Trematode Genomics and Proteomics.

Trematode infections stand out as one of the frequently overlooked tropical diseases, despite their wide global prevalence and remarkable capacity to parasitize diverse host species and tissues. Furthermore, these parasites hold significant socio-economic, medical, veterinary and agricultural implications. Over the past decades, substantial strides have been taken to bridge the information gap concerning various "omic" tools, such as proteomics and genomics, in this field. In this edition of the book, we highlight recent progress in genomics and proteomics concerning trematodes with a particular focus on the advances made in the past 5 years. Additionally, we present insights into cutting-edge technologies employed in studying trematode biology and shed light on the available resources for exploring the molecular facets of this particular group of parasitic helminths.

Identification and characterization of specific motifs in effector proteins of plant parasites using MOnSTER.

Plant pathogens cause billions of dollars of crop loss every year and are a major threat to global food security. Identifying and characterizing pathogens effectors is crucial towards their improved control. Because of their poor sequence conservation, effector identification is challenging, and current methods generate too many candidates without indication for prioritizing experimental studies. In most phyla, effectors contain specific sequence motifs which influence their localization and targets in the plant. Therefore, there is an urgent need to develop bioinformatics tools tailored for pathogen effectors. To circumvent these limitations, we have developed MOnSTER a specific tool that identifies clusters of motifs of protein sequences (CLUMPs). MOnSTER can be fed with motifs identified by de novo tools or from databases such as Pfam and InterProScan. The advantage of MOnSTER is the reduction of motif redundancy by clustering them and associating a score. This score encompasses the physicochemical properties of AAs and the motif occurrences. We built up our method to identify discriminant CLUMPs in oomycetes effectors. Consequently, we applied MOnSTER on plant parasitic nematodes and identified six CLUMPs in about 60% of the known nematode candidate parasitism proteins. Furthermore, we found co-occurrences of CLUMPs with protein domains important for invasion and pathogenicity. The potentiality of this tool goes beyond the effector characterization and can be used to easily cluster motifs and calculate the CLUMP-score on any set of protein sequences.

Inference of Essential Genes of the Parasite Haemonchus contortus via Machine Learning.

Over the years, comprehensive explorations of the model organisms Caenorhabditis elegans (elegant worm) and Drosophila melanogaster (vinegar fly) have contributed substantially to our understanding of complex biological processes and pathways in multicellular organisms generally. Extensive functional genomic-phenomic, genomic, transcriptomic, and proteomic data sets have enabled the discovery and characterisation of genes that are crucial for life, called 'essential genes'. Recently, we investigated the feasibility of inferring essential genes from such data sets using advanced bioinformatics and showed that a machine learning (ML)-based workflow could be used to extract or engineer features from DNA, RNA, protein, and/or cellular data/information to underpin the reliable prediction of essential genes both within and between C. elegans and D. melanogaster. As these are two distantly related species within the Ecdysozoa, we proposed that this ML approach would be particularly well suited for species that are within the same phylum or evolutionary clade. In the present study, we cross-predicted essential genes within the phylum Nematoda (evolutionary clade V)-between C. elegans and the pathogenic parasitic nematode H. contortus-and then ranked and prioritised H. contortus proteins encoded by these genes as intervention (e.g., drug) target candidates. Using strong, validated predictors, we inferred essential genes of H. contortus that are involved predominantly in crucial biological processes/pathways including ribosome biogenesis, translation, RNA binding/processing, and signalling and which are highly transcribed in the germline, somatic gonad precursors, sex myoblasts, vulva cell precursors, various nerve cells, glia, or hypodermis. The findings indicate that this in silico workflow provides a promising avenue to identify and prioritise panels/groups of drug target candidates in parasitic nematodes for experimental validation in vitro and/or in vivo.

Enhanced Genomic and Transcriptomic Resources for Trichinella pseudospiralis and T. spiralis to Underpin the Discovery of Molecular Differences between Stages and Species.

