Evolutionary analysis of species-specific duplications in flatworm genomes.

Platyhelminthes, also known as flatworms, is a phylum of bilaterian invertebrates infamous for their parasitic representatives. The classes Cestoda, Monogenea, and Trematoda comprise parasitic helminths inhabiting multiple hosts, including fishes, humans, and livestock, and are responsible for considerable economic damage and burden on human health. As in other animals, the genomes of flatworms have a wide variety of paralogs, genes related via duplication, whose origins could be mapped throughout the evolution of the phylum. Through in-silico analysis, we studied inparalogs, i.e., species-specific duplications, focusing on their biological functions, expression changes, and evolutionary rate. These genes are thought to be key players in the adaptation process of species to each particular niche. Our results showed that genes related with specific functional terms, such as response to stress, transferase activity, oxidoreductase activity, and peptidases, are overrepresented among inparalogs. This trend is conserved among species from different classes, including free-living species. Available expression data from Schistosoma mansoni, a parasite from the trematode class, demonstrated high conservation of expression patterns between inparalogs, but with notable exceptions, which also display evidence of rapid evolution. We discuss how natural selection may operate to maintain these genes and the particular duplication models that fit better to the observations. Our work supports the critical role of gene duplication in the evolution of flatworms, representing the first study of inparalogs evolution at the genome-wide level in this group.

Cell repertoire and proliferation of germinative cells of the model cestode Mesocestoides corti.

The phylum Platyhelminthes shares a unique population of undifferentiated cells responsible for the proliferation capacity needed for cell renewal, growth, tissue repair and regeneration. These cells have been extensively studied in free-living flatworms, whereas in cestodes the presence of a set of undifferentiated cells, known as germinative cells, has been demonstrated in classical morphology studies, but poorly characterized with molecular biology approaches. Furthermore, several genes have been identified as neoblast markers in free-living flatworms that deserve study in cestode models. Here, different cell types of the model cestode Mesocestoides corti were characterized, identifying differentiated and germinative cells. Muscle cells, tegumental cells, calcareous corpuscle precursor cells and excretory system cells were identified, all of which are non-proliferative, differentiated cell types. Besides those, germinative cells were identified as a population of small cells with proliferative capacity in vivo. Primary cell culture experiments in Dulbecco's Modified Eagle Medium (DMEM), Echinococcus hydatid fluid and hepatocyte conditioned media in non-reductive or reductive conditions confirmed that the germinative cells were the only ones with proliferative capacity. Since several genes have been identified as markers of undifferentiated neoblast cells in free-living flatworms, the expression of pumilio and pL10 genes was analysed by qPCR and in situ hybridization, showing that the expression of these genes was stronger in germinative cells but not restricted to this cell type. This study provides the first tools to analyse and further characterise undifferentiated cells in a model cestode.

Global analysis of neuropeptides in cestodes identifies Attachin, a SIFamide homolog, as a stimulant of parasite motility and attachment.

Many anthelmintics target the neuromuscular system, in particular by interfering with signaling mediated by classical neurotransmitters. Although peptidergic signaling has been proposed as a novel target for anthelmintics, current knowledge of the neuropeptide complement of many helminth groups is still limited, especially for parasitic flatworms (cestodes, trematodes, and monogeneans). In this work, we have characterized the neuropeptide complement of the model cestode Hymenolepis microstoma. Peptidomic characterization of adults of H. microstoma validated many of the neuropeptide precursor (npp) genes previously predicted in silico, and identified novel neuropeptides that are conserved in parasitic flatworms. Most neuropeptides from parasitic flatworms lack significant similarity to those from other animals, confirming the uniqueness of their peptidergic signaling. Analysis of gene expression of ten npp genes by in situ hybridization confirmed that all of them are expressed in the nervous system and identified cryptic features, including the first evidence of dorsoventral asymmetry, as well as a new population of peripheral peptidergic cells that appears to be conserved in the trematode Schistosoma mansoni. Finally, we characterized in greater detail Attachin, an SIFamide homolog. Although its expression is largely restricted to the longitudinal nerve cords and cerebral commissure in H. microstoma, it shows widespread localization in the larval nervous system of Echinococcus multilocularis and Mesocestoides corti. Exogenous addition of a peptide corresponding to the highly conserved C-terminus of Attachin stimulated motility and attachment of M. corti larvae. Altogether, this work provides a robust experimental foothold for the characterization of peptidergic signaling in parasitic flatworms. Cover Image for this issue: https://doi.org/10.1111/jnc.15418.

