Estudo morfológico e ecológico do filo Cycliophora

Doutoramento em BiologiaCycliophora é um filo animal descrito recentemente que acomoda, apenas, duas espécies: Symbion pandora Funch e Kristensen, 1995 e S. americanus Obst, Funch e Kristensen, 2006. Este filo é caracterizado por um ciclo de vida assaz complexo, cuja posição filogenética tem sido debatida desde a sua descoberta. Esta dissertação visa aprofundar o conhecimento geral existente acerca destes enigmáticos e pouco explorados metazoários. Assim, vários aspectos da morfologia e ecologia de ciclióforos foram estudados através de observações in vivo, técnicas de microscopia e reconstrução tridimensional. A mioanatomia de várias fases do ciclo de vida é descrita para S. pandora e S. americanus. Os nossos resultados revelam uma similaridade contundente entre a musculatura das duas espécies. A mioanatomia geral de Symbion é, ainda, comparada à de outros metazoários. A expressão de algumas substâncias imunorreactivas, como são exemplo a serotonina e as sinapsinas, é investigada em várias formas do ciclo de vida. Quando comparados com outros representantes de Spiralia, conclui-se que a neuroanatomia geral dos ciclióforos se assemelha mais às formas larvares do que aos adultos. Apesar de possuírem um plano corporal sofisticado, com extensas áreas ciliadas e uma mioanatomia complexa, descobrimos que o macho de ambas as espécies Symbion é composto por apenas algumas dezenas de células. Baseando-nos nestas observações, inferimos que a complexidade dos metazoários não se relaciona com o tamanho corporal nem com o número de células de um organismo. Estudos sobre a ultra-estrutura da fêmea revelaram, entre outras estruturas, um putativo poro genital, extensões citoplasmáticas do oócito e glândulas posteriores. Morfologia e implicações funcionais destas estruturas são aqui discutidas. A anatomia do protonefrídeo da larva cordóide é descrita. A arquitectura deste órgão diverge daquela presente noutros representantes de Nephrozoa, particularmente ao nível da área de filtração da célula terminal. As nossas observações são discutidas em termos filogenéticos. A maturação sexual em ciclióforos é investigada. Os nossos resultados sugerem que a transição de reprodução assexual a sexual se relacione com a idade da forma séssil, a “feeding stage”. A presença da larva Prometeus assente no tronco desta também poderá influenciar o processo, embora mais estudos sejam desejáveis para o comprovar. Os nossos resultados são discutidos integrativa e comparativamente com o conhecimento prévio sobre Cycliophora. A cumulação deste conhecimento será essencial para a compreensão da evolução e filogenia deste enigmático filo.As one of the most recently discovered animal phyla, Cycliophora to date comprises only two described species, Symbion pandora Funch and Kristensen, 1995 and S. americanus Obst, Funch and Kristensen, 2006. This enigmatic phylum is characterized by a complex life cycle and a phylogenetic position that has been under debate ever since its discovery. This dissertation aims to increase the general knowledge on these cryptic animals. Various aspects of the cycliophoran morphology and ecology are studied by a combination of in vivo observations, several microscopic techniques and 3D reconstruction imaging. Herein, we describe the muscle arrangement of the several life cycle stages of S. pandora and S. americanus. Overall, our results reveal striking similarities in the myoanatomy of both cycliophoran species. We discuss our findings with respect to muscle elements of other metazoan groups, as well. The distribution of a number of immunoreactive substances, e.g., serotonin and synapsin, is investigated in various life cycle stages. It is concluded that the overall neuroanatomical condition of all cycliophoran larval stages resembles much more the situation of adult rather than larval spiralians. Despite its sophisticated bodyplan, which includes extensive locomotory ciliated fields and a complex myoanatomy, we find that the cycliophoran dwarf male of both Symbion species is composed of solely a few dozens of cells. Based on these findings, we infer that complexity of metazoans is neither correlated to the body size nor the overall number of cells of an individual. Continued studies on the ultrastructure of the female revealed the existence of, among other features, a putative gonopore, cytoplasmic extensions of the oocyte and posterior glands. The morphology and the functional implications of these structures are discussed. The complete ultrastructural anatomy of the cycliophoran protonephridium, including the filtration area and the nephridiopore, is described herein. The cycliophoran protonephridial architecture diverges from that of all other nephrozoans mainly in the filtration area, in which no pores or clefts are found. Our findings are discussed in phylogenetic terms. Sexual maturation in the complex cycliophoran life cycle is investigated. Based on our findings we argue in favor of a shift to sexuality motivated by the age of the feeding stages, which is estimated by the number of cuticular wrinkles. The settled Prometheus larva may also induce the generation of a female by a feeding individual, though this needs to be further investigated. Our new findings are integrated and comprehensively discussed with the preexistent knowledge on Cycliophora. This accumulation of information will be essential to retrace the evolution of this enigmatic phylum and to make a final decision on its phylogenetic placement within Metazoa.FCT - SFRH/BD/24436/2005POPH/FS

