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.     

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.     

The ultrastructure of the mastax of Filinia longiseta (Flosculariaceae, Rotifera): Informational value of the trophi structure and mastax musculature

The study contributes to the discussion of mastax evolution within Rotifera by giving an insight into the ultrastructure of the mastax in the rotifer species Filinia longiseta (Flosculariacea) and additionally into the bdelloid rotifer species Adineta vaga and Zelinkiella synaptae. The existence of cuticularized jaw elements (trophi) in the mastax, a muscular pharynx, is one of the defining rotiferan characters and the basis on which the monophyletic taxon Gnathifera Ahlrichs 1995a, comprising Rotifera, Gnathostomulida, Micrognathozoa and Acanthocephala, was erected. By means of SEM observations of the trophi and ultrathin serial sections (TEM) of the mastax, the internal and external organization of the jaw elements of F. longiseta is reconstructed. TEM sections of the incus of Filinia demonstrate that the fulcrum and the rami are built up by multitudes of tiny cuticular tubes. While tubular substructures in the rotiferan fulcrum have been described previously, distinct cuticular tubes as a substructure of the ramus have only been described for species belonging to the taxa Seisonidea and Bdelloidea so far ( [Koehler and Hayes, 1969] and [Ahlrichs, 1995b]). By comparing the appearance and arrangement of the cuticular tubes in the rami of F. longiseta to those found in species of Seisonidea and Bdelloidea, a higher degree of resemblance between the structures in F. longiseta and Bdelloidea can be reported. The occurrence of the ramus substructures in species of Seisonidea (Paraseison annulatus and Seison nebaliae) is given consideration to represent an intermediate between the ramus substructure of Bdelloidea/Flosculariacea and Ploima. Additionally, the mastax musculature of F. longiseta, being associated with the trophi, is described: A total of seven muscles are found that directly insert the jaw elements or are indirectly associated with them via muscle-to-muscle connections.     

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.     

Trophi Structure in Bdelloid Rotifers

Bdelloids show a rather uniform morphology of jaws (trophi), named ramate. The most recognizable feature is the presence of a series of teeth forming unci plates. The unci are not uniform in size; each plate has 1–10 major median teeth. Using SEM pictures of trophi and data from the literature, we analyzed the number of major unci teeth in relation to trophi size, total number of teeth, and environmental features. Variability in the number of major unci teeth in bdelloids is not related to trophi size or to total number of unci teeth, while total number of unci teeth and trophi size seem to be related to each other: larger trophi in general have more teeth than smaller trophi. Few major teeth are more common in species living in water bodies where they possibly eat unicellular algae, while more major teeth are more common in species living outside water bodies, among mosses and lichens, where they possibly eat bacteria.     

Revisiting the Cephalodella trophi types

The six trophi types proposed by Wulfert (Archiv für Hydrobiologie 31:592–636, 1937) are used as one main character to identify Cephalodella species, although these trophi types were just based on the trophi of 31 species described after light microscopy findings. Given the 160 species nowadays valid and the possibility of examining trophi with scanning electron microscopy it is questionable if the definitions of the six types might or rather have to be improved in order to facilitate species identification. Here, a new even simpler definition scheme is proposed to identify the six trophi types.     

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.     

STRIP-LIKE COVERING REVEALED ON “WALL-LESS”ASTEROMONAS (CHLOROPHYCEAE)1

Asteromonas Artari previously described as a wall-less biflagellated unicell exhibits a strip-like cell covering on some cells. Both tannic acid fixation and freeze-etch reveal the covering of electron opaque strips outside the plasmalemma. Cross striations are apparent in the surface strips. Golgi vesicles contain material presumed to be precursor components of the covering strips. The crystalline pattern of the freeze-etched material is similar to that reported for other, walled volvocalean algae.     

A carnivorous bdelloid rotifer, Abrochtha carnivora n.sp.

