Comment on "Ultrastructure reveals ancestral vertebrate pharyngeal skeleton in yunnanozoans".

Tian et al. (Research Articles, 8 July 2022, abm2708) hypothesized that yunnanozoans are stem-group vertebrates on the basis of "cellular cartilage", "fibrillin microfibers", and "subchordal rod" associated with the branchial arches of yunnanozoans. However, we reject the presence of cellular cartilage, fibrillin, and the phylogenetic proposal of vertebrate affinities based on ultrastructure and morphology of yunnanozoans from more than 8000 specimens.

FOREGUT ULTRASTRUCTURE OF ADULT SANGUINICOLA VOLGENSIS (RASÍN, 1929) MCINTOSH, 1934 (DIGENEA: APOROCOTYLIDAE).

Herein, we use scanning and transmission electron microscopy to describe the foregut (mouth, pharyngeal canal, and associated epithelia and musculature) of an adult freshwater fish blood fluke, Sanguinicola volgensis (Rasín, 1929) McIntosh, 1934, infecting the blood of sabre, Pelecus cultratus Linnaeus, 1758 (Cypriniformes: Leuciscidae) from the Volga River, Russia. Our results indicate that S. volgensis has a pharynx and lacks an oral sucker and that its pharyngeal canal acts as a peristaltic pump that sucks blood into the esophagus, whereupon digestion commences with granules secreted from the esophageal epithelium. We saw no evidence of longitudinal muscle fibers beneath the pharyngeal canal epithelium, pharyngeal glands, or pharyngeal epithelial cells or muscle cells within the pharyngeal muscular complex; collectively indicating the presence of a pharynx rather than an oral sucker. The specialized epithelial lining associated with the mouth and pharyngeal canal evidently is unique among neodermatans; it is smooth, ∼40 nm thick anteriorly, and thickens (∼250-700 nm) posteriorly as the mouth cavity transitions into the pharyngeal canal. The pharyngeal canal epithelium has lumps of dense material resembling those of the basal lamina and fibrous coat of the tegument. The actin-like material within the pharyngeal cavity epithelium could provide structural support to the pharynx.

The remarkable larval anatomy of Proceratophrys minuta Napoli, Cruz, Abreu and Del-Grande, 2011 (Amphibia: Anura: Odontophrynidae).

The free living larvae of anurans (i.e., tadpoles) are a key element in the evolution and diversification of this group, and as such, their morphology is an important element to understand the phylogenetic relationships of frogs. However, the lack of data on larval morphology prevents us from fully understanding larval evolution in several lineages. The Neotropical genus Proceratophrys currently comprises 39 species, but descriptions of the internal morphology of larvae in this group are rare and restricted to few aspects of their buccopharyngeal cavity, chondrocranium, and muscles. In the present study, I describe the internal anatomy of the tadpole of P. minuta and report a new remarkable myological character state for the species. Given the rarity of this material, the description of this species' buccopharyngeal and musculo-skeletal elements is based on two tadpoles in developmental stages 30 and 31. Several new apomorphic character states are described: (a) the presence of a conical papilla in the interior of the nostril; (b) a row of five short, conical papilla preceding the tall, postnarial papilla; and (c) the m. mandibulolabialis inserting in the gular skin. This latter feature is a remarkable, newly discovered character state that had never been reported in the literature before and is probably related to a particular feeding habit of the tadpoles of this species. The function of the m. mandibulolabialis in P. minuta is unknown.

A comparative study of the pharyngeal plate of Apoidea (Hymenoptera: Aculeata), with implications for the understanding of phylogenetic relationships of bees.

The pharyngeal plate is a morphological complex with extensive anatomical variation among bees and, therefore, potential as a source of phylogenetic information. The pharyngeal plate of bees is divided into four morphologically distinct regions: sitophore, hypopharyngeal lobe, pharyngeal rods, and median oral plate. In this work we illustrate and document in detail for the first time the pharyngeal plate of 43 bee species, providing descriptions of the morphological variation and contrasting these findings with representatives of apoid wasps (Crabronidae and Sphecidae). We evaluate and discuss the potential of this structure as a rich source of morphological information in the context of bee phylogeny and any research potentially impacted by comparative morphological data. The shape of the hypopharyngeal lobe is highly variable among suprageneric taxa of bees and can be readily employed to characterise taxa at various levels. We argue that the global patterns in the variation of the pharyngeal plate can provide information for phylogenetic inference within bees and constructed and coded 10 characters that encompass the most noticeable morphological differences discussed herein.

