Substructure of the axoneme of pterygote insect spermatozoa: Phylogenetic considerations

Spermatozoa from a great number of insect species were fixed in a tannic acid-containing fixative and the ultrastructure of the flagellar axoneme was examined in a search for apomorphies. Most of the examined species, representing a majority of insect orders. have accessory tubules outside the axoneme (hence a 9 + 9 + 2 pattern), and these consist of 16 protofilaments. Some important apomorphies concern the number of protofilaments in the accessory tubules: 13 (plus 7 inner elements) in Ephemeroptera, 13 in the (elliptic) tubules of Psocoptera + Anoplura + Mallophaga (thus a synapomorphy), 13 in Tipulidae + Brachycera, 15 in the dipteran families Dixidae + Chironomidae (with a 9 + 9 + 2 axoneme) and Culicidae + Bibionidae (with a 9 + 9 + “1” axoneme), 17 in Phasmatodea, and 17–20 in Trichoptera. Other apomorphies concern the appearance of the so-called intertubular material outside the microtubular doublets, the appearance of the interior of the various microtubules, and the loss, in some taxa, of outer or inner dynein arms of both dynein arms. In some cases, the flagellum is completely abnormal; the sperm tail of Thysanoptera, for example, consists of 27 elements of 3 different kinds. The different taxa within orders Diptera and Trichoptera have sperm tail axonemes of different appearances, where those from other orders have a rather uniform appearance. The conclusions that can be drawn from this spermatological study, generally agree with data from classical studies, except with some variations, in some cases.     

Characteristics of the sperm flagellum in fungus gnats (Insecta,Diptera, Mycetophiloidea)

Spermatozoa from several members of the closely related Mycetophilidae and Keroplatidae were examined by electron microscopy using a fixative that contains glutaraldehyde and tannic acid, followed by a post-fixative that consists of uranyl acetate rather than osmium tetroxide. With this fixative, the detailed architecture of the flagellar axoneme and its various microtubules could be resolved. The so-called accessory tubules, which surround the central 9+2 unit of the sperm axoneme, were found to have 16 protofilaments in several examined Mycetophiloidea, but in no other Diptera. As 16 is the common number in holometabolic insects, it is presumably the plesiomorphic condition in Diptera. Other fungus gnats have accessory tubules with 15 or 14 protofilaments. The intertubular material situated between the accessory tubules is smaller in the examined members of the Mycetophilidae than in the Keroplatidae. The acrosome consists of an apical vesicle, which in one species, Macrorhyncha ancae, has three microtubular doublets in its anterior part and two large and three small extensions which extend posteriorly along the sperm axoneme.     

Sperm structure of Limoniidae and their phylogenetic relationship with Tipulidae (Diptera, Nematocera)

The sperm ultrastructure of a few species of Limoniidae (Limonia nigropunctata; L. nubeculosa; Chionea n. sp.; C. alpina; C. lutescens) was studied. The two species of Limonia have a monolayered acrosome with crystallized material, a three-lobed nucleus in cross section, a ring of centriole adjunct material and a flagellum which consists of a 9+9+1 axoneme and a single mitochondrial derivative. The central axonemal tubule is provided with 15 protofilaments in its tubular wall, while the accessory tubules have 13 protofilaments and are flanked by the electron-dense intertubular material. The three species of Chionea share a monolayered acrosome, a nucleus with two longitudinal grooves, a centriole adjunct material which surrounds the centriole and the initial part of the axoneme. The axoneme is of conventional type, with 9+9+2 microtubular pattern, with accessory tubules provided with 13 protofilaments and intertubular material. However, in C. lutescens the accessory tubules start with 15 protofilaments and transform into a tubule with 13 protofilaments. These data are discussed in the light of the phylogenetic relationship between Limoniidae and Tipulidae. For this purpose, the sperm ultrastructure of Nephrotoma appendiculata was also considered comparatively.     

