Kinetid structure in Choanocytes of Sponges (Heteroscleromorpha): Toward the ancestral Kinetid of Demospongiae

Every large clade of Eukarya has its own pattern of kinetid (flagellar apparatus) structure, which is stable and specific within the group, thereby being a good phylogenetic marker. The kinetid structure of sponge choanocytes might be a candidate for such marker for the phylogeny of Porifera. Kinetids of two heteroscleromorphs, Halichondria sp. (Suberitida) and Crellomima imparidens (Poecilosclerida), have been investigated here for the first time, and a reconstruction of the kinetid for each species is provided. The kinetids of both species comprise a flagellar kinetosome with a nuclear fibrillar root, a basal foot and satellite producing microtubules; a centriole is absent. Good resolution images reveal a new thin structure, the axial granule, in the flagellar transition zone which might be present in other sponges. The comparison of kinetids in investigated sponges revealed three types of kinetid in Demospongiae, and their distribution in the taxon has been shown on a molecular phylogenetic tree. Kinetid characters of the common ancestor of Demospongiae are discussed. J. Morphol. 277:925–934, 2016. © 2016 Wiley Periodicals, Inc.     

Structure of the choanocyte kinetid in the sponge <Emphasis Type="Italic">Haliclona</Emphasis> sp. (Demospongiae: Haplosclerida) and its implication for taxonomy and phylogeny of Demospongiae

Sponges are a group of primitive animals that are of particular interest in terms of evolutionary theory. Molecular and phylogenic studies have revealed the common origin of sponges and Metazoa and the relationship of Metazoa to Choanoflagellata. Comparative studies of the morphology have allowed reconstruction of the structure of ancestors common to Metazoa and Choanoflagellata, as well as for Porifera and other Metazoa. For this purpose, a comparative analysis of phylogenically informative structures, such as the kinetid, is needed. This article presents data on the structure of the choanocyte kinetid in Haliclona sp., which was investigated for the first time. The reconstruction of the kinetid based on ultrathin serial sections shows that it lacks an accessory centriole; it has fibrillar roots despite the basal position of the nucleus; two-to-three triplets of the kinetosome are longer than the others; bundles of lateral microtubules arise from fibrillar roots and reach the cytoplasmic membrane in the region of intercellular contact. The studies of the kinetid in the few representatives of Demospongiae investigated showed the compatibility of the data on the ultrastructure and molecular phylogeny.     

Comparative study of the choanosome of Porifera: 1. The Homoscleromorpha

The choanoderm and pinacoderm of representatives of the two families of Homoscleromorpha sponges, the Oscarellidae and Plakinidae, have been examined by transmission and scanning electron microscopy. Different fixative procedures have shown the dramatic influence of fixation conditions on the morphology of choanocytes. These two families of sponges have the following morphological features in common: flagellated endopinacocytes with short apical microvilli and basal pseudopods; the presence of a very thin and dense sheet of matrix material which limits the mesohyl. There are, however, only minor differences in the flagellar morphology, granule content, and anchoring system of their choanocytes. Two findings are of particular interest: (1) the presence of glycocalyx bridges between the microvilli of the choanocyte collar; and (2) the discovery of a new cell type, the apopylar cell, which has a morphology intermediate between that of pinacocytes and choanocytes. The apopylar cells limit the apopylar opening of the choanocyte chamber and indicate the transition between choanoderm and pinacoderm.     

Ultrastructural evidence for the biflagellate origin of the uniflagellate fungal zoospore

Summary The morphological similarities between the kinetosome and the second centriole of the zoospores of Phlyctochytrium kniepii and P. punctatum (Chytridiomycetes) suggest that the second centriole in the chytrid zoospore is a vestigial flagellum base. It is suggested that the term vestigial kinetosome may also be used when referring to the structure which is presently termed the second centriole of the chytrid zoospore. Morphological similarities between the chytrid zoospores of P. kniepii and P. punctatum and the zoospores of Rhizidiomyces apophysatus (Hyphochytridiomycetes) are noted. The possible biflagellate origin of fungi with uniflagellate zoospores is discussed. The third fiber (C fiber) of the kinetosome triplet is shown to form as an outgrowth of the B fiber of the kinetosome doublet.     

