Immunocytochemical study of tubulin in the 9+‘1’ sperm axoneme of a monogenean (Platyhelminthes), Pseudodactylogyrus sp.

The spermatozoon of the monopisthocotylean monogenean Pseudodactylogyrus sp. (a gill parasite of eels) has a single axoneme showing a 9+‘1’ pattern, a nucleus and a mitochondrion, but has no cortical microtubules. This species thus provides a very simple model for the study of tubulin in the 9+‘1’ axonemes of the Platyhelminthes, in contrast with digenean sperm which have a more complex spermatozoon with two such axonemes and cortical microtubules. Indirect immunofluorescence labelling of tubulin shows that the elongating spermatids, initially lying in all directions in the early stages, are arranged as parallel elements in further stages. The number of spermatids in an isogenic group could also be precisely counted and equals 32. Nuclear labelling with fluorescent dyes shows that the nuclei, first located in the common mass of the spermatids, later elongate and migrate into the growing spermatids, and that the nucleus is located in the central part of the mature spermatozoon, with the two extremities devoid of nucleus. Labelling with antibodies directed against acetylated, tyrosinated, and polyglutamylated tubulin gave positive results, thus indicating that these post-translational modifications of tubulin are present in the axoneme of spermatids and spermatozoa of monopisthocotylean monogeneans.     

Ultrastructure of Spermiogenesis in Acanthocotyle and Myxinidocotyle (Platyhelminthes,Monogenea, Acanthocotylidae)

Abstract Spermiogenesis was studied by transmission electron microscopy in the acanthocotylid monogeneans Myxinidocotyle californica (from Eptatretus stoutii) and Acanthocotyle lobianchi (from Raja clavata). In Myxinidocotyle and Acanthocotyle, the zone of differentiation shows two 9+‘1’ axonemes, the elongating nucleus and mitochondrion, and a single cortical cytoplasmic microtubule. This single microtubule is found in the mature spermatozoon of both species and was also noted in capsalids. This requires a modified definition of ‘pattern 2’ of spermatozoa which becomes: ‘spermatozoa with two axonemes and no cortical microtubules, except one single element much shorter than the spermatozoon’. A very unusual structure was found in Myxinidocotyle, but not in Acanthocotyle: the centriolar derivative of one of the 9+‘1’ axonemes is made up of 18 diverging singlets of unequal length associated with electron-dense cytoplasm. This seems to be the first case of a centriolar derivative without nine-fold symmetry associated with an axoneme with nine-fold symmetry.     

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.     

Phylogenesis of spermatogenesis in Annulipalpia caddisflies: An ultrastructural analysis on philopotamidae spermiogenesis

Characters currently used in phylogenetic analyses are insufficient for determining the status of several families of the order Trichoptera (caddisflies). Comparative spermatology can contribute to solving this problem. In the suborder Integripalpia, the sperm axoneme displays the pattern of 9 + 2 pairs of parallel microtubules found in many animal species. In the suborder Annulipalpia, however, axonemes bear remarkable aberrations. Consistently, in Chimarra florida (Philopotamidae, Annulipalpia) we found that the number of central microtubules varies from zero to four in axonemes of the spermatids and that spermatozoa lack axonemes. We propose that, similarly, the axoneme pattern became unstable in the Annulipalpia ancestor, from which branched two phylogenetic lines having different kinds of axoneme aberrations: (a) families in which the number of central microtubules of the axoneme exceeds two (e.g., Philopotamidae, Polycentropodidae) and (b) families in which the number of central microtubules of the axoneme does not exceed two (e.g., Hydropsychidae).     

Spermiogenesis and sperm ultrastructure in the monogenean parasite Acanthocotyle lobianchi

The ultrastructural events of spermiogenesis and the ultrastructure of the mature spermatozoon of an acanthocotylid monogenean, Acanthocotyle lobianchi, are described. The early zone of differentiation (ZD) contains two roughly perpendicular centrioles which become parallel and produce two free flagella, although these later become incorporated into the same body of cytoplasm. No cortical microtubules were found supporting the ZD at any stage of spermiogenesis. Much of the length of the thread-like sperm contains two axonemes of the 9 + '1' pattern together with a nuclear and mitochondrial profile but the 'posterior' region is occupied only by a single axoneme and the nucleus. A laterally situated electron-lucent vesicle with specialization of the adjacent surface membrane is found in the 'anterior' region of the sperm. The phylogenetic implications of these observations are discussed.     