New findings on sperm ultrastructure of Xenos vesparum (Rossi) (Strepsiptera,Insecta)

The systematic position of insect order Strepsiptera is still under debate. It was, therefore, thought of interest to examine the ultrastructure of a strepsipteran in a search for synapomorphies shared with Coleoptera, Diptera, or any other insect order. The fine structure of spermatozoa and the spermatid from Xenos vesparum (Rossi) was re-examined using scanning and transmission electron microscopy and a fixation technique that permits the visualization of the macromolecular organization of the organelles. The spermatozoon was shown to possess several traits that are characteristics of insects in general, such as a 9 + 9 + 2 axoneme, two mitochondrial derivatives containing a crystalline material and two 'zipper lines' present along the sperm tail. Seventeen protofilaments occurred along most of the accessory tubules, which reduced to 16 posteriorly. An acrosome is absent. The neck region contains a prominent centriolar adjunct, which gives rise to two accessory bodies which adhere to the mitochondrial derivatives, and to slender strands of the so-called intertubular material found between the accessory tubules. Of interest is the finding that the glycocalyx consists of prominent filamentous strands, similar to those found in siphonapterans, mecopterans and basal dipterans.     

Sperm structure of Trichoptera. I. Integripalpia : Limnephiloidea

Spermal ultrastructure in 16 caddisflies (Trichoptera) belonging to the suborder Integripalpia, superfamily Limnephiloidea, was examined in a search for apomorphic and plesiomorphic features. In all species examined, the sperm tail axoneme was of the 9 + 9 + 2 + type, the axonemal doublets lacked outer dynein arms but had inner ones, and the cell membrane was scalloped with a prominent glycocalyx. The number of protofilaments in the accessory tubules depended on the phylogenetic position: 18 in the family Leptoceridae, 19 in Limnephilidae, Goeridae and Odontoceridae (with a reduction in the number distally), and 20 in Sericostomatidae. Spermatozoa in Leptoccridae are further characterized by the 2 central microtubules being flattened and eccentric and not being surrounded by a central sheath. Spermatozoa of Sericostomatidae have an accessory body and a helicoidal array of the sperm tail.     

The mushroom bodies of the lower nematocera: A link between those of the higher diptera and other mecopteroids

Nematoceran Diptera are nonuniform in the structure of their mushroom bodies. Members of the more basal families (Ptychopteridae, Pediciidae, and Tipulidae) have bipartite mushroom bodies, characteristic of members of the other mecopteroid complex orders. In members of Bibionomorpha (Bibionidae and Anisopodidae), tripartite mushroom bodies have been found characteristic of Brachycera Orthorrhapha.     

Sperm structure of Trichoptera. IV. Rhyacophilidae and Glossosomatidae

The families Rhyacophilidae and Glossosomatidae (Trichoptera) are considered to be the most primitive ones within the order. We examined the spermatozoa of members of these families to see whether their ultrastructure is consistent with an ancestral position. Axonemal structures, after fixation with a tannic acid-containing fixative, have been shown to be particularly useful as taxonomical indicators. It was found that 4 members of Rhyacophilidae, representing 3 subgenera (Rhyacophila, Pararhyacophila, and Hyporhyacophila) all have motile spermatozoa, with a 9 + 9 + 2 axoneme in which inner (but no outer) dynein arms are present. The accessory tubules have a wall consisting of 17 protofilaments, decreasing to 16 near the distal end, whereas the examined member of Glossosomatidae, Catagapetus nigrans, has accessory tubules with 18 protofilaments and a 9 + 9 + 2 axoneme with inner dynein arms and with motility similar to the Rhyacophilidae. Sperm motility is consistent with the inclusion of these 2 families within the suborder Integripalpia, and the axonemal pattern 9 + 9 + 2 indicates that the families indeed occupy a primitive position within Trichoptera.     

Spermatozoa from the supertribes Lasiopteridi and Stomatosematidi (Insecta, Diptera, Cecidomyiidae): ultrastructure data and phylogeny of the subfamily Cecidomyiinae

Spermatozoa from seven gall-midge species, representing the supertribes Lasiopteridi and Stomatosematidi, have been examined by electron microscopy and compared to the spermatozoa of 30 previously examined gall-midge species of the subfamily Cecidomyiinae. Derived characteristics of all examined representatives of this subfamily are the lack of acrosome and the absence of accessory tubules in their axoneme. The sperm axoneme of Didactylomyia has a unique '9 + 5 pattern', i.e. with 9 outer doublets and 5 inner singlet microtubules. Unlike other members of Cecidomyiinae, Didactylomyia has retained some plesiomorphic traits, namely crystal containing mitochondria and a homogeneously condensed nucleus. Stomatosema has a 10 + 0 axoneme and an irregular nuclear condensation. Thus, Didactylomyia may serve as an useful outgroup for phylogenetic analysis within the subfamily Cecidomyiinae. The supertribe Stomatosematidi is a paraphyletic assemblage. Nearly all members of Lasiopteridi, Ozirhincus, Arnoldiola, Brachineura and Ledomyia , have a synapomorphic character in the great number of microtubules in parallel to the axoneme. A notable exception is Rhizomyia , which shares certain apomorphic traits with several genera that have previously been classified within the Cecidomyiidi, i.e. Contarinia, Allocontarinia, Lestodiplosis and Myricomyia. Moreover, these genera have a flat sperm tail and microtubular doublets located close to the cell membrane, both characteristics that differ markedly from the very peculiar sperm tail found in other members of Cecidomyiidi.
Cladistic analysis of sperm characters suggests that the supertribe Cecidomyiidi is a polyphyletic taxon and supports the existence of a sister-group relationship between the supertribe Lasiopteridi and the genera Rhizomyia, Contarinia, Allocontarinia, Lestodiplosis and Myricomyia.     