Inhibitors of protein kinases A and C impair the motility of oncospheres of the model tapeworm Hymenolepis microstoma.

The oncosphere larvae of tapeworms cyclically extend and retract their hooks during the penetration of the intestine of their intermediate hosts. The mechanisms regulating these movements are essentially unknown, in part due to the biohazardous nature of oncospheres from human pathogens. In this work, we standardized a method for the analysis of motility of hatched oncospheres (hexacanths) of the model tapeworm Hymenolepis microstoma. We used this assay to explore the relevance of protein kinases C (PKC) and A (PKA) in these processes. Pharmacological inhibition of the PKC pathway resulted in impaired larval motility. On the other hand, the PKA inhibitor H-89 potently blocked larval motility, as well as the motility of other life stages, although other inhibitors of the PKA pathway were not effective. This work represents the first study of the mechanisms that regulate the motility of oncospheres, and provides a path for further exploration.

Expression profiling of Echinococcus multilocularis miRNAs throughout metacestode development in vitro.

The neglected zoonotic disease alveolar echinococcosis (AE) is caused by the metacestode stage of the tapeworm parasite Echinococcus multilocularis. MicroRNAs (miRNAs) are small non-coding RNAs with a major role in regulating gene expression in key biological processes. We analyzed the expression profile of E. multilocularis miRNAs throughout metacestode development in vitro, determined the spatial expression of miR-71 in metacestodes cultured in vitro and predicted miRNA targets. Small cDNA libraries from different samples of E. multilocularis were sequenced. We confirmed the expression of 37 miRNAs in E. multilocularis being some of them absent in the host, such as miR-71. We found a few miRNAs highly expressed in all life cycle stages and conditions analyzed, whereas most miRNAs showed very low expression. The most expressed miRNAs were miR-71, miR-9, let-7, miR-10, miR-4989 and miR-1. The high expression of these miRNAs was conserved in other tapeworms, suggesting essential roles in development, survival, or host-parasite interaction. We found highly regulated miRNAs during the different transitions or cultured conditions analyzed, which might suggest a role in the regulation of developmental timing, host-parasite interaction, and/or in maintaining the unique developmental features of each developmental stage or condition. We determined that miR-71 is expressed in germinative cells and in other cell types of the germinal layer in E. multilocularis metacestodes cultured in vitro. MiRNA target prediction of the most highly expressed miRNAs and in silico functional analysis suggested conserved and essential roles for these miRNAs in parasite biology. We found relevant targets potentially involved in development, cell growth and death, lifespan regulation, transcription, signal transduction and cell motility. The evolutionary conservation and expression analyses of E. multilocularis miRNAs throughout metacestode development along with the in silico functional analyses of their predicted targets might help to identify selective therapeutic targets for treatment and control of AE.

Stage-specific transcriptomic analysis of the model cestode Hymenolepis microstoma.

Most parasitic flatworms go through different life stages with important physiological and morphological changes. In this work, we used a transcriptomic approach to analyze the main life-stages of the model tapeworm Hymenolepis microstoma (eggs, cysticercoids, and adults). Our results showed massive transcriptomic changes in this life cycle, including key gene families that contribute substantially to the expression load in each stage. In particular, different members of the cestode-specific hydrophobic ligand-binding protein (HLBP) family are among the most highly expressed genes in each life stage. We also found the transcriptomic signature of major metabolic changes during the transition from cysticercoids to adult worms. Thus, this work contributes to uncovering the gene expression changes that accompany the development of this important cestode model species, and to the best of our knowledge represents the first transcriptomic study with robust replicates spanning all of the main life stages of a tapeworm.

