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Echinococcus granulosus: Insights into the protoscolex F-actin cytoskeleton
Accepted Manuscript Title: Echinococcus granulosus: Insights into the protoscolex F-actin cytoskeleton Authors: Silvana La-Rocca, Joaquina Farias, Cora Chalar, Alejandra E. Kun, Veronica Fernandez PII: DOI: Article Number:
S0001-706X(19)30116-0 https://doi.org/10.1016/j.actatropica.2019.105122 105122
Reference:
ACTROP 105122
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
25 January 2019 29 July 2019 1 August 2019
Please cite this article as: La-Rocca S, Farias J, Chalar C, Kun AE, Fernandez V, Echinococcus granulosus: Insights into the protoscolex F-actin cytoskeleton, Acta Tropica (2019), https://doi.org/10.1016/j.actatropica.2019.105122 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Echinococcus granulosus: Insights into the protoscolex F-actin cytoskeleton. Silvana La-Roccaa, Joaquina Fariasb, Cora Chalarc, Alejandra E. Kunb,c, Veronica Fernandeza. a
Area Inmunología, Departamento de Biociencias, Facultad de Química, Universidad de la República,
Montevideo, Uruguay. b
Department of Proteins and Nucleic Acids, Instituto de Investigaciones Biológicas Clemente Estable,
Montevideo, Uruguay. C
Sección Bioquímica, Departamento de Biología celular y Molecular, Facultad de Ciencias, Universidad de la
República.
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Corresponding author:
Verónica FERNANDEZ MANCEBO, Laboratorio de Inmunología, Av. Alfredo Navarro 3051, piso 2;
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Montevideo, CP 11600; Uruguay. E-mail: [email protected]
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Graphical abstract
PSC has a highly organized F-actin cytoskeleton with unevenly distributed filaments
F-actin prevailed in the suckers and rostellum in both evaginated and invaginated PSC
E. granulosus evaginated PSC seem to retain their ability to reinvaginate in vitro
F-actin cytoskeleton seems to keep constant in the evagination/invagination process
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Abstract Echinococcus granulosus is a cestode parasite whose cytoskeleton plasticity allows it to enter and develop inside its hosts, completing thus its life cycle. We focused our attention on F-actin organization and distribution in E. granulosus protoscoleces (PSC) in order to contribute to the knowledge of the parasite cytoskeleton. In particular, we addressed some aspects of F-actin rearrangements in PSC at different stages of the evagination/invagination process. The use of light microscopy allowed us to identify different PSC structures and phalloidin staining displayed a parasite's highly organized F-actin cytoskeleton. Suckers exhibit an important musculature composed of a set of radial fibers. At the rostellum, the F-actin filaments
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are arranged in a bulbar shape with perforations that appear to be the attachment places for the hooks. Also, "circular" structures of F-actin were identified, which remind the flame cells. Furthermore, parasite Factin filaments, unevenly distributed, seem to have remained substantially unchanged during the
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evagination/invagination process. Finally, we showed that the scolex of an evaginated E. granulosus PSC
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reinvaginates in vitro without any treatment.
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Abbreviations
PSC: E. granulosus protoscoleces; PHEM buffer: 25 mM HEPES, 10 mM EGTA, 60 mM PIPES, 2 mM MgCl2
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(pH 7,3); RT: room temperature
Keywords
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Echinococcus granulosus; invagination/evagination process; protoscoleces; F-Actin cytoskeleton.
1.
Introduction
The cestode Echinococcus granulosus, like all parasites, presents a complex life cycle. It requires two mammalian hosts: a definitive host (always carnivore) in which the adult worm (strobilar stage) develops in the small intestine, and an intermediate host (for example, livestock or human) in which the cystic metacestode (larval stage) usually develops in the viscera (Agudelo Higuita et al., 2016; Jenkins et al., 2005; Thompson and Lymbery, 1995). The metacestode is a fluid-filled cystic structure that undergoes asexual multiplication to produce large numbers of scolices, termed protoscoleces (PSC) (Thompson, 2017). The
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PSC, which are juvenile scoleces, is the anterior end of the adult tapeworm (Koziol et al., 2016), carrying suckers and hooks by which it adheres to a host (Thompson and Lymbery, 1995). In the cyst, the apical region of the protoscolex (scolex) is invaginated within the mucopolysaccharide-coated basal region of the
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protoscolex tegument (invaginated PSC) (Thompson, 2017). When PSC are ingested by the definitive host, they mature towards the strobilar stage after an evagination process, in which the scolex is exposed
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outwardly (evaginated PSC) (Thompson, 2017). Then, in the intestine, each attached-parasite lengthens its
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body which becomes divided into three segments called proglottids. The last proglottid, containing embryonated eggs, detaches and, together with the host's feces, reaches the outside where the eggs are released (Thompson, 2017). The parasitic life cycle is completed when the eggs are ingested by
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intermediate hosts and the released oncospheres develop into PSC-containing hydatid cysts in their viscera (Jenkins et al., 2005; Thompson, 2017). On the other hand, PSC have also the ability to differentiate into
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fully developed cysts when they are released into a cavity of the intermediate host, for example, upon
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metacestode rupture (Thompson, 2017). For this transformation, the PSC must have their scolex exposed as well (Elissondo et al., 2004). Therefore, the scolex evagination step is a requirement for the PSC to differentiate into hydatid cysts (Elissondo et al., 2004) or into adult worms (Smyth et al., 1967). In the first half of the 20th century, a growing number of descriptions about the most distinctive characteristics of E. granulosus and its life cycle have been published where some structures were identified and their possible functions were speculated (reviewed by Thompson and Lymbery, 1995). Particularly, the PSC exhibit a protective covering (tegument) supported by a broad fibrous zone crossed by
muscle bundles arranged into two perpendicular muscle fibers layers, one circular and the other transversal (Morseth, 1967, 1966). Coutelen et al. (1952) (Coutelen et al., 1952) classify the musculature as a subcuticular set, a suckers-associated set, a rostellum-associated set and a deeper fibers set, although they suggest that the whole musculature would be associated with the movements of evagination and invagination. It has also been suggested, in Taenia solium, that this muscle system, with high F-actin expression, could define the movement direction and the required force in response to different physiological situations, such as scolex attachment in the definitive host gut (Ambrosio et al., 2003). The plasticity of the cytoskeleton has been acquired by parasite helminths as one of the strategies that
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allow them to enter, settle and develop in their hosts, thus completing their life cycle. Due to this, the cytoskeleton components have been proposed as target for therapeutic drugs and, in fact, the helminth cytoskeleton organization is affected for some anthelmintic drug (Conder et al., 1981; Cumino et al., 2009;
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Ingold et al., 1999; Markoski et al., 2006; Pérez-Serrano et al., 1995). Confocal laser scanning microscopy combined with phalloidin staining, which marks F-actin in muscle cells with high specificity, allows muscle
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systems to be viewed accurately in 3D for analysis. Recently, the three-dimensional muscular arrangement
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of helminthes has emerged as a useful morphological tool for taxonomic and phylogenetic analyzes (Adami et al., 2017; Petrov and Podvyaznaya, 2016). While this type of phylogeny is not yet available in Platyhelminthes, it seems to be a promising approach (Adami et al., 2017). For example, using this
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methodology, differences between three species of freshwater Macrostomum were observed, although the three shared a general muscular pattern (Adami et al., 2017). Here we focus our attention on the F-actin
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distribution in E. granulosus in order to contribute to the knowledge of its cytoskeleton. In particular, we
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address some morphological aspects of PSC by optical and fluorescence microscopy. The alterations in the F-actin distribution that accompany the evagination/invagination process allow us to infer the behavior of the cytoskeleton in such process.