Sperm Axoneme in Some Apterygote Insects Examined by Computer-aided Image Analysis

Sperm tail axonemes from three insect species, representing the three apterygote orders Diplura, Archeognatha and Zygentoma, have been examined by high resolution electron microscopy. The samples were fixed in a mixture of glutaraldehyde and tannic acid followed by uranyl acetate post-fixation and the electron micrographs were subjected to computer analysis in order better to characterize the minute details of the axonemal microtubules. The dipluran Campodea was seen to have a row of accessory microtubules composed of 13 protofilaments, the archeognath Machilinus to have two rows of accessory microtubules composed of 16 protofilaments. and the zygentoman Lepismodes to have accessory microtubules in contact with the flagellar doublets and with a wall containing 16 protofilaments and an electron-dense lumen. The axonemal structure in Zygentoma is the one most similar to those of the pterygote insects. The protofilaments of the axonemal doublets were seen to have a somewhat widened interspace between some pairs of protofilaments and these ‘gaps’ coincided with the location of the basal parts of the outer dynein arm, the inner dynein arm, the spoke, and the intertubular material. It hence seems that these microtubule-associated structures influence the architecture of the microtubular wall.     

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

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

Accessory tubules and axonemal microtubules of Apis mellifera sperm flagellum differ in their tubulin isoform content

In the insect sperm flagellum, an extra set of nine additional microtubules, named accessory tubules, is present surrounding the axoneme. Using a sarcosyl/urea extraction, we were able to fractionate the microtubular cytoskeleton of the sperm flagellum of the insect Apis mellifera resulting in the dissociation of the axonemal microtubule protein components and the accessory tubules. This has allowed us to compare the tubulin isoform content of axonemal microtubules and accessory tubules by immunoelectron microscopy and immunoblotting using a panel of monoclonal antibodies directed against different tubulin post-translational modifications (PTMs). All the PTMs occurring in axonemal tubulin are also present in accessory tubules, which indicates the close relativeness of accessory tubules to axonemal rather than to cytoplasmic microtubules. However, our results demonstrate the presence of significant differences in the tubulin isoform content of axonemal microtubules and accessory tubules. First, the tubulin tyrosination extent of accessory tubules is far lower than that of axonemal microtubules, thus confirming at the molecular level their morphogenetic origin as outgrowths from the B-subtubule of each microtubular doublet. Second, although polyglycylation seems to occurr at the same extent in both microtubular systems, alpha-tubulin exhibits a larger amount of monoglycylated sites in axonemal microtubules than in accessory tubules. Third, a greater amount of beta-tubulin molecules is glutamylated in axonemal microtubules than in accessory tubules. Moreover, highly acidic isoforms, likely molecules with longer polyglutamate side chains, are present only in axonemal microtubules. Taken together, our data are indicative of a higher level of tubulin heterogeneity in axonemal microtubules than in accessory tubules. They also show a segregation of post-translationally modified isoforms between accessory tubules and axonemal microtubules and suggest the implication of PTMs in the functional specialization of the two microtubular systems.     

Characteristics of the flagellar axoneme in Neuroptera,Coleoptera, and Strepsiptera

Spermatozoa from representatives of the five insect orders in superorder Neuropteroidea were examined by electron microscopy following a new fixation method that includes tannic acid in the primary fixative but has uranyl acetate rather than osmium tetroxide as the secondary fixative. The sperm axoneme was found to be similar in the four orders Megaloptera, Raphidioptera, Neuroptera, and Coleoptera, and is characterized above all by its so-called intertubular material being divided into two portions, one located outside, but in contact with the doublet, and the other projecting from the accessory tubule and having a beak-like shape. These features have not been seen in insects from other orders and may be a synapomorphy for these neuropteroid orders. The accessory tubules in these four orders have 16 protofilaments. The shape of the accessory bodies adjacent to the mitochondrial derivatives is nearly the same in insects from the more primitive neuropteroid orders and in Coleoptera. The sperm tail of the examined strepsipteran deviates in several respects from that of other neuropteroids: the particle row in the wall of accessory tubules is incomplete, an intertubular material is missing, and the mitochondria contain no crystal. © 1994 Wiley-Liss, Inc.     