A identidade do “Outro” colonizado à luz das reflexões dos estudos Pós-Coloniais

The present article include the construction and descostruction of identity of the colonized other envisoned by intellectuals whit Homí K.Bhabha, Frantz Fanon, Albert Memmi, V.S Naipaul, Spivak, Edward Said, in the others, trough the optics of post-colonial studies. For Bhabha, the representation of the difference shoud not be seen as reflections of cultural traits or ethical entered through discouses and strategies attempinting to establish by means of the exclusiornary binaries of the other`s identity. The mimicry was the appropriate ambivalent strategy of colonialism with the order to legitimize the discourse of superiority of the colonizer over the colonized.O presente artigo contempla a construção e desconstrução da identidade do “Outro” colonizado vislumbrada por intelectuais como Homí.K.Bhabha, Frantz Fanon, Albert Memmi, V.S Naipaul, Spivak, Edward Said, dentre outros, através da ótica dos estudos Pós-coloniais. Para Bhabha, a representação da diferença não deve ser vista como reflexos de traços culturais ou éticos inscritos por meio de discursos e estratégias que tentam fixar por meio de binarismos excludentes a identidade do Outro. A mímica constituiu-se em uma estratégia ambivalente apropriada pelo colonialismo com o intuito de legitimar o discurso de superioridade do colonizador sobre o colonizado

Comparative myoanatomy of Tardigrada:new insights from the heterotardigrades <i>Actinarctus doryphorus</i> (Tanarctidae) and <i>Echiniscoides sigismundi</i> (Echiniscoididae)

Abstract Background Tardigrada is a group of microscopic invertebrates distributed worldwide in permanent and temporal aquatic habitats. Famous for their extreme stress tolerance, tardigrades are also of interest due to their close relationship with Arthropoda and Cycloneuralia. Despite recent efforts in analyzing the musculature of a number of tardigrade species, data on the class Heterotardigrada remain scarce. Aiming to expand the current morphological framework, and to promote the use of muscular body plans in elucidating tardigrade phylogeny, the myoanatomy of two heterotardigrades, Actinarctus doryphorus and Echiniscoides sigismundi, was analyzed by cytochemistry, scanning electron and confocal laser scanning microscopy and 3D imaging. We discuss our findings with reference to other tardigrades and internal phylogenetic relationships of the phylum. Results We focus our analyses on the somatic musculature, which in tardigrades includes muscle groups spanning dorsal, ventral, and lateral body regions, with the legs being musculated by fibers belonging to all three groups. A pronounced reduction of the trunk musculature is seen in the dorsoventrally compressed A. doryphorus, a species that generally has fewer cuticle attachment sites as compared to E. sigismundi and members of the class Eutardigrada. Interestingly, F-actin positive signals were found in the head appendages of A. doryphorus. Our analyses further indicate that cross-striation is a feature common to the somatic muscles of heterotardigrades and that E. sigismundi—as previously proposed for other echiniscoidean heterotardigrades—has relatively thick somatic muscle fibers. Conclusions We provide new insights into the myoanatomical differences that characterize distinct evolutionary lineages within Tardigrada, highlighting characters that potentially can be informative in future phylogenetic analyses. We focus our current analyses on the ventral trunk musculature. Our observations suggest that seven paired ventromedian attachment sites anchoring a large number of muscles can be regarded as part of the ground pattern of Tardigrada and that fusion and reduction of cuticular attachment sites is a derived condition. Specifically, the pattern of these sites differs in particular details between tardigrade taxa. In the future, a deeper understanding of the tardigrade myoanatomical ground pattern will require more investigations in order to include all major tardigrade lineages