Abstract. Here we describe a new bdelloid rotifer, Abrochtha carnivora n.sp., that preys on other bdelloids and monogonont rotifers. This is the first report of predatory behavior in bdelloids. Despite this extraordinary behavior, A. carnivora displays no major structural deviation from the standard body plan for members of the bdelloid family Philodinavidae: body with head (with ciliated corona), trunk, and foot, mastax with ramate trophi. The finding of such a carnivorous species, unique for the class, appears to contradict the common assumption that the wholly parthenogenetic bdelloids are evolutionary dead-ends.     

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

Summary 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%).     

Do Rotifer Jaws Grow After Hatching?

The hard articulated jaws of some pseudocoelomate metazoans were recently used in reconstructing their phylogenetic relationships, but we still do not know if these structures could change in size and shape during the life of individuals, and experimental data are lacking on their post-embryonic development. Rotifers are one of the groups in which hard articulated jaws, called trophi, are well known, and are widely used taxonomically. Here we report on SEM study of trophi of rotifers of different ages, to determine if the trophi structures change in shape and/or in size during post-embryonic development. We used linear measurements and geometric morphometrics analyses from scanning electron microscopic pictures of trophi of Cupelopagis vorax, Dicranophorus forcipatus, Macrotrachela quadricornifera, Notommata glyphura, Rotaria macrura, R. neptunoida, and R. tardigrada. Results for these species show that trophi do not change after hatching, either in size or in shape. In contrast, data on Asplanchna priodonta reveal trophi growth after hatching.     

Body musculature of Beauchampiella eudactylota (Gosse, 1886) (Rotifera: Euchlanidae) with additional new data on its trophi and overall morphology

This study presents the results of confocal laser scanning microscopy and fluorescence‐labelled phalloidin used to visualize the system of body musculature in Beauchampiella eudactylota. Moreover, the poorly known trophi of B. eudactylota are described based on scanning electron microscopy. In total, four paired longitudinal muscles (musculi longitudinales I–IV) and three circular muscles (musculi circulares I–III) were identified. Among these are the musculus longitudinalis ventralis, the musculus longitudinalis dorsalis and the musculus circumpedalis as documented in previous studies for other rotifer species. Compared to other species, B. eudactylota is characterized by the low number of lateral longitudinal muscles and the absence of some longitudinal muscles (musculi longitudinales capitum) and circular muscles (corona sphincter, musculus pars coronalis). Moreover, scanning electron microscopic data on the trophi of B. eudactylota reveal a number of striking similarities to the trophi in some species of Epiphanidae. This suggests that either (1) these similarities represent plesiomorphic characters present both in Epiphanidae and B. eudactylota or (2) they are synapomorphic features of B. eudactylota and some species of Epiphanidae, which would question the monophyly of Euchlanidae.     

Study of architectonics of rotifer musculature by the method of fluorescence with use of confocal microscopy

Musculature of two species of rotifers Testudinella patina (Testudinellidae) and Platyias patulus (Brachiomidae) was studied in confocal laser scanning microscope (CLSM) using fluorescent-labeled phalloidin. It includes cutaneous, visceral, and cutaneus-visceral musculature. The common pattern of structure of the cutaneous musculature is represented by postcoronal circular or transverse muscles and connected with them 2–3 pairs of retractors of the trunk, dorsolateral muscles (17-4), two pairs or bundles of lateral retractors of the corona, circular muscles of the foot, and 10-2 retractors of the foot. Visceral musculature includes muscles of the mastax of both kinds. Spiral-like muscle of cloaca of the T. patina and associated with it V-shaped one as well as strong dorsolateral retractors consisting of 6 longitudinal muscle bundles are typical of Testudinellidae only. Three pairs of cutaneus-visceral muscles bind the musculature of mastax with the body surface in T. patina. Differences in localization and thickness of some elements of musculature of these species are determined by morphological peculiarities of structure of the corona, mastax, and foot, as well as by the rotifer body shape.     