Phytogeny of the nematocerous families of Diptera (insecta)

The relationships of the nematocerous families of Diptera are cladistieally analysed using the parsimony programs PAUP and Hennig86. An extensive review, as well as a data matrix, is presented for 98 almost exclusively morphological characters (larva, 56; pupa, 6; adult, 36). Four infraorders are recognized, viz , Ptychopteromorpha, Culicomorpha, Blephariceromorpha, Bibionomorpha, and a clade containing the 'higher Nematocera' and Brachycera. Traditionally the family Nymphomyiidae or the infraorder Tipulomorpha (=Tipulidae, with or without Trichoceridae) are considered the most basal clade of the extant Diptera. On the basis of our cladistic analysis it is suggested that the Ptychopteromorpha-Culicomorpha clade is the sister-group of all other extant Diptera. We provide evidence that the Axymyiidae are part of a monophyletic Bibionomorpha. The latter infraorder is proposed as the sister-group of the higher Nematocera and Brachycera. We transfer the Tipulidae (Tipulomorpha) to the higher Nematocera, at a position next to Trichoceridae and near the Anisopodidae-Brachycera lineage. Previous hypotheses concerning nematocerous relationships are reviewed.     

The male postabdomen and reproductive system of Bibio marci Linnaeus, 1758 (Hexapoda: Diptera: Bibionidae)

The male postabdomen and the internal parts of the male genital system of Bibio marci (Bibionomorpha) were examined and reconstructed 3‐dimensionally. Several features differ from the presumptive dipteran groundplan. The bases of the gonopods are fused with each other and with tergite IX. The penis is not tube‐shaped and only sclerotized on the ventral side. The vasa deferentia are S‐shaped, and two pairs of accessory glands are present. In contrast to these characteristics, the arrangement of the internal parts is probably close to the ancestral condition. With its specific shape, the penis is well suited for the transfer of a spermatophore. The dorsal sclerite of the copulatory organ probably represents the medially fused parameres. A cladistic analysis of 27 characters of the postabdomen yielded two most parsimonious trees, with the strict consensus as follows: Nannochoristidae (outgroup) + (Culicidae [Culicomorpha] + ((Nymphomyiidae + (Tipulidae + Trichoceridae)) + (Tabanidae [Brachycera] + (Bibionidae, Anisopodidae, Axymyiidae [Bibionomorpha])))). Potential synapomorphies of Bibionomorpha (including Axymyiidae) and Brachycera are the fusion of sternum IX with the gonocoxites, the fusion of the parameres forming the dorsal sclerite and the presence of an entire series of postabdominal muscles (M4, M20, M23, M26, M27, M31, M35 and M37). The results of the analysis are preliminary as it is based on a single‐character system with a limited taxon sampling. However, the main result – a clade Bibionomorpha + Brachycera – is fully compatible with current hypotheses on dipteran phylogeny.     

Sperm ultrastructure and spermiogenesis of Coniopterygidae (Neuroptera, Insecta)

The spermiogenesis and the sperm ultrastructure of several species of Coniopterygidae have been examined. The spermatozoa consist of a three-layered acrosome, an elongated elliptical nucleus, a long flagellum provided with a 9+9+3 axoneme and two mitochondrial derivatives. No accessory bodies were observed. The axoneme exhibits accessory microtubules provided with 13, rather than 16, protofilaments in their tubular wall; the intertubular material is reduced and distributed differently from that observed in other Neuropterida. Sperm axoneme organization supports the isolated position of the family previously proposed on the basis of morphological data.     

The sperm structure of Embioptera (Insecta) and phylogenetic considerations

The sperm structure of two species of Embioptera, Embia savignyi Westwood 1837 and Aposthonia japonica (Okajima 1926), was studied. Spermatozoa of both species exhibit a monolayered acrosome and a layer of material surrounding the sperm cells for most of their length. The presence of a 9+9+2 axoneme provided with accessory microtubules with 16 protofilaments, two accessory bodies and two crystallized mitochondrial derivatives are characters shared with other polyneopteran taxa. The supposed close relationship between Embioptera and Phasmatodea is not supported by characters of the sperm ultrastructure.     