The challenge of proving the existence of metazoan life in permanently anoxic deep-sea sediments

The demonstration of the existence of metazoan life in absence of free oxygen is one of the most fascinating and difficult challenges in biology. Danovaro et al. (2010) discovered three new species of the Phylum Loricifera, living in the anoxic sediments of the L’Atalante, a deep-hypersaline anoxic basin of the Mediterranean Sea. Multiple and independent analyses based on staining, incorporation of radio labeled substrates, CellTracker Green incorporation experiments and ultra-structure analyses, allowed Danovaro et al. (2010) to conclude that these animals were able to spend their entire life cycle under anoxic conditions. Bernhard et al. (2015) investigated the same basin. Due to technical difficulties in sampling operations, they could not collect samples from the permanently anoxic sediment, and sampled only the redoxcline portion of the L’Atalante basin. They found ten individuals of Loricifera and provided alternative interpretations of the results of Danovaro et al. (2010). Here we analyze these interpretations, and present additional evidence indicating that the Loricifera encountered in the anoxic basin L’Atalante were actually alive at the time of sampling. We also discuss the reliability of different methodologies and approaches in providing evidence of metazoans living in anoxic conditions, paving the way for future investigation

A first look at the biodiversity of Loricifera in the southern Gulf of Mexico

Loricifera is a group of marine, meiobenthic invertebrates of microscopic size that are found worldwide. The Loriciferan fauna of the Southern Gulf of Mexico (SGM) remains unknown. In the present study we report on the first exploration of the Loriciferans from the SGM, which is a strategic area for oil exploitation. The Loriciferan specimens were collected during deep-sea cruises XIXIMI-4 (August-September 2015) and XIXIMI-6 (August 2017), in which six out of 22 stations yielded specimens of Loricifera. A total of six specimens were found (although one of them was lost), namely two adults and four larval stages. Preliminary analyses based on the peculiarity of their morphological features indicate that the specimens include representatives of new species of Pliciloricus and Rugiloricus, an undescribed genus of Pliciloricidae and, possibly, an undescribed genus of Nanaloricidae. None of the specimens found could be assigned to previously described species. Since all specimens are either intermediate life cycle stages or poorly preserved, only a short description based on their morphological traits is provided here. Our findings are discussed from a comparative perspective with known Loriciferan species

Supercritical fluid (SCF)-assisted preparation of cyclodextrin-based poly(pseudo)rotaxanes for transdermal purposes

