Combining confocal laser scanning and transmission electron microscopy for revealing the mastax musculature in Bryceella stylata (Milne, 1886) (Rotifera: Monogononta)

The rotiferan jaw apparatus (mastax) is characterized by enormous plasticity and according to morphology and feeding strategy, different mastax types can be distinguished. The cuticular hard parts (trophi) of the mastax are often highly specialized and have both a major taxonomic and phylogenetic relevance. Owing to numerous light and scanning electron microscopic studies, the morphology of the trophi is well known but only few attempts have been made to analyze the morphology and functionality of the mastax as a whole. Particularly, the complex muscular system connecting the individual trophi elements and moving them against each other was disregarded in the past. Therefore, the subject of the present study is a detailed analysis of the mastax musculature of the proalid rotifer Bryceella stylata using a combination of transmission electron and confocal laser scanning microscopic techniques, previously applied for revealing the somatic musculature in rotifers exclusively. Based on ultrathin serial sections and phalloidin-dyed specimens, a total number of six paired and two unpaired individual mastax muscles have been identified for the modified malleate trophi system of B. stylata. Possibly homologous muscles in other, so far investigated rotifer species are discussed as well as functional considerations of the individual mastax muscles and their interaction when moving the trophi elements are suggested.     

Combining confocal laser scanning and transmission electron microscopy for revealing the mastax musculature in Bryceella stylata (Milne, 1886) (Rotifera: Monogononta)

The rotiferan jaw apparatus (mastax) is characterized by enormous plasticity and according to morphology and feeding strategy, different mastax types can be distinguished. The cuticular hard parts (trophi) of the mastax are often highly specialized and have both a major taxonomic and phylogenetic relevance. Owing to numerous light and scanning electron microscopic studies, the morphology of the trophi is well known but only few attempts have been made to analyze the morphology and functionality of the mastax as a whole. Particularly, the complex muscular system connecting the individual trophi elements and moving them against each other was disregarded in the past. Therefore, the subject of the present study is a detailed analysis of the mastax musculature of the proalid rotifer Bryceella stylata using a combination of transmission electron and confocal laser scanning microscopic techniques, previously applied for revealing the somatic musculature in rotifers exclusively. Based on ultrathin serial sections and phalloidin-dyed specimens, a total number of six paired and two unpaired individual mastax muscles have been identified for the modified malleate trophi system of B. stylata. Possibly homologous muscles in other, so far investigated rotifer species are discussed as well as functional considerations of the individual mastax muscles and their interaction when moving the trophi elements are suggested.     

Study of the Trophi of <Emphasis Type="Italic">Testudinella</Emphasis> Bory de St. Vincent and <Emphasis Type="Italic">Pompholyx</Emphasis> Gosse (Rotifera: Testudinellidae) by Scanning Electron Microscopy

The fine morphology of the trophi of Pompholyx sulcata and nine species of Testudinella (Rotifera, Monogononta, Flosculariacea, Testudinellidae) was studied by scanning electron microscopy. The number of unci teeth and arched rami scleropili, and the shape of the major unci teeth and fulcrum are considered to be reliable additional characters for identification.     

On the evolution and morphology of the rotiferan trophi, with a cladistic analysis of Rotifera

The phylogeny of Rotifera was examined in different computer‐generated cladistic analyses, including Seisonidea, Bdelloidea, Flosculariacea, Collothecacea and all ploimids treated on family level. The analyses were based on a character matrix solely dealing with morphological characters, primarily based on the trophi morphology. Limnognathia maerski (Micrognathozoa), Rastrognathia macrostoma and Gnathostomula paradoxa (Gnathostomulida) were used as outgroups. The cladistic analysis performed by paup produced 288 most parsimonious trees. peewee analyses produced between 140 and 432 trees, depending on the concavity value. The monophyly of Eurotatoria, Monogononta and Ploima was confirmed in all obtained trees. All analyses suggested a division of Ploima into major clades. One clade corresponded to Transversiramida while the other contained all other ploimid taxa and recognized Antrorsiramida as a monophylum. Based on the obtained results a scenario for the trophi evolution is proposed. The analyses suggested that the presence of an incus is synapomorphic for Gnathifera while mallei are synapomorphic for Micrognathozoa and Rotifera. The ancestral rotifer trophi probably resembled those in Harringia (Asplanchnidae).     

