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Los cambios fisiológicos producto del envejecimiento afectan al ser humano en todos sus sistemas, los cuales repercuten en su estilo de vida y deja al paciente adulto mayor en estado de vulnerabilidad. Con el paso del tiempo estos cambios ocasionan el deterioro físico, la exacerbación de enfermedades sistémicas y secuelas en el aparato bucal por la ingesta de los medicamentos prescritos para tratar enfermedades. Es por ello por lo que, se requiere una atención integral donde el médico y el odontólogo, con sus diferentes especialidades, atiendan las necesidades de este tipo de pacientes. Conocer las etapas de este proceso y su manejo multidisciplinario son importantes actualmente para el profesional de la salud. The physiological changes resulting from aging impacts the human being in all his systems, which affects his lifestyle and leaves the elderly patient in a state of vulnerability. Over time, these changes cause physical deterioration, exacerbation of systemic diseases and sequelae in the oral apparatus due to the intake of medications prescribed to treat diseases. That is why comprehensive care is required where the doctor and the dentist, with their different specialties, meet the needs of this type of patient. Knowing the stages of this process and its medical-dental management are important for the current health professional. 62-75 revodontlosandes@gmail.com od.andreinatejada@gmail.com dario.sosa@gmail.com Semestral http://erevistas.saber.ula.ve/odontoula

While the diversity of Scutiger is high in the central and eastern parts of the Himalaya-Tibet orogen, only a single species, Scutiger occidentalis Dubois, 1978, can be found in the westernmost regions, in Northwest India and Pakistan. Little is known about its genetic variation, ecology, and distribution and far less about its larval morphology. Here, we provide new records, detailed morphological data of tadpoles, and genetic data (mtDNA and nDNA) of the species from two, so far unknown localities in northern Pakistan. Molecular analysis confirmed the taxonomic identity of the tadpoles and indicated shallow intraspecific genetic differentiation. Tadpole characterizations were illustrated by detailed imagery of live and preserved specimens. Measurements and qualitative traits on oral apparatus provide relevant taxonomic characteristics to distinguish the tadpoles of this species from other lazy toads.

BACKGROUND The vertebrate jaw is thought to have evolved through developmental modification of the mandibular arch. An extant jawless vertebrate, the lamprey, possesses a structure called "velum"-a mandibular arch derivative-in addition to the oral apparatus. This leads us to assess the velum's possible contribution to the evolution of jaws. RESULTS The velar muscles develop from progenitor cells distinct from those from which the oral muscles develop. In addition, the oral and velar regions originate from the different sub-population of the trigeminal neural crest cells (NCCs): the former region receives NCCs from the midbrain, whereas the latter region receives NCCs from the anterior hindbrain. The expression of patterning genes (eg, DlxA and MsxA) is activated at a later developmental stage in the velum compared to the oral region, and more importantly, in different cells from those in the oral region. CONCLUSION The lamprey mandibular arch consists of two developmental units: the anterior oral unit and the posterior velar unit. Because structural elements of the lamprey velum may be homologous to the jaw, the evolution of vertebrate jaws may have occurred by the velum being released from its functional roles in feeding or respiration in jawless vertebrates.

Currently, most amphibian populations in the world exist under the influence of numerous stress factors. Among them, the main factors that affect almost all terrestrial animals, namely, the fragmentation of habitats, environmental pollution and anthropic transformation of landscapes. Moreover, those factors are joined by negative causes that affect only amphibians – specific viral and fungal infections: ranaviruses (Ambystoma tigrinum virus (ATV), Bohle iridovirus (BIV), and frog virus 3) (also dangerous for some reptiles) and chytrid fungi (Batrachochytrium dendrobatidis and B. salamandrivorans). All these factors are one of the main reasons for the current global decline of amphibian populations in the world. In today's world, agricultural chemicals are one of the most important in terms of toxicity, environmental emissions and total area of impact. Among them, a significant proportion are formed by synthetic insecticides, which include pyrethroids and neonicotinoids. Pollution by these substances has a negative impact on amphibian populations, despite the relatively short period of their life in the environment. The vast majority of studies on the effects of pyrethroid and neonicotinoid insecticides were made in laboratory experiments with the larval stages of tailless amphibians. Tadpoles are easy to get in sufficient quantities and kept in the laboratory. Cypermethrin reduces the viability of tadpoles and causes precocious metamorphosis of survived larvae. However, in a combination with other pesticides, it delays metamorphosis. In addition, embryos were more resistant to pyrethroids than tadpoles. Pyrethroid pesticides cause spasms indicating adverse neurological effects. Formation of oral apparatus abnormalities in tadpoles, anisochromasia and increasing number of immature erythrocytes are also caused by pyrethroids. Neonicotinoids show similar effects. Under the action of imidacloprid the erythrocytes are also disturbed that is showed up in the DNA damage and micronuclei formation. Neurological disorders are manifested in the ability to perceive or respond to a predator, disorientation, erratic movement and loss of balance. Pyrethroids and neonicotinoids have been shown cause a variety of disorders: increase mortality and reduce survival of tadpoles; have a teratogenic effect and affect the metamorphosis and morphological parameters of amphibians; change many biochemical parameters that characterize the protein metabolism and oxidative stress; have genotoxic effects and affect the state of the nervous system and animal behaviour. Most of these parameters are proposed for use as biomarkers of pesticide intoxication.

We report Armillifer moniliformis species infecting the endemic Sri Lankan brown palm civet (Paradoxurus montanus) from the Knuckles Range Forest Conservation Area, Sri Lanka. Larval stages of A. moniliformis were found during the postmortem of three civet cats found dead. Morphological studies were done by a light microscope and a scanning electron microscope (SEM). Histopathological examination was conducted using tissue samples obtained from the liver. For the molecular analysis, DNA was extracted from the isolated third-stage larvae. The NADH dehydrogenase subunit 5 (ND5) and the second internal transcribed spacer region (ITS-2), a portion of the large subunit nuclear ribosomal DNA (28S), a portion of 18S ribosomal rRNA gene (18S), and cytochrome c oxidase subunit 1 gene (COX1) were amplified using polymerase chain reaction (PCR). Excysted third-stage larvae were observed in the lungs, omentum, the pleural cavity, the abdominal cavity, and the surface of the spleen and the pericardium. Around 88 third-stage larvae were isolated from three civet cats. First-stage larvae in the liver were surrounded by outer fibrous layer over the inner germinal layer and filled with clear fluid. Slight hemorrhage, leukocyte infiltration, and mild hepatocellular degeneration in the liver were observed. The SEM examination indicated the unique oral apparatus comprises the oval-shaped mouth opening in between two pairs of curved, retractile hamuli. The sequences obtained for ND5, ITS-2, 28S, 18S, and COX1 were 301, 382, 325, 414, and 644 bp in length respectively. Morphology, sequence similarity search, sequence alignment, and phylogenetic analysis identified this parasite as A. moniliformis.