2.
Materials and methods
2.1.
Parasite cultures
Hydatid cysts were collected during the routine work of local abattoirs in Montevideo (Uruguay) from the lungs of naturally infected bovines. E. granulosus PSC were recovered from fertile cysts according to Baz et al. (Baz et al., 1995) and the viability was determined by eosin-exclusion method (Smyth and Barrett, 1980). Only PSC batches with over 95% viability were used. After washing with PBS, the PSC were incubated in Medium-199 with Earle's salts (M-199, GIBCO) supplemented with antibiotics (penicillin, streptomycin and gentamicin at 100 U/mL, 100 μg/mL and 30 μg/mL, respectively) containing 4 mg/mL glucose, at 37° and 5% CO2. The cultured PSC were examined by light microscopy using inverted microcopies (Nikon Eclipse Ti
processed by the softwares ImageJ or ZEN Version 2.0, respectively). 2.2.
Sample processing and phalloidin staining
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coupled to a Nikon Digital Sight DS-QiMc or Carl Zeiss coupled to AxioCam ERc5s and the images were
The samples were processed according to Kun et al., 2012 (Kun et al., 2012), with some modifications.
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Briefly, cultured PSC were fixed for 1 h at 4°C, in 3% w/v paraformaldehyde in PHEM (25 mM HEPES, 10 mM EGTA, 60 mM PIPES, 2 mM MgCl2, pH 7,3). Then, the samples were cryoprotected until PSC fall down to the
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bottom of the tube. Samples infiltration was done through progressive substitution (25%, 50%, 100%) of
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sucrose/PHEM by Tissue Tek (Sakura Finetek USA) at 4°C with stirring. Subsequently, the PSC were quickly frozen and cryosectioned at -20°C (10 µm) using a Cryostat (SLEE), adhered onto poly-D/L-lysine precoated slides and kept at -20ºC until use. To evidence filamentous actin (F-actin), the permeabilized cryosections
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were blocked using 1% w/v sodium borohydride in PHEM for 10 min at room temperature (RT). After washing with PHEM, the slides were incubated with Alexa Fluor 546-phalloidin (Invitrogen), in incubation
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buffer (0,1% w/v BSA, 75mM lysine, in PHEM), for 45 minutes, in darkness, at RT. Next, the slides were
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rinsed in PHEM, mounted in ProLong Gold Antifade Mountant (Thermo Fisher Scientific) and were kept in the darkness, at RT, 24 hours, and then at 4ºC until observation for confocal analyze. Images were collected with an Olympus FV300 or a Leica TCS SP5 II confocal microscope using a Plan Apo 60X/1.42 NA, a Plan Apo 63X/1.4 NA or a Plan Apo 100X/1.4 NA oil immersion objective, with or without digital zoom. Image-J free software was used for image processing (including brightness/contrast adjustment and Gaussian blur filtering). 3.
Results
We achieve evaginated parasites in vitro culturing fresh E. granulosus PSC without any previous treatment in M-199 supplemented with glucose and antibiotics. At the beginning, the invaginated PSC remain almost immobile, but a few hours later they become very active. They perform different types of movements where some PSC externalize and re-internalize their scolex in an asynchronous way (see supplementary material). Fig. 1 and 2 show PSC after 24 h of in vitro culture, where both invaginated and evaginated parasites co-exist. We recognized the parasite scolex (Sc) containing the attaching structures to the definitive host gut: the suckers (S) and the hooks (H) joined to the rostellum (R). The invaginated PSC show the classical spherical or oval shape (Fig. 1A-C); inside them, the subtegumentary muscle-fibers extend
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throughout the organism where longitudinal and transversal fibers are distinguished (Fig. 2A, D and E). The tunnel (t), generated by the introversion of the PSC apical portion, extends from the muscular pad (hook region) to the orifice (O) at the anterior region (Fig. 1A-C and 2A-D). A mesh of F-actin fibers, similar to the
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rest of the organism, forms the tunnel (t) and the orifice (O) (Fig. 2A-D). The suckers (S), located along the tunnel (t) (Fig. 1 and 2), exhibit an important musculature composed of a set of radial fibers (Fig. 2A, B, D
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and E). The hooks (H) are oriented with the blades towards the anterior region, whereas the structures
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through which they are anchored to the rostellum, handles and guards, are directed towards the posterior and lateral region, respectively (Fig. 1A, B and C). At the rostellum (R) area, a mesh of F-actin fibers is observed (Fig. 2D) and immediately below this mesh, a group of F-actin filaments in different directions
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could also belong to the rostellum (Fig. 2D and E). On the other hand, in the evaginated PSC stands out the scolex (Sc), the neck (N) and the body (B), as it is possible to see in Fig. 1A, F and 2H-I. In this stage, the
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hooks (H) remain anchored to the rostellum (R) but their blades now look towards the back of the parasite
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(Fig. 1A, F and 3A). The hooks are organized in two crowns where the upper row contains the largest hooks (Fig. 3A). They are in a region with abundant muscle fibers: the rostellum (R) (Fig. 3A) which would be acting as support. The four suckers (S) (Fig. 1A and F) have a set of both longitudinal and transverse muscle fibers as well as radial and circular ones. As we expected, that the suckers (S) and rostellum (R) maintain the same organization of F-actin fibers observed in the invaginated parasite (Fig. 2). Next to the scolex, the evaginated PSC show a narrowing that constitutes the neck (N) (Fig. 1A, F, 2H-I and 3A) which connects the scolex with the body. The parasite's neck (N) exhibits a mesh of actin F fibers (Fig. 2H-I and 3A) indicating an
important muscular system with transverse and longitudinal muscle fibers. The body (B), which represents approximately 50-60% of the total PSC length (Fig. 1A, F and 2H-I), is formed by different cell types and structures. For instance, it contains numerous calcareous corpuscles (Fig. 1) and the cells that form them. Another important structure is the parasitic protonephridial system constituted by the flame cells and the collecting ducts. It is necessary to point out that both evaginated and invaginated parasites display several “circular” F-actin organizations that resemble flame cells (Fig. 2 and 3B, arrowhead). Moreover, along the anterior-posterior axis of the evaginated PSC, it is possible to clearly visualize some structures that resemble the collecting ducts (Fig. 2 H and I). These structures can also be observed in some invaginated
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PSC (Fig. 3Ba), although possibly in fully invaginated ones they could be masked or displaced by the apical region.
For the scolex evagination process since each PSC progressed independently, we were able to record
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several of such moments by light and fluorescence microscopy (Fig. 1 and 2). In Fig. 1A-C and 2A-D the orifice and the tunnel through which the scolex is exteriorized are shown. Fig. 2E shows a PSC whose
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rostellum is directed towards the anterior region compressing the suckers. In Fig. 1D-E and 2F-G the
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emerging suckers, first as spherical dome-shaped bodies and then as cups are shown. The hooks, anchored to rostellum, maintain their original disposition with the blades pointing towards the orifice (Fig. 1D-E). Once outside, they are organized in two crowns where the blades now look towards the PSC body (Fig. 1A,
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F and 3A). The hook crowns are anchored between the F-actin filaments (Fig. 3A) and they reach their final arrangement when the rostellum assumes a bulbar shape (Fig. 3A) and the apical dome (rostellar pad)
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appears (Fig. 1A and F). No substantial changes in the F-actin filaments organization of rostellum and
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suckers have been perceived during scolex evagination steps (Fig. 2 and 3A). Additionally, our evidence indicates that E. granulosus PSC retain the ability to reinvaginate under our in vitro culture conditions. In this sense, we provide some movies that support this statement (Supplementary video A.1 shows at least one re-invaginating PSC, supplementary video A.2 shows a protoscolex that rearranges its hooks and internalizes its rostellum, and supplementary video A.3 shows a protoscolex entering its suckers, ending the reinvagination process at 4, 24 and 16 hour-cultures, respectively).