Ultrastructural Observations on Fertilization in Dionchus remorae (Platyhelminthes,Monogenea, Dionchidae)

Ultrastructural observations are presented for some of the stages occurring during fertilization in Dionchus remorae (a gill parasite of Echeneis naucrates) and are believed to be the first published concerning a monogenean. Fertilized female germ cells were found in the ovary. Several loops of the spermatozoon were present within the oocyte cytoplasm; the sperm nucleus became electron lucent and the parallel peripheral doublets of the axonemes became increasingly divergent. The cortical granules in the oocyte were not released immediately after penetration by the spermatozoon. The homogeneity apparently found in the oocyte ultrastructure and process of fertilization in the monogeneans and digeneans contrasts with the variety that exists in their sperm ultrastructure.     

Ultrastructural study of the male gamete of Glossobothrium sp. (Cestoda: Bothriocephalidea: Triaenophoridae) a parasite of Schedophilus velaini (Perciformes: Centrolophidae) in Senegal

This paper describes the ultrastructure of the male gamete of Glossobothrium sp. (Bothriocephalidea: Triaenophoridae). The mature spermatozoon of Glossobothrium sp. is filiform and possesses two axonemes, a single helicoidal crested body, a parallel nucleus, parallel cortical microtubules and granules of glycogen. In Glossobothrium sp. we describe for first time a 200-250 nm thick crest-like body in the Bothriocephalidean. The anterior part of the spermatozoon exhibits a ring of 27 electron-dense cortical microtubules encircling the first axoneme. This structure persists until the appearance of the second axoneme. When the ring of electron-dense cortical microtubules disappears, the spermatozoon exhibits two bundles of thin cortical microtubules. The posterior part of the spermatozoon contains the posterior extremity of the second axoneme, the posterior extremity of the nucleus and few cortical microtubules. Soon nucleus disappears and the axoneme is disorganized. Thus the posterior extremity of the spermatozoon of Glossobothrium sp. exhibits only singlets produced by the disorganization of the doublets of the second axoneme and few cortical microtubules. This type of posterior extremity of the mature spermatozoon has never been described previously in the Triaenophoridae.     

Ultrastructure de la spermiogenèse et du spermatozoïde de Loimosina wikoni et affinités phylétiques des Loimoidae (Plathelminthes, Monogenea, Monopisthocotylea)

In Loimosina, during spermiogenesis, the zone of differentiation of the spermatid contains two centrioles continued as two 9 +‘1’axonemes. One of the axonemes lengthens and will become the principal axoneme of the spermatozoon. The other axoneme is as long as the first one at the beginning of spermiogenesis, but is shorter in the mature sperm cell. The spermatozoon consists of several regions: (a) at the anterior end, the centriolar derivative of the principal axoneme; (b) a short region which shows cortical microtubules coinciding with external ornamentations; (c) a long uniflagellate region, with mitochondrion; (d) a biflagellate region containing the anterior slender part of the nucleus; (e) the posterior part of the nucleus, with no accompanying cytoplasmic organelles. Spermiogenesis and sperm structure in Loimosina differ from what is known in all other described monogeneans, excepting the monocotylid Heterocofyle to which they show close resemblances. However, the alteration of the second axoneme is more complete in Heterocofyle than in Loimosina. Comparative study of spermiogenesis and sperm ultrastructure thus shows an interesting coincidence with classical phylogenies of the monogeneans, in which the families Loimoidae and Monocotylidae are closely related. Chez Loimosina, pendant la spermiogenèse, la zone de différenciation de la spermatide contient deux centrioles prolongés par deux axonemes de type 9 +‘1′. L'un des axonemes s'allonge et deviendra l'axonème principal du spermatozoïde mûr. Le deuxième axontme est aussi long que le premier au début de la spermiogenèse, mais il est plus court dans le spermatozoïde mûr. Le spermatozoïde comprend: (a) à l'avant, le dérivé centriolaire de l'axonème principal; (b) une courte région contenant quelques microtubules corticaw longitudinaux coincidant avec des omementations extramembranaires; (c) une longue région miflagellée avec mitochondrie; (d) une région biflagellée contenant La partie antérieure effilée du noyau; (e) la région postérieure du noyau, sans organites cytoplasmiques accompagnateurs. La spermiogenèse et la structure du spermatozoïde de Loimosina sont différentes de ce qui est connu chez tous les autres Monogènes décrits, excepté le Monocotylidae Heterocofyle auquel elles ressemblent beaucoup. Toutefois, l'altération du deuxième axonème est moins complète chez Loimosina que chez Heterocotyle. L'étude comparée des spermatozoïdes et des spermiogenèses montre une bonne coincidence avec les phylogenèses classiques, dans lesquelles les familles Loimoidae et Monocotylidae sont proches.     