Old and new data on Seisonidea (Rotifera)

Class Seisonidea consists of one marine genus, Seison, with two species (S. nebaliae and S. annulatus) which are epizoic on the crustacean Nebalia. Seisonidea are the only rotifers whose reproduction is strictly bisexual. They also possess the unusual feature of having well-developed males. In this paper we review the literature concerning biogeographical distribution, relationships with their host, and morphology, and present new information obtained with SEM and optical microscopy of both living animals and serial sections.Some seisonid characters, such as the paired retrocerebral organ, nervous system, and trophi, suggest that they hold a primitive position within phylum Rotifera. However, they do share features with other rotifers: paired gonads and an unusual locomotory behavior with Bdelloidea; lateral antennae, cellular stomach wall, arrangement of intracytoplasmic lamina, and other characters with Monogononta. Some features are peculiar to Seison: obligatory amphimixis, endolecythal eggs, encysted spermatozoa, unique mastax, and absence of copulatory organ in males and of vitellaria in females. Reduction of the corona and absence of resistant stages in Seisonidea may be related to their habitat and to their life style. We propose a closer relationship of Seisonidea to Monogononta than previously asserted.     

A gentle method for the preparation of hard parts (trophi) of the mastax of rotifers and scanning electron microscopy of the trophi ofBrachionus plicatilis (Rotifera)

Summary Trophi of the rotiferBrachionus plicatilis were prepared by dissolving rotifer tissues and lorica with sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) and were examined by scanning electron microscopy in order to obtain information on the functional morphology of these structures. The trophi are not composed of distinct parts but form a continuous structure of rigid pieces and connecting membranous regions. The membranous components allow movements of the rigid parts against each other and/or restrict the extent of such movements. The main hinge for the movement of the trophi is the membranous connection between the two rami; movements of rami and unci occur together since these parts are tightly connected by two narrow membranes in the subuncus region. The subunci seem to constitute masticating devices which act against grooved ridges of the rami and might make it feasible to disintegrate nutrient particles to as small as 1 m. Crushing of coarser nutrients might be performed by the opposite surfaces of the two rami.Abbreviations br bulla rami - c crest along the ventral surface of the manubrium - cd distal part of the manubrium (cauda) - cv proximal part of the manubrium (clava) - d1 entrance from the bowl-shaped recess at the dorsal side of the manubrium into one of the (three) cavities within the proximal part of the manubrium - d2 second dorsal entrance into one of the (three) cavities within the proximal part of the manubrium - eu edge at the distal end of the uncus which fits into a notch at the proximal end of the manubrium - gr small, oblong grooves at the distal surface of the reinforced projecting ridges of the rami - f fulcrum - h hinge between the two rami - l ligaments extending from the central membranes to the dorsal surfaces of the unci - m central membranous structures - ma manubrium - p partition between the cavities within the ramus - ps large, proximal surfaces of the rami - r ramus - rd reinforced ridge on the anterio/dorsal part of the ramus - rv ventral, triangular surface of the ramus - su subuncus - sur root-like structures connecting the subuncus with the uncus plate - t teeth-like proximal endings of the uncus ridges - u uncus - um membranous connection between uncus and manubrium - ur membranous connection between uncus and ramus in the subuncus region - uvr ridges on the ventral side of the uncus - ve ventral entrance into one of the (three) cavities within the proximal part of manubrium     

The musculature of Squatinella rostrum (Milne, 1886) (Rotifera: Lepadellidae) as revealed by confocal laser scanning microscopy with additional new data on its trophi and overall morphology