Functional anatomy of the precibarial valve in Philaenus spumarius (L.).

In phytophagous sap-sucking insects, the precibarial valve plays an important role in sap ingestion. We used light and electron microspcopy to study the morphology and the ultrastructure of the precibarial valve of the meadow spittlebug, Philaenus spumarius (Hemiptera, Aphrophoridae), in order to better understand the operative mechanism of this structure. The precibarial valve revealed to be a complex structure with a bell-like invagination in the middle of the precibarium (on the epipharynx). Unlike the current hypothesis, we propose that the valve opens by dilator muscles and closes through cuticular and fluid tensions, the latter leading to morphological changes to the plane of the valve based on sap flow. Moreover, the presence of a precibarial secretory structure is described for the first time for auchenorrhynchan insects. In light of these observations, functions are hypothesized and discussed for this secretory structure.

Three-dimensional reconstruction of the pharyngeal gland cells in the predatory nematode Pristionchus pacificus.

Pristionchus pacificus is a model system in evolutionary biology and for comparison to Caenorhabditis elegans. As a necromenic nematode often found in association with scarab beetles, P. pacificus exhibits omnivorous feeding that is characterized by a mouth-form dimorphism, an example of phenotypic plasticity. Eurystomatous animals have a dorsal and a sub-ventral tooth enabling predatory feeding on other nematodes whereas stenostomatous animals have only a dorsal tooth and are microbivorous. Both mouth forms of P. pacificus, like all members of the Diplogastridae family, lack the grinder in the terminal bulb of the pharynx resulting in a fundamentally different organization of several pharynx-associated structures. Here, we describe the three-dimensional reconstruction of the pharyngeal gland cells in P. pacificus based on serial transmission electron microscopical analysis of 2527 sections of 50 nm thickness. In comparison to C. elegans, P. pacificus lacks two gland cells (g2) usually associated with grinder function, whereas the three gland cells of g1 (g1D, g1VL, and g1VR) are very prominent. The largest expansion is seen for g1D, which has an anterior process that opens into the buccal cavity through a canal in the dorsal tooth. We provide the morphological description and fine structural analysis of the P. pacificus gland cells, the behavior of the pharynx and preliminary insight into exocytosis of gland cell vesicles in P. pacificus.

"High-Throughput Characterization of Region-Specific Mitochondrial Function and Morphology".

The tissue-specific etiology of aging and stress has been elusive due to limitations in data processing of current techniques. Despite that many techniques are high-throughput, they usually use singular features of the data (e.g. whole fluorescence). One technology at the nexus of fluorescence-based screens is large particle flow cytometry ("biosorter"), capable of recording positional fluorescence and object granularity information from many individual live animals. Current processing of biosorter data, however, do not integrate positional information into their analysis and data visualization. Here, we present a bioanalytical platform for the quantification of positional information ("longitudinal profiling") of C. elegans, which we posit embodies the benefits of both high-throughput screening and high-resolution microscopy. We show the use of these techniques in (1) characterizing distinct responses of a transcriptional reporter to various stresses in defined anatomical regions, (2) identifying regions of high mitochondrial membrane potential in live animals, (3) monitoring regional mitochondrial activity in aging models and during development, and (4) screening for regulators of muscle mitochondrial dynamics in a high-throughput format. This platform offers a significant improvement in the quality of high-throughput biosorter data analysis and visualization, opening new options for region-specific phenotypic screening of complex physiological phenomena and mitochondrial biology.

Anterior regeneration after fission in the holothurian Cladolabes schmeltzii (Dendrochirotida: Holothuroidea).