Nematodes of the genus Trichinella are important pathogens of humans and animals. This study aimed to enhance the genomic and transcriptomic resources for T. pseudospiralis (non-encapsulated phenotype) and T. spiralis (encapsulated phenotype) and to explore transcriptional profiles. First, we improved the assemblies of the genomes of T. pseudospiralis (code ISS13) and T. spiralis (code ISS534), achieving genome sizes of 56.6 Mb (320 scaffolds, and an N50 of 1.02 Mb) and 63.5 Mb (568 scaffolds, and an N50 value of 0.44 Mb), respectively. Then, for each species, we produced RNA sequence data for three key developmental stages (first-stage muscle larvae [L1s], adults, and newborn larvae [NBLs]; three replicates for each stage), analysed differential transcription between stages, and explored enriched pathways and processes between species. Stage-specific upregulation was linked to cellular processes, metabolism, and host-parasite interactions, and pathway enrichment analysis showed distinctive biological processes and cellular localisations between species. Indeed, the secreted molecules calmodulin, calreticulin, and calsyntenin-with possible roles in modulating host immune responses and facilitating parasite survival-were unique to T. pseudospiralis and not detected in T. spiralis. These insights into the molecular mechanisms of Trichinella-host interactions might offer possible avenues for developing new interventions against trichinellosis.

The Bursaphelenchus xylophilus Effector BxNMP1 Targets PtTLP-L2 to Mediate PtGLU Promoting Parasitism and Virulence in Pinus thunbergii.

Pinus is an important economic tree species, but pine wilt disease (PWD) seriously threatens the survival of pine trees. PWD caused by Bursaphelenchus xylophilus is a major quarantine disease worldwide that causes significant economic losses. However, more information about its molecular pathogenesis is needed, resulting in a lack of effective prevention and treatment measures. In recent years, effectors have become a hot topic in exploring the molecular pathogenic mechanism of pathogens. Here, we identified a specific effector, BxNMP1, from B. xylophilus. In situ hybridization experiments revealed that BxNMP1 was specifically expressed in dorsal gland cells and intestinal cells, and RT-qPCR experiments revealed that BxNMP1 was upregulated in the early stage of infection. The sequence of BxNMP1 was different in the avirulent strain, and when BxNMP1-silenced B. xylophilus was inoculated into P. thunbergii seedlings, the disease severity significantly decreased. We demonstrated that BxNMP1 interacted with the thaumatin-like protein PtTLP-L2 in P. thunbergii. Additionally, we found that the ß-1,3-glucanase PtGLU interacted with PtTLP-L2. Therefore, we hypothesized that BxNMP1 might indirectly interact with PtGLU through PtTLP-L2 as an intermediate mediator. Both targets can respond to infection, and PtTLP-L2 can enhance the resistance of pine trees. Moreover, we detected increased salicylic acid contents in P. thunbergii seedlings inoculated with B. xylophilus when BxNMP1 was silenced or when the PtTLP-L2 recombinant protein was added. In summary, we identified a key virulence effector of PWNs, BxNMP1. It positively regulates the pathogenicity of B. xylophilus and interacts directly with PtTLP-L2 and indirectly with PtGLU. It also inhibits the expression of two targets and the host salicylic acid pathway. This study provides theoretical guidance and a practical basis for controlling PWD and breeding for disease resistance.

Molecular characterization of EcCLP1, a new putative cathepsin L protease from Echinococcus canadensis.

Echinococcus granulosus sensu lato is a platyhelminth parasite and the etiological cause of cystic echinococcosis (CE), a zoonotic and neglected disease that infects animals and humans worldwide. As a part of the biological arsenal of the parasite, cathepsin L proteases are a group of proteins that are believed to be essential for parasite penetration, immune evasion, and establishment in the tissues of the host. In this work, we have cloned and sequenced a new putative cathepsin L protease from Echinococcus canadensis (EcCLP1). The bioinformatic analysis suggests that EcCLP1 could be synthesized as a zymogen and activated after proteolytic cleavage. The multiple sequence alignment with other cathepsin proteases reveals important functional conserved features like a conserved active site, an N-linked glycosylation residue, a catalytic triad, an oxyanion hole, and three putative disulfide bonds. The phylogenetic analysis suggests that EcCLP1 could indeed be a cathepsin L cysteine protease from clade 1 as it grouped with cathepsins from other species in this clade. Modeling studies suggest that EcCLP1 has two domains forming a cleft where the active site is located and an occluding role for the propeptide. The transcriptomic analysis reveals different levels of cathepsin transcript expression along the different stages of the parasite life cycle. The whole-mount immunohistochemistry shows an interesting superficial punctate pattern of staining which suggests a secretory pattern of expression. The putative cathepsin L protease characterized here may represent an interesting tool for diagnostic purposes, vaccine design, or a new pharmacological target for antiparasitic intervention.