Wilts, E.F., Wulfken, D., Ahlrichs, W.H. and Martínez Arbizu, P. 2012. The musculature of Squatinella rostrum (Milne, 1886) (Rotifera: Lepadellidae) as revealed by confocal laser scanning microscopy with additional new data on its trophi and overall morphology.—Acta Zoologica (Stockholm) 93 : 14–27. The monogonont rotifer Squatinella rostrum was investigated with light, scanning electron and confocal laser scanning microscopy to reveal new morphological data on its inner and outer anatomy. In total, the visualized somatic musculature displays five paired longitudinal muscles (musculi longitudinales I–V) and nine circular muscles (musculi circulares I–IX). Compared to other species, S. rostrum is characterized by the absence of several longitudinal and circular muscles (e.g. musculus longitudinalis capitis, corona sphincter and pars coronalis). A reconstruction of the mastax musculature revealed a total number of seven paired and two unpaired mastax muscles. Possibly homologous somatic and mastax muscles in other, thus far investigated rotifers are discussed. Moreover, we provide a phylogenetic evaluation of the revealed morphological characters and suggest possible autapomorphic characters supporting Squatinella and Lepadellidae. Finally, we refer to some striking similarities in the morphology, ecology and way of movement of Squatinella and Bryceella that may indicate a closer relationship of both taxa.     

On the structure and function of the mastax ofBrachionus plicatilis (Rotifera), a scanning electron microscope analysis

Sectioned specimens ofBrachionus plicatilis were examined by scanning electron microscopy (SEM). This technique was used to characterize the out-side appearance as well as the position and arrangement of the hard parts (trophi) in the interior of the mastax. Two strips of muscle stretch dorsally across the width of the mastax. Below these muscle strips, all the other components of the mastax form a continuous structure which is smoothly locked to the outside. Some nerve structures and glands of the mastax are found on the dorsal side, outside these muscle strips. By comparing the shape of parts of the trophi, exposed by sectioning, with the views of preparations of isolated trophi, it was possible to estimate how the trophi are positioned in relation to other components of the mastax and in relation to the animal as a whole. The results are used to complement and modify statements and conclusion from a previous study.     

Ontogeny of the jaws of monogonont rotifers: the malleate trophi of Rhinoglena and Proalides (Ploima,Epiphanidae)

Information on the embryonic development of the malleate trophi in Epiphanidae (Rotifera, Monogononta, Ploima) is presented, based on scanning electron microscopy observations in Rhinoglena fertoeensis, R. frontalis, R. kutikovae, R. tokioensis, and Proalides tentaculatus, to contribute to the understanding of this structure of high evolutionary and functional relevance in Rotifera. The first observable and distinctly sclerotized structures were a double row of median transversal sclerites along the longitudinal axis, wherein the future unci, rostellar scleropili, cristae rami, and basal apophyses became recognizable. Fulcrum and manubria arose subsequently; the fulcrum sclerites were longitudinally ordered in a double layer. The rami chambers developed last as lamellar structures. Unci appeared as separate thin, elongate elements, the primary uncini, developing to uncus plates by transversal growth and apposition of sclerite material on the shafts of the uncini. The heads of the uncini showed their greatest development after fusion of their shafts into uncus plates. The interjacent spaces between the heads functioned as a mold, organizing bundles of sclerites which developed into the uniseriate, zigzag‐shaped cristae rami. The fulcrum attained its definite shape by elongation of the double layer of rod‐shaped sclerites into appressed sclerofibrillae. Manubria became visible as a proximal ridge of sclerites, whereupon a triangular lamella composed of crisscross‐oriented sclerites developed distally, growing out to the manubrial chambers. Ramus chambers originated from two longitudinal amorphous lamellae incorporating the median rami sclerites and closing from distal to proximal; subbasal chambers were formed before the basal chambers.     

EST based phylogenomics of Syndermata questions monophyly of Eurotatoria

Background  

The metazoan taxon Syndermata comprising Rotifera (in the classical sense of Monogononta+Bdelloidea+Seisonidea) and Acanthocephala has raised several hypotheses connected to the phylogeny of these animal groups and the included subtaxa. While the monophyletic origin of Syndermata and Acanthocephala is well established based on morphological and molecular data, the phylogenetic position of Syndermata within Spiralia, the monophyletic origin of Monogononta, Bdelloidea, and Seisonidea and the acanthocephalan sister group are still a matter of debate. The comparison of the alternative hypotheses suggests that testing the phylogenetic validity of Eurotatoria (Monogononta+Bdelloidea) is the key to unravel the phylogenetic relations within Syndermata. The syndermatan phylogeny in turn is a prerequisite for reconstructing the evolution of the acanthocephalan endoparasitism.     

