Ultrastructure of sperms in Acoela (Acoelomorpha) and its concordance with molecular systematics

Abstract. The sperms of the Acoela, a group of lower worms, are filiform cells with 2 flagella incorporated into the cell body. Their axonemes can variously have 9+2, 9+1, or 9+0 patterns of microtubules; and singlet microtubules in the cell body can be arranged in axial or cortical positions. An analysis of phylogenetic relationships of acoels based on molecular characters (18S rDNA sequence data) showed that these patterns of microtubules, where known, fell into discrete monophyletic groups. To test this hypothesis, we have expanded the database of sperm characters by examining the ultrastructure of a further 10 species representing 4 acoel families. As expected, the Convolutidae fell into 2 unrelated groups: “small‐bodied convolutids”(Convoluta pulchra, Praeconvoluta tigrina, Pseudaphanostoma smithrii) having 9+2 axonemes and cortical microtubules, and “large‐bodied convolutids” (including Wulguru cuspidata) having 9+0 axonemes and axial microtubules. Also, as expected, a member of the Mecynostomidae (Paedomecynostomum bruneum) has 9+1 axonemes and axial microtubules. Members of a family that appears intermediate by molecular characters, the Otocelididae, significantly have a variety of patterns: axonemes with both 9+2 and 9+0 patterns (Notocelis gullmarensis) or just 9+2 (the other species), and either axial (Philocelis brueggemanni), both axial and cortical (N. gullmarensis) microtubules, or microtubules that bend between axial and cortical positions along the length of the sperm (Otocelis sandara). Members of the Dakuidae (Daku woorimensis) also belong to this intermediate group, having 9+2 axonemes and axial microtubules, while in a fifth otocelidid (Stomatricha hochbergi), sperm characters are like those of the “large‐bodied convolutids” (9+0 axonemes and axial microtubules). Characters of sperm morphology generally support the molecular hypothesis of relationships and confirm a suspected polyphyly of the families Convolutidae, Otocelididae, and Actinoposthiidae.     

A new viviparous acoel Childia vivipara sp. nov. with observations on the developing embryos, sperm ultrastructure, body wall and stylet musculatures

A free‐living viviparous acoel, Childia vivipara sp. nov., from the Gullmar fjord of the Swedish coast is described. The new species is assigned to the taxon Childia based on histological, ultrastructural and molecular sequence similarities. All available molecular markers (18S rRNA, 28S rRNA and histone H3) and several morphological characters, obtained using transmission electron microscopy and confocal scanning laser microscopy of whole mount specimen stained with TRITC‐labelled phalloidin, support the placement of C. vivipara in the taxon Childia. Childia vivipara and other Childia species share the following morphological synapomorphies: well‐developed copulatory organs built of tightly packed stylet needles, proximal part of the stylet inserted into the seminal vesicle, reversed body‐wall musculature, absence of ventral diagonal muscles, presence of dorsal diagonal muscles, and presence of ventral straight longitudinal muscles between frontal pore and mouth, 9 + 1 sperm axoneme structure, six distal sperm cytoplasmic microtubules, and extensive overlap of axonemes and nucleus. The new species can be easily distinguished from other Childia species by its viviparous mode of reproduction and single curved stylet. Observations on late embryonic development based on the oldest developing embryos are discussed.     

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.     

The sperm structure of <Emphasis Type="Italic">Galloisiana yuasai</Emphasis> (Insecta,Grylloblattodea) and implications for the phylogenetic position of Grylloblattodea

The sperm ultrastructure of the Grylloblattodea Galloisiana yuasai was described and the sperm characters were comparatively examined in several orthopteroid insect orders for inferring the phylogenetic placement of the Grylloblattodea. The spermatozoa of G. yuasai are joined in bundles (spermatodesms) containing 200 units. Major features of these spermatozoa include a monolayered acrosome, a 9+9+2 axoneme with 16-pfs accessory microtubules and expanded intertubular material, and an evident “centriole adjunct”. The diffused material observed between the axoneme and the mitochondrial derivatives is considered to be an extension of the three connecting bands observed in other orthopteroid taxa, similar to what happens in some orthopteran lineages. The presence of the connecting bands, even though modified in G. yuasai, suggests that the Grylloblattodea are to be placed in a clade with Mantophasmatodea, Mantodea and Orthoptera.     

The Homology of Cortical Microtubules in Platyhelminth Spermatozoa: a Comparative Immunocytochemical Study of Acetylated Tubulin

Spermatozoa of certain acoels, a group of primitive Platyhelminthes, and spermatozoa of the most derived Platyhelminthes, the Cercomeridea (parasitic Platyhelminthes), show a general morphological resemblance in that they are long filiform cells with two incorporated axonemes and longitudinal cortical microtubules. A possible way to test the homology of these cortical microtubules in the different groups is to analyze the presence/absence of post-translational modifications of tubulin. An indirect immunofluorescence study showed that the doublet microtubules of the sperm axonemes are labelled by an anti acetylated-alpha-tubulin antibody in all groups, irrespective of the axoneme pattern (9 + 0, 9 + 2 and non-trepaxonematan 9 + “1” in various acoels, and trepaxonematan 9 + “1” of the temnocephalid Troglocaridicola sp., the digenean Echinostoma caproni and the monopisthocotylean monogenean Pseudodactylogyrus sp.). Significant differences are found in the sperm cortical microtubules: they are acetylated in the acoel Actinoposthia beklemischevi, but not in the digenean E. caproni and the temnocephalid Troglocaridicola sp. These results suggest that the sperm cortical microtubules of the acoels are not homologous with the morphologically similar elements found in the higher Platyhelminthes.     