Through in silico comparative E. granulosus transcriptomics, we study the differential profiles of F-actin gene expression between adult-PSC and PSC-hydatid cyst wall. The result is presented in table 1 where we highlight the expression of six actin genes from E. granulosus (EGR_05821, EGR_06859, EGR_08301, EGR_08341, EGR_09069 and EGR_10650). As it can see, the expression values of some of them vary significantly among parasite stages. 4.
Discussion
The PSC physiological evagination process is controlled by a diversity of factors which are not known exactly. Although the in vitro conditions are probably far from those that exist inside the host, evaginated
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parasites have been obtained by culturing PSC with or without previous enzyme-treatment, like trypsin, pancreatin, etc. (Elissondo et al., 2004; Smyth et al., 1967; Thompson and Lymbery, 1995). In our culture system, the PSC evagination was reached without any previous treatment. Since evagination was a non-
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synchronized process, we were able to record individual parasites at different times between the invaginated and evaginated PSC stages. It is probably a generalized fact among cestodes since it was also
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taurocholate (Marchiondo and Andersen, 1984).
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described for E. multilocularis, where the PSC were in vitro treated with trypsin, pancreatin and sodium
As we pointed out and others authors documented previously (Coutelen et al., 1952; Morseth, 1967), the invaginated PSC exhibits a subtegumentary musculature which is compatible with the different types of
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movements that the PSC display in a few hours-culture, particularly, the scolex protrusion and reinvagination. Regard the mesh of F-actin fibers that forms the tunnel and the orifice, their disposition
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suggests that it could also be contributing to the scolex evagination/invagination process. Since suckers
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move continuously, their muscles could also being involved to the evagination/invagination process. The Factin fibers at the rostellum area could be the hooks' supporting and could help in reorienting them once scolex has been evaginated, as well as they could also be relevant for the scolex exteriorization. Taken into account the F-actin distribution data in this stage, all the parasitic musculature could be involved in the evagination process. On the other hand, the evaginated PSC-main regions described by Galindo et al., 2008 (Galindo et al., 2008), are identified in our in vitro system. As well, the hooks organization is also consistent with the
literature (Antoniou and Tselentis, 1993). In addition to supporting the hooks, the rostellum muscles could cause the hooks extension-retraction movements, thus contributing to the helminth anchoring to the definitive host's gut. Likewise, the four suckers would also contribute to this fixation by suction. They have a set of both longitudinal and transverse muscle fibers as well as radial and circular ones, which is consistent with the vacuum generation by contraction and relaxation. Thus, in the definitive host, the scolex will keep the worm attached to the intestinal mucosa through the complete hook set and the four suckers (Antoniou and Tselentis, 1993). Morseth also described the mesh of F-actin fibers in the parasite’s neck that would be indicative of an important muscular system containing transverse and longitudinal
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fibers (Morseth, 1967). This musculature could be essential for the body to perform ascending, descending, pendular and rotational movements, which would be important during the parasite's fixation to the host's intestine (Galindo et al., 2008).
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The numerous calcareous corpuscles distributed in the body (Galindo et al., 2008) are mineralized structures which may be involved in the deposition of excess calcium, avoiding the parasite calcification
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(Chalar et al., 2016; Vargas-Parada and Laclette, 1999). They could also act as phosphate or carbonate
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deposits necessary to neutralize acids from its own metabolism and/or from the host’s stomach (Chalar et al., 2016; Vargas-Parada and Laclette, 1999). Another important body structure is the parasitic protonephridial system constituted by flame cells and collecting ducts (Galindo et al., 2008; Kutyrev et al.,
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2017; Morseth, 1967). This is an excretory system in phyla Platyhelminthes which is responsible for conserve water and eliminate salts in order to the parasite survives inside its hosts (Ambrosio et al., 2015,
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2014; Bahia et al., 2006; Galindo et al., 2008; Kutyrev et al., 2017). The cestode flame cells have at least
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three cytoskeletal proteins: actin, myosin and tubulin, all proteins associated with contractile movements in cells (Ambrosio et al., 2014; Kutyrev et al., 2017; Valverde-Islas et al., 2011), assembled as a protein complex responsible for the unique-shape to these cells (Ambrosio et al., 2015, 2014; Bahia et al., 2006; Koziol et al., 2011; Kutyrev et al., 2017; Rohde et al., 1992; Valverde-Islas et al., 2011; Wahlberg, 1998). The scolex evagination process for the closely related parasite, Echinococcus multilocuaris, has been detailed by light and scanning electron microscopy (Marchiondo and Andersen, 1984). Here, by light and fluorescence microscopy, we were able to record several of these moments for E. granulosus, although we think that our
parasites could be either in an evagination or invagination process rather than just evagination (see supplementary videos). We also put forward that the F-actin cytoskeleton could actively participate in both processes, scolex evagination and invagination, as a central structural element of the movement physiology in the PSC muscle fibers, as was suggested for other cestodes (Ambrosio et al., 2003; Reynoso-Ducoing et al., 2014; Wahlberg, 1998). Finally, several F-actin isoforms had been identified in E. granulosus (da Silva et al., 1993; Monteiro et al., 2010). With the advent of the genome projects, expression data for this parasite are available, for example, different-stage transcriptomes from E. granulosus were reported (Zheng et al., 2013). From this article, we
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underline that E. granulosus expresses six actin genes, where the expression values of some of them vary significantly according to the parasite stage. The data shown in Table 1 are consistent with the hypothesis that different actin organizations could be associated with the parasite development and/or the infection
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process as well as the host-parasite relationship. A PSC is an intermediate stage that can progress towards a hydatid cyst or an adult worm, depending on its environment, but in both cases, the PSC must have its
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scolex exposed. Therefore, we propose that the actin genes regulation should be occurring after the
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evagination step, although more studies are necessary.
In conclusion, our data describe the F-actin distribution contributing to the knowledge of the cytoskeleton of E. granulosus. The use of phalloidin-fluorescence and confocal microscopy as well as light microscopy
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allowed us to visualize in part the PSC morphology, identifying different structures and their distribution. The phalloidin labeling also allowed observing a highly organized cytoskeleton, whose filaments are
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distributed unevenly and arranged in different regions of the PSC. F-actin predominated in the suckers and
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rostellum regions, and seems to maintain the same fiber organization both the invaginated and the evaginated parasites. Similar organization of the actin filaments has been described in other parasite helminthes (Ambrosio et al., 2003; Bahia et al., 2006; Borges et al., 2017; Coutelen et al., 1952; Mair et al., 1998; Petrov and Podvyaznaya, 2016; Reynoso-Ducoing et al., 2014; Tansatit et al., 2006; Wahlberg, 1998). Note that in the present work we show parasites at different times in the evagination/invagination process that have not received any type of pre-treatment. The expression of E. granulosus F-actin genes seems to be modulated differently at each stage of its development. Therefore, if transcriptional modulation is also
confirmed at translational level would be supporting the relevance of the F-actin cytoskeleton as a possible therapeutic target. To achieve a better understanding of the E. granulosus cytoskeleton network during its life cycle and in its host interaction, the participation of microtubules as well as specific intermediate filaments should be considered in a future work. Finally, our evidence supports the idea that E. granulosus evaginated PSC retain the ability to internalize their scolex, at least in our working conditions.