Title: Caractérisation moléculaire d'EcCLP1, une nouvelle protéase putative de type cathepsine L d'Echinococcus canadensis. Abstract: Echinococcus granulosus sensu lato est un Plathelminthe parasite et la cause étiologique de l'échinococcose kystique (EK), une maladie zoonotique et négligée qui infecte les animaux et les humains dans le monde entier. En tant que partie de l'arsenal biologique du parasite, les protéases de type cathepsine L sont un groupe de protéines considérées comme essentielles à la pénétration du parasite, l'évasion immunitaire et son établissement dans les tissus de l'hôte. Dans ce travail, nous avons cloné et séquencé une nouvelle protéase putative de type cathepsine L d'Echinococcus canadensis (EcCLP1). L'analyse bioinformatique suggère qu'EcCLP1 pourrait être synthétisée sous forme de zymogène et activée après clivage protéolytique. L'alignement de séquences multiples avec d'autres protéases de type cathepsine révèle d'importantes caractéristiques fonctionnelles conservées telles qu'un site actif conservé, un résidu de glycosylation lié à N, une triade catalytique, un trou oxyanion et trois liaisons disulfure putatives. L'analyse phylogénétique suggère qu'EcCLP1 pourrait en effet être une protéase de type cathepsine L du clade 1 car elle se regroupe avec les cathepsines d'autres espèces de ce clade. Les études de modélisation suggèrent qu'EcCLP1 possède deux domaines formant une fente où se trouve le site actif et un rôle d'occlusion pour le propeptide. L'analyse transcriptomique révèle différents niveaux d'expression du transcrit de la cathepsine au cours des différentes étapes du cycle de vie du parasite. L'immunohistochimie de montages entiers montre un intéressant motif de coloration ponctuée superficielle qui suggère un modèle d'expression sécrétoire. La protéase putative de type cathepsine L caractérisée ici peut représenter un outil intéressant à des fins de diagnostic, de conception de vaccins ou une nouvelle cible pharmacologique pour une intervention antiparasitaire.

The root-knot nematode effector MiEFF12 targets the host ER quality control system to suppress immune responses and allow parasitism.

Root-knot nematodes (RKNs) are microscopic parasitic worms able to infest the roots of thousands of plant species, causing massive crop yield losses worldwide. They evade the plant's immune system and manipulate plant cell physiology and metabolism to transform a few root cells into giant cells, which serve as feeding sites for the nematode. RKN parasitism is facilitated by the secretion in planta of effector molecules, mostly proteins that hijack host cellular processes. We describe here a conserved RKN-specific effector, effector 12 (EFF12), that is synthesized exclusively in the oesophageal glands of the nematode, and we demonstrate its function in parasitism. In the plant, MiEFF12 localizes to the endoplasmic reticulum (ER). A combination of RNA-sequencing analysis and immunity-suppression bioassays revealed the contribution of MiEFF12 to the modulation of host immunity. Yeast two-hybrid, split luciferase and co-immunoprecipitation approaches identified an essential component of the ER quality control system, the Solanum lycopersicum plant bap-like (PBL), and basic leucine zipper 60 (BZIP60) proteins as host targets of MiEFF12. Finally, silencing the PBL genes in Nicotiana benthamiana decreased susceptibility to Meloidogyne incognita infection. Our results suggest that EFF12 manipulates PBL function to modify plant immune responses to allow parasitism.

Effect of Hydroxyurea on Morphology, Proliferation, and Protein Expression on Taenia crassiceps WFU Strain.

