Strobilar Development Research Articles

Genome-wide transcriptome analysis of the early developmental stages of Echinococcus granulosus protoscoleces reveals extensive alternative splicing events in the spliceosome pathway

BackgroundThe complex life cycle of Echinococcus granulosus involves numerous environmental conditions within different intermediate and definitive hosts. This requires adaptation at different levels of transcript regulation. Alternative splicing (AS) and the related cellular functions as one of the major fields of post-genomics has been poorly studied in tapeworms. In the present study, we investigated AS events and their potential biological effects in E. granulosus.MethodsWhole transcriptome sequencing data of four groups of protoscoleces were prepared for RNA-seq library construction. Fresh protoscoleces were either used as non-induced controls (NT group) or incubated for 15 min with pepsin (PEP group) and cultivated in a biphasic medium for 12 and 24 h (12 and 24 h groups). The frequency and different types of AS events were identified using rMATS software. Functional annotations and gene ontology of differential AS (DAS) genes were performed using Blast2GO software. AS events were experimentally validated by PCR on the protoscolex cDNAs using specific primers for each gene.ResultsAt least one AS event was found in 38.1% of the genes (3904 out of 10,245) in the protoscoleces during early strobilar development. The genes were associated primarily with cellular and metabolic processes and binding and catalytic activity. KEGG pathway analysis of DAS events revealed a number of genes belonging to different components of the spliceosome complex. These genes tended to belong to common SR proteins, U1-related factors, U2-related factors, complex A-specific factors and other splicing-related proteins.ConclusionsThe high number of AS events in the transcriptome regulatory mechanisms indicates the essential rapid molecular processes required by the parasite for adaptation in different environments.Graphical

Transcriptomic Analysis of the Early Strobilar Development of Echinococcus granulosus.

Echinococcus granulosus has a complex life cycle involving two mammalian hosts. The transition from one host to another is accompanied by changes in gene expression, and the transcriptional events that underlie this transition have not yet been fully characterized. In this study, RNA-seq was used to compare the transcription profiles of samples from E. granulosus protoscoleces induced in vitro to strobilar development at three time points. We identified 818 differentially expressed genes, which were divided into eight expression clusters formed over the entire 24 h period. An enrichment of gene transcripts with molecular functions of signal transduction, enzymes, and protein modifications was observed upon induction and developmental progression. This transcriptomic study provides insights for understanding the complex life cycle of E. granulosus and contributes for searching for the key genes correlating with the strobilar development, which can be used to identify potential candidates for the development of anthelmintic drugs.

Spatio-temporal expression of Mesocestoides corti McVAL2 during strobilar development

VAL proteins belong to a diverse superfamily containing the CAP domain, with members described for various eukaryotic organisms, including parasites. They are implicated in diverse biological activities and, as secreted proteins, may be related in host – parasite interactions. For this reason they have been proposed as vaccine candidates against nematode infections. However, little is known about their function in cestodes.In M. corti, four partial cDNA sequences coding for members of the CAP superfamily were previously isolated. In this work we characterize the expression of McVAL2 in the larvae and segmented worms of M. corti, describing mRNA and protein localization using fluorescent microscopy. We also optimized real time PCR analysis for quantification of mRNA expression through the different stages of strobilar development. We show that McVAL2 is differentially located, depending on the developmental stage, and can be used as a molecular marker for the neuroendocrine system in the larvae.The dynamic and stage-specific expression patterns of McVAL2, combined with the large number of VAL proteins found in the genomes of parasitic flatworms, suggest varied roles for the VAL protein family in the biology of these parasites.

Identification of Newly Synthesized Proteins by Echinococcus granulosus Protoscoleces upon Induction of Strobilation.

BackgroundThe proteins responsible for the key molecular events leading to the structural changes between the developmental stages of Echinococcus granulosus remain unknown. In this work, azidohomoalanine (AHA)-specific labeling was used to identify proteins expressed by E. granulosus protoscoleces (PSCs) upon the induction of strobilar development.Methodology/Principal FindingsThe in vitro incorporation of AHA with different tags into newly synthesized proteins (NSPs) by PSCs was analyzed using SDS-PAGE and confocal microscopy. The LC-MS/MS analysis of AHA-labeled NSPs by PSCs undergoing strobilation allowed for the identification of 365 proteins, of which 75 were differentially expressed in comparison between the presence or absence of strobilation stimuli and 51 were expressed exclusively in either condition. These proteins were mainly involved in metabolic, regulatory and signaling processes.Conclusions/SignificanceAfter the controlled-labeling of proteins during the induction of strobilar development, we identified modifications in protein expression. The changes in the metabolism and the activation of control and signaling pathways may be important for the correct parasite development and be target for further studies.