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Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Acknowledgements
This work was supported by grants from Universidad de la Republica (CSIC-034 and -334, Uruguay) and
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PEDECIBA (Uruguay).
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Supplementary material.
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Supplementary videos related to this article can be found in supplemental files.
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Galindo, M., Schadebrodt, G., Galanti, N., 2008. Echinococcus granulosus: Cellular territories and morphological regions in mature protoscoleces. Exp. Parasitol. 119, 524–533. https://doi.org/10.1016/j.exppara.2008.04.013
Ingold, K., Bigler, P., Thormann, W., Cavaliero, T., Gottstein, B., Hemphill, A., 1999. Efficacies of Albendazole Sulfoxide and Albendazole Sulfone against In Vitro-Cultivated Echinococcus multilocularis Metacestodes. Antimicrob. Agents Chemother. 43, 1052–1061. Jenkins, D.J., Romig, T., Thompson, R.C.A., 2005. Emergence/re-emergence of Echinococcus spp.—a global
update. Int. J. Parasitol. 35, 1205–1219. https://doi.org/10.1016/j.ijpara.2005.07.014 Koziol, U., Costabile, A., Domínguez, M.F., Iriarte, A., Alvite, G., Kun, A.E., Castillo, E., 2011. Developmental expression of high molecular weight tropomyosin isoforms in Mesocestoides corti. Mol. Biochem. Parasitol. 175, 181–191. https://doi.org/10.1016/j.molbiopara.2010.11.009 Koziol, U., Jarero, F., Olson, P.D., Brehm, K., 2016. Comparative analysis of Wnt expression identifies a highly conserved developmental transition in flatworms. BMC Biol. 14, 1–16. https://doi.org/10.1186/s12915-016-0233-x Kun, A.E., Canclini, L., Rosso, G., Bresque, M., Romeo, C., Hanusz, A., Cal, K., Calliari, A., Sotelo Silveira, J.,
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Sotelo, J.R., 2012. F-actin distribution at nodes of Ranvier and Schmidt-Lanterman incisures in mammalian sciatic nerves. Cytoskeleton 69, 486–495. https://doi.org/10.1002/cm.21011
Kutyrev, I.A., Biserova, N.M., Olennikov, D.N., Korneva, J. V., Mazur, O.E., 2017. Prostaglandins E2 and D2–
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regulators of host immunity in the model parasite Diphyllobothrium dendriticum: An immunocytochemical and biochemical study. Mol. Biochem. Parasitol. 212, 33–45.
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Mair, G.R., Maule, A.G., Shaw, C., Johnston, C.F., Halton, D.W., 1998. Gross anatomy of the muscle systems of Fasciola hepatica as visualized by phalloidin-fluorescence and confocal microscopy. Parasitology 117, 75–82. https://doi.org/10.1017/S0031182098002807
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Marchiondo, A.A., Andersen, F.L., 1984. Light microscopy and scanning electron microscopy of the In vitro evagination process of Echinococcus multilocularis protoscolices. Int. J. Parasitol. 14, 151–157.
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Markoski, M.M., Trindade, E.S., Cabrera, G., Laschuk, A., Galanti, N., Zaha, A., Nader, H.B., Ferreira, H.B., 2006. Praziquantel and albendazole damaging action on in vitro developing Mesocestoides corti (Platyhelminthes: Cestoda). Parasitol. Int. 55, 51–61. https://doi.org/10.1016/j.parint.2005.09.005
Monteiro, K.M., De Carvalho, M.O., Zaha, A., Ferreira, H.B., 2010. Proteomic analysis of the Echinococcus granulosus metacestode during infection of its intermediate host. Proteomics 10, 1985–1999. https://doi.org/10.1002/pmic.200900506 Morseth, D.J., 1967. Fine Structure of the Hydatid Cyst and Protoscolex of Echinococcus granulosus. J.
Parasitol. 53, 312–325. Morseth, D.J., 1966. The Fine Structure of the Tegument of Adult Echinococcus granulosus, Taenia hydatigena, and Taenia pisiformis. J. Parasitol. 52, 1074–1085. Pérez-Serrano, J., Denegri, G., Casado, N., Bodega, G., Rodríguez-Caabeiro, F., 1995. Anti-tubulin immunohistochemistry study of Echinococcus granulosus protoscolices incubated with albendazole and albendazole sulphoxide in vitro. Parasitol. Res. 81, 438–440. https://doi.org/10.1007/BF00931507 Petrov, A., Podvyaznaya, I., 2016. Muscle architecture during the course of development of Diplostomum pseudospathaceum Niewiadomska, 1984 (Trematoda, Diplostomidae) from cercariae to
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metacercariae. J. Helminthol. 90, 321–336. https://doi.org/10.1017/S0022149X15000310
Reynoso-Ducoing, O., Valverde-Islas, L., Paredes-Salomon, C., Pérez-Reyes, A., Landa, A., Robert, L.,
Mendoza, G., Ambrosio, J.R., 2014. Analysis of the expression of cytoskeletal proteins of Taenia
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crassiceps ORF strain cysticerci (Cestoda). Parasitol. Res. 113, 1955–1969. https://doi.org/10.1007/s00436-014-3846-4
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Rohde, K., Watson, N.A., Roubal, F.R., 1992. Ultrastructure of the protonephridial system of Anoplodiscus
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cirrusspiralis (Monogenea Monopisthocotylea). Int. J. Parasitol. 22, 443–457. Smyth, J.D., Barrett, N.J., 1980. Procedures for testing the viability of human hydatid cysts following surgical removal, especially after chemotherapy. Trans. R. Soc. Trop. Med. Hyg. 74, 649–652.
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Smyth, J.D., Miller, J., Howkins, A.B., 1967. Further Analysis of the Factors Controlling and Maturation of
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Echinococcus Differentiation , in vitro. Exp. Parasitol. 21, 31–41.
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Tansatit, T., Sahaphong, S., Riengrojpitak, S., Viyanant, V., Sobhon, P., 2006. Immunolocalization of cytoskeletal components in the tegument of the 3-week-old juvenile and adult Fasciola gigantica. Vet. Parasitol. 135, 269–278. https://doi.org/10.1016/J.VETPAR.2005.10.018
Thompson, R., 2017. Biology and Systematics of Echinococcus. Adv. Parasitol. 95, 65–109. https://doi.org/10.1016/bs.apar.2016.07.001 Thompson, R.C.A., Lymbery, A.J., 1995. Echinococcus and hydatid disease. CAB International, Wallingford, Oxon, UK.
Valverde-Islas, L.E., Arrangoiz, E., Vega, E., Robert, L., Villanueva, R., Reynoso-Ducoing, O., Willms, K., Zepeda-Rodríguez, A., Fortoul, T.I., Ambrosio, J.R., 2011. Visualization and 3d reconstruction of flame cells of Taenia solium (Cestoda). PLoS One 6, e14754. https://doi.org/10.1371/journal.pone.0014754 Vargas-Parada, L., Laclette, J.P., 1999. Role of the calcareous corpuscles in cestode physiology: a review. Rev. Latinoam. Microbiol. 41, 303–307. Wahlberg, M.H., 1998. The distribution of F-actin during the development of Diphyllobothrium dendriticum (Cestoda). Cell Tissue Res. 291, 561–570. https://doi.org/10.1007/s004410051025 Zheng, H., Zhang, W., Zhang, L., Zhang, Z., Li, J., Lu, G., Zhu, Y., Wang, Y., Huang, Y., Liu, J., Kang, H., Chen, J.,
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Wang, L., Chen, A., Yu, S., Gao, Z., Jin, L., Gu, W., Wang, Z., Zhao, L., Shi, B., Wen, H., Lin, R., Jones, M.K., Brejova, B., Vinar, T., Zhao, G., McManus, D.P., Chen, Z., Zhou, Y., Wang, S., 2013. The genome of the hydatid tapeworm Echinococcus granulosus. Nat. Genet. 45, 1168–1175.