Flatworms are known for their remarkable regenerative ability, one which depends on totipotent cells known as germinative cells in cestodes. Depletion of germinative cells with hydroxyurea (HU) affects the regeneration of the parasite. Here, we studied the reduction and recovery of germinative cells in T. crassiceps cysticerci after HU treatment (25 mM and 40 mM of HU for 6 days) through in vitro assays. Viability and morphological changes were evaluated. The recovery of cysticerci's mobility and morphology was evaluated at 3 and 6 days, after 6 days of treatment. The number of proliferative cells was evaluated using EdU. Our results show morphological changes in the size, shape, and number of evaginated cysticerci at the 40 mM dose. The mobility of cysticerci was lower after 6 days of HU treatment at both concentrations. On days 3 and 6 of recovery after 25 mM of HU treatment, a partial recovery of the proliferative cells was observed. Proteomic and Gene Ontology analyses identified modifications in protein groups related to DNA binding, DNA damage, glycolytic enzymes, cytoskeleton, skeletal muscle, and RNA binding.

Spirocerca lupi draft genome, vaccine and anthelmintic targets.

Spirocerca lupi is a parasitic nematode affecting predominantly domestic dogs. It causes spirocercosis, a disease that is often fatal. The assembled draft genome of S. lupi consists of 13,627 predicted protein-coding genes and is approximately 150 Mb in length. Several known anthelmintic gene targets such as for ß-Tubulin, glutamate, and GABA receptors as well as known vaccine gene targets such as cysteine protease inhibitor and cytokines were identified in S. lupi by comparing orthologs of C. elegans anthelmintic gene targets as well as orthologs to known vaccine candidates. New anthelmintic targets were predicted through an inclusion-exclusion strategy and new vaccine targets were predicted through an immunoinformatics approach. New anthelminthic targets include DNA-directed RNA polymerases, chitin synthase, polymerases, and other enzymes. New vaccine targets include cuticle collagens. These gene targets provide a starting platform for new drug identification and vaccine design.

Unraveling the Roles of Neuropeptides in the Chemosensation of the Root-Knot Nematode Meloidogyne javanica.

The identification of novel drug targets in plant-parasitic nematodes (PPNs) is imperative due to the loss of traditional nematicides and a lack of replacements. Chemosensation, which is pivotal for PPNs in locating host roots, has become a focus in nematode behavioral research. However, its underlying molecular basis is still indistinct in such a diverse group of PPNs. To characterize genes participating in chemosensation in the Javanese root-knot nematode Meloidogyne javanica, RNA-sequencing of the second-stage juveniles (J2s) treated with tomato root exudate (TRE) for 1 h and 6 h was performed. Genes related to chemosensation in M. javanica mainly responded to TRE treatment at 1 h. Moreover, a gene ontology (GO) analysis underscored the significance of the neuropeptide G protein-coupled receptor signaling pathway. Consequently, the repertoire of putative neuropeptides in M. javanica, including FMRFamide-like peptides (FLPs), insulin-like peptides (ILPs), and neuropeptide-like peptides (NLPs), were outlined based on a homology analysis. The gene Mjflp-14a, harboring two neuropeptides, was significantly up-regulated at 1 h TRE treatment. Through peptide synthesis and J2 treatment, one of the two neuropeptides (MjFLP-14-2) was proven to influence the J2 chemotaxis towards tomato root tips. Overall, our study reinforces the potential of nematode neuropeptides as novel targets and tools for root-knot nematode control.

Molecular insights and antibody response to Dr20/22 in dogs naturally infected with Dirofilaria repens.

Subcutaneous dirofilariasis, caused by the parasitic nematode Dirofilaria repens, is a growing concern in Europe, affecting both dogs and humans. This study focused on D. repens Dr20/22, a protein encoded by an alt (abundant larval transcript) gene family. While well-documented in L3 larvae of other filariae species, this gene family had not been explored in dirofilariasis. The research involved cloning Dr20/22 cDNA, molecular characterization, and evaluating its potential application in the diagnosis of dirofilariasis. Although Real-Time analysis revealed mRNA expression in both adult worms and microfilariae, the native protein remained undetected in lysates from both developmental stages. This suggests the protein's specificity for L3 larvae and may be related to a process called SLTS (spliced leader trans-splicing), contributing to stage-specific gene expression. The specificity of the antigen for invasive larvae positions it as a promising early marker for dirofilariasis. However, ELISA tests using sera from infected and uninfected dogs indicated limited diagnostic utility. While further research is required, our findings contribute to a deeper understanding of the molecular and immunological aspects of host-parasite interactions and could offer insights into the parasite's strategies for evading the immune system.

An insulin-like signalling pathway model for Fasciola gigantica.