A Mitogenomic Re-Evaluation of the Bdelloid Phylogeny and Relationships among the Syndermata

Molecular and morphological data regarding the relationships among the three classes of Rotifera (Bdelloidea, Seisonidea, and Monogononta) and the phylum Acanthocephala are inconclusive. In particular, Bdelloidea lacks molecular-based phylogenetic appraisal. I obtained coding sequences from the mitochondrial genomes of twelve bdelloids and two monogononts to explore the molecular phylogeny of Bdelloidea and provide insight into the relationships among lineages of Syndermata (Rotifera + Acanthocephala). With additional sequences taken from previously published mitochondrial genomes, the total dataset included nine species of bdelloids, three species of monogononts, and two species of acanthocephalans. A supermatrix of these 10-12 mitochondrial proteins consistently recovered a bdelloid phylogeny that questions the validity of a generally accepted classification scheme despite different methods of inference and various parameter adjustments. Specifically, results showed that neither the family Philodinidae nor the order Philodinida are monophyletic as currently defined. The application of a similar analytical strategy to assess syndermate relationships recovered either a tree with Bdelloidea and Monogononta as sister taxa (Eurotatoria) or Bdelloidea and Acanthocephala as sister taxa (Lemniscea). Both outgroup choice and method of inference affected the topological outcome emphasizing the need for sequences from more closely related outgroups and more sophisticated methods of analysis that can account for the complexity of the data.     

Ultrastructure and function of the mastax in Dicranophorus forcipatus (Rotifera: Monogononta)

Rotifers are characterized by a complex set of cuticularized jaw elements in the pharynx. The fine structure of the jaw elements has been the subject of SEM studies for some time, but only very limited information exists on the ultrastructure of the jaw elements and their function beyond taxonomic considerations. Drawing on SEM and TEM techniques, the present study presents a detailed analysis of the mastax in Dicranophorus forcipatus, a carnivorous monogonont rotifer species from freshwater habitats characterized by an extrusible, grasping jaw apparatus. Based on ultrathin serial sections, the jaw elements are reconstructed and, in total, nine paired and two unpaired muscles identified. Possibly homologous muscles in other rotifer species are discussed and functional considerations of the forcipate mastax are suggested.     

Micrognathozoa: a new class with complicated jaws like those of Rotifera and Gnathostomulida

A new microscopic aschelminth-like animal, Limnognathia maerski nov. gen. et sp., is described from a cold spring at Disko Island, West Greenland, and assigned to Micrognathozoa nov. class. It has a complex of jaws in its pharynx, and the ultrastructure of the main jaws is similar to that of the jaws of advanced scleroperalian gnathostomulids. However, other jaw elements appear also to have characteristics of the trophi of Rotifera. Jaw-like structures are found in other protostome taxa as well-for instance, in proboscises of kalyptorhynch platyhelminths, in dorvilleid polychaetes and aplacophoran mollusks-but studies of their ultrastructure show that none of these jaws is homologous with jaws found in Gnathostomulida, Rotifera, and Micrognathozoa. The latter three groups have recently been joined into the monophylum Gnathifera Ahlrichs, 1995, an interpretation supported by the presence of jaw elements with cuticular rods with osmiophilic cores in all three groups. Such tubular structures are found in the fulcrum of all Rotifera and in several cuticular sclerites of both Gnathostomulida and Micrognathozoa. The gross morphology of the pharyngeal apparatus is similar in the three groups. It consists of a ventral pharyngeal bulb and a dorsal pharyngeal lumen. The absence of pharyngeal ciliation cannot be used as an autapomorphy in the ground pattern of the Gnathifera because the Micrognathozoa has the plesiomorphic alternative with a ciliated pharyngeal epithelium. The body of Limnognathia maerski nov. gen. et sp. consists of a head, thorax, and abdomen. The dorsal and lateral epidermis have plates formed by an intracellular matrix, as in Rotifera and Acanthocephala; however, the epidermis is not syncytial. The ventral epidermis lacks internal plates, but has a cuticular oral plate without ciliary structures. Two ventral rows of multiciliated cells form a locomotory organ. These ciliated cells resemble the ciliophores present in some interstitial annelids. An adhesive ciliated pad is located ventrally close to a caudal plate. As in many marine interstitial animals-e.g., gnathostomulids, gastrotrichs, and polychaetes-a special form of tactile bristles or sensoria is found on the body. Two pairs of protonephridia with unicellular terminal cells are found in the trunk; this unicellular condition may be the plesiomorphic condition in Bilateria. Only specimens with the female reproductive system have been found, indicating that all adult animals are parthenogenetic females. We suggest that 1) jaws of Gnathostomulida, Rotifera, and the new taxon, Micrognathozoa, are homologous structures; 2) Rotifera (including Acanthocephala) and the new group might be sister groups, while Gnathostomulida could be the sister-group to this assemblage; and 3) the similarities to certain gastrotrichs and interstitial polychaetes are convergent.     