Tamoxifen Treatment in Hamsters Induces Protection during Taeniosis byTaenia solium

Human neurocysticercosis by Taenia solium is considered an emergent severe brain disorder in developing and developed countries. Discovery of new antiparasitic drugs has been recently aimed to restrain differentiation and establishment of the T. solium adult tapeworm, for being considered a central node in the disease propagation to both pigs and humans. Tamoxifen is an antiestrogenic drug with cysticidal action on Taenia crassiceps, a close relative of T. solium. Thus, we evaluated the effect of tamoxifen on the in vitro evagination and the in vivo establishment of T. solium. In vitro, tamoxifen inhibited evagination of T. solium cysticerci in a dose-time dependent manner. In vivo, administration of tamoxifen to hamsters decreased the intestinal establishment of the parasite by 70%, while recovered tapeworms showed an 80% reduction in length, appearing as scolices without strobilar development. Since tamoxifen did not show any significant effect on the proliferation of antigen-specific immune cells, intestinal inflammation, and expression of Th1/Th2 cytokines in spleen and duodenum, this drug could exert its antiparasite actions by having direct detrimental effects upon the adult tapeworm. These results demonstrate that tamoxifen exhibits a strong cysticidal and antitaeniasic effect on T. solium that should be further explored in humans and livestock.

Proteomic survey of the cestode Mesocestoides corti during the first 24 hours of strobilar development

Despite the fact that cestodes represent major etiological agents of both human and domestic animal diseases, little is known about the molecular aspects of cestode development. In this work, Mesocestoides corti, a model cestode species, was studied from the early development of its larval form (tetrathyridium) into adult worms (strobilation) using different proteomic approaches. The protein profiles of M. corti tetrathyridia induced or not induced to undergo strobilation were compared. Proteomic mapping by two-dimensional gel electrophoresis showed the resolution of 248 and 154 spots from tetrathyridia that were subjected or not subjected to strobilation induction, respectively, allowing for the detection of at least nine spots exclusive to each group. Spot analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) or MALDI-TOF MS/MS identified four reference proteins (six spots). LC-MS/MS analyses of protein extracts identified 66 proteins, eight of which were found exclusively in non-induced tetrathyridia, while 13 were found exclusively in strobilation-induced tetrathyridia. Among the proteins exclusively identified in strobilation-induced worms, there was a predominance of proteins with functions relating to chaperone activity and protein synthesis and turnover. Quantitative differential expression analysis between M. corti tetrathyridia prior to and after strobilation induction revealed six proteins upregulated in strobilation-induced worms; these proteins were involved in metabolic pathways, cell proliferation, and cytoskeletal rearrangement. Overall, despite the absence of a sequenced M. corti genome, using sequences from other platyhelminthes, we were able to establish comprehensive protein profiles for tetrathyridia prior to and after strobilation induction and identify several proteins potentially involved in the early events leading to strobilation.

Neoskrjabinolepis nuda n. sp. from shrews on Sakhalin Island, Russia, with a taxonomic review of Neoskrjabinolepis Spasskii, 1947 (Cestoda: Cyclophyllidea: Hymenolepididae)

Neoskrjabinolepis (Neoskrjabinolepidoides) nuda n. sp. is described from the shrews Sorex unguiculatus (type-host), S. gracillimus, S. isodon and S. caecutiens on Sakhalin Island, Russia. The new species is characterised by: rostellar hooks 40-44 microm long and provided with small epiphyseal thickening of the handle; a long (95-100 microm) cirrus consisting of basal region with claw-shaped spines, a parabasal region with thin needle-shaped spines and an unarmed distal region; a cirrus-sac extending well into the median field; and 15-22 eggs per gravid uterus. A review of the species of Neoskrjabinolepis Spasskii, 1947 is presented. Currently, this genus includes nine species and is divided in two subgenera on the basis of strobilar development, which is gradual in the subgenus Neoskrjabinolepis (four species) and serial in the subgenus Neoskrjabinolepidoides Kornienko, Gulyaev & Mel'nikova, 2006 (five species). An amended generic diagnosis and an identification key to Neoskrjabinolepis spp. are presented.