Ultrastructural and immunocytochemical investigation of acoel sperms with 9 + 1 axoneme structure: new sperm characters for unraveling phylogeny in Acoela

Acoel sperm characters proved useful in deciphering acoel taxonomy. The phylogenetic value of sperm characters in closely related sub-groups or in a monophyletic taxon has not yet been assessed. We have investigated sperm ultrastructure in seven members of the monophyletic taxon Childia sensu (Tekle et al. J Zool Sys Evol Res 43(1):72–90, 2005) and in their closest relatives, the Mecynostomidae (four taxa). All members of Childia examined show little variation in their sperm ultrastructure. The common characters of Childia taxa are: 9 + 1 axoneme structure, the presence of six distal cytoplasmic microtubules in the absence of axial or cortical ones, long nucleus and extensive nucleus–flagella overlap. We have identified a new set of cytoplasmic microtubules lying in the centriolar end of the sperm cell, distal microtubules. The origin and phylogenetic significance of this character is discussed. The types and arrangement of cytoplasmic granules could be used as phylogenetic characters at a low taxonomic level. A loose membrane amorphous core type of granule was found to be a synapomorphy for the following clade within the taxon Childia: C. crassum + C. groenlandica + C. vivipara + C. brachyposthium + C. macroposthium. Sausage shaped granules are plesiomorphic among the taxa examined. The rest of the granule characters were found to be homoplasious. Sperm ultrastructural characters have again proven their concordance with molecular phylogeny. The only morphological synapomorphies known for the sister taxa Childia–Mecynostomidae, in the molecular phylogeny, are characters derived from sperm ultrastructure: distal microtubules arranged in two groups of three microtubules each and a 9 + 1 axoneme structure. The spermatozoa of Childia and Mecynostomidae show 9 + 1 axoneme configuration, seemingly similar to the 9 + ‘1’ axoneme pattern of the Platyhelminthes—Trepaxonemata. Using electron-microscope immunocytochemistry, we have demonstrated that, unlike the central cylinder of trepaxonematans, the central cylinder of the 9 + 1 axonemal pattern in acoels is immunoreactive to tubulin and contains a single central microtubule. Therefore, the 9 + 1 patterns in acoels and trepaxonematans are homoplasious.     

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.     

Phylogenetic position of rhyacophiloid caddisflies (Insecta, Trichoptera): a spermatological analysis of Rhyacophilidae and Glossosomatidae

Most caddisflies (Insecta, Trichoptera) are classified into two suborders, Annulipalpia and Integripalpia. However, the use of the derived characters that are regularly applied in systematic and phylogenetic analyses of Trichoptera is insufficient to determine with certainty the position of the families belonging to Rhyacophiloidea, which are considered by different authors to be either Annulipalpia, or Integripalpia, or even a separate suborder. Rhyacophiloidea comprise four overall similar families: free-living Rhyacophilidae and Hydrobiosidae, saddle-case making Glossosomatidae, and purse-case making Hydroptilidae. It was previously found that Annulipalpia spermatozoa have aberrant axonemes while Integripalpia spermatozoa display the plesiomorph 9 + 2 axoneme. The present spermatological analysis of the families Rhyacophilidae and Glossosomatidae shows that both have spermatozoa with aberrant axonemes lacking the two central microtubules found in the typical axoneme of insect spermatozoa. This is an apomorphic character shared with the superfamily Hydropsychoidea, indicating that from this point of view, Rhyacophiloidae are more closely related to Annulipalpia than to Integripalpia.     