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https://doi.org/10.1038/ng.2757
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Figure 1 – Light micrographs of cultured E. granulosus PSC in different moments of evagination/invagination process. E. granulosus PSC were incubated in M-199 medium containing glucose and antibiotics at 37°C without any treatment. At 24 hours, they were recorded and the pictures were
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processed using ImageJ software. A-F: PSC in various steps of evagination/invagination process. A: invaginated and evaginated PSC coexisting. B-C: Invaginated PSC; D-E: PSC showing their rostellums and
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hooks inside but their suckers outside, as spherical dome-shaped bodies (D) and as cups ornaments (E). F:
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Four PSC with their scolex fully exposed (evaginated PSC). B: body; CC: calcareous corpuscles; H: hooks; N: neck; O: orifice; PAD: rostellar pad; R: rostellum; S: suckers; Sc: scolex; t: tunnel. Scale bar: indicate 20 µm
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Figure 2 – Distribution of F-actin in different moments of E. granulosus PSC evagination/invagination
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process. PSC cryosections were stained with Alexa Fluor 546-phalloidin probe and recorded in an Olympus FV300 or a Leica TCS SP5 II confocal microscope with or without digital zoom.
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A: Invaginated PSC showing the suckers (S) and the orifice (O). B: Invaginated PSC showing the suckers (S)
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along the evagination channel (t). C: Invaginated PSC showing the orifice (O) and an evaginated PSC showing the rostellum (R) and the four suckers (S). D: Invaginated PSC showing the tunnel (t) and the pad region (PAD) of the rostellum (R). E: Invaginated PSC where the rostellum (R) is at the middle of the PSC. F: PSC showing its rostellum inside but its spherical dome-shaped suckers outside. G: External cup-shaped suckers. H-I: Evaginated PSC. B: body; N: neck; O: orificie; PAD: rostellar pad; R: rostellum; S: suckers; Sc: scolex; t: tunnel; Arrowhead: “circular” actin organizations. Scale bar: indicate 20 µm
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Figure 3 – Actin organizations in PSC.
A – Hooks localizations in an evaginated PSC: Evaginated PSC cryosections were stained by Alexa Fluor
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546-phalloidin (a) and mounted in a medium containing the nuclear stain DAPI (ProLong Gold Antifade reagent, Invitrogen) (b) which specifically stained the cell nuclei and nonspecifically to the hooks. (c) Image superposition (merge). Images were recorded in an Olympus confocal microscope and were processed by ImageJ free software. B – Parasite “circular” F-actin organizations: Several “circular” F-actin structures in both invaginated (a) and evaginated (b) PSC are shown (arrowheads). Inset: higher magnification (200%) of “circular” actin organizations.
B: body; H: hooks (b: blade; g: guard; h: handle); N: neck; R: rostellum; S: suckers; Sc: scolex. Scale bar:
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indicate 20 µm.
Table 1 – Comparative profiles of F-actin genes transcription between adult-PSC and PSC-hydatid cyst wall (Cyst) of E. granulosus (data extracted from Zheng et al., 2012).
Table 1
PSC-Cyst log2(Fold_change) p-value normalized 1,2 1,5386E-10 ns -2,4 4,208E-208 ns ns
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Adult-PSC log2(Fold_change) p-value normalized EG_05821 ns EG_06859 -1,7 0,00423583 EG_08301 ns EG_08341 2,4 2,7865E-17 EG_09069 2,1 3,6752E-58 Note: (ns) No significant change
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Gene ID
S0001-706X(19)30116-0 https://doi.org/10.1016/j.actatropica.2019.105122 105122
Reference:
ACTROP 105122
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
25 January 2019 29 July 2019 1 August 2019
Please cite this article as: La-Rocca S, Farias J, Chalar C, Kun AE, Fernandez V, Echinococcus granulosus: Insights into the protoscolex F-actin cytoskeleton, Acta Tropica (2019), https://doi.org/10.1016/j.actatropica.2019.105122 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Echinococcus granulosus: Insights into the protoscolex F-actin cytoskeleton. Silvana La-Roccaa, Joaquina Fariasb, Cora Chalarc, Alejandra E. Kunb,c, Veronica Fernandeza. a
Area Inmunología, Departamento de Biociencias, Facultad de Química, Universidad de la República,
Montevideo, Uruguay. b
Department of Proteins and Nucleic Acids, Instituto de Investigaciones Biológicas Clemente Estable,
Montevideo, Uruguay. C
Sección Bioquímica, Departamento de Biología celular y Molecular, Facultad de Ciencias, Universidad de la
República.
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Corresponding author:
Verónica FERNANDEZ MANCEBO, Laboratorio de Inmunología, Av. Alfredo Navarro 3051, piso 2;
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Montevideo, CP 11600; Uruguay. E-mail: [email protected]
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Graphical abstract
PSC has a highly organized F-actin cytoskeleton with unevenly distributed filaments
F-actin prevailed in the suckers and rostellum in both evaginated and invaginated PSC
E. granulosus evaginated PSC seem to retain their ability to reinvaginate in vitro
F-actin cytoskeleton seems to keep constant in the evagination/invagination process
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Abstract Echinococcus granulosus is a cestode parasite whose cytoskeleton plasticity allows it to enter and develop inside its hosts, completing thus its life cycle. We focused our attention on F-actin organization and distribution in E. granulosus protoscoleces (PSC) in order to contribute to the knowledge of the parasite cytoskeleton. In particular, we addressed some aspects of F-actin rearrangements in PSC at different stages of the evagination/invagination process. The use of light microscopy allowed us to identify different PSC structures and phalloidin staining displayed a parasite's highly organized F-actin cytoskeleton. Suckers exhibit an important musculature composed of a set of radial fibers. At the rostellum, the F-actin filaments
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are arranged in a bulbar shape with perforations that appear to be the attachment places for the hooks. Also, "circular" structures of F-actin were identified, which remind the flame cells. Furthermore, parasite Factin filaments, unevenly distributed, seem to have remained substantially unchanged during the
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evagination/invagination process. Finally, we showed that the scolex of an evaginated E. granulosus PSC
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reinvaginates in vitro without any treatment.
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Abbreviations
PSC: E. granulosus protoscoleces; PHEM buffer: 25 mM HEPES, 10 mM EGTA, 60 mM PIPES, 2 mM MgCl2
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(pH 7,3); RT: room temperature
Keywords
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Echinococcus granulosus; invagination/evagination process; protoscoleces; F-Actin cytoskeleton.
1.