The regeneration of the anterior portion of the body after fission was studied in the holothurian Cladolabes schmeltzii using electron microscopy methods. Following fission, the posterior portion of the digestive tube, cloaca, and respiratory trees remain in the posterior fragment of the body. The regeneration comprises five stages. In the first stage, connective-tissue thickening (an anlage of the aquapharyngeal bulb) occurs on the anterior end between the torn-off ends of the ambulacra. Most of the lost anterior organs developed in the second and third stages. The structures of water-vascular system and nerve ring form through dedifferentiation, proliferation, and migration of cells of the radial water-vascular canals and the radial nerve cords, correspondently. The lost digestive system portion is restored through the formation and merging of two anlagen. The digestive epithelium of the esophagus and pharynx develops from lining cells of microcavities near the central portion of the connective-tissue thickening, which probably migrate from the epidermis. The second gut anlage develops through transformation of the anterior gut remnant portion. The enterocytes partly dedifferentiate, but the epithelium retains integrity. The gut anlage grows down the mesentery and joins the regenerating aquapharyngeal bulb. In the fourth and fifth stages, all lost organs are formed and have nearly normal structure. The regeneration was concluded to occur through morphallactic rearrangements of the remaining parts of organs. Epithelial morphogenesis is the key development mechanism of the digestive, water-vascular, and nervous systems.

[Heterochronies in the Formation of the Nervous and Digestive Systems in Early Postlarval Development of Opistobranch Mollusks: Organization of Basic Functional Systems of the Arctic Dorid Cadlina laevis].

For the first time using laser confocal microscopy and histochemical and immunocytochemical methods (detection of F-actine, catecholamines, acetylcholintransferase, substances of P and FM RFamide) in combination with classical histological methods and electron microscopy of total preparations, the general structure and regularities of formation of the main organs and the nervous, muscular, and digestive systems in early postlarval development (2 to 4 months) in the opistobranch mollusk Cadlina laevis were studied. Heterochronies manifested in positive allometry of the sensory organs, ganglia of the central nervous system, and the pharyngeal region of the digestive system in relation to general body sizes in juvenile individuals compared to adult animals were detected.

Distinct Neural Circuits Control Rhythm Inhibition and Spitting by the Myogenic Pharynx of C. elegans.

Neural circuits have long been known to modulate myogenic muscles such as the heart, yet a mechanistic understanding at the cellular and molecular levels remains limited. We studied how light inhibits pumping of the Caenorhabditis elegans pharynx, a myogenic muscular pump for feeding, and found three neural circuits that alter pumping. First, light inhibits pumping via the I2 neuron monosynaptic circuit. Our electron microscopic reconstruction of the anterior pharynx revealed evidence for synapses from I2 onto muscle that were missing from the published connectome, and we show that these "missed synapses" are likely functional. Second, light inhibits pumping through the RIP-I1-MC neuron polysynaptic circuit, in which an inhibitory signal is likely transmitted from outside the pharynx into the pharynx in a manner analogous to how the mammalian autonomic nervous system controls the heart. Third, light causes a novel pharyngeal behavior, reversal of flow or "spitting," which is induced by the M1 neuron. These three neural circuits show that neurons can control a myogenic muscle organ not only by changing the contraction rate but also by altering the functional consequences of the contraction itself, transforming swallowing into spitting. Our observations also illustrate why connectome builders and users should be cognizant that functional synaptic connections might exist despite the absence of a declared synapse in the connectome.

Hallucigenia's head and the pharyngeal armature of early ecdysozoans.

The molecularly defined clade Ecdysozoa comprises the panarthropods (Euarthropoda, Onychophora and Tardigrada) and the cycloneuralian worms (Nematoda, Nematomorpha, Priapulida, Loricifera and Kinorhyncha). These disparate phyla are united by their means of moulting, but otherwise share few morphological characters--none of which has a meaningful fossilization potential. As such, the early evolutionary history of the group as a whole is largely uncharted. Here we redescribe the 508-million-year-old stem-group onychophoran Hallucigenia sparsa from the mid-Cambrian Burgess Shale. We document an elongate head with a pair of simple eyes, a terminal buccal chamber containing a radial array of sclerotized elements, and a differentiated foregut that is lined with acicular teeth. The radial elements and pharyngeal teeth resemble the sclerotized circumoral elements and pharyngeal teeth expressed in tardigrades, stem-group euarthropods and cycloneuralian worms. Phylogenetic results indicate that equivalent structures characterized the ancestral panarthropod and, seemingly, the ancestral ecdysozoan, demonstrating the deep homology of panarthropod and cycloneuralian mouthparts, and providing an anatomical synapomorphy for the ecdysozoan supergroup.