The genus Synchaeta (rotifers) in a north-western Mediterranean coastal lagoon (Etang de Thau, France): taxonomical and ecological remarks

During a survey of a north-western Mediterranean coastal lagoon, (the Etang de Thau, and at a nearby marine station, 1994–1998), six species of the genus Synchaetawere identified. The systematics of this illoricate genus is difficult and identification is virtually impossible in preserved material, although some indication may be obtained from an examination of the trophi after treatment with sodium hypochlorite. In this study, taxonomic characters (mastax, body morphology and egg size) were obtained from living material prior to preservation. We add some ecological observations: distribution of species (neither temperature nor salinity were found to be determining factors), co-existence (up to five species can co-occur) and consideration of trophic links (competition with tintinnids, copepod nauplii and cirripedes).     

Stomach stress and strain depend on location, direction and the layered structure

The stomach is as other parts of the gastrointestinal tract functionally subjected to dimensional change. Hence, the biomechanical properties are of functional importance. Our group has previously demonstrated that the stress–strain properties of the rat and rabbit stomach wall were species-, location- and direction-dependent. We further wanted to study the anisotropic biomechanical properties of the stomach wall in pigs. Furthermore, we made an in-depth biomechanical test on the layered wall of the stomach in different regions. Two stomach strips were cut both in longitudinal direction (parallel with the greater curvature) and circumferential direction (perpendicular to the greater curvature) from the gastric fundus, corpus and antrum. One strip was used for the non-separated (intact) wall test and the other one was separated for the test on the mucosa–submucosa and muscle layers individually. The length, thickness and width of each strip were measured from digital images. The uni-axial stress and strain were computed from the force generation and the tissue strip deformation during stretching. The muscle layer was the thickest in the antrum whereas the mucosal–submucosal layer was the thickest in the corpus of the stomach (P<0.01). The strips from the corpus were stiffest among the three regions in both longitudinal and circumferential directions (P<0.001). The longitudinal strips was stiffer than the circumferential strips in all three regions (P<0.001) and the mucosa–submucosa strips was stiffer than the intact wall and the muscle layer in both directions for the fundus and the corpus (P<0.001). The constant a of the intact wall and mucosa–submucosa layer was in both directions linearly associated with the mucosa–submucosa thickness. In conclusion, the uni-axial stress–strain curves of pig stomach were location-, direction- and layer-dependent. The stiffer wall in the corpus is likely due to its thicker mucosa, i.e., the stiffness of the mucosa–submucosa layer seems can explain the intact wall stiffness. Since the structure and function of the pig stomach are similar to the human stomach, we believe that the data obtained from this study can be extended to humans. Detailed biomechanical mapping of the stomach will likely help us to understand physiological functions of the different parts of the human stomach, such as gastric accommodation and mechanosensation.     

Rhinoglena frontalis (Rotifera,Monogononta): a scanning electron microscopic study

Females and males of Rhinoglena frontalis (Monogononta, Epiphanidae) are observed by SEM and their external morphologies are compared. The two sexes differ in size and shape of the body. The female body is fusiform with a short, conical foot, while the male body is more slender and has a rather long foot. The rotatory apparatus (or corona) of both sexes is similar with only minor differences and consists of rows and tufts of cilia arranged around the mouth opening. The corona is made of two paired lobes lateral to the mouth and of a third prominent dorsal lobe, usually called proboscis. The three lobes are lined externally by dense rows of cilia, which constitute the cingulum, used for swimming. The central surface of the proboscis is covered with numerous longitudinal rows of cilia bent towards the mouth. The lateral lobes show, on their central surfaces, two concentric arcs of cirri (made of tightly packed cilia) bent towards the mouth. The similar organization of the rotatory apparatus of both sexes is related to the fact that the male, in this species, is able to feed and has a developed mastax and digestive system. The trophi of both sexes are illustrated and compared.     