Axoneme-dependent tubulin modifications in singlet microtubules of the Drosophila sperm tail

Drosophila melanogaster sperm tubulins are posttranslationally glutamylated and glycylated. We show here that axonemes are the substrate for these tubulin C-terminal modifications. Axoneme architecture is required, but full length, motile axonemes are not necessary. Tubulin glutamylation occurs during or shortly after assembly into the axoneme; only glutamylated tubulins are glycylated. Tubulins in other testis microtubules are not modified. Only a small subset of total Drosophila sperm axoneme tubulins have these modifications. Biochemical fractionation of Drosophila sperm showed that central pair and accessory microtubules have the majority of poly-modified tubulins, whereas doublet microtubules have only small amounts of mono- and oligo-modified tubulins. Glutamylation patterns for different beta-tubulins experimentally assembled into axonemes were consistent with utilization of modification sites corresponding to those identified in other organisms, but surrounding sequence context was also important. We compared tubulin modifications in the 9 + 9 + 2 insect sperm tail axonemes of Drosophila with the canonical 9 + 2 axonemes of sperm of the sea urchin Lytichinus pictus and the 9 + 0 motile sperm axonemes of the eel Anguilla japonica. In contrast to Drosophila sperm, L. pictus sperm have equivalent levels of modified tubulins in both doublet and central pair microtubule fractions, whereas the doublets of A. japonica sperm exhibit little glutamylation but extensive glycylation. Tubulin C-terminal modifications are a prevalent feature of motile axonemes, but there is no conserved pattern for placement or amount of these     

Evolution and phylogeny of the Diptera: a molecular phylogenetic analysis using 28S rDNA sequences

Portions of the large ribosomal subunit RNA gene (28S rDNA) encompassing the D1 and the D7 region were obtained from 16 dipteran species and families to reconstruct early phylogenetic events in the order Diptera. For outgroup comparison, the corresponding sequences were used from representative taxa of the Siphonaptera, Mecoptera, and Lepidoptera. A subset of 488 unambiguously alignable sites was analyzed with respect to important sequence evolution parameters. We found (1) sequence variability is significantly higher in double-stranded sites than in single-stranded sites, (2) transitions are close to saturation in most pairwise sequence comparisons, (3) significant substitution rate heterogeneity exists across sites, and (4) significant substitution rate heterogeneity exists among lineages. Tree reconstruction was carried out with the neighbor joining, maximum parsimony, and maximum likelihood methods. Four major subgroups are consistently and robustly supported: the Brachycera, the Culicomorpha, the Tipulomorpha sensu stricto, and the hitherto controversial Bibionomorpha sensu lato, which includes the families Sciaridae, Mycetophilidae, Cecidomyiidae, Bibionidae, Scatopsidae, and Anisopodidae. The phylogenetic relationships within or among these subclades and the positions of the families Psychodidae and Trichoceridae were not robustly resolved. These results support the view that the mouthparts of extant dipteran larvae evolved from a derived ground state characterized by subdivided and obliquely moving mandibles. Furthermore, sequence divergence and the paleontological record consistently indicate that a period of rapid cladogenesis gave rise to the major dipteran subgroups.     

Sperm ultrastructure and spermiogenesis in the relic species Tricholepidion gertschi Wygodzinsky (Insecta, Zygentoma)

The silverfish Tricholepidion gertschi is of interest in that it is the most basal representative of Zygentoma. An ultrastructural study of its spermiogenesis was performed to find out whether there are traits which resemble those of other, more advanced insects. This was found to be the case; spermiogenesis can be considered to be of a common insectan type, leading to the formation of elongated sperm cells with acrosome, nucleus, neck region and a tail with axoneme and two mitochondrial derivatives. Total cell length, 50 microm, is short for an insect. There are some specializations, which probably represent autapomorphies. The acrosome has a posterior canal or cleft that makes a U-turn. The centriole adjunct forms a prominent post-nuclear ring surrounding the centriole and have a posterior extension, and further originates nine intertubular fibers with a longitudinal periodicity and two accessory bodies. The mitochondrial derivatives have five rows of regularly spaced cristae within a crystalline matrix. The axoneme has accessory tubules consisting of 16 protofilaments, formed at the B-tubules of the doublets and placed at some distance from them in the posterior part of the sperm tail.