Little is known about the life history, ecology, and distribution of the genus Allopaa (Dicroglossidae) and far less recent data are available about the larvae of this taxon. Here, we provide data on the larval stage of Allopaa hazarensis (Dubois & Khan, 1979) from northern Pakistan based on the examination of three tadpoles. Specimens were obtained from two sites in Buner, Khyber Pakhtunkhwa province, Pakistan. Morphological and genetic analysis (mtDNA and nDNA) confirmed the identity of the tadpoles as A. hazarensis. Tadpole characterizations were illustrated by detailed imagery. Basic measurements and details on oral apparatus provide relevant taxonomic characteristics to distinguish the tadpoles of this species from other spiny frogs. The illustration and description of the tadpole of A. hazarensis should facilitate the identification of this species in the field.

The giant ciliate Stentor coeruleus is a classical model system for studying regeneration and morphogenesis at the level of a single cell. The anterior of the cell is marked by an array of cilia, known as the oral apparatus, which can be induced to shed and regenerate in a series of reproducible morphological steps, previously shown to require transcription. If a cell is cut in half, each half will regenerate an intact cell, including a new oral apparatus in the posterior half. We used RNAseq to assay the dynamic changes in Stentor’s transcriptome during regeneration, after both oral apparatus shedding and bisection, allowing us to identify distinct temporal waves of gene expression including kinases, RNA binding proteins, centriole biogenesis factors, and orthologs of human ciliopathy genes implicated in Meckel and Joubert syndromes. By comparing transcriptional profiles of different regeneration events in the same species, we were able to identify distinct modules of gene expression corresponding to oral apparatus regeneration, posterior holdfast regeneration, and recovery after wounding. By measuring gene expression in cells in which translation is blocked, we show that the sequential waves of gene expression involve a cascade mechanism in which later waves of expression are triggered by translation products of early-expressed genes. Among the early-expressed genes, we identified an E2F transcription factor and the conserved RNA binding protein Pumilio as potential regulators of regeneration based on the expression pattern of their predicted target genes. RNAi mediated knockdown experiments indicate that Pumilio is required for regenerating oral structures of the correct size. We show that E2F is involved in the completion of regeneration but is dispensable for earlier steps. This work allows us to classify regeneration genes into groups based on their potential role for regeneration in distinct cell regeneration paradigms, and provides insight into how a single cell can coordinate complex morphogenetic pathways to regenerate missing structures.

Made available in DSpace on 2019-10-06T16:18:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-04-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Objective: The present study aimed to describe the morphology of oral apparatus and oral cavity of bullfrog tadpoles during their development and metamorphosis. Design: The oral apparatus and oropharyngeal cavity of tadpoles from hatching up to metamorphosis stage was dissected for further analysis. These structures were fixed in Karnovsky solution, afterwards in osmium tetroxide and metalized in palladium gold and electron-micrographed using the scanning electron microscope. Results: The development of oral apparatus started with the formation and keratinization of the jaw sheaths and labial teeth followed by the formation of marginal and sub-marginal papillae. Degeneration of oral apparatus and formation of mouth was observed during metamorphosis. From stage-42 (metamorphic climax) to stage-43, the jaw sheath and labial tooth rows were disappeared progressively while the size and number of labial papillae were decreased. At stage-44, mouth formation started with the development of anterior and posterior labium though the labial papillae were still present. At stage-45 and 46, mouth was already formed, being very similar to the adult and characterized by the progressive increase in size. Conclusion: The sequence of events that happen during the development of oral apparatus of Lithobates catesbeianus Shaw, 1802 tadpoles follows the same pattern as occur in other anuran species but metamorphic atrophy of the oral apparatus follows the sequence of morphogenesis. Department of Animal Morphology and Physiology Faculty of Agricultural and Veterinary Sciences São Paulo State University (UNESP), Via de Acesso Paulo Donato Castellane s/n Institute of Biological Sciences Federal University of Pará, Avenida Augusto Correa n1 Aquaculture Center São Paulo State University (Unesp), Via de Acesso Prof. Paulo Donato Castellane s/n Department of Technology Faculty of Agricultural and Veterinary Sciences São Paulo State University (UNESP), Via de Acesso Paulo Donato Castellane s/n Brazil University (Univ. Brasil), Campus Fernandópolis - Est. Projetada F-1, s/n - Fazenda Santa Rita Department of Animal Morphology and Physiology Faculty of Agricultural and Veterinary Sciences São Paulo State University (UNESP), Via de Acesso Paulo Donato Castellane s/n Aquaculture Center São Paulo State University (Unesp), Via de Acesso Prof. Paulo Donato Castellane s/n Department of Technology Faculty of Agricultural and Veterinary Sciences São Paulo State University (UNESP), Via de Acesso Paulo Donato Castellane s/n FAPESP: 03/12575-6 FAPESP: 04/15415-2

Stentor coeruleus is a well-known model organism for the study of unicellular regeneration. Transcriptomic analysis of individual cells revealed hundreds of genes-many not associated with the oral apparatus (OA)-that are differentially regulated in phases throughout the regeneration process. It was hypothesized that this systemic reorganization and mobilization of cellular resources towards growth of a new OA will lead to observable changes in movement and behavior corresponding in time to the phases of differential gene expression. However, the morphological complexity of S. coeruleus necessitated the development of an assay to capture the statistics and timescale. A custom script was used to track cells in short videos, and statistics were compiled over a large population (N ~100). Upon loss of the OA, S. coeruleus initially loses the ability for directed motion; then starting at ~4 h, it exhibits a significant drop in speed until ~8 h. This assay provides a useful tool for the screening of motility phenotypes and can be adapted for the investigation of other organisms.