A simplified method for preparing rotifer trophi for scanning electron microscopy

A simple, quick technique for the preparation of rotifer trophi for scanning electron microscopy is described. The method permits visual monitoring of the extraction process and does not require critical point drying of the specimens. Micrographs showing fine, structural detail of the hard parts of the mastax of representatives of the following genera are presented:Asplanchna, Conochilus, Filinia, Hexarthra, Keratella, Proalides, Synchaeta, andTrichocerca.     

Morphology of Floscularia ringens (Rotifera, Monogononta) from egg to adult

Abstract. Floscularia ringens is a cosmopolitan, sessile rotifer (class Monogononta) that lives inside a tube it constructs from numerous small, rounded pellets. Adults of F. ringens produce parthenogenetic eggs that are retained within the tube. Upon hatching, juveniles remain within the maternal tube for a short time completing their development before swimming away. The free-swimming juvenile has a conical body, short foot, small corona, and mastax with trophi, but appears unable to feed. After a short time (<1 day), the young rotifer attaches permanently to a substrate and its morphology changes radically: the corona develops 4 wide lobes and the foot elongates, becoming slender and retractable. Once the corona has developed, the young animal begins to feed by producing filtering currents, and also starts to build its own tube. Here we report 4 new morphological details regarding this species. (1) A specialized epidermal groove is present on the trunk in front of the cloaca. (2) A small hole is located in the center of the inner surface of each pellet of the tube. (3) The muscles inside the foot are U-shaped in transverse section. (4) The size of the trophi remains unchanged during growth of the juvenile into an adult.     

First description of the serotonergic nervous system in a bdelloid rotifer: Macrotrachela quadricornifera Milne 1886 (Philodinidae)

Class Bdelloidea of phylum Rotifera comprises aquatic microinvertebrates that are known for both obligate parthenogenesis and for resisting desiccation through a dormant reversible state. In the frame of an investigation about the role of the nervous system in controlling life cycle, reproduction and dormancy, we describe the serotonergic system of a bdelloid, Macrotrachela quadricornifera, using serotonin immunohistochemistry and confocal laser scanning microscopy. Serotonin immunoreactivity is present in the cerebral ganglion, lateral nerve cords and peripheral neurites. The cerebral ganglion consists of perikarya that send neurites cephalically to the rostrum and corona. A pair of neurites exits the cerebral ganglion as lateral nerve cords, and proceeds caudally to the pedal ganglion where additional neurites enter the foot. Based on the location of serotonergic immunoreactivity, we hypothesize that the neurotransmitter is involved in both motor activity (e.g., ciliary beating, inchworm-like locomotion) and sensory activity. A comparison between the serotonergic nervous systems of M. quadricornifera and species of Monogononta reveals differences in the numbers and patterns of cerebral perikarya, peripheral perikarya, and periperhal neurites. These differences may have functional significance for understanding adaptations to specific environments and/or systematic significance for reconstructing the rotiferan ground pattern.     