Mesocestoides corti: a LIM-homeobox gene upregulated during strobilar development

To understand the molecular processes regulating morphological changes during cestode life histories we focused on homeodomain (HD) proteins, a family of transcription factors essential for pattern formation during development. In this study we report the isolation of the partial sequence of MvLim, a LIM-HD gene of Mesocestoides corti. Other members of this gene family, characterized in Drosophila melanogaster, Caenorhabditis elegans and vertebrates contribute to cell fate determination of various neuronal subtypes. Phylogenetic analyses showed that MvLim clusters with members of the LIN-11 group and that platyhelminths have at least two different LIM-HD genes. By real time PCR we determined that MvLim expression is 20-fold greater in segmented worms than in tetrathyridia. The enhancement of MvLim expression during strobilation could be associated to changes in the innervation pattern occurring in proglottids development.

Effect of praziquantel on the strobilar development of Mesocestoides corti in vitro

The effect of praziquantel (PZQ) on the strobilar development of the cyclophyllidean cestode Mesocestoides corti was explored. Mesocestoides corti larvae were cultivated under conditions reported to favour their differentiation to the adult stage. Parasites were exposed to 0.1 microg ml(-1) PZQ for 16 h and subsequently transferred to drug-free medium. The ocurrence of segmentation--an early event of the larval somatic differentiation to the adult worm-- was considered as quantitative data. This phenomenon was evidenced earlier in worms transiently exposed to PZQ with respect to control cultures. Moreover, the rate of segmentation of drug-treated worms at the end of the experiment almost doubled that of control worms. To date, no similar effect on any cestode developmental process has been reported for an anthelmintic drug. In the light of the existing knowledge and understanding of PZQ mechanisms of action, the proposed experimental approach could contribute to the elucidation of pathways and mechanisms involved in cestode strobilar development.

Effect of praziquantel on the strobilar development of Mesocestoides corti in vitro

AbstractThe effect of praziquantel (PZQ) on the strobilar development of the cyclophyllidean cestode Mesocestoides corti was explored. Mesocestoides corti larvae were cultivated under conditions reported to favour their differentiation to the adult stage. Parasites were exposed to 0.1 μg ml-1 PZQ for 16 h and subsequently transferred to drug-free medium. The ocurrence of segmentation — an early event of the larval somatic differentiation to the adult worm — was considered as quantitative data. This phenomenon was evidenced earlier in worms transiently exposed to PZQ with respect to control cultures. Moreover, the rate of segmentation of drug-treated worms at the end of the experiment almost doubled that of control worms. To date, no similar effect on any cestode developmental process has been reported for an anthelmintic drug. In the light of the existing knowledge and understanding of PZQ mechanisms of action, the proposed experimental approach could contribute to the elucidation of pathways and mechanisms involved in cestode strobilar development.

Some studies on the growth and development of Echinococcus granulosus, camel origin in experimentally infected dogs

The development of Echinococcus granulosus of camel subspecies in 35 experimentally infected dogs was studied at 3, 7, 10, 13, 18, 23, 35 and 56 days post-infection (d.p.i.). The morphological characteristics of each developmental stage is studied and discussed. Morphological characters included number, total length, shape, arrangement of rosteller hooks and blade length to total length percentage (Bl/Tl%). In addition, total worm length, scolex, suckers, rostellum, neck and segments were measured and described. Other useful characters are considered to be the position of common genital pore, number and distribution of testes, uterus, shape of ovary and vitelline glands. Generally, the results indicated that the segmentation first appears at 18 d.p.i. Moreover, male and female genitalia could be detected at the same period. However, completely developed genitalia appeared at 56 d.p.i. Histological examination of the small intestine of experimentally infected dogs revealed that the parasites were found in distended and thin wall crypts of Liberkuhn at the periods of 3,7 and 10 d.p.i, while the parasite scoleces were found embeded in the mucosa at the periods of 13, 18 and 23 d.p.i. No significant pathological changes were encountered in both infected and control dogs. The data presented here are the first report of strobilar development of E. granulosus of camel origin in experimentally infected dogs.

Echinococcus granulosus: observations on strobilar development in in vitro monophasic culture.

The in vitro cultivation technique of Echinococcus granulosus protoscoleces usually states the necessity of a biphasic medium with a solid protein substrate for strobilar development to take place; otherwise, in a monophasic medium, protoscoleces follow a vesicular development. However, in some monophasic cultures, the development of several strobilate individuals (in different quantities and stages of development, depending on the culture) were observed. The only known difference from cultures made previously and since, where the development was vesicular, was the batch of foetal calf serum used in the constitution of the liquid medium, and this is presumed to be the cause of this unexpected strobilar development.

