organization of the female genital system of the diving beetle Stictonectes optatus was studied, clarifying the complex structure of the spermatheca and spermathecal gland. The two structures adhere closely to each other, sharing a small area of their cuticular epithelium. A long duct connects the bursa copulatrix to the spermatheca, where the sperm are stored. The sperm reach the common oviduct, where egg fertilization occurs, via a fertilization duct. The spermathecal gland cells have extracellular cisterns where secretions are stored. Thin ducts composed of duct-forming cells transport these secretions to the apical gland region and into the spermathecal lumen. Soon after mating, the bursa copulatrix is almost completely occupied by a plug secreted by the male accessory glands. The secretions of the bursa epithelium seem to contribute to plug formation. Later this plug becomes large and spherical, obstructing the bursa copulatrix.

The Structure of the Female Genital System of the Diving Beetle Scarodytes halensis (Fabricius, 1787) (Hydroporinae, Dytiscidae), and the Organization of the Spermatheca and the Spermathecal Gland Complex.

The fine structure of the female reproductive organs of the diving beetle Scarodytes halensis has been described, with particular attention to the complex organization of the spermatheca and the spermathecal gland. These organs are fused in a single structure whose epithelium is involved in a quite different activity. The secretory cells of the spermathecal gland have a large extracellular cistern with secretions; duct-forming cells, by their efferent duct, transport the secretions up to the apical cell region where they are discharged into the gland lumen. On the contrary, the spermatheca, filled with sperm, has a quite simple epithelium, apparently not involved in secretory activity. The ultrastructure of the spermatheca is almost identical to that described in a closely related species Stictonectes optatus. Sc. halensis has a long spermathecal duct connecting the bursa copulatrix to the spermatheca-spermathecal gland complex. This duct has a thick outer layer of muscle cells. Through muscle contractions, sperm can be pushed forwarding up to the complex of the two organs. A short fertilization duct allows sperm to reach the common oviduct where eggs will be fertilized. The different organization of the genital systems of Sc. halensis and S. optatus might be related to a different reproductive strategy of the two species.

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.

Applying Satyrization to Insect Pest Control: The Case of the Spotted Wing Drosophila, Drosophila suzukii Matsumura.

Drosophila suzukii represents one of the major agricultural pests worldwide. The identification of safety and long-lasting tools to suppress its populations is therefore crucial to mitigate the environmental and economic damages due to its occurrence. Here, we explore the possibility of using satyrization as a tool to control the abundance of D. suzukii. By using males of D. melanogaster, we realized courtship tests, spermathecae analysis, and multiple-choice experiments to assess the occurrence and extent of pre- and post-zygotic isolation between the two species, as well as the occurrence of fitness costs in D. suzukii females due to satyrization. Our results showed that: (i) D. melanogaster males successfully courted D. suzukii females; (ii) D. melanogaster males significantly affected the total courtship time of D. suzukii males, which reduced from 22.6% to 6.4%; (iii) D. melanogaster males were able to inseminate D. suzukii and reduce their offspring, inducing a high fitness cost. Reproductive interference occurs at different steps between D. melanogaster and D. suzukii, both alone and in combination with other area-wide control approaches.

Ofd1 is required in limb bud patterning and endochondral bone development.

Oral-facial-digital type I (OFDI) syndrome is an X-linked male lethal developmental disorder. It is ascribed to ciliary dysfunction and characterized by malformation of the face, oral cavity, and digits. Conditional inactivation using different Cre lines allowed us to study the role of the Ofd1 transcript in limb development. Immunofluorescence and ultrastructural studies showed that Ofd1 is necessary for correct ciliogenesis in the limb bud but not for cilia outgrowth, in contrast to what was previously shown for the embryonic node. Mutants with mesenchymal Ofd1 inactivation display severe polydactyly with loss of antero-posterior (A/P) digit patterning and shortened long bones. Loss of digit identity was found to be associated with a progressive loss of Shh signaling and an impaired processing of Gli3, whereas defects in limb outgrowth were due to defective Ihh signaling and to mineralization defects during endochondral bone formation. Our data demonstrate that Ofd1 plays a role in regulating digit number and identity during limb and skeletal patterning increasing insight on the functional role of primary cilia during development.

Isomin: a novel cytoplasmic intermediate filament protein from an arthropod species.

BACKGROUND: The expression of intermediate filaments (IFs) is a hallmark feature of metazoan cells. IFs play a central role in cell organization and function, acting mainly as structural stress-absorbing elements. There is growing evidence to suggest that these cytoskeletal elements are also involved in the integration of signalling networks. According to their fundamental functions, IFs show a widespread phylogenetic expression, from simple diblastic animals up to mammals, and their constituent proteins share the same molecular organization in all species so far analysed. Arthropods represent a major exception in this scenario. Only lamins, the nuclear IF proteins, have so far been identified in the model organisms analysed; on this basis, it has been considered that arthropods do not express cytoplasmic IFs. RESULTS: Here, we report the first evidence for the expression of a cytoplasmic IF protein in an arthropod - the basal hexapod Isotomurus maculatus. This new protein, we named it isomin, is a component of the intestinal terminal web and shares with IFs typical biochemical properties, molecular features and reassembly capability. Sequence analysis indicates that isomin is mostly related to the Intermediate Filament protein C (IFC) subfamily of Caenorhabditis elegans IF proteins, which are molecular constituents of the nematode intestinal terminal web. This finding is coherent with, and provides further support to, the most recent phylogenetic views of arthropod ancestry. Interestingly, the coil 1a domain of isomin appears to have been influenced by a substantial molecular drift and only the aminoterminal part of this domain, containing the so-called helix initiation motif, has been conserved. CONCLUSIONS: Our results set a new basis for the analysis of IF protein evolution during arthropod phylogeny. In the light of this new information, the statement that the arthropod phylum lacks cytoplasmic IFs is no longer tenable.See commentary article: http://www.biomedcentral.com/1741-7007-9-16.

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.

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.