BACKGROUND: The insulin/insulin-like signalling (IIS) pathway is common in mammals and invertebrates, and the IIS pathway is unknown in Fasciola gigantica. In the present study, the IIS pathway was reconstructed in F. gigantica. We defined the components involved in the IIS pathway and investigated the transcription profiles of these genes for all developmental stages of F. gigantica. In addition, the presence of these components in excretory and secretory products (ESPs) was predicted via signal peptide annotation. RESULTS: The core components of the IIS pathway were detected in F. gigantica. Among these proteins, one ligand (FgILP) and one insulin-like molecule binding protein (FgIGFBP) were analysed. Interestingly, three receptors (FgIR-1/FgIR-2/FgIR-3) were detected, and a novel receptor, FgIR-3, was screened, suggesting novel functions. Fg14-3-3ζ, Fgirs, and Fgpp2a exhibited increased transcription in 42-day-old juveniles and 70-day-old juveniles, while Fgilp, Fgigfb, Fgsgk-1, Fgakt-1, Fgir-3, Fgpten, and Fgaap-1 exhibited increased transcription in metacercariae. FgILP, FgIGFBP, FgIR-2, FgIR-3, and two transcription factors (FgHSF-1 and FgSKN-1) were predicted to be present in FgESPs, indicating their exogenous roles. CONCLUSIONS: This study helps to elucidate the signal transduction pathway of IIS in F. gigantica, which will aid in understanding the interaction between flukes and hosts, as well as in understanding fluke developmental regulation, and will also lay a foundation for further characterisation of the IIS pathways of trematodes.

A reanalysis and integration of transcriptomics and proteomics datasets unveil novel drug targets for Mekong schistosomiasis.

Schistosomiasis, caused by Schistosoma trematodes, is a significant global health concern, particularly affecting millions in Africa and Southeast Asia. Despite efforts to combat it, the rise of praziquantel (PZQ) resistance underscores the need for new treatment options. Protein kinases (PKs) are vital in cellular signaling and offer potential as drug targets. This study focused on focal adhesion kinase (FAK) as a candidate for anti-schistosomal therapy. Transcriptomic and proteomic analyses of adult S. mekongi worms identified FAK as a promising target due to its upregulation and essential role in cellular processes. Molecular docking simulations assessed the binding energy of FAK inhibitors to Schistosoma FAK versus human FAK. FAK inhibitor 14 and PF-03814735 exhibited strong binding to Schistosoma FAK with minimal binding for human FAK. In vitro assays confirmed significant anti-parasitic activity against S. mekongi, S. mansoni, and S. japonicum, comparable to PZQ, with low toxicity in human cells, indicating potential safety. These findings highlight FAK as a promising target for novel anti-schistosomal therapies. However, further research, including in vivo studies, is necessary to validate efficacy and safety before clinical use. This study offers a hopeful strategy to combat schistosomiasis and reduce its global impact.

Annexin gene family in Spirometra mansoni (Cestoda: Diphyllobothriidae) and its phylogenetic pattern among Platyhelminthes of medical interest.

The plerocercoid larvae of Spirometra mansoni are etiological agents of human and animal sparganosis. Annexins are proteins with important roles in parasites. However, our knowledge of annexins in S. mansoni is still inadequate. In this study, 18 new members of the Annexin (ANX) family were characterized in S. mansoni. The clustering analysis demonstrated that all the SmANXs were divided into two main classes, consistent with the patterns of conserved motif organization. The 18 SmANXs were detected at all developmental stages (plerocercoid, adult, and egg) and displayed ubiquitous but highly variable expression patterns in all tissues/organs studied. The representative member rSmANX18 was successfully cloned and expressed. The protein was immunolocalized in the tegument and parenchyma of the plerocercoid and in the tegument, parenchyma, uterus and egg shell of adult worms. The recombinant protein can bind phospholipids with high affinity in a Ca2+-dependent manner, shows high anticoagulant activity and combines with FITC to recognize apoptotic cells. Annexin gene polymorphism and conservative core motif permutation were found in both cestodes and trematodes. SmANXs also revealed high genetic diversity among Platyhelminthes of medical interest. Our findings lay a foundation for further studies on the biological functions of ANXs in S. mansoni as well as other taxa in which ANXs occur.