Comparative spermatology of selected polyclad flatworms (platyhelminthes)

Sperm ultrastructure of four acotylean (Idioplana atlantica, Armatoplana leptalea, Styloplanocera fasciata, Melloplana ferruginea) and three cotylean polyclads (Pseudoceros bicolor, Phrikoceros mopsus, Enchiridium evelinae) was investigated. All spermatozoa are biflagellate, exhibiting a 9+"1" axoneme pattern. All acotylean axonemes originate and extend within the sperm shaft, and once exiting the shaft, remain attached to it. The flagella of all cotylean spermatozoa exit the shaft immediately and remain free. Structures shared by all species include: an elongated nucleus, in acotyleans located only in the posterior part of the shaft, whereas in cotyleans it extends along the entire sperm body; mitochondria along with small and large dense bodies arranged in a specific pattern; and a ring of microtubules that extends along the entire sperm shaft just beneath the cell membrane. A unique spermatozoon has been found in E. evelinae, where round vesicle-like structures fill the anterior part of the nucleus, and a different type of large dense bodies is present. The spermatozoa of all studied species exhibit numerous characters (axoneme/flagella position, distribution and position of large and small dense bodies, of mitochondria, presence of nuclear vesicles) that may be of phylogenetic value at the family and higher taxonomic levels.     

滇蛙多盘吸虫精子超微结构

利用透射电镜研究多盘吸虫精子的形态和结构。它属于单殖类中Ｐｏｌｙｏｐｉｓｔｈｏｃｏｔｙｌｅａ类型。精子细线形,含两根融入精子内的９＋１型轴丝,细长的细胞核,纤细的线粒体和微管。在第二根轴丝出现前的区段无微管的分布,具两根轴丝的主干段轴丝外无外侧管;细胞核消失后微管呈整圈排列;精子后部含糖原颗粒,背腹面中央向髓布内凹呈哑铃状并两侧向外突出形成波动膜;由精细胞则来的区段具外饰物。     

Sperm structure of Trichoptera. III. Hydropsychidae,polycentropodidae and philopotamidae (Annulipalpia)

Spermatozoa from 9 species belonging to 3 families of trichopteran suborder Annulipalpia (Philopotamidae : Philopotamus ludificatus, P. montanus, Wormaldia occipitalis, W. copiosa; Polycentropodidae: Plectrocnemia geniculata, Polycentropus mortoni, P. irroratus, Cyrnus trimaculatus; Hydropsychidae: Hydropsyche pellucidula) were studied by light, scanning, and transmission electron microscopy. The absence of axonemal dynein arms and a consequent sperm immotility are characteristic of the species studied. The presence of 7, rather than 2, central microtubules is shared by Polycentropodidae and some Philopotamidae. A greater variability in the sperm axoneme was found within Philopotamidae than in the other families. The species of the genus Wormaldia have an axial vesicle rather than microtubules and their axonemes have a 9 + 9 + 0 or 13 + 13 + 0 pattern. Hydropsychidae have spermatozoa provided with numerous finger-like appendages containing microtubular doublets. The progressive loss of sperm flagellum and motility within the group is discussed.     

Ultrastructure of the spermatozoa of the flatworms Phaenocelis peleca and Boninia divae (Platyhelminthes, Polycladida)

The ultrastructure of spermatozoa of the acotylean Phaenocelis peleca and the cotylean Boninia divae is described. All spermatozoa are filiform and biflagellate with a 9+"1" microtubular pattern in the axoneme. Sperm characters in P. peleca follow the morphologies described for other acotyleans, with axonemes exiting the sperm shaft at the distal end and remaining in close contact with the sperm membrane. The nucleus occupies the proximal region of the shaft, and two types of dense bodies and mitochondria are located at the distal end. Unlike other members of the Cotylea, the axonemes of B. divae spermatozoa are incorporated into the sperm shaft, leaving the shaft at some distance from the distal end and then remaining free. This type of morphology is characteristic for acotyleans. Additionally, the spermatozoa of B. divae contain only one type of dense bodies plus a unique structure, which we call a central core. The nucleus in this species is unique as well; it shows periodic constrictions and rings of electron-dense granules, characters that further contribute to the distinct status of Boniniidae.     