A lack of effective intervention in addressing patient non-adherence and the acceptability of solid oral dosage forms combined with the clinical consequences of swallowing problems in an ageing world population highlight the need for developing methods to study the swallowability of tablets. Due to the absence of suitable techniques, this study developed various in vitro analytical tools to assess physical properties governing the swallowing process of tablets by mimicking static and dynamic stages of time-independent oral transitioning events. Non-anatomical models with oral mucosa-mimicking surfaces were developed to assess the swallowability of tablets, an SLA 3D printed in vitro oral apparatus derived the coefficient of sliding friction and a friction sledge for a modified tensometer measured the shear adhesion profile. Film coat hydration and in vitro wettability was evaluated using a high-speed recording camera that provided quantitative measurements of micro-thickness changes, simulating static in vivo tablet–mucosa oral processing stages with artificial saliva. In order to ascertain the discriminatory power and validate the multianalytical framework, a range of commonly available tablet coating solutions and new compositions developed in our lab were comparatively evaluated according to a quantitative swallowability index that describes the mathematical relationship between the critical physical forces governing swallowability. This study showed that the absence of a film coat significantly impeded the ease of tablet gliding properties and formed chalky residues caused by immediate tablet surface erosion. Novel gelatin- and λ-carrageenan-based film coats exhibited an enhanced lubricity, lesser resistance to tangential motion, and reduced stickiness than polyvinyl alcohol (PVA)–PEG graft copolymer, hydroxypropyl methylcellulose (HPMC), and PVA-coated tablets, however, Opadry® EZ possessed the lowest friction–adhesion profile at 1.53 a.u., with the lowest work of adhesion profile at 1.28 J/mm2. For the first time, the in vitro analytical framework in this study provides a fast, cost-effective, and repeatable swallowability ranking method to screen the in vitro swallowability of solid oral medicines in an effort to aid formulators and the pharmaceutical industry to develop easy-to-swallow formulations.

In the giant ciliate Stentor coeruleus, oral apparatus (OA) regeneration is an experimentally tractable regeneration paradigm that occurs via a series of morphological steps. OA regeneration is thought to be driven by a complex regulatory system that orchestrates the temporal expression of conserved and specific genes. We previously identified a S. coeruleus-specific gene (named SCSG1) that was significantly upregulated during the ciliogenesis stages of OA regeneration, with an expression peak at the stage of the first OA cilia appearance. We established a novel RNA interference (RNAi) method through cyanobacteria Synechocystis sp. PCC6803 feeding in S. coeruleus. The expression of SCSG1 gene was significantly knocked down by using this method and induced abnormal ciliogenesis of OA regeneration in S. coeruleus, suggesting that SCSG1 is essential for OA regeneration in S. coeruleus. This novel RNAi method by cyanobacterial feeding has potential utility for studying other ciliates.

We often think about regeneration in terms of replacing missing structures, such as organs or tissues, with new structures generated via cell proliferation and differentiation. But at a smaller scale, single cells, themselves, are capable of regenerating when part of the cell has been removed. A classic model organism that facilitates the study of cellular regeneration in the giant ciliate Stentor coeruleus. These cells, which can grow to more than a millimeter in size, have the ability to survive after extensive wounding of their surface, and are able to regenerate missing structures. Even a small piece of a cell can regenerate a whole cell with normal geometry, in a matter of hours. Such regeneration requires cells to be able to trigger organelle biogenesis in response to loss of structures. But subcellular regeneration also relies on intracellular mechanisms to create and maintain global patterning within the cell. These mechanisms are not understood, but at a conceptual level they involve processes that resemble those seen in animal development and regeneration. Here we discuss single-celled regeneration in Stentor from the viewpoint of standard regeneration paradigms in animals. For example, there is evidence that regeneration of the oral apparatus in Stentor follows a sender-receiver model similar to crustacean eyestalk regeneration. By drawing these analogies, we find that many of the concepts already known from the study of animal-scale regeneration and development can be applied to the study of regeneration at the cellular level, such as the concepts of determination, induction, mosaic vs. regulative development, and epimorphosis vs. morphallaxis. We propose that the similarities may go beyond analogy, and that some aspects of animal development and regeneration may have evolved by exploiting pre-existing subcellular developmental strategies from unicellular ancestors.

The morphology, ontogenesis, and molecular phylogeny of a new ciliate, Gonostomum koreanum n. sp., discovered in a terrestrial moss sample from South Korea, were investigated. Morphologically, it is characterized by a gonostomatid oral apparatus, two macronuclear nodules, six frontoventral rows, the two rightmost of which (frontoventral rows V and VI) extend posteriorly to near pretransverse and transverse cirri, and three dorsal kineties each with a single caudal cirrus posteriorly. The new species is easily confused with members of the genus Metagonostomum because of the long frontoterminal cirral row but differs mainly in the de novo (vs. intrakinetal) origin of anlage VI, a character found only in Gonostomum and Paragonostomum. To solve the discrepancy between the interphasic and ontogenetic patterns, we additionally performed morphological and multigene analyses on three gonostomatid species, namely Gonostomum koreanum n. sp. and its morphologically (M. gonostomoida) and ontogenetically (G. kuehnelti) most similar species. The multigene analyses show that the new species is closely related to G. kuehnelti and the core gonostomatids consists of five groups based on the origin of the frontoventral rows.

In the present study, based on both morphologic and phylogenetic analyses, a new genus, Monourostylopsis n. gen., and new species, Metaurostylopsis alrasheidi n. sp. as well as a new combination, Monourostylopsis antarctica (Jung et al., 2011) n. comb. (original combination: Metaurostylopsis antarcticaJung et al., 2011), are suggested. The new genus is diagnosed mainly by having three or more frontoterminal cirri, a midventral complex with midventral pairs and a single midventral row, one right marginal row and two or more left marginal rows. The new genus can be easily separated from the morphologically similar genera mainly by having single right marginal row (vs. two or more right marginal rows). Based on live observation and protargol staining, the morphology and morphogenesis of a new species, M. alrasheidi n. sp. isolated from China, were investigated. The new species can be characterized by: two types of cortical granules; about 22 adoral membranelles; three or four frontoterminal, four or five transverse cirri; about eight midventral pairs and a midventral row of three or four unpaired midventral cirri; three or four left and right marginal rows. The main morphogenetic features of Metaurostylopsis alrasheidi n. sp. can be summarized as: (1) the entire parental ciliature, including the oral apparatus, is renewed; (2) the oral primordium of the proter probably originates within a pouch; (3) the oral primordium of the opisthe forms de novo on the cell surface; (4) the anlagen of marginal rows and dorsal kineties are formed intrakinetally, and (5) the fusion of macronuclear nodules results in an irregular branched mass prior to karyokinesis. In the phylogenetic trees, all the available Metaurostylopsis sequences cluster together in a clade with full support (ML/BI: 100/1.00) revealing that the genus is monophyletic within the large group of core urostylids.

