Short communication

Differentially expressed sequences from a cestode parasite reveals conserved developmental genes in platyhelminthes

Mesocestoides corti (syn. vogae) is a useful model for developmental studies of platyhelminth parasites of the Cestoda class, such as Taenia spp. or Echinococcus spp. It has been used in studies to characterize cestode strobilation, i.e. the development of larvae into adult worms. So far, little is known about the initial molecular events involved in cestode strobilation and, therefore, we carried out a study to characterize newly synthesized (NS) proteins upon strobilation induction. An approach based on bioorthogonal noncanonical amino acid tagging and mass spectrometry was used to label, isolate, identify, and quantify NS proteins in the initial steps of M. corti strobilation. Overall, 121 NS proteins were detected exclusively after induction of strobilation, including proteins related to development pathways, such as insulin and notch signaling. Metabolic changes that take place in the transition from the larval stage to adult worm were noted in special NS protein subsets related to developmental processes, such as focal adhesion, cell leading edge, and maintenance of location. The data shed light on mechanisms underlying early steps of cestode strobilation and enabled identification of possible developmental markers. We also consider the use of developmental responsive proteins as potential drug targets for developing novel anthelmintics.

Larval cestodiases are life-threatening parasitic diseases that affect both man and domestic animals worldwide. Cestode parasites present complex life cycles, in which they undergo major morphological and physiological changes in the transition from one life-stage to the next. One of these transitions occurs during cestode strobilation, when the mostly undifferentiated and non-segmented larval or pre-adult form develops into a fully segmented and sexually differentiated (strobilated) adult worm. Although the proteomes of bona fide larvae and strobialted adults have been previously characterized for a few cestode species, little is still known about the dynamic of protein synthesis during the early steps of cestode strobilation. Now, the assessment of newly synthesized (NS) proteins within the first 48 h of strobilation the model cestode M. corti allowed to shed light on molecular mechanisms that are triggered by strobilation induction. The functional analyses of this repertoire of over a hundred NS proteins pointed out to changes in metabolism and activation of classical developmental signaling pathways in early strobilation. Many of the identified NS proteins may become valuable cestode developmental markers and their involvement in vital processes make them also good candidate targets for novel anthelmintic drugs.

Cestode development involves complex morphological and physiological changes. Here, we performed a differential expression analysis of gene transcripts between two developmental stages of the model cestode Mesocestoides corti. A RNA-seq-based approach was used to compare the transcriptomes of the tetrathyridium (larval, TT) and strobilated worm (ST) stages of the parasite. We found 19,053 transcripts, from which ∼45% were complete matches to genes previously annotated in the available M. corti draft genome sequence, ∼24% were considered novel isoforms, and ∼24% were considered potential novel transcripts. Stage-specific transcripts were found for both TTs (66) and STs (136), along with shared transcripts significantly overrepresented in one stage (342 in TTs, and 559 in STs). Differential expression and Gene Ontology term enrichment analyzes provided evidence of upregulation of different sets of transcripts associated with ‘cytoskeleton’, ‘metabolism’ and ‘oxidation-reduction’ processes in each stage, suggesting functional involvement of the corresponding genes with stage-specific features. Transcripts and processes enriched in the TT reflect typical larval processes that occur with the parasite in the intermediate host, such as asexual reproduction and budding, as well as active migration from the peritoneum to the liver and vice versa. In STs, transcripts associated with ‘development’, ‘cell growth’, and ‘morphogenesis’ were enriched, along with processes related to sexual reproduction, represented by the upregulation of numerous transcription factors, protein kinases, and histones. Overall, our results contributed to significantly increase the knowledge on the M. corti gene repertoire and expression profile in two developmental stages. Functional implications for the biology of larval and adult cestode parasites and for host-parasite interactions are discussed.

Mesocestoides corti is a widely used model for the study of cestode biology, and its transition from the larval tetrathyridium (TT) stage to the strobilated, adult worm (ST) stage can be induced and followed in vitro. Here, a proteomic approach was used to describe and compare M. corti TT and ST protein repertories. Overall, 571 proteins were identified, 238 proteins in TT samples and 333 proteins in ST samples. Among the identified proteins, 207 proteins were shared by TTs and STs, while 157 were stage-specific, being 31 exclusive from TTs, and 126 from STs. Functional annotation revealed fundamental metabolic differences between the TT and the ST stages. TTs perform functions related mainly to basic metabolism, responsible for growth and vegetative development by asexual reproduction. STs, in contrast, perform a wider range of functions, including macromolecule biosynthetic processes, gene expression and control pathways, which may be associated to its proglottization/segmentation, sexual differentiation and more complex physiology. Furthermore, the generated results provided an extensive list of cestode proteins of interest for functional studies in M. corti. Many of these proteins are novel candidate diagnostic antigens, and/or potential targets for the development of new and more effective antihelminthic drugs.

