RESUMEN
Tissue clearing is an old-fashioned method developed in the 1900's and used to turn an opaque biological object into a 3D visualizable transparent structure. Developed and diversified over the last decade, this method is most of the time applied to mammals' tissues, and especially mouse and human tissues for cytological, histological and pathophysiological studies. Through autofluorescence, immunofluorescence, in situ hybridization, intercalating agents, fluorescent transfection markers or fluorescent particle uptake, optically cleared samples can be monitored to discover new biological structures and cellular interactions through 3D-visualization, which can be more challenging in some extend through classical histological methods. Most of the tissue clearing procedures have been developed for specific applications like endogenous fluorescence visualization, immunolabeling or for revealing specific organs. Thus, choosing the adapted protocol may be empirical for non-model species, especially for mollusks for which very little related literature is available. Herein, we suggest an effective optical tissue clearing procedure for the freshwater snail Biomphalaria glabrata, known as the intermediate host of the human parasite Schistosoma mansoni. This clearing procedure involves solvents with a minimal toxicity, preserves the endogenous fluorescence of labeled parasites inside snail tissues and is compatible with an immunolabeling procedure.
Asunto(s)
Biomphalaria , Schistosoma mansoni , Animales , Biomphalaria/parasitología , FluorescenciaRESUMEN
Schistosomiasis is considered as a significant public health problem, imposing a deeper understanding of the intricate interplay between parasites and their hosts. Unfortunately, current invasive methodologies employed to study the compatibility and the parasite development impose limitations on exploring diverse strains under various environmental conditions, thereby impeding progress in the field. In this study, we demonstrate the usefulness for the trematode parasite Schistosma mansoni, leveranging a fluorescence-imaging-based approach that employs fluorescein 5-chloromethylfluorescein diacetate (CMFDA) and 5-chloromethylfluorescein diacetate (CMAC) as organism tracker for intramolluscan studies involving the host snail Biomphalaria glabrata. These probes represent key tools for qualitatively assessing snail infections with unmatched accuracy and precision. By monitoring the fluorescence of parasites within the snail vector, our method exposes an unprecedented glimpse into the host-parasite compatibility landscape. The simplicity and sensitivity of our approach render it an ideal choice for evolutionary studies, as it sheds light on the intricate mechanisms governing host-parasite interactions. Fluorescent probe-based methods play a pivotal role in characterizing factors influencing parasite development and phenotype of compatibility, paving the way for innovative, effective, and sustainable solutions to enhance our understanding host-parasite immunobiological interaction and compatibility.
Asunto(s)
Biomphalaria , Parásitos , Animales , Schistosoma mansoni/genética , Biomphalaria/parasitología , Caracoles , FenotipoRESUMEN
The transformation of Schistosoma mansoni miracidia into mother sporocysts is induced, either in vivo by the penetration of the free-living larval stage, the miracidium, in the snail Biomphalaria glabrata or in vitro following the incubation of the miracidium in Chernin's Balanced Salt Solution (CBSS) or Bge (B. glabrata embryonic cell line) culture medium. The in vitro development of S. mansoni miracidium into mother sporocyst was monitored by Scanning Electron Microscopy (SEM) from 2.5 h to 120 h in CBSS. The transformation starts when the miracidium ciliate plates detach due to the proliferation of the intercellular ridge associated with the degeneration of mid-body papillae of the miracidium. The loss of ciliated plates causes the appearing of scars, filled across time by the proliferation of a new tegument originating from the interplate ridge. This new tegument covers the entire body of the metamorphosing parasite and differentiates over time, allowing some exchanges (uptakes or secretion/excretion) between the parasite and its host. In contrast to the well-described development of adult and free-living larval stages of S. mansoni using SEM, the developmental transformation of intramolluscan stages, especially tegumental changes in the mother sporocyst, has been sparcely documented at the ultrastructural level. In addition, taking into account the latest literature on miracidium electron microscopy and the advances in SEM technologies over the last thirty years, the present study gathers three main objectives: (i) Fill the gap of tegument scanning electron micrographs of in vitro transforming sporocysts; (ii) Update the current bibliographic miracidia and sporocysts image bank due to rapid evolution of SEM technology; (iii) Understand and describe the critical steps and duration of the in vitro miracidium-to-sporocyst transformation process to assist in understanding the interaction between the larval surface and snail immune factors.
Asunto(s)
Biomphalaria , Parásitos , Animales , Femenino , Humanos , Schistosoma mansoni , Oocistos , Factores de Tiempo , Madres , Biomphalaria/parasitología , LarvaRESUMEN
Aerolysins initially characterized as virulence factors in bacteria are increasingly found in massive genome and transcriptome sequencing data from metazoans. Horizontal gene transfer has been demonstrated as the main way of aerolysin-related toxins acquisition in metazoans. However, only few studies have focused on their potential biological functions in such organisms. Herein, we present an extensive characterization of a multigene family encoding aerolysins - named biomphalysin - in Biomphalaria glabrata snail, the intermediate host of the trematode Schistosoma mansoni. Our results highlight that duplication and domestication of an acquired bacterial toxin gene in the snail genome result in the acquisition of a novel and diversified toxin family. Twenty-three biomphalysin genes were identified. All are expressed and exhibited a tissue-specific expression pattern. An in silico structural analysis was performed to highlight the central role played by two distinct domains i) a large lobe involved in the lytic function of these snail toxins which constrained their evolution and ii) a small lobe which is structurally variable between biomphalysin toxins and that matched to various functional domains involved in moiety recognition of targets cells. A functional approach suggests that the repertoire of biomphalysins that bind to pathogens, depends on the type of pathogen encountered. These results underline a neo-and sub-functionalization of the biomphalysin toxins, which have the potential to increase the range of effectors in the snail's immune arsenal.