Made available in DSpace on 2021-06-25T12:18:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-08-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) UNILA Fundacao Parque Tecnologico Itaipu-BR Bokermannohyla ahenea is endemic to the Serra da Bocaina, a portion of the Serra do Mar in the Atlantic Forest of Southeastern Brazil. Little information is currently available about this species. Herein, we describe the larval morphology of B. ahenea, including internal oral features, chondrocranium, and hyobranchial apparatus, along with its advertisement call and natural history notes. This is the first description of the chondrocranium and hyobranchial apparatus for a member of the genus Bokermannohyla. The larval external morphology of B. ahenea is similar to those of other species of the B. circumdata group, but it can be distinguished from those of other species of the genus by its oral apparatus. The advertisement call of B. ahenea is complex with many numbers of notes. Also, spectral and temporal parameters of the advertisement call can be used to distinguish B. ahenea from its congeners. Univ Fed Integracao Latino Amer, Inst Latino Americano Ciencias Vida & Nat, Foz Do Iguacu, Parana, Brazil Univ Fed Mato Grosso do Sul, Setor Ecol, Inst Biociencias, POB 549, BR-79070900 Campo Grande, MS, Brazil CUNY City Coll, Dept Biol, New York, NY USA Univ Estadzzal Paulista, Dept Biodiversidade, Inst Biociencias, Campus Rio Claro, Rio Claro, SP, Brazil Univ Estadzzal Paulista, Ctr Aquicultura, Campus Rio Claro, Rio Claro, SP, Brazil Gothenburg Global Biodivers Ctr, Box 461, SE-40530 Gothenburg, Sweden Univ Estadual Paulista, Dept Zool & Bot, Campus Sao Jose do Rio Preto, Sao Jose Do Rio Preto, SP, Brazil Univ Fed Integracao Latino Amer, Programa Posgrad Biodiversidade Neotrop, Inst Latino Amer Ciencias Vida & Nat, Foz Do Iguacu, Parana, Brazil Univ Estadual Paulista, Dept Zool & Bot, Campus Sao Jose do Rio Preto, Sao Jose Do Rio Preto, SP, Brazil FAPESP: 2008/55744-6 FAPESP: 2016/13949-7 CNPq: 422537/2016-0 CNPq: 370419/2011-1 CNPq: 302518/2013-4 CNPq: 303522/2013-5 UNILA: PRPPG 110/2018 UNILA: 80/2019 FAPESP: 2008/50575-1 FAPESP: 2012/09243-0 FAPESP: 2013/26101-8 FAPESP: 2008/50928-1 FAPESP: 2013/50741-7 Fundacao Parque Tecnologico Itaipu-BR: 902443/2019 FAPESP: 563075/2010-4 FAPESP: 2010/52321-7

Algunas especies del género Espeletia sp. (Asteraceae) son afectados por hongos fitopatógenos e insectos en páramos de los Andes del Norte, amenazando su permanencia y la prestación de servicios que se les atribuye, como la regulación del ciclo hídrico. La escaza información sobre la afectación en el páramo de Paja Blanca (Nariño, Colombia), dificulta la comprensión de la dinámica del fenómeno y la formulación de estrategias de manejo. El objetivo de esta investigación fue identificar cambios en la composición de las comunidades de insectos asociados a E. pycnophylla durante el declive de las poblaciones de frailejones y evaluar si reflejan modificaciones en los hongos, asociados a dichos insectos. Para ello, se colectaron insectos en plantas de E. pycnophylla sanas y afectadas, a los que se aplicó índices ecológicos, para identificar posibles modificaciones en la diversidad y la composición; además, se aislaron e identificaron morfológica y molecularmente hongos, a partir de estructuras corporales de los insectos. Como resultado, se identificó una diversidad de insectos considerable y que no hay modificación en su composición de insectos entre los dos estados de frailejón analizados. Se logró aislar hongos, como Fusarium oxysporum, Botrytis sp., Epicoccum nigrum, Cladosporium sp., a partir de estructuras de los géneros de insectos Neomyopites sp., Diabrotica sp., Bradysia sp. y Dyscolus sp. Este estudio aporta información sobre la entomofauna presente en E. pycnopylla del páramo de Paja Blanca y hongos asociados a estructuras corporales, como el canal alimentario y aparato bucal de los insectos. Some species of the genus Espeletia sp. (Asteraceae) are affected by phytopathogenic fungi and insects in the paramos of the Northern Andes, threatening their permanence and the provision of services attributed to them such as the regulation of the water cycle. The scarce information about the impact on the Paja Blanca páramo (Nariño, Colombia) makes it difficult to understand the dynamics of the phenomenon and the formulation of management strategies. The goal of this research was to identify changes in the composition of the insect communities associated to E. pycnophylla during the decline of the frailejon populations and to evaluate if these reflect modifications in the fungi associated with those insects. For this, insects were collected in healthy and affected E. pycnophylla plants to which ecological indices were applied to identify possible changes in diversity and composition, Also fungi were isolated and identified morphologically and molecularly from body structures of insects. As result, a considerable diversity of insects was identified and no modification in their composition of insects between the two states of frailejón were found. It was possible to isolate fungi such as Fusarium oxysporum, Botrytis sp., Epicoccum nigrum, Cladosporium sp. from structures of the insect genera Neomyopites sp., Diabrotica sp., Bradysiasp. and Dyscolus sp. This study provides information about the entomofauna present in E. pycnopylla from the Paja Blanca páramo and fungi associated with body structures such as the alimentary canal and the oral system of insects. Incluye referencias bibliográficas

The ultrastructure of the oral apparatus is supposed to be significant for elucidating more recent common ancestry and might thus provide support for particular groupings of oligotrichean ciliates. The transmission electron microscopical study on mainly cryofixed Schmidingerella meunieri specimens provides the first detailed data for tintinnids and Oligotrichea in general. Ten new characters are included into the cladistic analysis. These features together with the very limited body of literature suggest that substantial changes in the oral ultrastructure correlate only with the formation of a circular adoral zone in choreotrichids. Despite homoplasious morphological and ontogenetic adaptations to the planktonic lifestyle in halteriid hypotrichs and oligotrichids, their oral apparatuses generally retain the plesiomorphic ultrastructure of the Perilemmaphora. The highly complex ultrastructure of the adoral zone is thus able to accomplish an extension in the zone's functionality without obvious changes; only the position of the adoral zone at the apical cell portion together with a globular to obconical cell shape are apparently crucial. Merely, minute apomorphies characterise the Oligotrichea and tintinnids, respectively. Tintinnids with derived somatic ciliary patterns possess distinct microtubular bundles connecting the oral apparatus with the myoneme in the peduncle.