Introduction
The cestode Echinococcus granulosus, like all parasites, presents a complex life cycle. It requires two mammalian hosts: a definitive host (always carnivore) in which the adult worm (strobilar stage) develops in the small intestine, and an intermediate host (for example, livestock or human) in which the cystic metacestode (larval stage) usually develops in the viscera (Agudelo Higuita et al., 2016; Jenkins et al., 2005; Thompson and Lymbery, 1995). The metacestode is a fluid-filled cystic structure that undergoes asexual multiplication to produce large numbers of scolices, termed protoscoleces (PSC) (Thompson, 2017). The
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PSC, which are juvenile scoleces, is the anterior end of the adult tapeworm (Koziol et al., 2016), carrying suckers and hooks by which it adheres to a host (Thompson and Lymbery, 1995). In the cyst, the apical region of the protoscolex (scolex) is invaginated within the mucopolysaccharide-coated basal region of the
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protoscolex tegument (invaginated PSC) (Thompson, 2017). When PSC are ingested by the definitive host, they mature towards the strobilar stage after an evagination process, in which the scolex is exposed
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outwardly (evaginated PSC) (Thompson, 2017). Then, in the intestine, each attached-parasite lengthens its
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body which becomes divided into three segments called proglottids. The last proglottid, containing embryonated eggs, detaches and, together with the host's feces, reaches the outside where the eggs are released (Thompson, 2017). The parasitic life cycle is completed when the eggs are ingested by
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intermediate hosts and the released oncospheres develop into PSC-containing hydatid cysts in their viscera (Jenkins et al., 2005; Thompson, 2017). On the other hand, PSC have also the ability to differentiate into
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fully developed cysts when they are released into a cavity of the intermediate host, for example, upon
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metacestode rupture (Thompson, 2017). For this transformation, the PSC must have their scolex exposed as well (Elissondo et al., 2004). Therefore, the scolex evagination step is a requirement for the PSC to differentiate into hydatid cysts (Elissondo et al., 2004) or into adult worms (Smyth et al., 1967). In the first half of the 20th century, a growing number of descriptions about the most distinctive characteristics of E. granulosus and its life cycle have been published where some structures were identified and their possible functions were speculated (reviewed by Thompson and Lymbery, 1995). Particularly, the PSC exhibit a protective covering (tegument) supported by a broad fibrous zone crossed by
muscle bundles arranged into two perpendicular muscle fibers layers, one circular and the other transversal (Morseth, 1967, 1966). Coutelen et al. (1952) (Coutelen et al., 1952) classify the musculature as a subcuticular set, a suckers-associated set, a rostellum-associated set and a deeper fibers set, although they suggest that the whole musculature would be associated with the movements of evagination and invagination. It has also been suggested, in Taenia solium, that this muscle system, with high F-actin expression, could define the movement direction and the required force in response to different physiological situations, such as scolex attachment in the definitive host gut (Ambrosio et al., 2003). The plasticity of the cytoskeleton has been acquired by parasite helminths as one of the strategies that
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allow them to enter, settle and develop in their hosts, thus completing their life cycle. Due to this, the cytoskeleton components have been proposed as target for therapeutic drugs and, in fact, the helminth cytoskeleton organization is affected for some anthelmintic drug (Conder et al., 1981; Cumino et al., 2009;
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Ingold et al., 1999; Markoski et al., 2006; Pérez-Serrano et al., 1995). Confocal laser scanning microscopy combined with phalloidin staining, which marks F-actin in muscle cells with high specificity, allows muscle
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systems to be viewed accurately in 3D for analysis. Recently, the three-dimensional muscular arrangement
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of helminthes has emerged as a useful morphological tool for taxonomic and phylogenetic analyzes (Adami et al., 2017; Petrov and Podvyaznaya, 2016). While this type of phylogeny is not yet available in Platyhelminthes, it seems to be a promising approach (Adami et al., 2017). For example, using this
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methodology, differences between three species of freshwater Macrostomum were observed, although the three shared a general muscular pattern (Adami et al., 2017). Here we focus our attention on the F-actin
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distribution in E. granulosus in order to contribute to the knowledge of its cytoskeleton. In particular, we
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address some morphological aspects of PSC by optical and fluorescence microscopy. The alterations in the F-actin distribution that accompany the evagination/invagination process allow us to infer the behavior of the cytoskeleton in such process.
2.
Materials and methods
2.1.
Parasite cultures
Hydatid cysts were collected during the routine work of local abattoirs in Montevideo (Uruguay) from the lungs of naturally infected bovines. E. granulosus PSC were recovered from fertile cysts according to Baz et al. (Baz et al., 1995) and the viability was determined by eosin-exclusion method (Smyth and Barrett, 1980). Only PSC batches with over 95% viability were used. After washing with PBS, the PSC were incubated in Medium-199 with Earle's salts (M-199, GIBCO) supplemented with antibiotics (penicillin, streptomycin and gentamicin at 100 U/mL, 100 μg/mL and 30 μg/mL, respectively) containing 4 mg/mL glucose, at 37° and 5% CO2. The cultured PSC were examined by light microscopy using inverted microcopies (Nikon Eclipse Ti
processed by the softwares ImageJ or ZEN Version 2.0, respectively). 2.2.
Sample processing and phalloidin staining
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coupled to a Nikon Digital Sight DS-QiMc or Carl Zeiss coupled to AxioCam ERc5s and the images were
The samples were processed according to Kun et al., 2012 (Kun et al., 2012), with some modifications.
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Briefly, cultured PSC were fixed for 1 h at 4°C, in 3% w/v paraformaldehyde in PHEM (25 mM HEPES, 10 mM EGTA, 60 mM PIPES, 2 mM MgCl2, pH 7,3). Then, the samples were cryoprotected until PSC fall down to the
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bottom of the tube. Samples infiltration was done through progressive substitution (25%, 50%, 100%) of
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sucrose/PHEM by Tissue Tek (Sakura Finetek USA) at 4°C with stirring. Subsequently, the PSC were quickly frozen and cryosectioned at -20°C (10 µm) using a Cryostat (SLEE), adhered onto poly-D/L-lysine precoated slides and kept at -20ºC until use. To evidence filamentous actin (F-actin), the permeabilized cryosections
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were blocked using 1% w/v sodium borohydride in PHEM for 10 min at room temperature (RT). After washing with PHEM, the slides were incubated with Alexa Fluor 546-phalloidin (Invitrogen), in incubation
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buffer (0,1% w/v BSA, 75mM lysine, in PHEM), for 45 minutes, in darkness, at RT. Next, the slides were
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rinsed in PHEM, mounted in ProLong Gold Antifade Mountant (Thermo Fisher Scientific) and were kept in the darkness, at RT, 24 hours, and then at 4ºC until observation for confocal analyze. Images were collected with an Olympus FV300 or a Leica TCS SP5 II confocal microscope using a Plan Apo 60X/1.42 NA, a Plan Apo 63X/1.4 NA or a Plan Apo 100X/1.4 NA oil immersion objective, with or without digital zoom. Image-J free software was used for image processing (including brightness/contrast adjustment and Gaussian blur filtering). 3.