Building the embryo of Developmental Biology in Uruguay.

In Uruguay, a country with a small population, and hence a small scientific community, there were no classical embryologists as such in the past. However, in the decade of the 1950s, a cumulus of favorable conditions gave rise to highly active and modern research groups in the fields of cytology and physiology, which eventually contributed to developmental biology. The advent of a long dictatorship between the 1970's and 1980's caused two things: a strong lag in local research and the migration of young investigators who learned abroad new disciplines and technologies. The coming back to democracy allowed for the return of some, now as solid researchers, and together with those who stayed, built a previously inexistent postgraduate training program and a globally-integrated academy that fostered diversity of research disciplines, including developmental biology. In this paper, we highlight the key contributions of pioneer researchers and the significant role played by academic and funding national institutions in the growth and consolidation of developmental biology in our country.

Evaluation of the effectiveness of transcutaneous nerve stimulation during labor in breech presentation: a case series.

Objective: To analyze the effectiveness of pain relief with transcutaneous electrical nerve stimulation (TENS) dispositive during labor in breech vaginal delivery and also to describe its tolerance and satisfaction.Design: A randomized, double-blind, and placebo-controlled trial was conducted.Setting: Labor room of Complejo Hospitalario Universitario Insular-Materno Infantil (Spain).Participants: Ten women who met the inclusion criteria of the clinical trial and also had a fetus breech presentation were randomly assigned to three groups.Interventions: A portable TENS, Cefar Rehab 2pro®, unit was used to apply the experimental intervention, with different doses in the three groups in women during labor. The device intensity (amplitude) was individually adjusted to each participant's maximum sensory level. The pain was evaluated with visual analog scale (VAS). COMFORTS scale was used to measure women's satisfaction.Results: A significant association of VAS was detected depending on the type of TENS over time. Active TENS2 was the only group that obtained an improvement with clinically significant results. In connection with women satisfaction, active TENS2 group was also the group that presented higher values.Conclusions: We can recommend the use of TENS dispositive to relieve pain during labor, also in those women with breech presentation.

Gene Silencing in the Liver Fluke Fasciola hepatica: RNA Interference.

The chronic infection with the liver fluke of the genus Fasciola spp. is the most prevalent foodborne trematodiasis, affecting at least one-fourth of the world livestock grazing in areas where the parasite is present. Moreover, fascioliasis is considered a major zoonosis mainly in rural areas of central South America, Northern Africa, and Central Asia. Increasing evidences of resistance against triclabendazole may compromise its use as drug of choice; thus, novel control strategies are desperately needed. Functional genomic approaches play a key role in the validation and characterization of new targets for drug and vaccine development. So far, RNA interference has been the only gene silencing approach successfully employed in liver flukes of the genus Fasciola spp. Herein, we describe a detailed step-by-step protocol to perform gene silencing mediated by RNAi in Fasciola hepatica.

Analysis of Gene Expression in Fasciola hepatica Juveniles and Adults by In Situ Hybridization.

In situ hybridization (ISH) is a technique used for the spatial localization of nucleic acids within tissues and cells. It is based on the ability of labeled nucleic acids (probes) to hybridize under the right conditions with the nucleic acids present in fixed biological specimens. In this chapter, we describe protocols for detection of RNA by ISH using digoxigenin (DIG)-labeled probes for Fasciola hepatica adults (in cryosections, given their large size) and for newly excysted juveniles (NEJs, which are ideally suited given their small size for whole-mount ISH). We describe fluorogenic and chromogenic protocols, respectively, but the detection methods can be easily interchanged by using the appropriate enzyme-conjugated antibodies and detection solutions.

Divergent Axin and GSK-3 paralogs in the beta-catenin destruction complexes of tapeworms.