The karyorelictean ciliate Kentrophoros lacks a defined oral apparatus but has a dense coat of symbiotic bacteria that it consumes by phagocytosis. Body size, shape, and nuclear characters are variable in this genus. We formally describe a new species, K. magnus from Elba (Italy), which has unusual folding of its symbiont-bearing surface into pouch-like compartments, a body form that we term “pseudotrophosomal”. K. magnus cells are large (2100 ± 700 × 170 ± 23 μm in vivo), but contain only one micronucleus and two macronuclei, although these are much bigger than other Kentrophoros (widths 20 ± 2.5 and 31 ± 4.0 μm respectively in K. magnus). We also present morphological observations on a close relative from Twin Cayes (Belize), which also has relatively large nuclei (micronuclei 13 ± 1.5 μm, mature macronuclei 20 ± 2.8 μm), but unlike K. magnus, it has on average 22 nuclei per cell, with different developmental stages of the macronuclei present simultaneously, and lacks pouch-like folding. Nuclear number and arrangement are important characters for karyorelicts. We suggest the use of a “nuclear formula” to simplify descriptions. Our discovery of large and morphologically distinctive new species underlines the incompleteness of our knowledge about meiofaunal ciliates.

One of the largest known amphibian tadpoles was identified based on its morphological characters. The tadpoles of Leptobrachium hasseltii Tschudi. 1838 specimens collected from Kembang Soka Waterfall Tourism Park. Kulon Progo Regency. Daerah Istimewa Yogyakarta. were described in detail using morphometric data and the larval developmental stage of its labial tooth row formula. This study aimed to identify the development of morphology. morphometry. and oral apparatus of Leptobrachium hasseltii tadpoles. Eighty seven specimens were observed in this study. Observations were performed on specimens between Gosner stage of stage 25 and stage 40. Leptobrachium hasseltii underwent a drastic morphometry development from the initial stage 25 to the final stage 25. Morphologically. myotom and myosepta that were not seen at the initial stage 25 were clearly visible in the final stage 25. In the oral apparatus it was found that in stage 25 the initial papilla and tooth series were not fully developed. In addition. there was a variation in the labial tooth row formula at the initial stage 25. The tooth formula between final stage 25 and stage 40 was found to be a stable and could be used as an identification character of the species during larval stage.

Food conditions are changing due to anthropogenic activities and natural sources and thus, many species are exposed to new challenges. Animals might cope with altered quantitative and qualitative composition [i.e. variable protein, nitrogen (N) and energy content] of food by exhibiting trophic and digestive plasticity. We examined experimentally whether tadpoles of the common frog (Rana temporaria) exhibit phenotypic plasticity of the oral apparatus and intestinal morphology when raised on a diet of either low (i.e. Spirulina algae) or high protein, N and energy content (i.e. Daphnia pulex). Whereas intestinal morphology was highly plastic, oral morphology did not respond plastically to different chemical compositions of food. Tadpoles that were fed food with low protein and N content and low-energy density developed significantly longer guts and a larger larval stomachs than tadpoles raised on high protein, N and an energetically dense diet, and developed a different intestinal surface morphology. Body sizes of the treatment groups were similar, indicating that tadpoles fully compensated for low protein, N and energy diet by developing longer intestines. The ability of a species, R. temporaria, to respond plastically to environmental variation indicates that this species might have the potential to cope with new conditions during climate change.

With the potential continuous application of mono- or few-layered black phosphorus (BP) in electronic, photonic, therapeutic, and environmental fields, the possible side effects of BP on aquatic organisms after release into natural water are of great concern. We investigated the potential toxicity of BP on the unicellular organism, Tetrahymena thermophila. After the exposure for 8 h at 10 μg/mL, the reproduction of T. thermophila significantly decreased by 46.3%. Severe cell membrane and cilium damage were observed by scanning electron microscopy (SEM) upon treatment with BP. Based on bright-field microscopy and three-dimensional Raman imaging, we investigated the cellular uptake and translocation of BP within T. thermophila. It was observed that the engulfment of BP by T. thermophila was oral apparatus dependent, through which intracellular BP was then transported to the posterior end of T. thermophila by food vacuole packaging. Our study also revealed that BP induced the increase of intracellular reactive oxidant species and formed oxidative stress-dependent toxicity to T. thermophila. Our findings paved a way for better understanding the BP toxicityon aquatic organisms and its potential ecological risks.

Diverse inner arm dyneins cooperate with outer arm dyneins to produce ciliary beating. This study demonstrates an expression system for inner arm dyneins in Tetrahymena. The motor domain of inner arm dynein (Dyh8p or Dyh12p) was fused with the tail of outer arm dynein (Dyh3p) and expressed in viable DYH3-knockout (vKO-DYH3) cells. The chimeric dyneins were observed in the oral apparatus and cilia on the cell bodies, and did not change the swimming speed of vKO-DYH3 cells. In a gliding assay, the motor domains of Dyh8p and Dyh12p moved toward the minus ends of microtubules at 0.8 and 0.3 μm/s, respectively. The gliding velocities of Dyh8p and Dyh12p were decreased in 5 mM ATP but not increased in 0.1 or 0.5 mM ADP. This expression system will be useful for molecular studies on diverse inner arm dyneins.

The hypotrich Schmidingerothrix elongata spec. nov., discovered in saline (20‰) soil of the Longfeng Wetland, Daqing, northern China, was studied using live observation and protargol impregnation. It is characterized, inter alia, by colorless cortical granules arranged in short rows, three frontoventral cirral rows with the rightmost extending far posteriorly, and 4-8, usually six macronuclear nodules. Cell division proceeds as in congeners and confirms the lack of dorsal ciliature. In phylogenetic analyses based on SSU rDNA, S. elongata is sister of S. salinarum+Paracladotricha salina. A re-investigation of the type slides of P. salina, type of Paracladotricha, revealed a misobservation in the original description. Since P. salina lacks, like Schmidingerothrix spp., a dorsal ciliature, Paracladotricha becomes a junior, subjective synonym of Schmidingerothrix with S. salina comb. nov. as fourth species. A review of the phylogenetic analyses dealing with Schmidingerothrix shows that its position is variable. However, together with the gonostomatid oral apparatus it can be hypothesized that Schmidingerothrix is a member of the Gonostomatidae or a close relative. A list of genera (14) and species (58) which have - like Schmidingerothrix - a gonostomatid oral apparatus, as well as a key to these genera are provided.