Results
We achieve evaginated parasites in vitro culturing fresh E. granulosus PSC without any previous treatment in M-199 supplemented with glucose and antibiotics. At the beginning, the invaginated PSC remain almost immobile, but a few hours later they become very active. They perform different types of movements where some PSC externalize and re-internalize their scolex in an asynchronous way (see supplementary material). Fig. 1 and 2 show PSC after 24 h of in vitro culture, where both invaginated and evaginated parasites co-exist. We recognized the parasite scolex (Sc) containing the attaching structures to the definitive host gut: the suckers (S) and the hooks (H) joined to the rostellum (R). The invaginated PSC show the classical spherical or oval shape (Fig. 1A-C); inside them, the subtegumentary muscle-fibers extend
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throughout the organism where longitudinal and transversal fibers are distinguished (Fig. 2A, D and E). The tunnel (t), generated by the introversion of the PSC apical portion, extends from the muscular pad (hook region) to the orifice (O) at the anterior region (Fig. 1A-C and 2A-D). A mesh of F-actin fibers, similar to the
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rest of the organism, forms the tunnel (t) and the orifice (O) (Fig. 2A-D). The suckers (S), located along the tunnel (t) (Fig. 1 and 2), exhibit an important musculature composed of a set of radial fibers (Fig. 2A, B, D
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and E). The hooks (H) are oriented with the blades towards the anterior region, whereas the structures
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through which they are anchored to the rostellum, handles and guards, are directed towards the posterior and lateral region, respectively (Fig. 1A, B and C). At the rostellum (R) area, a mesh of F-actin fibers is observed (Fig. 2D) and immediately below this mesh, a group of F-actin filaments in different directions
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could also belong to the rostellum (Fig. 2D and E). On the other hand, in the evaginated PSC stands out the scolex (Sc), the neck (N) and the body (B), as it is possible to see in Fig. 1A, F and 2H-I. In this stage, the
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hooks (H) remain anchored to the rostellum (R) but their blades now look towards the back of the parasite
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(Fig. 1A, F and 3A). The hooks are organized in two crowns where the upper row contains the largest hooks (Fig. 3A). They are in a region with abundant muscle fibers: the rostellum (R) (Fig. 3A) which would be acting as support. The four suckers (S) (Fig. 1A and F) have a set of both longitudinal and transverse muscle fibers as well as radial and circular ones. As we expected, that the suckers (S) and rostellum (R) maintain the same organization of F-actin fibers observed in the invaginated parasite (Fig. 2). Next to the scolex, the evaginated PSC show a narrowing that constitutes the neck (N) (Fig. 1A, F, 2H-I and 3A) which connects the scolex with the body. The parasite's neck (N) exhibits a mesh of actin F fibers (Fig. 2H-I and 3A) indicating an
important muscular system with transverse and longitudinal muscle fibers. The body (B), which represents approximately 50-60% of the total PSC length (Fig. 1A, F and 2H-I), is formed by different cell types and structures. For instance, it contains numerous calcareous corpuscles (Fig. 1) and the cells that form them. Another important structure is the parasitic protonephridial system constituted by the flame cells and the collecting ducts. It is necessary to point out that both evaginated and invaginated parasites display several “circular” F-actin organizations that resemble flame cells (Fig. 2 and 3B, arrowhead). Moreover, along the anterior-posterior axis of the evaginated PSC, it is possible to clearly visualize some structures that resemble the collecting ducts (Fig. 2 H and I). These structures can also be observed in some invaginated
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PSC (Fig. 3Ba), although possibly in fully invaginated ones they could be masked or displaced by the apical region.
For the scolex evagination process since each PSC progressed independently, we were able to record
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several of such moments by light and fluorescence microscopy (Fig. 1 and 2). In Fig. 1A-C and 2A-D the orifice and the tunnel through which the scolex is exteriorized are shown. Fig. 2E shows a PSC whose
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rostellum is directed towards the anterior region compressing the suckers. In Fig. 1D-E and 2F-G the
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emerging suckers, first as spherical dome-shaped bodies and then as cups are shown. The hooks, anchored to rostellum, maintain their original disposition with the blades pointing towards the orifice (Fig. 1D-E). Once outside, they are organized in two crowns where the blades now look towards the PSC body (Fig. 1A,
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F and 3A). The hook crowns are anchored between the F-actin filaments (Fig. 3A) and they reach their final arrangement when the rostellum assumes a bulbar shape (Fig. 3A) and the apical dome (rostellar pad)
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appears (Fig. 1A and F). No substantial changes in the F-actin filaments organization of rostellum and
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suckers have been perceived during scolex evagination steps (Fig. 2 and 3A). Additionally, our evidence indicates that E. granulosus PSC retain the ability to reinvaginate under our in vitro culture conditions. In this sense, we provide some movies that support this statement (Supplementary video A.1 shows at least one re-invaginating PSC, supplementary video A.2 shows a protoscolex that rearranges its hooks and internalizes its rostellum, and supplementary video A.3 shows a protoscolex entering its suckers, ending the reinvagination process at 4, 24 and 16 hour-cultures, respectively).
Through in silico comparative E. granulosus transcriptomics, we study the differential profiles of F-actin gene expression between adult-PSC and PSC-hydatid cyst wall. The result is presented in table 1 where we highlight the expression of six actin genes from E. granulosus (EGR_05821, EGR_06859, EGR_08301, EGR_08341, EGR_09069 and EGR_10650). As it can see, the expression values of some of them vary significantly among parasite stages. 4.
Discussion
The PSC physiological evagination process is controlled by a diversity of factors which are not known exactly. Although the in vitro conditions are probably far from those that exist inside the host, evaginated
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parasites have been obtained by culturing PSC with or without previous enzyme-treatment, like trypsin, pancreatin, etc. (Elissondo et al., 2004; Smyth et al., 1967; Thompson and Lymbery, 1995). In our culture system, the PSC evagination was reached without any previous treatment. Since evagination was a non-
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synchronized process, we were able to record individual parasites at different times between the invaginated and evaginated PSC stages. It is probably a generalized fact among cestodes since it was also
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taurocholate (Marchiondo and Andersen, 1984).
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described for E. multilocularis, where the PSC were in vitro treated with trypsin, pancreatin and sodium
As we pointed out and others authors documented previously (Coutelen et al., 1952; Morseth, 1967), the invaginated PSC exhibits a subtegumentary musculature which is compatible with the different types of
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movements that the PSC display in a few hours-culture, particularly, the scolex protrusion and reinvagination. Regard the mesh of F-actin fibers that forms the tunnel and the orifice, their disposition
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suggests that it could also be contributing to the scolex evagination/invagination process. Since suckers
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move continuously, their muscles could also being involved to the evagination/invagination process. The Factin fibers at the rostellum area could be the hooks' supporting and could help in reorienting them once scolex has been evaginated, as well as they could also be relevant for the scolex exteriorization. Taken into account the F-actin distribution data in this stage, all the parasitic musculature could be involved in the evagination process. On the other hand, the evaginated PSC-main regions described by Galindo et al., 2008 (Galindo et al., 2008), are identified in our in vitro system. As well, the hooks organization is also consistent with the
literature (Antoniou and Tselentis, 1993). In addition to supporting the hooks, the rostellum muscles could cause the hooks extension-retraction movements, thus contributing to the helminth anchoring to the definitive host's gut. Likewise, the four suckers would also contribute to this fixation by suction. They have a set of both longitudinal and transverse muscle fibers as well as radial and circular ones, which is consistent with the vacuum generation by contraction and relaxation. Thus, in the definitive host, the scolex will keep the worm attached to the intestinal mucosa through the complete hook set and the four suckers (Antoniou and Tselentis, 1993). Morseth also described the mesh of F-actin fibers in the parasite’s neck that would be indicative of an important muscular system containing transverse and longitudinal
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fibers (Morseth, 1967). This musculature could be essential for the body to perform ascending, descending, pendular and rotational movements, which would be important during the parasite's fixation to the host's intestine (Galindo et al., 2008).