Cestodiases are parasitic diseases with serious impact on human and animal health. Efforts to develop more effective strategies for diagnosis, treatment or control of cestodiases are impaired by the still limited knowledge on many aspects of cestode biology, including the complex developmental processes that occur in the life cycles of these parasites. Mesocestoides corti is a good experimental model to study the transition from the larval to the adult stage, called strobilation, which occur in typical cestode life-cycles. The performed proteomics approach provided large-scale identification and quantification of M. corti proteins. Many stage-specific or differentially expressed proteins were detected in the larval tetrathyridium (TT) stage and in the strobilated, adult worm (ST) stage. Functional comparative analyses of the described protein repertoires shed light on function and processes associated to specific features of both stages, such as less differentiation and asexual reproduction in TTs, and proglottization/segmentation and sexual differentiation in ST. Moreover, many of the identified stage-specific proteins are useful as cestode developmental markers, and are potential targets for development of novel diagnostic methods and therapeutic drugs for cestodiases.

MicroRNAs (miRNAs), a class of small non-coding RNAs, are key regulators of gene expression at post-transcriptional level and play essential roles in fundamental biological processes such as metabolism and development. The particular developmental characteristics of cestode parasites highlight the importance of studying miRNA gene regulation in these organisms. Here, we performed a comprehensive analysis of miRNAs in two developmental stages of the model cestode Mesocestoides corti. Using a high-throughput sequencing approach, we found transcriptional evidence of 42 miRNA loci in tetrathyridia larvae and strobilated worms. Tetrathyridium and strobilated worm-specific miRNAs were found, as well as differentialy expressed miRNAs between these developmental stages, suggesting miRNA regulation of stage-specific features. Moreover, it was shown that uridylation is a differential mechanism of post-transcriptional modification of M. corti miRNAs. The whole set of M. corti miRNAs represent 33 unique miRNA families, and confirm the remarkable loss of conserved miRNA families within platyhelminth parasites, reflecting their relatively low morphological complexity and high adaptation to parasitism. Overall, the presented results provide a valuable platform to studies aiming to identify and characterize novel miRNA-based molecular mechanisms of post-transcriptional gene regulation in cestodes, necessary for the elucidation of developmental aspects of the complex biology of these parasites.

Hox genes form a multigenic family that play a fundamental role during the early stages of development. They are organised in a single cluster and share a 60 amino acid conserved sequence that corresponds to the DNA binding domain, i.e. the homeodomain. Sequence conservation in this region has allowed investigators to explore Hox diversity in the metazoan lineages. Within parasitic flatworms only homeobox sequences of parasite species from the Cestoda and Digenea have been reported. In the present study we surveyed species of the Polyopisthocotylea (Monogenea) in order to clarify Hox identification and diversification processes in the neodermatan lineage. From cloning of degenerative PCR products of the central region of the homeobox, we report one ParaHox and 25 new Hox sequences from 10 species of the Polystomatidae and one species of the Diclidophoridae, which extend Hox gene diversity from 46 to 72 within Neodermata. Hox sequences from the Polyopisthocotylea were annotated and classified from sequence alignments and Bayesian inferences of 178 Hox, ParaHox and related gene families recovered from all available parasitic platyhelminths and other bilaterian taxa. Our results are discussed in the light of the recent Hox evolutionary schemes. They may provide new perspectives to study the transition from turbellarians to parasitic flatworms with complex life-cycles and outline the first steps for evolutionary developmental biological approaches within platyhelminth parasites.

Research into the roles played by Hox and related homeotic gene families in the diverse and complex developmental programmes exhibited by parasitic flatworms (Platyhelminthes) can hardly be said to have begun, and thus presents considerable opportunity for new research. Although featured in some of the earliest screens for homeotic genes outside Drosophila and mice, surveys in parasitic flatworms are few in number and almost nothing is yet known of where or when the genes are expressed during ontogeny. This contrasts sharply with a significant body of literature concerning Hox genes in free-living flatworms which have long served as models for the study of regeneration and the maintenance of omnipotent cell lines. Nevertheless, available information suggests that the complement of Hox genes and other classes of homeobox-containing genes in parasitic flatworms is typical of their free-living cousins and of other members of the Lophotrochozoa. Recent work on Schistosoma combined with information on Hox gene expression in planarians indicates that at least some disruption of the clustered genomic arrangement of the genes, as well as of the strict spatial and temporal colinear patterns of expression typical in other groups, may be characteristic of flatworms. However, available data on the genomic arrangement and expression of flatworm Hox genes is so limited at present that such generalities are highly tenuous. Moreover, a basic underlying pattern of colinearity is still observed in their spatial expression patterns making them suitable as cell or region-specific markers. I discuss a number of fundamental developmental questions and some of the challenges to addressing them in relation to each of the major parasitic lineages. In addition, I present newly characterized Hox genes from the model tapeworm Hymenolepis and analyze these by Bayesian inference together with > 100 Hox and ParaHox homeodomains of flatworms and select lophotrochozoan taxa, providing a phylogenetic scaffold for their identification.