Ultrastructural study of the spermatozoon of Heterolebes maculosus (Digenea,Opistholebetidae), a parasite of the porcupinefish Diodon hystrix (Pisces,Teleostei)

This paper describes the ultrastructure of the mature spermatozoon of Heterolebes maculosus. It is the first study of this kind concerning the Opistholebetidae (Platyhelminthes, Digenea). The ultrastructural elements observed in the spermatozoon are: two axonemes with 9+“1” pattern of Trepaxonemata and their attachment zones, two mitochondria, a nucleus, cortical microtubules, external ornamentation of the plasma membrane and spine-like bodies. The number and the disposition of cortical microtubules, the organisation of 11 cortical microtubules disposed in semi-circle around the first mitochondrion in the external ornamentation region and the organisation of the posterior part of the spermatozoon are discussed. Three principal types of posterior part of digenean spermatozoa are proposed. The similarity between the spermatozoon of the Opistholebetidae H. maculosus and Opecoelidae enables us to confirm that these two families are closely related.     

Ultrastructure of the spermatozoon of Bothriocotyle sp. (Cestoda: Bothriocephalidea), a parasite of Schedophilus velaini (Sauvage, 1879) (Perciformes: Centrolophidae) in Senegal

The mature spermatozoon of Bothriocotyle sp. is filiform and tapered at both extremities. It possesses 2 axonemes of unequal length, showing the 9 + "1" pattern of Trepaxonemata. The anterior extremity exhibits a crest-like body. Thereafter, the crest-like body disappears, and the first axoneme is surrounded by a ring of cortical microtubules (about 27 units) that persist until the appearance of the second axoneme. This ring of cortical microtubules is characteristic only for species of Bothriocephalidea and represents a very useful phylogenetic character. The spermatozoon cytoplasm is slightly electron-dense and contains numerous electron-dense granules of glycogen in several regions. The anterior and posterior extremities of the spermatozoon lack cortical microtubules. The posterior extremity of the spermatozoon of Bothriocotyle sp. possesses a nucleus and a disorganized axoneme, which also characterizes spermatozoa of the Echinophallidae studied to date.     

Ultrastructural characters of the spermatozoon of the digenean Hypocreadium caputvadum Kacem et al., 2011 (Lepocreadioidea: Lepocreadiidae), an intestinal parasite of Balistes capriscus in Tunisia

The ultrastructural organization of the spermatozoon of the digenean Hypocreadium caputvadum (Lepocreadioidea: Lepocreadiidae) is described. Live digeneans were collected from Balistes capriscus (Teleostei: Balistidae) from the Gulf of Gabès, Tunisia (Eastern Mediterranean Sea). The mature spermatozoon of H. caputvadum shows several ultrastructural characters such as two axonemes of different lengths exhibiting the classical 9 + “1” trepaxonematan pattern, a nucleus, two mitochondria, granules of glycogen, external ornamentation of the plasma membrane and two bundles of parallel cortical microtubules. Moreover, in the anterior extremity, the second axoneme is partly surrounded by a discontinuous and submembranous layer of electron-dense material. Our study provides new data on the spermatozoon of H. caputvadum in order to improve the understanding of phylogenetic relationships in the Digenea, particularly in the superfamily Lepocreadioidea. In this context, the electron-dense material surrounding one of the axonemes in the anterior spermatozoon extremity constitutes the unique distinguishing ultrastructural character of lepocreadioideans, and it is present in spermatozoa of lepocreadiids, aephnidiogenids and gyliauchenids.     

Axoneme beta-tubulin sequence determines attachment of outer dynein arms

Axonemes of motile eukaryotic cilia and flagella have a conserved structure of nine doublet microtubules surrounding a central pair of microtubules. Outer and inner dynein arms on the doublets mediate axoneme motility [1]. Outer dynein arms (ODAs) attach to the doublets at specific interfaces [2-5]. However, the molecular contacts of ODA-associated proteins with tubulins of the doublet microtubules are not known. We report here that attachment of ODAs requires glycine 56 in the beta-tubulin internal variable region (IVR). We show that in Drosophila spermatogenesis, a single amino acid change at this position results in sperm axonemes markedly deficient in ODAs. Moreover, we found that axonemal beta-tubulins throughout the phylogeny have invariant glycine 56 and a strongly conserved IVR, whereas nonaxonemal beta-tubulins vary widely in IVR sequences. Our data reveal a deeply conserved physical requirement for assembly of the macromolecular architecture of the motile axoneme. Amino acid 56 projects into the microtubule lumen [6]. Imaging studies of axonemes indicate that several proteins may interact with the doublet-microtubule lumen [3, 4, 7, 8]. This region of beta-tubulin may determine the conformation necessary for correct attachment of ODAs, or there may be sequence-specific interaction between beta-tubulin and a protein involved in ODA attachment or stabilization.