The transitory dermal infoldings are described for the first time in front and behind the upper jaw in the larvae and postlarvae of the American paddlefish, Polyodon spathula Walbaum, 1858. In sturgeons these infoldings are necessary for the jaw protraction. In the paddlefishes, they, probably recapitulate the ancestral state. At the same time, the presence of these folds at the larval stages might indicate that the paddlefish larvae possess the protractive jaws. Further developmental studies of the jaw kinematics in Polyodon spathula are needed to test both hypotheses.

A primary hypothesis for the evolution of mandibular symphyseal fusion in some mammals is that it functions to resist loads incurred during routine mastication. Anecdotal support for this hypothesis is based on the fact that when the symphysis fuses, it typically does so early during postnatal ontogeny prior to or around the time of weaning. However, little is known about the process of fusion, particularly relative to feeding behaviors and the dynamics of mastication, including occlusion and masticatory loading. In the present study, we investigate the timing and process of symphyseal fusion in alpacas (Vicugna pacos) in the context of maturation of the oral apparatus and oral behavior. We also report on in vivo strains from the symphysis and corpus in young alpacas prior to and following full fusion and M1 occlusion. Results show that fusion begins rostrally by 1 month and is complete by 6-7 months whereas all deciduous premolars and M1 come into occlusion by 6 months. Although symphyseal loading patterns are maintained throughout ontogeny, in young alpacas symphyseal strain magnitudes are low compared with adults but corpus strain magnitudes are comparable to those found in adults. Reduced symphyseal loading in young individuals is contrary to what might be predicted given that the symphysis is still fusing. When considered in light of the development of occlusion and rumination, strain magnitudes may be necessarily low and reflect an overall delay in the maturation of masticatory dynamics.

Tetrahymena and Paramecium are highly differentiated unicellular organisms with elaborated cortical patterns showing a regular arrangement of hundreds to thousands of basal bodies in longitudinal rows that extend from the anterior to the posterior region of the cell. Thus both ciliates exhibit a permanent antero–posterior axis and left–right asymmetry. This cell polarity is reflected in the direction of the structures nucleated around each basal body such as the ciliary rootlets. Studies in these ciliates showed that basal bodies assemble two types of cilia, the cortical cilia and the cilia of the oral apparatus, a complex structure specialized in food capture. These two cilia types display structural differences at their tip domain. Basal bodies possessing distinct compositions creating specialized landmarks are also present. Cilia might be expected to express and transmit polarities throughout signaling pathways given their recognized role in signal transduction. This review will focus on how local polarities in basal bodies/cilia are regulated and transmitted through cell division in order to maintain the global polarity and shape of these cells and locally constrain the interpretation of signals by different cilia. We will also discuss ciliates as excellent biological models to study development and morphogenetic mechanisms and their relationship with cilia diversity and function in metazoans.

Background The giant ciliate Stentor coeruleus is a suitable model organism for studying morphogenesis and regeneration at the single-cell level. The anterior end of the cell is dominated by thousands of cilia organized into a ciliated band of membranelles, known as the oral apparatus (OA). This feeding organelle can be induced to shed by urea treatment. The OA then regenerates via a series of defined morphological events and the cell resumes normal feeding activity. These defined events provide an ideal opportunity to identify the protein constituents of OA and their expression patterns in OA regeneration. Results We identified OA constituents in S. coeruleus by mass spectrometry. A total of 882 OA-associated proteins were identified; the homologs of 181 of these are known OA constituents in other organisms. The expression pattern of OA-associated genes during regeneration was investigated using single-cell transcriptome sequencing. The expression of most OA-associated genes was high during regeneration, indicating their stable expression after OA shedding. We also identified OA-associated differentially expressed genes that may be involved in regulating OA reconstruction. This information enabled us to hypothesize the architecture of OA of S. coeruleus WHEL. Conclusions We have identified protein components of the OA and determined the temporal changes in their expression during OA regeneration in S. coeruleus WHEL. Although our results are preliminary, they form the basis of further studies into the molecular mechanisms of OA development in S. coeruleus. In addition, as S. coeruleus is an important model organism for studying morphogenesis, our findings may help to improve the understanding of OA formation in ciliates.

Cryptosporidium parvum is a protozoan parasite of the phylum Apicomplexa responsible for cryptosporidiosis in calves, a disease that causes significant diarrhea and impairs gain of body weight, generating important production losses. As to now, no effective drugs or vaccines are available for the treatment or prevention of bovine cryptosporidiosis. Several reports suggest that development of a vaccine to prevent cryptosporidiosis is feasible, but relatively few vaccine candidates have been characterized and tested. The most prominent C. parvum antigen is gp60, an O-glycosylated mucin-like protein tethered to the parasite membrane by a glycosylphosphatidylinositol (GPI) anchor. Gp60 has been shown to be involved in essential mechanisms for the survival of C. parvum, such as recognition, adhesion to, and invasion of host cells. This work was aimed at expressing gp60 in Tetrahymena thermophila, a ciliated protozoon with numerous advantages for the heterologous expression of eukaryotic proteins, as a first approach for the development of a recombinant vaccine for bovine cryptosporidiosis. T. thermophila-expressed gp60 localized to the protozoon cell surface and oral apparatus, and partitioned into the Triton X-114 detergent phase. This indicates that the protein entered the reticuloendothelial system of the ciliate, and suggests it contains a GPI-anchor. Homogenates of gp60-expressing T. thermophila cells were recognized by sera from calves naturally infected with C. parvum demonstrating their immunoreactivity. In summary, the heterologous expression of gp60, a C. parvum-encoded GPI-anchored protein, has been successfully demonstrated in the ciliate T. thermophila. Fil: Elguero, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentina Fil: Tomazic, Mariela Luján. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Patobiologia Veterinaria. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Patobiologia Veterinaria.; Argentina Fil: Montes, María Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentina Fil: Florin Christensen, Mónica. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Patobiologia Veterinaria. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Patobiologia Veterinaria.; Argentina Fil: Schnittger, Leonhard. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Patobiologia Veterinaria. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Patobiologia Veterinaria.; Argentina Fil: Nusblat, Alejandro David. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentina

The testate amoeba Leptogromia operculata was described by Valkanov in 1970 from marine waters. We re-discovered this species in brackish water along the North Sea in the Netherlands. Based on detailed comparison of morphology and SSU rDNA phylogeny we conclude that this species represents a sister clade to the Euglyphida (Imbricatea, Cercozoa). We further describe a similar species Trivalvularis immunda gen. nov., sp. nov. from freshwater in France and the Netherlands on basis of morphological data. Trivalvularis and Leptogromia share a unique oral apparatus with three valves that can close the aperture of the shell. Due to this unique morphological character and the phylogenetic analysis of L. operculata we place both species in a new family Trivalvulariidae in the new order Trivalvulariida.

