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The ubiquitin-mediated pathway has been comprehensively explored in the free-living nematode Caenorhabditis elegans, but very little is known about this pathway in parasitic nematodes. Here, we inferred the ubiquitination pathway for an economically significant and pathogenic nematode - Haemonchus contortus - using abundant resources available for C. elegans. We identified 215 genes encoding ubiquitin (Ub; n = 3 genes), ubiquitin-activating enzyme (E1; one), -conjugating enzymes (E2s; 21), ligases (E3s; 157) and deubiquitinating enzymes (DUBs; 33). With reference to C. elegans, Ub, E1 and E2 were relatively conserved in sequence and structure, and E3s and DUBs were divergent, likely reflecting functional and biological uniqueness in H. contortus. Most genes encoding ubiquitination pathway components exhibit high transcription in the egg compared with other stages, indicating marked protein homeostasis in this early developmental stage. The ubiquitination pathway model constructed for H. contortus provides a foundation to explore the ubiquitin-proteasome system, crosstalk between autophagy and the proteasome system, and the parasite-host interactions. Selected E3 and DUB proteins which are very divergent in sequence and structure from host homologues or entirely unique to H. contortus and related parasitic nematodes may represent possible anthelmintic targets.

In this study, we explored the molecular alterations in the developmental switch from the L3 to the exsheathed L3 (xL3) and to the L4 stage of Haemonchus contortus in vitro using an integrated transcriptomic, proteomic and bioinformatic approach. Totals of 9,754 mRNAs, 88 microRNAs (miRNAs) and 1,591 proteins were identified, and 6,686 miRNA-mRNA pairs inferred in all larval stages studied. Approximately 16% of transcripts in the combined transcriptome (representing all three larval stages) were expressed as proteins, and there were positive correlations (r = 0.39-0.44) between mRNA transcription and protein expression in the three distinct developmental stages of the parasite. Of the predicted targets, 1,019 (27.0%) mRNA transcripts were expressed as proteins, and there was a negative correlation (r = -0.60 to -0.50) in the differential mRNA transcription and protein expression between developmental stages upon pairwise comparison. The changes in transcription (mRNA and miRNA) and protein expression from the free-living to the parasitic life cycle phase of H. contortus related to enrichments in biological pathways associated with metabolism (e.g., carbohydrate and lipid degradation, and amino acid metabolism), environmental information processing (e.g., signal transduction, signalling molecules and interactions) and/or genetic information processing (e.g., transcription and translation). Specifically, fatty acid degradation, steroid hormone biosynthesis and the Rap1 signalling pathway were suppressed, whereas transcription, translation and protein processing in the endoplasmic reticulum were upregulated during the transition from the free-living L3 to the parasitic xL3 and L4 stages of the nematode in vitro. Dominant post-transcriptional regulation was inferred to elicit these changes, and particular miRNAs (e.g., hco-miR-34 and hco-miR-252) appear to play roles in stress responses and/or environmental adaptations during developmental transitions of H. contortus. Taken together, these integrated results provide a comprehensive insight into the developmental biology of this important parasite at the molecular level in vitro. The approach applied here to H. contortus can be readily applied to other parasitic nematodes.

Roundworms belong to a diverse phylum (Nematoda) which is comprised of many parasitic species including whipworms (genus Trichuris). These worms have adapted to a biological niche within the host and exhibit unique morphological characteristics compared with other nematodes. Although these adaptations are known, the underlying molecular mechanisms remain elusive. The availability of genomes and transcriptomes of some whipworms now enables detailed studies of their molecular biology. Here, we defined and curated the full complement of an important class of enzymes, the protein kinases (kinomes) of two species of Trichuris, using an advanced and integrated bioinformatic pipeline. We investigated the transcription of Trichuris suis kinase genes across developmental stages, sexes and tissues, and reveal that selectively transcribed genes can be linked to central roles in developmental and reproductive processes. We also classified and functionally annotated the curated kinomes by integrating evidence from structural modelling and pathway analyses, and compared them with other curated kinomes of phylogenetically diverse nematode species. Our findings suggest unique adaptations in signalling processes governing worm morphology and biology, and provide an important resource that should facilitate experimental investigations of kinases and the biology of signalling pathways in nematodes.

We explored molecules involved in in vitro exsheathment of Oesophagostomum dentatum L3s using a proteomic-transcriptomic-bioinformatic approach. Analysis of L3s before, during and after exsheathment identified 11 proteins that were over-expressed exclusively during exsheathment. These proteins (including key enzymes, heat shock, structural and nematode-specific proteins) were inferred to be involved in development, metabolism, structure, motility and/or host-parasite interactions. Some of these molecules represented homologues linked to entry into and exit from the dauer stage in the free-living nematode Caenorhabditis elegans. The approach established here provides a basis for investigations of ecdysis in other strongylid nematodes.