THE FINE STRUCTURE OF BLASTOCLADIELLA EMERSONII ZOOSPORES

The zoospore of Blastocladiella emersonii has been re-examined with the electron microscope. The following new findings were made. A double unit-membrane system surrounds all cell organelles except γ-bodies, vacuoles and a few fragments of membranes. Lipid granules on one side of the large mitochondrion alternate with vesicles. The kinetosome of the posterior flagellum does not have any central fibrils as previously reported; a small, cylindrical structure is found within its anterior end. An associated centriole is located next to the kinetosome. Three striated rootlets pass from the kinetosome by separate channels through the mitochondrion. There appears to be no connection between the striated rootlets and the mitochondrion. Microtubules originating at the anterior end of the kinetosome pass into the cytoplasm between the mitochondrion and the nuclear cap. Long, dense strands were observed in some nuclei. The axoneme is taken up into the spore during encystment and is found in the freshly encysted spore. No trace of the flagellar sheath has been found in the encysted spore.     

Choanocyte ultrastructure in Halisarca dujardini (Demospongiae,Halisarcida)

Understanding poriferan choanocyte ultrastructure is crucial if we are to unravel the steps of a putative evolutionary transition between choanoflagellate protists and early metazoans. Surprisingly, some aspects of choanocyte cytology still remain little investigated. This study of choanocyte ultrastructure in the halisarcid demosponge Halisarca dujardini revealed a combination of minor and major distinctive traits, some of them unknown in Porifera so far. Most significant features were 1) an asymmetrical periflagellar sleeve, 2) a battery of specialized intercellular junctions at the lateral cell surface complemented with an array of lateral interdigitations between adjacent choanocytes that provides a particular sealing system of the choanoderm, and 3) a unique, unexpectedly complex, basal apparatus. The basal apparatus consists of a basal body provided with a small basal foot and an intricate transverse skeleton of microtubules. An accessory centriole, which is not perpendicular to the basal body, is about 45°. In addition, a system of short striated rootlets (periodicity = 50–60 nm) arises from the proximal edge of the basal body and runs longitudinally to contact the nuclear apex. This is the first flagellar rootlet system ever found in a choanocyte. The accessory centriole, the rootlet system, and the nuclear apex are all encircled by a large Golgi apparatus, adding another distinctive feature to the choanocyte cytology. The set of distinct features discovered in the choanocyte of H. dujardini indicates that the ultrastructure of the poriferan choanocyte may vary substantially between sponge groups. It is necessary to improve understanding of such variation, as the cytological features of choanocytes are often coded as characters both for formulation of hypotheses on the origin of animals and inference of phylogenetic relationships at the base of the metazoan tree. J. Morphol., 2009. © 2008 Wiley‐Liss, Inc.     

Fine structure of the spermatozoon of the viviparous teleost, Cymatogaster aggregata

The approximately 50 μm long sperm of Cymatoguster aggregata is composed of an elongate head (4 μm), an elongate mitochondria1 midpiece (3.5 μm) and a tail flagellum (roughly 40 μm). The sperm lacks an acrosome. Contained within depressions on one surface of the compressed head are a proximal centriole and a distal centriole separated by an electron dense, intercentriolar body. The anterior portion of the tail flagellum originates at the basal body (distal centriole) and is contained within an extracellular, flagellar tunnel within the mitochondria1 midpiece. The morphological similarity of C. uggregutu sperm to sperm of other internally fertilizing fishes supports the hypothesis that spermatozoan morphology is related to the mode of fertilization and that an elongate head and midpiece are specializations for internal fertilization.     

THE ORGANIZATION AND EVOLUTION OF MICROTUBULAR ORGANELLES IN CILIATED PROTOZOA

(1) Ciliated protozoa are viewed as unicellular organisms structured in a hierarchy of organizational levels that include the macromolecular, suborganellar, unit organellar, organellar complex, and organellar system. (2) The ciliate cortex is divided into two major functional regions, the somatic region and the oral region. The fundamental component of the cortex is an organellar complex, the kinetid, whose organizing centre is the kinetosome with which are associated three fibrillar associates diagnostic of ciliates. These three fibrillar associates are the periodically striated kinetodesmal fibril and two microtubular ribbons, the transverse and postciliary ribbons. (3) Somatic and oral kinetids are found to be of three major types: monokinetids are composed of one kinetosome and its fibrillar associates; dikinetids are composed of two kinetosomes and their fibrillar associates; polykinetids are composed of more than two kinetosomes and their fibrillar associptes. (4) The mechanisms underlying kinetid function and development remain largely unexplored. Research into the molecular biology and ultrastructure, especially of mutant forms, should provide basic insights in the near future. (5) The conservation of kinetid structure across major phyla of organisms suggests that this subcellular structure should be useful in phylogenetic analysis despite the concepts of ‘chemical identity’ and ‘organic design’. (6) The evolutionary rate of change of oral features is greater than that of somatic features, probably due to developmental and ecological factors. Nevertheless both cortical regions are constrained by the phenomenon of structural conservatism; that is, the conservation of structure through time is inversely related to the level of biological organization. (7) Eight major groupings of ciliate species are recognized, based on ultrastruc-tural features of the cortex. Several examples of differences between these eight groups and the groups presently recognized are discussed.     