Uniform Strobilar Development of Echinococcus multilocularis In vitro from Protoscolex to Immature Stages

The aim of this study was to obtain uniformity in strobilar development of Echinococcus multilocularis from protoscoleces in vitro. The isolate of E. multilocularis used was derived initially from a human case in France and subsequently maintained in the laboratory by intraperitoneal passage in Meriones unguiculatus. Protoscoleces used for culture were obtained using preparative procedures in which parasite tissue was disrupted gently with minimal exposure to pepsin and acidic conditions followed by immediate exposure to pancreatin in alkaline solution. Resultant cultures contained large numbers of evaginated, active, vermiform stages, which exhibited uniform strobilar development with formation of the first proglottid and segment and limited maturation of the first proglottid. All worms that exhibited proglottization subsequently segmented. Further proglottization did not occur and all worms degenerated within a few days following segmentation. The results are discussed in light of current knowledge of the relationships of somatic and germinal processes in Echinococcus. In view of these results, further studies should be encouraged to improve strobilar development of E. multilocularis in vitro.

A comparison of Echinococcus granulosus from different geographical areas of Australia using secondary cyst development in mice

Significant differences in the rate of development of secondary hydatid cysts of Echinococcus granulosus in the same strains of mice were demonstrated between the parasite of Tasmanian sheep origin and that from sheep in Eastern and Western Australia. Similar differences were also found between E. granulosus of macropod and sheep origin, from Eastern Australia. These results complement previous comparative studies on strobilar development and support the suggestion that at least three distinct strains of E. granulosus exist in Australia. Two are found in sheep, one on the mainland and the other in Tasmania and the third is perpetuated amongst macropods on the mainland. The value of secondary hydatidosis as a criterion for strain differentiation of Echinococcus is emphasised.

Comparative strobilar development of Echinococcus granulosus of sheep origin from different geographical areas of Australia in vivo and in vitro

Consistent differences in the growth, segmentation and maturation of Echinococcus granulosus in dogs were demonstrated between the parasite of Tasmanian sheep origin and that of Eastern and Western Australian sheep origin. These results were complemented by in vitro studies which found that Tasmanian E. granulosus produced eggs approx. 7 days earlier than the parasite of Eastern and Western Australian origin. These results support the suggestion that at least two distinct strains of E. granulosus of domestic animal origin exist in Australia. The significance of the differences in development demonstrated in this study is discussed.

Studies on tapeworm physiology

1. A technique is described for the preparation of axenic cultures of protoscolices of Echinococcus granulosus.2. Protoscolices removed aseptically from hydatid cysts are treated with 0·02% crystalline pepsin at pH 2·0 for 10 min. at 38°C. in order to remove (a) daughter cyst germinal membranes, and (b) dead or degenerating protoscolices.3. Protoscolices may be maintained for more than 100 days in a variety of media. These include media containing various proportions of some of the following: chick embryo extract, beef embryo extract, hydatid fluid, bovine amniotic fluid, Parker 199, bovine serum.4. 10–20 ml. of culture media was used at 38°C., either in roller tubes or shaken in a water-bath. Media were renewed every 48 hr.5. Protoscolices developed into cysts by two routes, either (a) by becoming vesicular, gradually growing in size, and finally becoming enclosed in a laminated envelope, or (b) by forming a ‘posterior bladder’ which became relatively enormous and ultimately absorbed the scolex region, the whole forming a cyst within an envelope as in (a).6. In the majority of media, after about 31–48 days' culture, a thin envelope was secreted which developed later into a thick laminated membrane.7. Cysts with laminated membranes ultimately underwent fatty degeneration and became cytologically abnormal.8. In the most successful experiments, cells or clusters of cells formed within the cysts and these may have represented the anlagen of the protoscolices.9. In no case was segmentation, or any other sign of strobilar development, obtained.10. These results show that the laminated membrane in E. granulosus is initially of parasite origin and not of host origin as believed by some workers to be the case.11. Since there is evidence from the literature that E. multilocularis does not form such an envelope in vitro, a clear developmental difference appears to exist between the two species.12. It is suggested that this difference offers an explanation for the formation of unilocular or multilocular cysts. Since E. granulosus secretes a laminated envelope, its development is limited to a single or unilocular type of cyst. In the case of E. multilocularis no such limitation occurs, so that the invasive multilocular type of cyst could more easily develop.