Publications by authors named "Pasi K Korhonen"

74 Publications

Harnessing model organism genomics to underpin the machine learning-based prediction of essential genes in eukaryotes - Biotechnological implications.

Biotechnol Adv 2021 Aug 27:107822. Epub 2021 Aug 27.

Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria 3010, Australia. Electronic address:

The availability of high-quality genomes and advances in functional genomics have enabled large-scale studies of essential genes in model eukaryotes, including the 'elegant worm' (Caenorhabditis elegans; Nematoda) and the 'vinegar fly' (Drosophila melanogaster; Arthropoda). However, this is not the case for other, much less-studied organisms, such as socioeconomically important parasites, for which functional genomic platforms usually do not exist. Thus, there is a need to develop innovative techniques or approaches for the prediction, identification and investigation of essential genes. A key approach that could enable the prediction of such genes is machine learning (ML). Here, we undertake an historical review of experimental and computational approaches employed for the characterisation of essential genes in eukaryotes, with a particular focus on model ecdysozoans (C. elegans and D. melanogaster), and discuss the possible applicability of ML-approaches to organisms such as socioeconomically important parasites. We highlight some recent results showing that high-performance ML, combined with feature engineering, allows a reliable prediction of essential genes from extensive, publicly available 'omic data sets, with major potential to prioritise such genes (with statistical confidence) for subsequent functional genomic validation. These findings could 'open the door' to fundamental and applied research areas. Evidence of some commonality in the essential gene-complement between these two organisms indicates that an ML-engineering approach could find broader applicability to ecdysozoans such as parasitic nematodes or arthropods, provided that suitably large and informative data sets become/are available for proper feature engineering, and for the robust training and validation of algorithms. This area warrants detailed exploration to, for example, facilitate the identification and characterisation of essential molecules as novel targets for drugs and vaccines against parasitic diseases. This focus is particularly important, given the substantial impact that such diseases have worldwide, and the current challenges associated with their prevention and control and with drug resistance in parasite populations.
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http://dx.doi.org/10.1016/j.biotechadv.2021.107822DOI Listing
August 2021

High-Throughput Phenotypic Assay to Screen for Anthelmintic Activity on .

Pharmaceuticals (Basel) 2021 Jun 26;14(7). Epub 2021 Jun 26.

Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC 3010, Australia.

Parasitic worms cause very significant diseases in animals and humans worldwide, and their control is critical to enhance health, well-being and productivity. Due to widespread drug resistance in many parasitic worms of animals globally, there is a major, continuing demand for the discovery and development of anthelmintic drugs for use to control these worms. Here, we established a practical, cost-effective and semi-automated high throughput screening (HTS) assay, which relies on the measurement of motility of larvae of the barber's pole worm () using infrared light-interference. Using this assay, we screened 80,500 small molecules and achieved a hit rate of 0.05%. We identified three small molecules that reproducibly inhibited larval motility and/or development (IC values of ~4 to 41 µM). Future work will critically assess the potential of selected hits as candidates for subsequent optimisation or repurposing against parasitic nematodes. This HTS assay has a major advantage over most previous assays in that it achieves a ≥ 10-times higher throughput (i.e., 10,000 compounds per week), and is thus suited to the screening of libraries of tens of thousands to hundreds of thousands of compounds for subsequent hit-to-lead optimisation or effective repurposing and development. The current assay should be adaptable to many socioeconomically important parasitic nematodes, including those that cause neglected tropical diseases (NTDs). This aspect is of relevance, given the goals of the World Health Organization (WHO) Roadmap for NTDs 2021-2030, to develop more effective drugs and drug combinations to improve patient outcomes and circumvent the ineffectiveness of some current anthelmintic drugs and possible drug resistance.
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http://dx.doi.org/10.3390/ph14070616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8308562PMC
June 2021

Cross-Predicting Essential Genes between Two Model Eukaryotic Species Using Machine Learning.

Int J Mol Sci 2021 May 11;22(10). Epub 2021 May 11.

Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC 3010, Australia.

Experimental studies of and have contributed substantially to our understanding of molecular and cellular processes in metazoans at large. Since the publication of their genomes, functional genomic investigations have identified genes that are essential or non-essential for survival in each species. Recently, a range of features linked to gene essentiality have been inferred using a machine learning (ML)-based approach, allowing essentiality predictions within a species. Nevertheless, predictions between species are still elusive. Here, we undertake a comprehensive study using ML to discover and validate features of essential genes common to both and . We demonstrate that the cross-species prediction of gene essentiality is possible using a subset of features linked to nucleotide/protein sequences, protein orthology and subcellular localisation, single-cell RNA-seq, and histone methylation markers. Complementary analyses showed that essential genes are enriched for transcription and translation functions and are preferentially located away from heterochromatin regions of and chromosomes. The present work should enable the cross-prediction of essential genes between model and non-model metazoans.
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http://dx.doi.org/10.3390/ijms22105056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8150380PMC
May 2021

Dipylidium caninum draft genome - a new resource for comparative genomic and genetic explorations of flatworms.

Genomics 2021 May 4;113(3):1272-1280. Epub 2021 Mar 4.