Despite right open reading frame kinases being essential for life, their functions, substrates and cellular pathways remain enigmatic. In the present study, gene structures were characterised for 26 right open reading frame kinase from draft genomes of parasitic and free-living nematodes. RNA-seq transcription profiles of right open reading frame kinase genes were investigated for selected parasitic nematodes and showed that these kinases are transcribed in developmental stages that infect their mammalian host. Three-dimensional structural models of Caenorhabditis elegans right open reading frame kinases were predicted, and elucidated functional domains and conserved regions in nematode homologs. These findings provide prospects for functional studies of RIO kinase genes in C. elegans and an opportunity for the design and validation of nematode-specific inhibitors of these enzymes in socioeconomic parasitic worms.

A more thorough understanding of the immunological interactions between Fasciola spp. and their hosts is required if we are to develop new immunotherapies to control fasciolosis. Deeper knowledge of the antigens that are the target of the acquired immune responses of definitive hosts against both Fasciola hepatica and Fasciola gigantica will potentially identify candidate vaccine antigens. Indonesian Thin Tail sheep express a high level of acquired immunity to infection by F. gigantica within 4weeks of infection and antibodies in Indonesian Thin Tail sera can promote antibody-dependent cell-mediated cytotoxicity against the surface tegument of juvenile F. gigantica in vitro. Given the high protein sequence similarity between F. hepatica and F. gigantica, we hypothesised that antibody from F. gigantica-infected sheep could be used to identify the orthologous proteins in the tegument of F. hepatica. Purified IgG from the sera of F. gigantica-infected Indonesian Thin Tail sheep collected pre-infection and 4weeks p.i. were incubated with live adult F. hepatica ex vivo and the immunosloughate (immunoprecipitate) formed was isolated and analysed via liquid chromatography-electrospray ionisation-tandem mass spectrometry to identify proteins involved in the immune response. A total of 38 proteins were identified at a significantly higher abundance in the immunosloughate using week 4 IgG, including eight predicted membrane proteins, 20 secreted proteins, nine proteins predicted to be associated with either the lysosomes, the cytoplasm or the cytoskeleton and one protein with an unknown cellular localization. Three of the membrane proteins are transporters including a multidrug resistance protein, an amino acid permease and a glucose transporter. Interestingly, a total of 21 of the 38 proteins matched with proteins recently reported to be associated with the proposed small exosome-like extracellular vesicles of adult F. hepatica, suggesting that the Indonesian Thin Tail week 4 IgG is either recognising individual proteins released from extracellular vesicles or is immunoprecipitating intact exosome-like extracellular vesicles. Five extracellular vesicle membrane proteins were identified including two proteins predicted to be associated with vesicle transport/ exocytosis (VPS4, vacuolar protein sorting-associated protein 4b and the Niemann-Pick C1 protein). RNAseq analysis of the developmental transcription of the 38 immunosloughate proteins showed that the sequences are expressed over a wide abundance range with 21/38 transcripts expressed at a relatively high level from metacercariae to the adult life cycle stage. A notable feature of the immunosloughates was the absence of cytosolic proteins which have been reported to be secreted markers for damage to adult flukes incubated in vitro, suggesting that the proteins observed are not inadvertent contaminants leaking from damaged flukes ex vivo. The identification of tegument protein antigens shared between F. gigantica and F. hepatica is beneficial in terms of the possible development of a dual purpose vaccine effective against both fluke species.

In the present study we sequenced or re-sequenced, assembled and annotated 15 mitochondrial genomes representing the 12 currently recognised taxa of Trichinella using a deep sequencing-coupled approach. We then defined and compared the gene order in individual mitochondrial genomes (∼14 to 17.7 kb), evaluated genetic differences among species/genotypes and re-assessed the relationships among these taxa using the mitochondrial nucleic acid or amino acid sequence data sets. In addition, a rich source of mitochondrial genetic markers was defined that could be used in future systematic, epidemiological and population genetic studies of Trichinella. The sequencing-bioinformatic approach employed herein should be applicable to a wide range of eukaryotic parasites.

Amoebic gill disease (AGD) is a potentially fatal disease of some marine fish. Two amphizoic amoebae Neoparamoeba pemaquidensis and Neoparamoeba branchiphila have been cultured from AGD-affected fish, yet it is not known if one or both are aetiological agents. Here, we PCR amplified the 18S rRNA gene of non-cultured, gill-derived (NCGD) amoebae from AGD-affected Atlantic salmon (Salmo salar) using N. pemaquidensis and N. branchiphila-specific oligonucleotides. Variability in PCR amplification led to comparisons of 18S rRNA and 28S rRNA gene sequences from NCGD and clonal cultured, gill-derived (CCGD) N. pemaquidensis and N. branchiphila. Phylogenetic analyses inferred from either 18S or 28S rRNA gene sequences unambiguously segregated a lineage consisting of NCGD amoebae from other members of the genus Neoparamoeba. Species-specific oligonucleotide probes that hybridise 18S rRNA were designed, validated and used to probe gill tissue from AGD-affected Atlantic salmon. The NCGD amoebae-specific probe bound AGD-associated amoebae while neither N. pemaquidensis nor N. branchiphila were associated with AGD-lesions. Together, these data indicate that NCGD amoebae are a new species, designated Neoparamoeba perurans n.sp. and this is the predominant aetiological agent of AGD of Atlantic salmon cultured in Tasmania, Australia.