The Wnt/beta-catenin pathway has many key roles in the development of animals, including a conserved and central role in the specification of the primary (antero-posterior) body axis. The posterior expression of Wnt ligands and the anterior expression of secreted Wnt inhibitors are known to be conserved during the larval metamorphosis of tapeworms. However, their downstream signaling components for Wnt/beta-catenin signaling have not been characterized. In this work, we have studied the core components of the beta-catenin destruction complex of the human pathogen Echinococcus multilocularis, the causative agent of alveolar echinococcosis. We focused on two Axin paralogs that are conserved in tapeworms and other flatworm parasites. Despite their divergent sequences, both Axins could robustly interact with one E. multilocularis beta-catenin paralog and limited its accumulation in a heterologous mammalian expression system. Similarly to what has been described in planarians (free-living flatworms), other beta-catenin paralogs showed limited or no interaction with either Axin and are unlikely to function as effectors in Wnt signaling. Additionally, both Axins interacted with three divergent GSK-3 paralogs that are conserved in free-living and parasitic flatworms. Axin paralogs have highly segregated expression patterns along the antero-posterior axis in the tapeworms E. multilocularis and Hymenolepis microstoma, indicating that different beta-catenin destruction complexes may operate in different regions during their larval metamorphosis.

Evaluation of different doses of transcutaneous nerve stimulation for pain relief during labour: a randomized controlled trial.

BACKGROUND: Pain during labour is one of the most intense pain that women may experience in their lifetime. There are several non-pharmacological analgesic methods to relieve pain during labour, among them transcutaneous electrical nerve stimulation (TENS). TENS is a low-frequency electrotherapy technique, analgesic type, generally used in musculoskeletal pathology, but it has also come to be used as an alternative treatment during labour. The purpose of this study is to investigate the pain-relieving effect of a TENS application during labour and to find out the most effective dose. METHODS: This study is a randomized, double-blind, placebo-controlled trial. TENS therapy was initiated at the beginning of the active phase of labour. Participants were randomly assigned to three groups (21 per group: two active TENS and one placebo). Active TENS 1 intervention consisted in a constant frequency of 100-Hz, 100-µs, active TENS 2 intervention consisted in a varying high-frequency (80-100 Hz), 350 µs, and in a placebo group, participants were connected to the TENS unit without electrical stimulation. TENS was applied with two self-adhesive electrodes placed parallel to the spinal cord (T10-L1 and S2-S4 levels). The primary outcome was pain intensity (0-10 cm) measured on a visual analogue scale (VAS) at several stages (at baseline and at 10 and 30 min later). Secondary outcomes included women's satisfaction (via the Care in Obstetrics: Measure for Testing Satisfaction scale). RESULTS: Sixty-three women participated. Regarding baseline characteristics, no differences were found among the three groups. The active TENS 2 group obtained an improvement with clinically significant VAS results (- 2.9, 95% confidence interval - 4.1 to - 1.6, p <  0.001). Regarding satisfaction, the results also revealed better results in the active TENS than in the placebo group. CONCLUSIONS: TENS with high frequencies modified in time as well as high pulse width are effective for relieving labour pain, and they are well considered by pregnant participants. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03137251 . Registered on 2 May 2017.

Analysis of classical neurotransmitter markers in tapeworms: Evidence for extensive loss of neurotransmitter pathways.

Parasitic flatworms have complex neuromuscular systems that serve important functions in their life cycles. However, our understanding of neurotransmission in parasitic flatworms is limited. Pioneering studies have suggested the presence of several classical neurotransmitter systems, but their molecular components have not been characterized in most cases. Because these components are conserved in bilaterian animals, we searched the genomes of parasitic flatworms for orthologs of genes required for neurotransmitter synthesis, vesicular transport, reuptake, and reception. Our results indicate that tapeworms have lost the genes that are specifically required in other animals for synaptic signaling using the classical neurotransmitters dopamine, tyramine, octopamine, histamine and gamma-aminobutyric acid (GABA). These results imply that these signaling pathways are either absent in these parasites, or that they require completely different molecular components in comparison with other animals. The orthologs of genes related to histaminergic and GABA signaling are also missing in trematodes (although Schistosoma-specific histaminergic receptors have been previously described). In contrast, conserved genes required for glutamatergic, serotonergic and cholinergic signaling could be found in all analyzed flatworms. We analyzed the expression of selected markers of each pathway in the tapeworm Hymenolepis microstoma by whole-mount in situ hybridization. Each marker was specifically expressed in the nervous system, although with different patterns. In addition, we analyzed the expression of proprotein convertase 2 as a marker of peptidergic cells. This gene showed the widest expression in the nervous system, but was also expressed in other tissues, suggesting additional roles of peptidergic signaling in tapeworm development and reproduction.