Scanning electron microscopy of the tongue, pharynx, and larynx of rats exposed to cigarette smoke.

OBJECTIVE: To examine, by using scanning electron microscopy, the surface of the tongue, pharynx, and larynx of animals exposed to tobacco. STUDY DESIGN: Experimental study. METHODS: Twenty rats were allocated to two groups: group I, control group: 10 rats not exposed to any inhaling pollutant and group II, tobacco group: 10 rats exposed to smoke from 10 cigarettes twice a day for 260 days. Animals of both groups had no restriction of food or water. After those 260 days, their aerodigestive segment was removed, and fragments of their tongue, hypopharynx, and right vocal fold were immersed in 2.5% glutaraldehyde and prepared for scanning electron microscopy. RESULTS: The filiform tongue papillae of the tobacco group were irregularly displayed, flattened, and adhered to each other. The hypopharynx mucosa was highly irregular, thickened, rough and had increased superficial peeling. The mucosa of the vocal folds had deep furrows surrounding the cells. These alterations were not identified for the control group. CONCLUSION: Several changes were recorded for the tongue, pharynx, and larynx of tobacco group animals, confirming the harmful effects of smoking to the respiratory and digestive epithelium.

The morphology of the digestive tract and respiratory organs of the holothurian Cladolabes schmeltzii (Holothuroidea, Dendrochirotida).

The microanatomy of the digestive and respiratory systems of the holothurian Cladolabes schmeltzii was studied. The digestive tube of C. schmeltzii is divided into seven parts. The pharynx, esophagus, and stomach are lined with cuticular immersed epithelium. In these regions, the epithelial cells are connected via desmosomes, septate junctions, and rivet-like structures. The presence of the cuticle and rivet-like structures suggests an ectodermal origin for these parts of the digestive tube. The luminal intestinal epithelium is formed by vesicular enterocytes, which have different structures in different intestinal regions. Moreover, the epithelium of the first descending part of the intestine contains the granular enterocytes. The respiratory system consists of paired respiratory trees lined by a luminal epithelium that is formed by cells of irregular shape. The apical surface of these epithelial cells has few lamellae. The cells are connected to each other through a system of intercellular junctions, consisting of both desmosomes and well-developed septate junctions. The coelomic epithelium of the intestine and the respiratory trees consists of peritoneal and myoepithelial cells.

Methods for studying programmed cell death in C. elegans.

Programmed cell death or apoptosis is a common cell fate during the development of multicellular organisms, including C. elegans. C. elegans has been proven to be an excellent model organism for studies of programmed cell death. Its transparency and the knowledge of its cell lineage, including its invariant life vs. death fate of all cells, allow programmed cell death to be studied in vivo at single cell resolution in a way that no other systems can currently match. This advantage, in combination with sophisticated genetic manipulations, has facilitated the identification and characterization of many genes important for different aspects of programmed cell death. In addition to the ability to observe life and death of cells directly under Nomarski optics or by using fluorescent transgene products in living animals, several methods have been developed to study other aspects of apoptosis. For example, fragmentation of chromosomal DNA and the identity of phagocytic cells that engulf cell corpses can be analyzed using specific staining methods. Proteases and nucleases involved in cell killing and DNA degradation, respectively, can be studied using specific biochemical assays. These cell death assays have been instrumental in elucidating the functions of cell death-related genes and the mechanisms by which they affect cell death. In this chapter, we will review and describe these methods in detail.

Gross morphology, histology, and ultrastructure of the alimentary system of Ricinulei (Arachnida) with emphasis on functional and phylogenetic implications.