Morphogenesis accompanying larval metamorphosis in Plakina trilopha (Porifera, Homoscleromorpha)

Morphological investigations of morphogenesis accompanying the metamorphosis of the cinctoblastula larva of poriferan Plakina trilopha (Homoscleromorpha) have been made. The larva possesses a distinct columnar epithelium which subdivides into three cellular areas: antero-lateral, postero-lateral, and posterior one. Characteristic morphological features of the cells in each area can be used as natural markers when tracing the fate of larval cells during metamorphosis. The ciliated epithelium of the larva is transformed directly into choanoderm and pinacoderm, without losing its organization. This transformation is a peculiar feature of the metamorphosis in Homoscleromorpha. Metamorphosis in P. trilopha is based on epithelial morphogenesis and includes the mechanisms of flattening of the exopinacoderm, evagination and invagination of larval epithelium in the course of the development of the rhagon aquiferous system. The flattening of larval cells during exopinacoderm formation in metamorphosing P. trilopha is a common change of cell shape during epithelial morphogenesis of this species. The separation of proximal fragments of cells has been observed here. This phenomenon, that we have called “cytoplasmic shedding”, appears to play an important role in the change of epithelial cell shape in P. trilopha. Mechanism of epithelial–mesenchymal transition, i.e., ingression of epithelial ciliated cells into the cavity of the metamorphosing larva of P. trilopha participates in mesohylar cell origin.     

Spermatogenesis and sperm structure of Acerella muscorum, (Ionescu, 1930) (Hexapoda, Protura)

The general organization of the male genital system, the spermatogenesis and the sperm structure of the proturan Acerella muscorum have been described. At the apex of testis apical huge cells are present; their cytoplasm contains a conventional centriole, a large amount of dense material and several less electron-dense masses surrounded by mitochondria. Spermatocytes have normal centrioles and are interconnected by cytoplasmic bridges. Such bridges seem to be absent between spermatid cells and justify the lack of synchronization of cell maturation. Spermatids are almost globular cells with a spheroidal nucleus and a large mass of dense material corresponding to the centriole adjunct. Within this mass a centriole is preserved. Mitochondria of normal structure are located between the nucleus and the plasma membrane. The spermatids are surrounded by a thick membrane. No flagellar structure is formed. Sperm have a compact spheroidal nucleus, a large cap of centriole adjunct material within which a centriole is still visible. A layer of mitochondria is located over the nucleus. The cytoplasm is reduced in comparison to spermatids; many dense bodies are interspersed with sperm in the testicular lumen. The sperm are small, immotile cells of about 2.5-3 μm in diameter.     

Spatial relationships between the anterior centriole and the mitotic center during interphase in the amoebae of the myxomycete Physarum polycephalum

Amoebae of the Myxomycete Physarum polycephalum in the interphase state typically contain only one proflagellar apparatus in which the anterior kinetosome (anterior centriole) is attached to the microtubule organizing center 1 (mtoc 1). We built strains possessing more than one mtoc 1 and a variable number of anterior centrioles to allow the appearance of new structures. In 8% of the amoebae of these strains, the 1:1 attachment between the anterior centriole and the mtoc 1 is not always respected. In nine cases studied using tridimensional reconstructions from ultrastructural thin sections, the pattern of attachment was more complex. A mtoc 1 could be linked to several anterior centrioles, and/or reciprocally an anterior centriole could be linked to several mtoc 1. In one case, an anterior centriole was not linked to a mtoc 1 and in three cases, a single centriole exhibited anterior and posterior characteristics. These observations suggest that (1) each pair of centrioles constitutes a morphological and physiological entity that is distinct from the mitotic center (mtoc 1); (2) the attachment of the anterior centriole to the mtoc 1 occurs at the end of each mitosis; (3) there is an inductory process during the morphogenesis of the link between the anterior centriole and the mtoc 1; (4) the anterior characteristics of a centriole can be present in the absence of the link with the mtoc 1; (5) the anterior and posterior characteristics of a centriole are not exclusive of each other, ruling out the existence of a lineage corresponding to the anterior centriole and a lineage corresponding to the posterior centriole; and (6) the differences between anterior and posterior centrioles result from a maturation process.     