Title: La famille des gènes des annexines chez Spirometra mansoni (Cestoda : Diphyllobothriidae) et son schéma phylogénétique parmi les Plathelminthes d'intérêt médical. Abstract: Les larves plérocercoïdes de Spirometra mansoni sont des agents étiologiques de la sparganose humaine et animale. Les annexines sont des protéines jouant un rôle important chez les parasites. Cependant, nos connaissances sur les annexines chez S. mansoni sont encore insuffisantes. Dans cette étude, 18 nouveaux membres de la famille des annexines (ANX) ont été caractérisés chez S. mansoni. L'analyse de regroupement a démontré que tous les SmANX étaient divisées en deux classes principales, ce qui correspond aux modèles d'organisation des motifs conservés. Les 18 SmANX ont été détectées à tous les stades de développement (plérocercoïde, adulte et œuf) et présentaient des modèles d'expression omniprésents mais très variables dans tous les tissus/organes étudiés. Le membre représentatif rSmANX18 a été cloné et exprimé avec succès. La protéine a été immunolocalisée dans le tégument et le parenchyme du plérocercoïde ainsi que dans le tégument, le parenchyme, l'utérus et la coquille d'œuf des vers adultes. La protéine recombinante peut se lier aux phospholipides avec une affinité élevée de manière dépendante du Ca2+, présente une activité anticoagulante élevée et se combine avec le FITC pour reconnaître les cellules apoptotiques. Un polymorphisme du gène de l'annexine et une permutation conservatrice du motif central ont été trouvés chez les cestodes et les trématodes. Les SmANX ont également révélé une grande diversité génétique parmi les Plathelminthes d'intérêt médical. Nos résultats jettent les bases pour des études plus approfondies sur les fonctions biologiques des ANX chez S. mansoni ainsi que dans d'autres taxons dans lesquels les ANX sont présents.

Regulatory effects of Trichinella spiralis serpin-type serine protease inhibitor on endoplasmic reticulum stress and oxidative stress in host intestinal epithelial cells.

Endoplasmic reticulum stress (ERS) and oxidative stress (OS) are adaptive responses of the body to stressor stimulation. Although it has been verified that Trichinella spiralis (T. spiralis) can induce ERS and OS in the host, their association is still unclear. Therefore, this study explored whether T. spiralis-secreted serpin-type serine protease inhibitor (TsAdSPI) is involved in regulating the relationship between ERS and OS in the host intestine. In this study, mice jejunum and porcine small intestinal epithelial cells (IECs) were detected using qPCR, western blotting, immunohistochemistry (IHC), immunofluorescence (IF), and detection kits. The results showed that ERS- and OS-related indexes changed significantly after TsAdSPI stimulation, and Bip was located in IECs, indicating that TsAdSPI could induce ERS and OS in IECs. After the use of an ERS inhibitor, OS-related indexes were inhibited, suggesting that TsAdSPI-induced OS depends on ERS. When the three ERS signalling pathways, ATF6, IRE1, and PERK, were sequentially suppressed, OS was only regulated by the PERK pathway, and the PERK-eif2α-CHOP-ERO1α axis played a key role. Similarly, the expression of ERS-related indexes and the level of intracellular Ca2+ were inhibited after adding the OS inhibitor, and the expression of ERS-related indexes decreased significantly after inhibiting calcium transfer. This finding indicated that TsAdSPI-induced OS could affect ERS by promoting Ca2+ efflux from the endoplasmic reticulum. The detection of the ERS and OS sequences revealed that OS occurred before ERS. Finally, changes in apoptosis-related indexes were detected, and the results indicated that TsAdSPI-induced ERS and OS could regulate IEC apoptosis. In conclusion, TsAdSPI induced OS after entering IECs, OS promoted ERS by enhancing Ca2+ efflux, and ERS subsequently strengthened OS by activating the PERK-eif2α-CHOP-ERO1α axis. ERS and OS induced by TsAdSPI synergistically promoted IEC apoptosis. This study provides a foundation for exploring the invasion mechanism of T. spiralis and the pathogenesis of host intestinal dysfunction after invasion.

Silencing Ditylenchus destructor cathepsin L-like cysteine protease has negative pleiotropic effect on nematode ontogenesis.