Ricinuleid functional mouthparts are the cucullus, the chelicerae, the pedipalps, and the labrum. These structures are movably jointed to the rest of the prosoma, most likely protruded upon hydrostatic hemolymph pressure and retracted by prosomal muscles. Seta-like protrusions from the labrum and the pedipalpal coxae form a sieve-like filter inside the preoral cavity and the mouth. Although the tip of the labrum can be elevated upon muscle constriction, ingestion of large, solid food particles is unlikely. The mouth has a crescent-shaped cross section. The cuticle-lined, also crescent-shaped pharynx is equipped with a large dilator muscle but lacks antagonistic constrictor muscles. It represents a precerebral sucking pump. The triangular to Y-shaped, cuticle-lined esophagus is equipped with constrictor and dilator muscles. Its posterior part represents a postcerebral sucking pump. Four blind ending diverticula ramify from the anterior prosomal part of the entodermal midgut tube. Two of these diverticula remain inside the prosoma and form few short branches. The other two extend through the pedicel into the opisthosoma and ramify and coil there. A stercoral pocket protrudes ventrally out of the midgut tube. The most distal part of the midgut tube is modified into a contractile rectal gland. Its secretions may have defensive or physiological functions. A short anal atrium is formed by the cuticle-lined ectodermal hindgut which opens at the end of the three-segmented metasoma. The telescoping segments of the metasoma are protruded by hemolymph pressure and retracted by muscles.

The pharyngeal organ in the buccal cavity of the male Siamese fighting fish, Betta splendens, supplies mucus for building bubble nests.

The male Siamese fighting fish, Betta splendens, builds a bubble nest on the water surface to care for offspring during the reproductive period. To our knowledge, this study is the first to determine the composition of the bubble nest and to compare the pharyngeal organs of male and female Siamese fighting fish to determine the relationship between the pharyngeal organ and the ability to make bubble nests. Dot blots of the bubble nest probed with periodic acid-Schiff's (PAS) staining and Ponceau S solution revealed that the contents of the nest are glycoprotein rich. Dissection of the heads of Siamese fighting fish showed that the pharyngeal organ is located in the position through which inhaled air passes. The epithelial structure of the pharyngeal organ of the Siamese fighting fish, like that of other teleosts, has numerous wrinkles and papillae. Mucous goblet cells were observed on the epithelium of pharyngeal organs in male and female fish. The pharyngeal organ was found to be larger in male than in female fish. In addition, the epithelium of the pharyngeal organ in male fish has a greater number of mucous goblet cells than that in female fish. In Siamese fighting fish, this sexual dimorphism of the pharyngeal organ suggests that the male fish secretes more glycoprotein-rich mucus to build the bubble nest. Future work will focus on the type of mucous cells found in the epithelium of the pharyngeal organ that contributes to bubble formation and will determine the components of the mucus in the bubble nest.

Fine needle aspiration of parapharyngeal space adult rhabdomyoma: a case report.

BACKGROUND: Rhabdomyomas are rare benign tumors of striated muscle and include cardiac and extracardiac types. Extracardiac rhabdomyomas are divided in three subtypes (adult, fetal, genital). The adult type is usually found in the head and neck regions of elderly persons. Misinterpretations in initial diagnosis of adult rhabdomyomas on fine needle aspiration have been reported. CASE: A 64-year-old man presented with gurgling and difficulty swallowing for approximately 3 months. Computed tomography and magnetic resonance imaging showed a 5.8-cm solid mass located in the right parapharyngeal space. Fine needle aspiration smears were cellular, showing cohesive clusters of cells with scattered individual cells. Cells had abundant eosinophilic glassy cytoplasm, peripherally placed round nuclei, and prominent nucleoli. Many traversing vessels were noted, but cross-striations were not seen. The cell block demonstrated clusters of cells with abundant eosinophilic granular cytoplasm, some with clear and/or vacuolated cytoplasm, and possible cross-striations. Tumor cells were positive for desmin. The lesion closely resembled normal muscle tissue. Electron microscopy showed many cells containing actin and myosin filaments with Z-band material. CONCLUSION: Correct diagnosis can be achieved with a combination of awareness of the lesion, familiarity with the characteristic cytologic features, and application of appropriate immunohistochemistry markers. Classic electron microscopic findings can support the diagnosis.

Ultramicroscopic examination of the ovine tonsillar epithelia.