Zur Morphologie der Malacostraca: Der Kaumagen der Mysidacea im Vergleich zu dem verschiedener Peracarida und Eucarida

Zusammenfassung 1. Der Kaumagen vonNeomysis vulgaris wird als Totalpräparat seiner Chitinintima dargestellt und im einzelnen beschrieben.2. Zur Erfassung des Oberflächenreliefs im Hinblick auf die Darstellung der Magenmuskulatur wurde das Chitinpräparat in einer Silbersalzlösung durch Reduktion mit einem Überzug aus feinsten Silberpartikeln versehen und damit undurchsichtig gemacht.3. Die Muskulatur des Labrum wird beschrieben und in Übersichtsbildern dargestellt. Ein Vergleich mit der Oberlippenmuskulatur vonDiastylis rathkei (Cumacea) ergibt Übereinstimmung für mehrere Muskelzüge.4. Die Muskulatur des Kaumagens wird beschrieben und in Übersichtsbildern dargestellt. Ein Vergleich mit der entsprechenden Muskulatur der Decapoda ist nur in sehr beschränktem Umfange möglich; das Ergebnis wurde in Tabelle 3 zusammengestellt.5. Unter Verwendung von Totalpräparaten der Chintinintima werden die Kaumägen der verschiedenen Vertreter der Eucarida und Peracarida verglichen. Es ergibt sich, daß im Rahmen des gemeinsamen Bauplanes ein Isopodentyp und ein Dekopadentyp ausgeprägt ist, die durch einen Zwischentyp verbunden sind: Die Mägen von Amphipoden, Cumaceen, Tanaidaceen und Isopoden einerseits und die Mägen von Mysideen, Euphausiaceen und Dekapoden andererseits sind durch gemeinsame Merkmale gekennzeichnet, während die Mägen der Lophogastriden Merkmale beider Gruppen vereinigen.6. Die Stücke, welche die Magenmühle der Dekapoden zusammensetzen, sind keine Sonderbildung dieser Gruppe, sondern gehören wie die übrigen Teile des Dekapodenmagens dem allgemeinen Bauplan des Malakostrakenmagens an.

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On the morphology of the Malacostraca: the mastax of the mysidacea as compared to that of various Peracarida and Eucarida

Anatomy and muscle arrangement of the mastax and the labrum ofNeomysis vulgaris are described. For this purpose the mastax as a whole was made transparent and its chitin preparation then covered with minute silver particles (transfer into a silver salt solution; reduction of the silver) and thus made opaque. A comparison of the upper lip muscular system with that ofDiastylis rathkei resulted in agreement with respect to several muscle groups. A comparison of the stomach muscles with corresponding muscles of the decapods — which is possible only to a limited extent — is given in Table 3. On the basis of total preparations of the chitinintima of the stomachs of different eucarid and peracarid representatives, a direct comparison was possible. Within the bounds of the common general morphology of the malacostracan stomach, an isopod type (Amphipoda, Cumacea, Tanaidacea, Isopoda) and a decapod type (Mysidacea, Euphausiacea, Decapoda) are distinguishable. These two types are connected by an intermediate type, which reveals characteristics of both (Lophogastrida). The parts of the stomach which compose the grinding mechanism of the decapods do not represent a special feature of this group, but belong, as do the other parts of the decapod stomach, to the general morphological design of the malacostracan stomach.

     

Disentangling the morphological stasis in two rotifer species of the <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> species complex

The taxonomic uncertainty surrounding cryptic species complexes has traditionally been resolved using lengthy experimental approaches, while, since the advent of PCR based techniques the number of cryptic species described in a variety of taxa is increasing steadily. Here we formally describe a new rotifer species of the Brachionus plicatilis complex: Brachionus manjavacas n.sp., disentangling what was known as a morphological stasis. Detailed morphological analyses demonstrated significant differences in body shape and size between B. manjavacas and B. plicatilis s.s., analysed by geometric morphometrics; unfortunately these statistical differences are not taxonomically reliable because of wide overlaps. Size and asymmetry of masticatory apparatus, named trophi, observed by SEM, gave similar results, with taxonomic ambiguity. Only the shape of small pieces of the trophi, named satellites, were consistently different between the species. On a strictly classical taxonomical basis it is absolutely useful to name new species on morphological bases, as we did, and to assess their status as distinct entities. Nevertheless, the two species are broadly similar; therefore, we do not suggest using the small differences in shape of satellites of trophi to identify the species for further ecological studies, but to continue discriminating them on genetic marker bases. Handling editor: K. Martens