Electron Microscopic Studies on the Basal Apparatus of the Flagellum in the Protozoon, Leishmania donovani

SYNOPSIS. The basal apparatus of the flagella and kinetoplast in Leishmania donovani have been studied with the electron microscope. The flagellar fibrils extend into the body of the protozoan to form the kinetosome. At the point of origin of the flagellum, the pellicle invaginates to form a kinetosomal vacuole around the kinetosome. The kinetoplast is formed by a transversely elongated banded structure, surrounded at some distance by a double layered kinetoplast membrane. There is no apparent connection between the kinetosome and the kinetoplast.     

NK homeobox genes with choanocyte-specific expression in homoscleromorph sponges

Data on nonbilaterian animals (sponges, cnidarians, and ctenophores) have suggested that Antennapedia (ANTP) class homeobox genes played a crucial role in the early diversification of animal body plans. Estimates of ancestral gene diversity within this important class of developmental regulators have been mostly based on recent analyses of the complete genome of a demosponge species, leading to the proposal that all ANTP families found in nonsponges animals (eumetazoans) derived from an ancestral "proto-NK" six-gene cluster. However, a single sponge species cannot reveal ancestral metazoan traits, in particular because lineage-specific gene duplications or losses are likely to have occurred during the long history of the Porifera. We thus looked for ANTP genes by degenerate polymerase chain reaction search in five species belonging to the Homoscleromorpha, a sponge lineage recently phylogenetically classified outside demosponges and characterized by unique histological features. We identified new genes of the ANTP class called HomoNK. Our phylogenetic analyses placed HomoNK (without significant support) close to the NK6 and NK7 families of cnidarian and bilaterian ANTP genes and did not recover the monophyly of the proposed "proto-NK" cluster. Our expression analyses of the HomoNK gene OlobNK in adult Oscarella lobularis showed that this gene is a strict marker of choanocytes, the most typical sponge cell type characterized by an apical flagellum surrounded by a collar of microvilli. These results are discussed in the light of the predominant neurosensory expression of NK6 and NK7 genes in bilaterians and of the recent proposal that choanocytes could be the sponge homologs of sensory cells.     

Piwi expression in archeocytes and choanocytes in demosponges: insights into the stem cell system in demosponges

SUMMARY Little is known about the stem cells of organisms early in metazoan evolution. To characterize the stem cell system in demosponges, we identified Piwi homologs of a freshwater sponge, Ephydatia fluviatilis, as candidate stem cell (archeocyte) markers. EfPiwiA mRNA was expressed in cells with archeocyte cell morphological features. We demonstrated that these EfPiwiA‐expressing cells were indeed stem cells by showing their ability to proliferate, as indicated by BrdU‐incorporation, and to differentiate, as indicated by the coexpression of EfPiwiA with cell‐lineage‐specific genes in presumptive committed archeocytes. EfPiwiA mRNA expression was maintained in mature choanocytes forming chambers, in contrast to the transition of gene expression from EfPiwiA to cell‐lineage‐specific markers during archeocyte differentiation into other cell types. Choanocytes are food‐entrapping cells with morphological features similar to those of choanoflagellates (microvillus collar and a flagellum). Their known abilities to transform into archeocytes under specific circumstances and to give rise to gametes (mostly sperm) indicate that even when they are fully differentiated, choanocytes maintain pluripotent stem cell‐like potential. Based on the specific expression of EfPiwiA in archeocytes and choanocytes, combined with previous studies, we propose that both archeocytes and choanocytes are components of the demosponge stem cell system. We discuss the possibility that choanocytes might represent the ancestral stem cells, whereas archeocytes might represent stem cells that further evolved in ancestral multicellular organisms.     

Phyllactinia fraxinicola,another Asian fungal pathogen on Fraxinus excelsior (common ash) introduced to Europe?