This study aims to investigate the effect of the preparation of solid dispersions using supercritical CO2 (scCO2) on the physicochemical properties and the performance of supramolecular gels based on polymer-cyclodextrin (CD) interactions (named poly(pseudo)rotaxanes, PPR) envisaging a transdermal administration. Solid dispersions containing Soluplus®, the antihypertensive drug carvedilol (CAR), and CD (αCD or HPβCD) were prepared and characterized by HPLC, XRPD, FTIR, and DSC. PPRs prepared from solid dispersions (SCF gels) and the corresponding physical mixtures (PM gels) were analyzed regarding rheology, morphology, in vitro drug diffusion, and ex vivo drug skin permeation. The application of scCO2 led to the loss of the crystalline lattice of CAR while preserving its chemical identity. On the contrary, αCD crystals were still present in the SCF solid dispersions. SCF gels were more uniform than their corresponding PM, and the supercritical treatment resulted in changes in the rheological behavior, reducing the viscosity. CAR in vitro diffusion was significantly higher (p < 0.05) for the αCD-based SCF gel than its corresponding PM gel. Drug skin permeation showed a significant increase in drug flux from CD-based SCF gels (containing αCD or HPβCD) compared to corresponding PM gels. Additionally, the pretreatment of the skin with αCD exhibited increased CAR permeation, suggesting an interaction between αCD and the skin membrane. Results evidenced that SCF processing decisively modified the properties of the supramolecular gels, particularly those prepared with αCDOpen Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This research was partially supported by the Brazilian agency Fundação de Apoio à Pesquisa do Estado de Goiás (FAPEG). The work was supported by MCIN/AEI/10.13039/501100011033 (PID 2020-113881RB-I00), Spain, Xunta de Galicia (ED431C 2020/17), and FEDERS

Rhamdia quelen's semen pre-cryopreservation processing and sperm viability time after thawing / Processo de pré-criopreservação de sêmen de Rhamdia quelen e tempo de viabilidade do esperma após o descongelamento

The study’s aim was to evaluate the sperm movement in the semen of Rhamdia quelen, cryopreserved at different times of exposure to nitrogen vapor, besides different equilibration times and with different concentrations of methanol, and in thawed semen kept at 25ºC for different periods of time. Three distinct experiments were carried out. In the first experiment, fresh semen aliquots were diluted in a solution containing 5% D-Fructose, 5% milk powder (Nestle®, Ninho Fortificado®) and 10% methanol, filled into 0.25 mL straws and immediately kept in nitrogen vapor by 0,5; 1.0; 4.0; 8.0; 12.0 and 18.0h. After these times, the straws were transferred to the liquid nitrogen (-196°C). After 72 h, the semen was thawed by immersing the straws in water at (25 ° C) for 10 seconds. The sperm activation was evaluated by diluting the thawed semen in distilled water in the proportion of 1:250 (semen:activating solution; v/v). Motility and spermatic velocity in thawed semen were measured using the CASA system. In the second experiment, the fresh semen was diluted in solutions with glucose and powdered milk (Nestle®, Ninho Fortificado®) and 5.0; 7.5; 10.0; 12.5% methanol. Immediately after, it was filled into 0.25mL straws and kept at 25ºC by equilibrium times corresponding to 0, 20, 40 and 60 minutes. Subsequently, part of the straws was used for spermatic evaluation before cryopreservation and others were submitted to cryopreservation in nitrogen vapor for 30 minutes and after transferred to liquid nitrogen (-196°C). The processes of thawing and sperm evaluation were performed as described above. In the third experiment, the cryopreserved semen was thawed and kept at 25°C for 0.0; 7,5; 15,0; 22.5 and 30.0 minutes. After each time, the spermatic movement was evaluated as described above. The exposure time of straws to nitrogen vapor and the kept time of the thawed semen at 25ºC did not affect (p &gt; 0.05) the sperm movement. On the other hand, methanol concentrations and equilibrium time (p &lt; 0.05) influenced the spermatic movement interactively both, before and after cryopreservation. The best results were achieved when the semen was cryopreserved immediately after dilution. The cryopreservation of Rhamdia quelen semen can be successfully performed when the semen is diluted in a solution containing 5% D-Fructose, 5% milk powder and 11.66% of methanol and immediately filled into 0.25 mL straws and kept in nitrogen vapor by 30 min before being transferred to liquid nitrogen. In addition, after thawing, the semen can be kept at 25ºC for up to 30 minutes without damage to spermatic movement