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The numerous calcareous corpuscles distributed in the body (Galindo et al., 2008) are mineralized structures which may be involved in the deposition of excess calcium, avoiding the parasite calcification
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(Chalar et al., 2016; Vargas-Parada and Laclette, 1999). They could also act as phosphate or carbonate
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deposits necessary to neutralize acids from its own metabolism and/or from the host’s stomach (Chalar et al., 2016; Vargas-Parada and Laclette, 1999). Another important body structure is the parasitic protonephridial system constituted by flame cells and collecting ducts (Galindo et al., 2008; Kutyrev et al.,
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2017; Morseth, 1967). This is an excretory system in phyla Platyhelminthes which is responsible for conserve water and eliminate salts in order to the parasite survives inside its hosts (Ambrosio et al., 2015,
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2014; Bahia et al., 2006; Galindo et al., 2008; Kutyrev et al., 2017). The cestode flame cells have at least
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three cytoskeletal proteins: actin, myosin and tubulin, all proteins associated with contractile movements in cells (Ambrosio et al., 2014; Kutyrev et al., 2017; Valverde-Islas et al., 2011), assembled as a protein complex responsible for the unique-shape to these cells (Ambrosio et al., 2015, 2014; Bahia et al., 2006; Koziol et al., 2011; Kutyrev et al., 2017; Rohde et al., 1992; Valverde-Islas et al., 2011; Wahlberg, 1998). The scolex evagination process for the closely related parasite, Echinococcus multilocuaris, has been detailed by light and scanning electron microscopy (Marchiondo and Andersen, 1984). Here, by light and fluorescence microscopy, we were able to record several of these moments for E. granulosus, although we think that our
parasites could be either in an evagination or invagination process rather than just evagination (see supplementary videos). We also put forward that the F-actin cytoskeleton could actively participate in both processes, scolex evagination and invagination, as a central structural element of the movement physiology in the PSC muscle fibers, as was suggested for other cestodes (Ambrosio et al., 2003; Reynoso-Ducoing et al., 2014; Wahlberg, 1998). Finally, several F-actin isoforms had been identified in E. granulosus (da Silva et al., 1993; Monteiro et al., 2010). With the advent of the genome projects, expression data for this parasite are available, for example, different-stage transcriptomes from E. granulosus were reported (Zheng et al., 2013). From this article, we
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underline that E. granulosus expresses six actin genes, where the expression values of some of them vary significantly according to the parasite stage. The data shown in Table 1 are consistent with the hypothesis that different actin organizations could be associated with the parasite development and/or the infection
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process as well as the host-parasite relationship. A PSC is an intermediate stage that can progress towards a hydatid cyst or an adult worm, depending on its environment, but in both cases, the PSC must have its
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scolex exposed. Therefore, we propose that the actin genes regulation should be occurring after the
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evagination step, although more studies are necessary.
In conclusion, our data describe the F-actin distribution contributing to the knowledge of the cytoskeleton of E. granulosus. The use of phalloidin-fluorescence and confocal microscopy as well as light microscopy
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allowed us to visualize in part the PSC morphology, identifying different structures and their distribution. The phalloidin labeling also allowed observing a highly organized cytoskeleton, whose filaments are
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distributed unevenly and arranged in different regions of the PSC. F-actin predominated in the suckers and
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rostellum regions, and seems to maintain the same fiber organization both the invaginated and the evaginated parasites. Similar organization of the actin filaments has been described in other parasite helminthes (Ambrosio et al., 2003; Bahia et al., 2006; Borges et al., 2017; Coutelen et al., 1952; Mair et al., 1998; Petrov and Podvyaznaya, 2016; Reynoso-Ducoing et al., 2014; Tansatit et al., 2006; Wahlberg, 1998). Note that in the present work we show parasites at different times in the evagination/invagination process that have not received any type of pre-treatment. The expression of E. granulosus F-actin genes seems to be modulated differently at each stage of its development. Therefore, if transcriptional modulation is also
confirmed at translational level would be supporting the relevance of the F-actin cytoskeleton as a possible therapeutic target. To achieve a better understanding of the E. granulosus cytoskeleton network during its life cycle and in its host interaction, the participation of microtubules as well as specific intermediate filaments should be considered in a future work. Finally, our evidence supports the idea that E. granulosus evaginated PSC retain the ability to internalize their scolex, at least in our working conditions.
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Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Acknowledgements
This work was supported by grants from Universidad de la Republica (CSIC-034 and -334, Uruguay) and
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PEDECIBA (Uruguay).
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Supplementary material.
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Supplementary videos related to this article can be found in supplemental files.
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Figure 1 – Light micrographs of cultured E. granulosus PSC in different moments of evagination/invagination process. E. granulosus PSC were incubated in M-199 medium containing glucose and antibiotics at 37°C without any treatment. At 24 hours, they were recorded and the pictures were
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processed using ImageJ software. A-F: PSC in various steps of evagination/invagination process. A: invaginated and evaginated PSC coexisting. B-C: Invaginated PSC; D-E: PSC showing their rostellums and
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hooks inside but their suckers outside, as spherical dome-shaped bodies (D) and as cups ornaments (E). F:
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Four PSC with their scolex fully exposed (evaginated PSC). B: body; CC: calcareous corpuscles; H: hooks; N: neck; O: orifice; PAD: rostellar pad; R: rostellum; S: suckers; Sc: scolex; t: tunnel. Scale bar: indicate 20 µm
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Figure 2 – Distribution of F-actin in different moments of E. granulosus PSC evagination/invagination
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process. PSC cryosections were stained with Alexa Fluor 546-phalloidin probe and recorded in an Olympus FV300 or a Leica TCS SP5 II confocal microscope with or without digital zoom.
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A: Invaginated PSC showing the suckers (S) and the orifice (O). B: Invaginated PSC showing the suckers (S)
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along the evagination channel (t). C: Invaginated PSC showing the orifice (O) and an evaginated PSC showing the rostellum (R) and the four suckers (S). D: Invaginated PSC showing the tunnel (t) and the pad region (PAD) of the rostellum (R). E: Invaginated PSC where the rostellum (R) is at the middle of the PSC. F: PSC showing its rostellum inside but its spherical dome-shaped suckers outside. G: External cup-shaped suckers. H-I: Evaginated PSC. B: body; N: neck; O: orificie; PAD: rostellar pad; R: rostellum; S: suckers; Sc: scolex; t: tunnel; Arrowhead: “circular” actin organizations. Scale bar: indicate 20 µm
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Figure 3 – Actin organizations in PSC.
A – Hooks localizations in an evaginated PSC: Evaginated PSC cryosections were stained by Alexa Fluor
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546-phalloidin (a) and mounted in a medium containing the nuclear stain DAPI (ProLong Gold Antifade reagent, Invitrogen) (b) which specifically stained the cell nuclei and nonspecifically to the hooks. (c) Image superposition (merge). Images were recorded in an Olympus confocal microscope and were processed by ImageJ free software. B – Parasite “circular” F-actin organizations: Several “circular” F-actin structures in both invaginated (a) and evaginated (b) PSC are shown (arrowheads). Inset: higher magnification (200%) of “circular” actin organizations.
B: body; H: hooks (b: blade; g: guard; h: handle); N: neck; R: rostellum; S: suckers; Sc: scolex. Scale bar:
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indicate 20 µm.
Table 1 – Comparative profiles of F-actin genes transcription between adult-PSC and PSC-hydatid cyst wall (Cyst) of E. granulosus (data extracted from Zheng et al., 2012).
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PSC-Cyst log2(Fold_change) p-value normalized 1,2 1,5386E-10 ns -2,4 4,208E-208 ns ns
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Adult-PSC log2(Fold_change) p-value normalized EG_05821 ns EG_06859 -1,7 0,00423583 EG_08301 ns EG_08341 2,4 2,7865E-17 EG_09069 2,1 3,6752E-58 Note: (ns) No significant change
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Gene ID