Constantly, aquatic ecosystems are under pressure by complex mixtures of contaminants whose effects are not always easy to evaluate. Due to this, organisms are sought in which early warning signs may be detected upon the presence of potentially toxic xenobiotic substances. Thereby, the study evaluated the incidence of deformities and other morphometric variations in the mentum and wing of Chironomus columbiensis exposed to water from some of the Colombian Andes affected by mining, agriculture, and cattle raising. Populations of C. columbiensis were subjected throughout their life cycle (24 days) for two generations (F1 and F2). Five treatments were carried out in controlled laboratory conditions (water from the site without impact, site of mining mercury, mining mercury + cyanide, cattle raising, and agriculture) and the respective control (reconstituted water). Thereafter, the percentage of deformities in the mentum was calculated, and for the morphometric analysis 29 landmarks were digitized for the mentum and 12 for the wing. As a result, four types of deformities were registered in the C. columbiensis mentum, like absence of teeth, increased number of teeth, fusion and space between teeth, none of them detected in the individuals from the control. Additionally, the highest incidence of deformity in F1 occurred in the treatment of mining mercury, while for F2 this took place in the treatments of mining mercury + cyanide, cattle raising and agriculture. Differences were also found with respect to the morphometric variations of the mentum and wing of C. columbiensis among the control and the treatments with water from the creeks intervened. The treatments of mining mercury + cyanide and agriculture had the highest morphological variation in the mentum and wing of C. columbiensis. The results suggest that the anthropogenic impacts evaluated generate alterations in the oral apparatus of the larval state of C. columbiensis and in the adult state provoke alterations in the wing shape (increased width and reduced basal area). These deformities may be related to multiple stress factors, among them the xenobiotics metabolized by the organisms under conditions of environmental contamination.

Developmental constraints can have significant influence on the magnitude and direction of evolutionary change, and many studies have demonstrated that these effects are manifested on macroevolutionary scales. Phenotypic integration, or the strong interactions among traits, has been similarly invoked as a major influence on morphological variation, and many studies have demonstrated that trait integration changes through ontogeny, in many cases decreasing with age. Here, we unify these perspectives in a case study of the ontogeny of the mammalian cranium, focusing on a comparison between marsupials and placentals. Marsupials are born at an extremely altricial state, requiring, in most cases, the use of the forelimbs to climb to the pouch, and, in all cases, an extended period of continuous suckling, during which most of their development occurs. Previous work has shown that marsupials are less disparate in adult cranial form than are placentals, particularly in the oral apparatus, and in forelimb ontogeny and adult morphology, presumably due to functional selection pressures on these two systems during early postnatal development. Using phenotypic trajectory analysis to quantify prenatal and early postnatal cranial ontogeny in 10 species of therian mammals, we demonstrate that this pattern of limited variation is also apparent in the development of the oral apparatus of marsupials, relative to placentals, but not in the skull more generally. Combined with the observation that marsupials show extremely high integration of the oral apparatus in early postnatal ontogeny, while other cranial regions show similar levels of integration to that observed in placentals, we suggest that high integration may compound the effects of the functional constraints for continuous suckling to ultimately limit the ontogenetic and adult disparity of the marsupial oral apparatus throughout their evolutionary history.

During routine histological examination of oysters (Crassostrea virginica) from Great Bay, New Hampshire, USA, a high prevalence and intensity of ciliate xenomas has been noted since sampling began in 1997. Xenomas are hypertrophic lesions on the gills of bivalve molluscs caused by intracellular ciliates, likely Sphenophrya sp. Although not known to cause mortality in oysters, xenomas have not previously been reported at this high abundance. The objectives of this study were to characterize the xenomas, describe the ciliates, and gather baseline epizootiological data with correlations to environmental and biological parameters. Upon gross examination, xenomas appeared as white nodules, up to 3mm in diameter, located in the gill tissue and occasionally fusing into large masses along the gill filaments. Light microscopy of histological sections revealed xenomas located in the gill water tubes, which they often completely blocked. Higher magnification revealed dual nuclei, eight kineties, and conjugation of the ciliates. Transmission electron microscopy revealed dual nuclei that varied in density, a maximum of twenty cilia in each kinety radiating from the oral apparatus to the posterior, and a 9+2 axoneme structure within the cilia. These traits place the ciliates into the Order Rhynchodida, but insufficient molecular data exist to confirm classification of this ciliate to the Genus Sphenophrya. Since 1997, xenoma prevalence has fluctuated with peaks in 2000, 2004, and 2011. Infected oysters generally contained30 xenomas, but 2.1% contained100, sharply contrasting the rare prevalence and low intensity reported elsewhere. Prevalence increased with oyster size, leveling off near 50% in oysters60mm. Infection intensity peaked in 70-90mm oysters and declined in larger oysters. Individual oyster condition was not associated with xenoma intensity, but sites with oysters in higher condition generally had a greater prevalence and intensity of xenoma infections. Seasonal data indicated an infection cycle increasing from summer to fall, peaking at 55-65% in November and dropping to10% by spring. The oyster population in Great Bay, NH warrants further examination to understand the mechanisms and conditions controlling xenoma formation.

O percevejo-preto, Cyrtomenus mirabilis (Perty, 1830) (Hemiptera: Cydnidae) é considerada atualmente como a mais importante praga de solo em amendoim no Brasil. Seus danos estão relacionados ao ataque em vagens na fase de desenvolvimento dos grãos, na qual ninfas e adultos inserem o estilete de seu aparelho bucal, atingindo os grãos em desenvolvimento. Há a hipótese de que o enxofre possa ser útil atuando como inseticida e/ou repelente desta praga em amendoim. O objetivo do trabalho foi identificar possíveis fontes de enxofre capazes de repelir ou controlar o percevejo-preto diminuindo os danos seus ocasionados. Para isso, foram instalados dois experimentos em área do Polo Centro Norte em Pindorama, SP, e no Centro de Cana do IAC em Ribeirão Preto, SP. Os experimentos foram em blocos casualizados com oito tratamentos, e quatro repetições. Ao final do ciclo do amendoim foi contabilizado o número de percevejos por trincheira e avaliado os danos causados nos diferentes tratamentos. Em Pindorama a baixa ocorrência do inseto impediu as comparações. Já em Ribeirão Preto foi possível observar diferenças entre os tratamentos. Com base nos resultados obtidos no experimento em Ribeirão Preto pode-se concluir que: O uso de enxofre reduz acima de 25% os danos ocasionados pelo percevejo-preto; Sulfurgran B-Max na dosagem de 70 Kg ha-1 aplicado no sulco de semeadura associado à gessagem na dosagem de 1.000 Kg ha-1 aos 92 DAS reduz a população de percevejos e seus danos ocasionados.