Dynamic Proteome Alteration and Functional Modulation of Human Saliva Induced by Dietary Chemosensory Stimuli.

Saliva flow measurements and SDS-PAGE separation of human whole saliva freshly collected after oral stimulation with citric acid (sour), aspartame (sweet), iso-α-acids (bitter), mono sodium l-glutamate (umami), NaCl (salty), 6-gingerol (pungent), hydroxy-α-sanshool (tingling), and hydroxy-ß-sanshool (numbing), followed by tryptic digestion, nano-HPLC-MS/MS, and label-free protein quantitation demonstrated a stimulus- and time-dependent influence of the dietary chemosensates on salivation and the salivary proteome composition. Gene ontology enrichment analysis showed evidence for stimulus-induced alterations of the saliva proteome to boot an efficient molecular defense network of the oral cavity, e.g., 6-gingerol increased salivary lactoperoxidase activity, catalyzing the oxidation of thiocyanate to produce the antimicrobial and antifungal hypothiocyanate, from 0.37 ± 0.02 to 0.91 ± 0.05 mU/mL 45 s after stimulation. In comparison, oral citric acid stimulation induced an increase of myeloperoxidase activity, catalyzing the chloride oxidation to generate antimicrobial hypochloride in saliva, from 0.24 ± 0.04 to 0.70 ± 0.1 mU/mL as well as an increase of salivary levels of lysozyme, exhibiting antimicrobial activity on Gram-positive bacteria, from 6.0-10 to 100-150 µg/mL. Finally, microbial growth experiments clearly demonstrated for the first time that the increase of the salivary lysozyme abundance upon oral citric acid stimulation translates into an enhanced biological function, that is an almost complete growth inhibition of the two lysozyme-sensitive Gram-positive bacteria tested.

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.

De novo discovery of neuropeptides in the genomes of parasitic flatworms using a novel comparative approach.

Neuropeptide mediated signalling is an ancient mechanism found in almost all animals and has been proposed as a promising target for the development of novel drugs against helminths. However, identification of neuropeptides from genomic data is challenging, and knowledge of the neuropeptide complement of parasitic flatworms is still fragmentary. In this work, we have developed an evolution-based strategy for the de novo discovery of neuropeptide precursors, based on the detection of localised sequence conservation between possible prohormone convertase cleavage sites. The method detected known neuropeptide precursors with good precision and specificity in the models Drosophila melanogaster and Caenorhabditis elegans. Furthermore, it identified novel putative neuropeptide precursors in nematodes, including the first description of allatotropin homologues in this phylum. Our search for neuropeptide precursors in the genomes of parasitic flatworms resulted in the description of 34 conserved neuropeptide precursor families, including 13 new ones, and of hundreds of new homologues of known neuropeptide precursor families. Most neuropeptide precursor families show a wide phylogenetic distribution among parasitic flatworms and show little similarity to neuropeptide precursors of other bilaterian animals. However, we could also find orthologs of some conserved bilaterian neuropeptides including pyrokinin, crustacean cardioactive peptide, myomodulin, neuropeptide-Y, neuropeptide KY and SIF-amide. Finally, we determined the expression patterns of seven putative neuropeptide precursor genes in the protoscolex of Echinococcus multilocularis. All genes were expressed in the nervous system with different patterns, indicating a hidden complexity of peptidergic signalling in cestodes.

Identification of novel CAP superfamily protein members of Echinococcus granulosus protoscoleces.