Ditylenchus destructor is a migratory plant-parasitic nematode that severely harms many agriculturally important crops. The control of this pest is difficult, thus efficient strategies for its management in agricultural production are urgently required. Cathepsin L-like cysteine protease (CPL) is one important protease that has been shown to participate in various physiological and pathological processes. Here we decided to characterize the CPL gene (Dd-cpl-1) from D. destructor. Analysis of Dd-cpl-1 gene showed that Dd-cpl-1 gene contains a signal peptide, an I29 inhibitor domain with ERFNIN and GNFD motifs, and a peptidase C1 domain with four conserved active residues, showing evolutionary conservation with other nematode CPLs. RT-qPCR revealed that Dd-cpl-1 gene displayed high expression in third-stage juveniles (J3s) and female adults. In situ hybridization analysis demonstrated that Dd-cpl-1 was expressed in the digestive system and reproductive organs. Silencing Dd-cpl-1 in 1-cell stage eggs of D. destructor by RNAi resulted in a severely delay in development or even in abortive morphogenesis during embryogenesis. The RNAi-mediated silencing of Dd-cpl-1 in J2s and J3s resulted in a developmental arrest phenotype in J3 stage. In addition, silencing Dd-cpl-1 gene expression in female adults led to a 57.43% decrease in egg production. Finally, Dd-cpl-1 RNAi-treated nematodes showed a significant reduction in host colonization and infection. Overall, our results indicate that Dd-CPL-1 plays multiple roles in D. destructor ontogenesis and could serve as a new potential target for controlling D. destructor.

Multi-epitope protein production and its application in the diagnosis of opisthorchiasis.

BACKGROUND: Opisthorchiasis and cholangiocarcinoma (CCA) continue to be public health concerns in many Southeast Asian countries. Although the prevalence of opisthorchiasis is declining, reported cases tend to have a light-intensity infection. Therefore, early detection by using sensitive methods is necessary. Several sensitive methods have been developed to detect opisthorchiasis. The immunological detection of antigenic proteins has been proposed as a sensitive method for examining opisthorchiasis. METHODS: The Opisthorchis viverrini antigenic proteins, including cathepsin B (OvCB), asparaginyl endopeptidase (OvAEP), and cathepsin F (OvCF), were used to construct multi-antigenic proteins. The protein sequences of OvCB, OvAEP, and OvCF, with a high probability of B cell epitopes, were selected using BepiPred 1.0 and the IEDB Analysis Resource. These protein fragments were combined to form OvCB_OvAEP_OvCF recombinant DNA, which was then used to produce a recombinant protein in Escherichia coli strain BL21(DE3). The potency of the recombinant protein as a diagnostic target for opisthorchiasis was assessed using immunoblotting and compared with that of the gold standard method, the modified formalin-ether concentration technique. RESULTS: The recombinant OvCB_OvAEP_OvCF protein showed strong reactivity with total immunoglobulin G (IgG) antibodies against light-intensity O. viverrini infections in the endemic areas. Consequently, a high sensitivity (100%) for diagnosing opisthorchiasis was reported. However, cross-reactivity with sera from other helminth and protozoan infections (including taeniasis, strongyloidiasis, giardiasis, E. coli infection, enterobiasis, and mixed infection of Echinostome spp. and Taenia spp.) and no reactivity with sera from patients with non-parasitic infections led to a reduced specificity of 78.4%. In addition, the false negative rate (FNR), false positive rate (FPR), positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy were 0%, 21.6%, 81.4%, 100%, and 88.9%, respectively. CONCLUSIONS: The high sensitivity of the recombinant OvCB_OvAEP_OvCF protein in detecting opisthorchiasis demonstrates its potential as an opisthorchiasis screening target. Nonetheless, research on reducing cross-reactivity should be undertaken by detecting other antibodies in other sample types, such as saliva, urine, and feces.

Temperature Effects on Expression Levels of hsp Genes in Eggs and Second-Stage Juveniles of Meloidogyne hapla Chitwood, 1949.