The number of dams has had a significant increase in the last six decades, altering the physiography of rivers and creating systems of reservoirs in series. Therefore, it is expected that in a cascade of reservoirs, detritivorous fish will be especially affected due to the loss of nutritional quality in these regions, resulted from the high sedimentation of nutrients and organic matter. The objective of this study was to evaluate whether changes in the abundance of detritivorous fish, throughout a reservoir cascade in Brazil, resulted from trophic and limnological variations associated with sediment and nutrient retention. We used the Structural Equation Models approach (SEM) to test hypotheses. The six reservoirs studied are located in the Sao Francisco River, in the Northeast Brazil. The variables used to index latent variable oligotrophication were divided into trophic (total phosphate, nitrate and chlorophyll-a) and limnological variables (turbidity and water residence time accumulated), and the abundance of the detritivore guild was indexed by the catch per unit effort (CPUE). Our results indicate a significant negative impact of the both trophic (total phosphate, chlorophyll-a and nitrate) and limnological indicators (turbidity) on the oligotrophication process. The longer residence times of the water accumulated along the reservoir cascade augment the oligotrophication process, consequently affecting negatively all the trophic and limnological indicators analysed. Finally, the latent variable oligotrophication had a significant negative effect on the latent variable detritivorous fish abundance throughout the cascade. The Curimatidae family was the most affected by oligotrophication along the cascade, followed by Prochilodontidae. These different responses to the detritivorous fish families reflect different patterns in the morphology of the oral apparatus and forms of food apprehension. Our findings are of particular importance for the management of fish species in highly fragmented rivers.

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Mutational analyses of axonemal dyneins are useful for elucidating the molecular mechanism of ciliary motility. This study demonstrates a mutation system for characterizing lethal P-loop mutations in Tetrahymena outer arm dynein (Dyh3p). The viable DYH3-knockout (vKO-DYH3) cells isolated in this study enabled the examination of lethal mutations in P-loops 1 and 2. The P1 mutant dynein localized in the oral apparatus and the proximal region of the cilia, and the P2 mutant dynein localized only in the oral apparatus. Both results are different from the localization of wild-type Dyh3p. In addition, a co-precipitation assay showed that the P1 and P2 mutant dyneins did not dissociate from microtubules in ATP plus vanadate or in no-ATP conditions, in contrast to wild-type Dyh3p. This mutation system is useful for further molecular studies of axonemal dyneins and ciliary motility.

While ciliates of the genus Stentor are known for their ability to regenerate when their cells are damaged or even fragmented, the physical and molecular mechanisms underlying this process are poorly understood. To identify genes involved in the regenerative capability of Stentor cells, RNA sequencing of individual Stentor polymorphus cell fragments was performed. After splitting a cell over the anterior-posterior axis, the posterior fragment has to regenerate the oral apparatus, while the anterior part needs to regenerate the hold fast. Altogether, differential expression analysis of both posterior and anterior S. polymorphus cell fragments for four different post-split time points revealed over 10,000 upregulated genes throughout the regeneration process. Among these, genes involved in cell signaling, microtubule-based movement, and cell cycle regulation seemed to be particularly important during cellular regeneration. We identified roughly nine times as many upregulated genes in regenerating S. polymorphus posterior fragments as compared to anterior fragments, indicating that regeneration of the anterior oral apparatus is a complex process that involves many genes. Our analyses identified several expanded groups of genes, such as dual-specific tyrosine-(Y)-phosphorylation-regulated kinases and MORN domain-containing proteins that seemingly act as key regulators of cellular regeneration. In agreement with earlier morphological and cell biological studies [1, 2], our differential expression analyses indicate that cellular regeneration and vegetative division share many similarities. Onsbring et al. sequence transcriptomes of individual bisections of regenerating cells of the giant heterotrichous ciliate Stentor polymorphus. Their differential expression analysis reveals that protein phosporylation, microtubule-based processes, and genes involved in the cell cycle are important for cellular regeneration.

Cranium of jawed vertebrates is composed of dorsal moiety that encapsulates the brain, or the neurocranium, and the is called the neurocranium, and the ventral moiety, the viscerocranium, that supports the pharynx. In modern jawed vertebrates (crown gnathostomes), the viscerocranium is predominantly of neural crest origin, and for the neurocranium, the rostral part is derived from neural crest cells, whereas the posterior part from the mesoderm. In the cyclostome cranium, the mesoderm/neural crest boundary of the neurocranium used to be enigmatic, let alone the morphological comparison of neurocranial between two cyclostome groups, lampreys and hagfishes. By examining the hagfish development it has become clear that cyclostomes share a common craniofacial embryonic pattern that is not shared by modern gnathostomes, and cyclostome cranium can be compared among the group as developmental modular units with comparable mesoderm/neural crest boundary within the neuroranium. Also, the dual origin of the jawed vertebrate neurocranium has now turned out to represent a derived condition, and ancestrally, the neurocranium would likely have been predominantly of mesodermal origin. Enlargement of the forebrain and reorganization of the oral apparatus seem to have led to the involvement of the neural crest in the rostral neurocranium.

SummaryWhile ciliates of the genus Stentor are known for their ability to regenerate when their cells are damaged or even fragmented, the physical and molecular mechanisms underlying this process are poorly understood. To identify genes involved in the regenerative capability of Stentor cells, RNA sequencing of individual Stentor polymorphus cell fragments was performed. After splitting a cell over the anterior-posterior axis, the posterior fragment has to regenerate the oral apparatus, while the anterior part needs to regenerate the hold fast. Altogether, differential expression analysis of both posterior and anterior S. polymorphus cell fragments for four different post-split time points revealed over 10,000 up-regulated genes throughout the regeneration process. Among these, genes involved in cell signaling, microtubule-based movement and cell cycle regulation seemed to be particularly important during cellular regeneration. We identified roughly nine times as many up-regulated genes in regenerating S. polymorphus posterior fragments as compared to anterior fragments, indicating that regeneration of the anterior oral apparatus is a complex process that involves many genes. Our analyses identified several expanded groups of genes such as dual-specific tyrosine-(Y)-phosphorylation regulated kinases and MORN domain containing proteins that seemingly act as key-regulators of cellular regeneration. In agreement with earlier morphological and cell biological studies, our differential expression analyses indicate that cellular regeneration and vegetative division share many similarities.