Characteristics of sperm ultrastructure in the gall midges Porricondylinae (Insecta,Diptera, Cecidomyiidae), with phylogenetic considerations on the subfamily

 Sperm ultrastructure was studied in ten genera of the Porricondylinae (Cecidomyiidae). Sperm structure is remarkably simplified by the absence of the acrosome and the accessory tubules, as happens in all the cecidomyiid flies. All genera of the Porricondylinae show a peculiar 9+3 axonemal model except Diallactes, which retains the plesiomorphic condition of a 9+2 axoneme, and Winnertzia, which appears to have secondarily acquired a 9+0 model. A cladistic analysis of relevant sperm characters (based on the axonemal model, the number of mitochondrial derivatives and the size and structure of the centriolar adjunct) was performed to infer phylogenetic relationships among six tribes of the Porricondylinae. In this cladogram, the Porricondylini are the sister group to the Asynaptini, Heteropezini and Winnertzini and these four taxa form the sister group to the Dicerurini. The tribe Diallactini are regarded as the group with the most plesiomorphic characters within the family. Accepted: 15 March 1996     

The spermatogenesis and sperm structure of Acerentomon microrhinus (Protura, Hexapoda) with considerations on the phylogenetic position of the taxon

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.     

ULTRASTRUCTURE OF SWARMERS IN THE LAMINARIALES (PHAEOPHYCEAE). II. SPERM1

The ultrastructure of sperm from 13 species in 11 genera of Laminariales collected in the northeast Pacific Ocean is unique in the brown algae. The sperm are elongate, and possess a nucleus, several mitochondria and two or three chloroplasts, but no eyespot. The anterior flagellum bears mastigonemes on the proximal half of its length; a distal “whiplash” portion lacks mastigonemes and is an extension of only the two central singlet microtubules of the axoneme. A peculiar feature of these sperm is the posterior flagellum, which is longer than the anterior flagellum and tapers distally as the doublet microtubules become singlets and decrease in number. This feature contrasts with the laminarialean zoospore, which possesses a short posterior flagellum with the usual “9 + 2” axoneme. The structure of these sperm differs from that reported for Chorda, the sperm of which resembles a primitive brown algal zoospore. The facts support the concept that Chorda is the most primitive member of the Laminariales.     

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. J. Morphol. 276:361–369, 2015. © 2014 Wiley Periodicals, Inc.     

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.     

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.     

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.     

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.     

ULTRASTRUCTURE OF THE FLAGELLUM IN SPERM OF CKCINELLA SEPTEMPUNCTATA

Abstract  Using cell whole mount preparation and ultrathin section technique, the ultrastructure of the flagellum in the sperm of Coccinella septempunctata L. was examined with transmission electron microscope. The flagellum is made up of a classic 9+9+2 axoneme containing two similar crystallized mitochondria1 derivatives, two accessory bodies, which are divided in to two portions, an osmiophilic dense crescent and a spongy one, and a non-crystalline body. At the end of the flagellum, only the axoneme is present, it loses the two central microtubules but retains the nine doublets with dynein arms and the nine accessory microtubules.     

The ultrastructure of the spermatozoon of Neochasmus sp. (Cryptogonimidae, Digenea, Trematoda) and its phylogenetic significance

Jamieson B. G. M. and Daddow L. M. 1979. The ultrastructure of the spermatozoon of Neochasmus sp. (Cryptogonimidae, Digenea, Trematoda) and its phylogenetic significance. International Journal for Parasitology12: 547–559. The typical spermatozoon is filiform, monopartite and internally strongly asymmetrical, with a mean length of 254 μm. Its two axonemes are wholly incorporated though not deeply. The six major regions, in anteroposterior (proximal-distal) sequence, are: acrosomoid, prenuclear, uniaxonemal, nuclear, intermediate (amitochondrial), posterior mitochondrial and posterior amitochondrial regions. Centrioles and flagellar rootlets, present in the spermatid, are absent from the spermatozoon. The right axoneme (axial unit) originates more anteriorly than the left and anteriorly bears an acrosome-like projection (acrosomoid). Spinose regions carrying epiplasmalemmal projections are reported for the first time in the Digenea. Dimorphism exists as replacement of the typical posterior mitochondrial and amitochondrial regions by an amitochondrial region characterized by asymmetrical distribution of spines around one axoneme. Excepting known schistosomatid sperm, the digenean sperm is diagnosed by the combination of three characters: cortical microtubules, two fully incorporated 9 + 1 axonemes and presence of mitochondria although no one of these characters is limited to the group. Subtle intertaxon variations occur.     