Meloidogyne hapla is one of the most important nematode pathogens. It is a sedentary, biotrophic parasite of plants that overwinters in the soil or in diseased roots. The development of M. hapla is temperature dependent. Numerous studies have been performed on the effect of temperature on the development of M. hapla, but only a few of them analyzed the heat shock protein (hsp) genes. The aim of the study was to perform expression profiling of eight hsp genes (Mh-hsp90, Mh-hsp1, Mh-hsp4, Mh-hsp6, Mh-hsp60, Mh-dnj19, Mh-hsp43, and Mh-hsp12.2) at two development stages of M. hapla, i.e., in eggs and second-stage juveniles (J2). The eggs and J2 were incubated under cold stress (5 °C), heat stress (35 °C, 40 °C), and non-stress (10 °C, 20 °C, and 30 °C) conditions. Expression profiling was performed by qPCR. It was demonstrated that only two genes, Mh-hsp60 and Mh-dnj19, have been upregulated by heat and cold stress at both development stages. Heat stress upregulated the expression of more hsp genes than cold stress did. The level of upregulation of most hsp genes was more marked in J2 than in eggs. The obtained results suggest that the Mh-hsp90 and Mh-hsp1 genes can be used as bioindicators of environmental impacts on nematodes of the Meloidogyne genus.

Genome-wide RNA interference of the nhr gene family in barber's pole worm identified members crucial for larval viability in vitro.

Nuclear hormone receptors (NHRs) are emerging target candidates against nematode infection and resistance. However, there is a lack of comprehensive information on NHR-coding genes in parasitic nematodes. In this study, we curated the nhr gene family for 60 major parasitic nematodes from humans and animals. Compared with the free-living model organism Caenorhabditis elegans, a remarkable contraction of the nhr family was revealed in parasitic species, with genetic diversification and conservation unveiled among nematode Clades I (10-13), III (16-42), IV (33-35) and V (25-64). Using an in vitro biosystem, we demonstrated that 40 nhr genes in a blood-feeding nematode Haemonchus contortus (clade V; barber's pole worm) were responsive to host serum and one nhr gene (i.e., nhr-64) was consistently stimulated by anthelmintics (i.e., ivermectin, thiabendazole and levamisole); Using a high-throughput RNA interference platform, we knocked down 43 nhr genes of H. contortus and identified at least two genes that are required for the viability (i.e., nhr-105) and development (i.e., nhr-17) of the infective larvae of this parasitic nematode in vitro. Harnessing this preliminary functional atlas of nhr genes for H. contortus will prime the biological studies of this gene family in nematode genetics, infection, and anthelmintic metabolism within host animals, as well as the promising discovery of novel intervention targets.

Proteomic analysis of Neobenedenia sp. and Rhabdosynochus viridisi (Monogenea, Monopisthocotylea): Insights into potential vaccine targets and diagnostic markers for finfish aquaculture.

Monogeneans are parasitic flatworms that represent a significant threat to the aquaculture industry. Species like Neobenedenia melleni (Capsalidae) and Rhabdosynochus viridisi (Diplectanidae) have been identified as causing diseases in farmed fish. In the past years, molecular research on monogeneans of the subclass Monopisthocotylea has focused on the generation of genomic and transcriptomic information and the identification in silico of some protein families of veterinary interest. Proteomic analysis has been suggested as a powerful tool to investigate proteins in parasites and identify potential targets for vaccine development and diagnosis. To date, the proteomic dataset for monogeneans has been restricted to a species of the subclass Polyopisthocotylea, while in monopisthocotyleans there is no proteomic data. In this study, we present the first proteomic data on two monopisthocotylean species, Neobenedenia sp. and R. viridisi, obtained from three distinct sample types: tissue, excretory-secretory products (ESPs), and eggs. A total of 1691 and 1846 expressed proteins were identified in Neobenedenia sp. and R. viridisi, respectively. The actin family was the largest protein family, followed by the tubulin family and the heat shock protein 70 (HSP70) family. We focused mainly on ESPs because they are important to modulate the host immune system. We identified proteins of the actin, tubulin, HSP70 and HSP90 families in both tissue and ESPs, which have been recognized for their antigenic activities in parasitic flatworms. Furthermore, our study uncovered the presence of proteins within ESPs, such as annexin, calcium-binding protein, fructose bisphosphate aldolase, glutamate dehydrogenase, myoferlin, and paramyosin, that are targets for immunodiagnostic and vaccine development and hold paramount relevance in veterinary medicine. This study expands our knowledge of monogeneans and identified proteins that, in other platyhelminths are potential targets for vaccines and drug discovery.