As solid morphological knowledge of ovine tonsillar epithelia might contribute to a better understanding of the pathogenesis of several diseases including prion diseases, the epithelia of all tonsils of 7 one-year-old Texel sheep were examined using scanning and transmission electron microscopy. Major parts of the pharyngeal and tubal tonsils were covered by pseudostratified columnar ciliated epithelia that were interrupted by patches of epithelium containing cells with densely packed microfolds or microvilli, and cells with both microvilli and cilia. Smaller parts were covered by either flattened polygonal cells with densely packed microvilli or microfolds, squamous epithelial cells, or patches of reticular epithelium. The palatine and paraepiglottic tonsils were mainly lined by squamous epithelial cells with apical microplicae or short knobs. Additionally, regions of reticular epithelium containing epithelial cells with apical microvilli were seen. The lingual tonsil was uniformly covered by a keratinized squamous epithelium and devoid of microvillous cells and patches of reticular epithelium. The rostral half of the tonsil of the soft palate was lined by a pseudostratified columnar ciliated epithelium with characteristics of the pharyngeal and tubal tonsils. The epithelium of the caudal part resembled the epithelia of the palatine and paraepiglottic tonsils. Putative M cells, mainly characterized by apical microvilli or microfolds and a close association with lymphoid cells, seem manifestly present on the nasopharyngeal tonsils. The reticular epithelium of the palatine and paraepiglottic tonsils also harbor cells with small apical microvilli. The exact nature of these presumptive M cells should, however, be elucidated in functional studies.

Structure and evolution of the pharynx simplex in acoel flatworms (Acoela).

The homology of pharynges within the mostly pharynx-less Acoela has been a matter of discussion for decades and even the basic question of whether a pharynx is a primitive trait within the Acoela and homologous to the pharynx of platyhelminth turbellarians is open. By using fluorescence staining of musculature, as well as conventional histological techniques and transmission electron microscopy, the present study sets focus on the mouth and pharynx (where present) of seven species of Acoela within Paratomellidae, Solenofilomorphidae, Hofsteniidae, Proporidae, and Convolutidae, as well as one species of Nemertodermatida and Catenulida, respectively. It is shown that among the investigated families of acoels there is a great variability in muscle systems associated with the mouth and pharynx and that pharynx histology and ultrastructural characters are widely diverse. There are no close similarities between the acoel pharynges and the catenulid pharynx but there is a general resemblance of the musculature associated with the mouth in the representatives of Paratomellidae and Nemertodermatida. On the basis of the profound differences in pharynx morphology, three major conclusions are drawn: 1) the pharynges as present in Recent acoels are not homologous to the pharynx simplex characteristic for Catenulida and Macrostomida within the Platyhelminthes; 2) the different muscular pharynx types of acoels are not homologous between higher taxa and thus a single acoel-type pharynx simplex cannot be defined; 3) the presence of a muscular pharynx most likely does not represent the ancestral state.

Gross morphology and surface ultrastructure of the gills of Odontesthes argentinensis (Actinopterygii, Atherinopsidae) from a Southwestern Atlantic coastal lagoon.

Odontesthes argentinensis was collected from Mar Chiquita Coastal Lagoon, the Southernmost coastal Atlantic Lagoon of Argentina. The morphology of the gills was analyzed by scanning electron microscopy. The morphology of the superficial structures of the gill filaments and pharyngeal region of the gill arch was discussed and related to their functional aspects. The gills arches are structurally similar to those of other teleosts and bring out the osmoregulatory capacity of this species. The epithelium that covers the surface of the filaments and the pharyngeal region of the gill arch is formed by polygonal pavement cells with conspicuous microridges. These folds in the membrane are not denoted in the epithelium of the respiratory lamellae. Apical crypts of chloride cells are present on the afferent and interlamellar filament surfaces, but are absent elsewhere on the gill arch. The highest density of mucous cells is observed into the gill filament and the pharyngeal region which indicates the existence of a protective strategy of the respiratory lamellae and the pharynx. The epithelium of the gill arches and the rakers is studded with spines. There are taste buds along the whole pharyngeal region that may be associated with their participation in tasting at this zone.