Echinoccocus granulosus is the causative agent of Cyst Echinococcosis, a zoonotic infection affecting humans and livestock representing a public health and an economic burden for several countries. Despite decades of investigation an effective vaccine still remains to be found. Parasitic cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 proteins (CAPs) have been proposed as vaccine candidates against helmith's infection. In this work we have identified two novel proteins of this superfamily expressed at the protoescoleces larval stage named EgVAL1 and EgVAL2. The open reading frame sequences were deduced. The aminoacidic sequence was analyzed and confronted against already known vertebrate' and helminth's proteins sequences in order to infer putative functions. Immunolocalization studies were also performed. The obtained data supported by immunolocalization studies and homology models suggest that these proteins could be involved in protease activity inhibition.

Expression of the histone chaperone SET/TAF-Iß during the strobilation process of Mesocestoides corti (Platyhelminthes, Cestoda).

The histone chaperone SET/TAF-Iß is implicated in processes of chromatin remodelling and gene expression regulation. It has been associated with the control of developmental processes, but little is known about its function in helminth parasites. In Mesocestoides corti, a partial cDNA sequence related to SET/TAF-Iß was isolated in a screening for genes differentially expressed in larvae (tetrathyridia) and adult worms. Here, the full-length coding sequence of the M. corti SET/TAF-Iß gene was analysed and the encoded protein (McSET/TAF) was compared with orthologous sequences, showing that McSET/TAF can be regarded as a SET/TAF-Iß family member, with a typical nucleosome-assembly protein (NAP) domain and an acidic tail. The expression patterns of the McSET/TAF gene and protein were investigated during the strobilation process by RT-qPCR, using a set of five reference genes, and by immunoblot and immunofluorescence, using monospecific polyclonal antibodies. A gradual increase in McSET/TAF transcripts and McSET/TAF protein was observed upon development induction by trypsin, demonstrating McSET/TAF differential expression during strobilation. These results provided the first evidence for the involvement of a protein from the NAP family of epigenetic effectors in the regulation of cestode development.

RNA interference in Fasciola hepatica newly excysted juveniles: long dsRNA induces more persistent silencing than siRNA.

In trematodes RNA interference is the current tool of choice for functional analysis of genes since classical reverse genetic approaches remain unavailable. Whereas this approach has been optimized in schistosomes, few reports are available for other trematodes, likely reflecting the difficulties in the establishment of the technology. Here we standardized conditions for RNAi in the liver fluke Fasciola hepatica, the causative agent of fasciolosis, one of the most problematic infections affecting livestock worldwide. Targeting a single copy gene, encoding leucine aminopeptidase (LAP) as a model, we refined delivery conditions which identified electro-soaking, i.e. electroporation and subsequent incubation as efficient for introduction of small RNAs into the fluke. Knock down of LAP was achieved with as little as 2.5 µg/ml dsRNA concentrations, which may reduce or obviate off-target effects. However, at these concentrations, tracking incorporation by fluorescent labeling was difficult. While both long dsRNA and short interfering RNA (siRNA) are equally effective at inducing a short-term knock down, dsRNA induced persistent silencing up to 21 days after treatment, suggesting that mechanisms of amplification of the interfering signal can be present in this pathogen. Persistent silencing of the invasive stage for up to 3 weeks (close to what it takes for the fluke to reach the liver) opens the possibility of using RNAi for the validation of putative therapeutic targets.

Comprehensive proteome analysis in Cenococcum geophilum Fr. as a tool to discover drought-related proteins.

Cenococcum geophilum is a widely distributed ectomycorrhizal fungus potentially playing a significant role in resistance and resilience mechanisms of its tree hosts exposed to drought stress. In this study, we performed a large scale protein analysis in pure cultures of C. geophilum in order to gain first global insights into the proteome assembly of this fungus. Using 1-D gel electrophoresis coupled with ESI-MS/MS, we indentified 638 unique proteins. Most of these proteins were related to the metabolic and cellular processes, and the transport machinery of cells. In a second step, we examined the influence of water deprivation on the proteome of C. geophilum pure cultures at three time points of gradually imposed drought. The results indicated that 12 proteins were differentially abundant in mycelia subjected to drought compared to controls. The induced responses in C. geophilum point towards regulation of osmotic stress, maintainance of cell integrity, and counteracting increased levels of reactive oxygen species formed during water deprivation.