The syndermatan phylogeny and the evolution of acanthocephalan endoparasitism as inferred from 18S rDNA sequences

The phylogeny of the Syndermata (Rotifera: Monogononta, Bdelloidea, Seisonidea; Acanthocephala: Palaeacanthocephala, Eoacanthocephala, Archiacanthocephala) is key to understanding the evolution of acanthocephalan endoparasitism from free-living ancestors. In the present study, maximum likelihood, distance/neighbor-joining, and maximum parsimony analyses have been carried out based on 18S rDNA data of 22 species (four new sequences). The results suggest a monophyletic origin of the Eurotatoria (Monogononta+Bdelloidea). Seison appears as the acanthocephalan sistergroup. Palaeacanthocephala split into an "Echinorhynchus"-and a "Leptorhynchoides"-group, the latter sharing a monophyletic origin with the Eoacanthocephala and Archiacanthocephala. As inferred from the phylogeny obtained acanthocephalan endoparasitism evolved from a common ancestor of Seison and Acanthocephala that lived epizoically on an early mandibulate. Probably, an acanthocephalan stem species invaded the mandibulate host, thus establishing an endoparasitic lifestyle. Subsequently, vertebrates (or gnathostomes) became part of the parasite's life cycle. In the stem line of the Archiacanthocephala, a terrestrial life cycle has evolved, with an ancestor of the Tracheata (Insecta, Myriapoda) acting as intermediate host.     

The hard parts (trophi) of the rotifer mastax do contain chitin: evidence from studies on Brachionus plicatilis

The jaws (trophi) of the rotifer Brachionus plicatilis are soluble in strong acids but are resistant to long treatments by strong alkali. They show the same buoyant density as chitin and also as the chitin-containing layers of rotifer egg-shells. The presence of chitin in these structures was confirmed using the following techniques: chitosan-tests, thin-layer chromatography of trophi-hydrolysates which revealed glucosamine, by dissolving trophi with chitinase and electron microscopic WGA/gold-labelling. The content of chitin in the trophi was estimated by two different methods to be approx. 64% (50-75%).     

Cuticular hydrocarbon pattern as a chemotaxonomy marker to assess six species of thrips

Thrips constitute several families of slender insects with fringed wings and unique asymmetrical mouthparts. They have become globally important pests, infesting a variety of agriculturally important crops. Species of thrips are difficult to identify due to their small size and similarities in morphology. Recently, in addition to morphology, both molecular and non-molecular taxonomic tools have been used to identify species differences. Insect cuticular hydrocarbons have been widely used in chemotaxonomy. In this study, a Thermal Separation Probe was used to identify the cuticular hydrocarbons of Frankliniella occidentalis, Frankliniella intonsa, Thrips palmi, Thrips hawaiiensis, Haplothrips chinensis and Gynaikothrips ficorum. We analyzed the hydrocarbon composition of adults in all 6 species, and in the larvae of F. occidentalis, T. hawaiiensis and T. palmi. The results showed that the composition of cuticular hydrocarbons differed between species. All 6 species of adults and 3 species of larvae were easily distinguishable by quantitative analysis of hydrocarbon profiles. These results provide a possible method for the identification of thrips.     

Broad taxonomic sampling of mitochondrial cytochrome c oxidase subunit I does not solve the relationships between Rotifera and Acanthocephala

The phylogenetic relationships within Syndermata (Acanthocephala + Rotifera) are still unresolved. Cladistic morphological analyses support monophyly of Rotifera and Eurotatoria (Bdelloidea + Monogononta), while molecular phylogenies of 18S, 28S, COI, hsp82 and EST propose different topologies, with at least six contrasting scenarios. All these phylogenies are characterized by poor taxon sampling; thus, our aim is to solve the relationships within Syndermata sampling as many sequences as possible from one single locus. We reconstructed phylogenetic relationship using more than 1000 sequences of COI. We performed Maximum Likelihood and Bayesian phylogenetic reconstructions on amino acid alignments, using either Gnathostomulida or Platyhelminthes as an outgroup, and then we performed SH tests to provide confidence on the best phylogenetic hypotheses. All four major clades (Acanthocephala, Bdelloidea, Monogononta and Seisonidea) are always highly supported. The basal relationship among the four clades is not consistently resolved by any of the phylogenetic reconstructions; nevertheless, there is a strong support for a clade of Acanthocephala + Bdelloidea from the SH tests, in agreement with other phylogenies from ribosomal genes and EST analyses.