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.     

Ultrastructural observations of spermatozoa and spermiogenesis in Wandella orana Gray, 1994 (Araneae: Filistatidae) with notes on their phylogenetic implications

Spermatozoa and spermiogenesis of the prithine filistatid spider Wandella orana are described. The spider produces coenospermia, i.e. sperm aggregations that include several single sperm cells commonly surrounded by a secretion sheath. One sectioned coenospermium in W. orana contains at least five spermatozoa. During copulation many coenospermia are transferred into the female. Coenospermia are regarded as a peculiar transfer form of sperm which occurs in early derivative spiders such as Liphistiomorphae and Mygalomorphae. The only exception which was found in Araneomorphae until now was the filistatine spider Filistata insidiatrix. Our observation is the second case and supports the view that Filistatidae represent an early derivative taxon. Furthermore, the individual sperm cells show characteristics which also may be regarded as being plesiomorphic. There is a cone-shaped acrosomal vacuole, a very long acrosomal filament, a rather stout nucleus and a small implantation fossa. The axoneme shows the 9x2+3 pattern of microtubules which is synapomorphic in Megoperculata (Uropygi, Amblypygi and Araneae). The finding of coenospermia in two distant taxa of Filistatidae may have consequences for phylogenetic and systematic considerations.     

Systematic revision of acoels with 9+0 sperm ultrastructure (Convolutida) and the influence of sexual conflict on morphology

We have used newly discerned morphological characters as well as molecular‐sequence data from 18S and 28S rDNA to revise the families recently designated as the ‘9+0’ acoels – what we call Convolutida. Characters from the ultrastructure of sperm, with their ‘9+0’ axonemes, are useful in delineating the Convolutida, but are either species‐specific or too conserved within the group to be used to infer relationships within it. Male genital organs, prostatoid organs, and sagittocysts, on the other hand, give a good phylogenetic signal for reconstructing relationships of such genera as Conaperta, Anaperus, and Achoerus; some features of the reproductive organs correlate with habitat and show how the Convolutida probably originated as epiphytic predators and radiated into the mesopsammon, pelagic, and coral‐associated realms. In this revision of the Convolutida we provide revised synopses of its families – which we restrict to the Anaperidae, Convolutidae, and Sagittiferidae – and describe a new species, Polychoerus gordoni, from New Zealand. We transfer the genus Adenopea from the Antroposthiidae to the Convolutidae; Conaperta, Neochildia, and Oxyposthia from the Convolutidae to the Anaperidae; Paranaperus and Praeanaperus from the Anaperidae to the Haploposthiidae. Convoluta aegyptica is synonymized with Convoluta boehmigi, Convoluta lacazii with Convoluta sordida, and the genus Picola (Convolutidae) with Deuterogonaria (Haploposthiidae). Amphiscolops blumi, A. carvalhoi, and A. langerhansi, all of which possess a cellular seminal bursa, are transferred to the genus Heterochaerus. Convoluta elegans and Pseudanaperus tinctus are classified as nomina nuda. We use our findings on the ultrastructure of female genital organs and spermatozoa to show that sexual conflict plays a major role in the evolution of diversity of these structures and that the phylogeny of the Acoela would comprise early forms without female genital organs and hyper‐ or hypodermal transfer of sperm through advanced forms with ever longer and narrower bursal nozzles and sperm with axial microtubules. Moreover, our results show that the acquisition of endosymbiotic algae happened at least twice within the Acoela.     

Anatomy and ultrastructure of the male reproductive system in Pleioplana atomata (Platyhelminthes: Polycladida)

Abstract. The ultrastructure of the male reproductive system in the polyclad flatworm Pleioplana atomata is described. Numerous testes are scattered throughout the entire body but are heavily concentrated on the ventral side. All stages of differentiating sperm cells are present in all testes follicles. Intercellular bridges connect spermatocytes and spermatids derived from a single spermatogonium. In the distal part of spermatids, a zone of differentiation develops with a row of microtubules beneath the plasmalemma. Adjacent to these microtubules, an intercentriolar body is flanked by two basal bodies that give rise to two axonemes (each with a 9+“1” microtubular pattern) that face in opposite directions. The Golgi complex appears in the central portion of the spermatid and produces numerous small and large electron-dense bodies. The small bodies surround the nucleus, whereas the large bodies cluster along with the mitochondria in the central part of the spermatid. Development of the spermatid leads to cell elongation and formation of a filiform, biflagellate mature spermatozoon with cortical microtubules all along the sperm shaft. The male canal system consists of paired vasa deferentia that separately enter a single seminal vesicle. A single prostatic canal connects the seminal vesicle to the prostatic vesicle. Ultrastructurally, the seminal vesicle and prostatic canal are very similar, and along with the prostatic vesicle and stylet pocket, are lined by a ciliated epithelium. The ultrastructure of the prostatic vesicle indicates that it probably produces a large volume of seminal fluid that, along with spermatozoa, is transferred to the mating partner through a stylet. Some of the findings, particularly on sperm ultrastructure, may provide characters useful for phylogenetic analysis.