Since the mid-nineties Phyllactinia fraxini has become frequent in Germany. This species has hitherto been characterized by having straight conidiophore foot cells. However, we found that recent collections from Germany have conidiophores with sinuated and twisted foot cells. So far sinuated foot cells were only known from the related P. fraxinicola, another species with Eastern Asian origin. We thus hypothesized that recent collections from Germany belong to P. fraxinicola which might have been introduced to Europe. Using morphological and molecular rDNA data we found that no introduction took place and that there is only P. fraxini in Germany.     

Analysis of cytoplasmic microtubules and flagellar roots in the zoospores ofAllomyces macrogynus

Summary Cytoskeletal and flagellar microtubules in the zoospores of the aquatic fungusAllomyces macrogynus are resistant to microtubule depolymerizing drugs. Consequently, we have analyzed the partial composition and organization of microtubules (Mts) in the cytoplasm and flagellar apparatus in the zoospores ofA. macrogynus. Evidence from two-dimensional gel electrophoresis demonstrated the presence of two -tubulin isoforms in axonemal and cytoplasmic Mts. In addition, a monoclonal antibody specific for acetylated -tubulin was used on one-dimensional protein blots to show that acetylated -tubulins are present in isolated zoospore cell bodies and axonemes. Immunofluorescence microscopy observations using this monoclonal antibody demonstrated that flagellar, kinetosomal, and cytoplasmic Mts were labeled. The nature of Mts in the flagellar apparatus was studied ultrastructurally. InA. macrogynus, the flagellar apparatus consists of the kinetosome, rhizopolast (striated flagellar rootlet), axoneme, and 9 sets of triplet Mts which radiate anteriorly from the proximal end of the kinetosome (microtubular rootlet), Analysis of the rhizoplast indicated that this structure does not contain Mts. The rhizoplast, which connects the functional kinetosome with a single, large basal mitochrondrion, consists of four electron-opaque bands. Serial-sectioning indicated that the rhizoplast is always adjacent to kinetosome triplets 1, 2, and 9, and thus lies perpendicular to the plane of flagellar beat. These results suggest that the primary function of the rhizoplast is to organize the kinetosome and mitochondrion with respect to one another and to bias flagellar beat in the appropriate orientation for cell motility.Abbreviations BSA bovine serum albumin - BCA bicinchoninic acid - DS dilute salts - EGTA ethylene glycol-bis-(-aminoethyl ether)-N,N-tetracetic acid - EM electron microscopy - Mes 2-(N-morpholinomethane sulfonic acid - Mt microtubule - NP-40 Nonidet P-40 - 1-D PAGE one-dimensional polyacrylamide gel electrophoresis - PBS phosphate-buffered saline - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate - 2-D PAGE two-dimensional polyacrylamide gel electrophoresis - Tween-20 polyoxyethylenesorbitan monolaurate     

Gametogenesis inCoelomomyces dodgei Couch (Blastocladiales,Chytridiomycetes)

Summary The ultrastructure of gametogenesis was studied inCoelomomyces dodgei Couch (Blastocladiales, Chytridiomycetes), an obligate parasite of anopheline mosquito larvae and the copepod,Acanthocyclops vernalis. In infected copepods reared under a 16/8 hours light/dark photoperiod at 25 +2 °C., the gametophyte develops over a period of approximately seven days, and gametogenesis is triggered by the onset of the dark period during the last day of development. The initial step of gametogenesis is the elongation of the centriole to form the kinetosome, and measuring time from the onset of the final dark period (0 hours), this occurs prior to the beginning of the light period (8 hours). Subsequently, small vesicles that appear to originate from elements of the rough endoplasmic reticulum (rER) fuse at the distal end of the kinetosome forming the flagellar vesicle into which the axonemal microtubules elongate to form the flagellum (8–12 hours). Similar small vesicles apparently also derived from rER align in planes and fuse to form cleavage furrows which delineate the gamete initials (12–14 hours). As the gamete initials begin forming, the mitochondria within each initial fuse to form a single mitochondrion that associates with the lipid globules and microbodies forming the microbody-lipid globule complex (12–16 hours). The time elapsed between the formation of the flagellar vesicle to the release of mature gametes from the copepod host is about 8.5 hours. No differences were observed in the processes or timing of gametogenesis in male and female gametophytes.