State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China; College of Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, China. Electronic address:

Here, we present a draft genome of the tapeworm Dipylidium caninum (family Dipylidiidae) and compare it with other cestode genomes. This draft genome of D. caninum is 110 Mb in size, has a repeat content of ~13.4% and is predicted to encode ~10,000 protein-coding genes. We inferred excretory/secretory molecules (representing the secretome), other key groups of proteins (including peptidases, kinases, phosphatases, GTPases, receptors, transporters and ion-channels) and predicted potential intervention targets for future evaluation. Using 144 shared single-copy orthologous sequences, we investigated the genetic relationships of cestodes for which nuclear genomes are available. This study provides first insights into the molecular biology of D. caninum and a new resource for comparative genomic and genetic explorations of this and other flatworms.
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http://dx.doi.org/10.1016/j.ygeno.2021.02.019DOI Listing
May 2021

High-quality reference genome for Clonorchis sinensis.

Genomics 2021 May 4;113(3):1605-1615. Epub 2021 Mar 4.

Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria 3010, Australia.

The Chinese liver fluke, Clonorchis sinensis, causes the disease clonorchiasis, affecting ~35 million people in regions of China, Vietnam, Korea and the Russian Far East. Chronic clonorchiasis causes cholangitis and can induce a malignant cancer, called cholangiocarcinoma, in the biliary system. Control in endemic regions is challenging, and often relies largely on chemotherapy with one anthelmintic, called praziquantel. Routine treatment carries a significant risk of inducing resistance to this anthelmintic in the fluke, such that the discovery of new interventions is considered important. It is hoped that the use of molecular technologies will assist this endeavour by enabling the identification of drug or vaccine targets involved in crucial biological processes and/or pathways in the parasite. Although draft genomes of C. sinensis have been published, their assemblies are fragmented. In the present study, we tackle this genome fragmentation issue by utilising, in an integrated way, advanced (second- and third-generation) DNA sequencing and informatic approaches to build a high-quality reference genome for C. sinensis, with chromosome-level contiguity and curated gene models. This substantially-enhanced genome provides a resource that could accelerate fundamental and applied molecular investigations of C. sinensis, clonorchiasis and/or cholangiocarcinoma, and assist in the discovery of new interventions against what is a highly significant, but neglected disease-complex.
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http://dx.doi.org/10.1016/j.ygeno.2021.03.001DOI Listing
May 2021

Nanopore Sequencing Resolves Elusive Long Tandem-Repeat Regions in Mitochondrial Genomes.

Int J Mol Sci 2021 Feb 11;22(4). Epub 2021 Feb 11.

Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC 3010, Australia.

Long non-coding, tandem-repetitive regions in mitochondrial (mt) genomes of many metazoans have been notoriously difficult to characterise accurately using conventional sequencing methods. Here, we show how the use of a third-generation (long-read) sequencing and informatic approach can overcome this problem. We employed Oxford Nanopore technology to sequence genomic DNAs from a pool of adult worms of the carcinogenic parasite, , and used an informatic workflow to define the complete mt non-coding region(s). Using long-read data of high coverage, we defined six dominant mt genomes of 33.4 kb to 22.6 kb. Although no variation was detected in the order or lengths of the protein-coding genes, there was marked length (18.5 kb to 7.6 kb) and structural variation in the non-coding region, raising questions about the evolution and function of what might be a control region that regulates mt transcription and/or replication. The discovery here of the largest tandem-repetitive, non-coding region (18.5 kb) in a metazoan organism also raises a question about the completeness of some of the mt genomes of animals reported to date, and stimulates further explorations using a Nanopore-informatic workflow.
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http://dx.doi.org/10.3390/ijms22041811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918261PMC
February 2021

Molecular diagnosis of scabies using a novel probe-based polymerase chain reaction assay targeting high-copy number repetitive sequences in the Sarcoptes scabiei genome.

PLoS Negl Trop Dis 2021 02 24;15(2):e0009149. Epub 2021 Feb 24.

QIMR Berghofer Medical Research Institute, Brisbane, Australia.

Background: The suboptimal sensitivity and specificity of available diagnostic methods for scabies hampers clinical management, trials of new therapies and epidemiologic studies. Additionally, parasitologic diagnosis by microscopic examination of skin scrapings requires sample collection with a sharp scalpel blade, causing discomfort to patients and difficulty in children. Polymerase chain reaction (PCR)-based diagnostic assays, combined with non-invasive sampling methods, represent an attractive approach. In this study, we aimed to develop a real-time probe-based PCR test for scabies, test a non-invasive sampling method and evaluate its diagnostic performance in two clinical settings.

Methodology/principal Findings: High copy-number repetitive DNA elements were identified in draft Sarcoptes scabiei genome sequences and used as assay targets for diagnostic PCR. Two suitable repetitive DNA sequences, a 375 base pair microsatellite (SSR5) and a 606 base pair long tandem repeat (SSR6), were identified. Diagnostic sensitivity and specificity were tested using relevant positive and negative control materials and compared to a published assay targeting the mitochondrial cox1 gene. Both assays were positive at a 1:100 dilution of DNA from a single mite; no amplification was observed in DNA from samples from 19 patients with other skin conditions nor from house dust, sheep or dog mites, head and body lice or from six common skin bacterial and fungal species. Moderate sensitivity of the assays was achieved in a pilot study, detecting 5/7 (71.4% [95% CI: 29.0% - 96.3%]) of clinically diagnosed untreated scabies patients). Greater sensitivity was observed in samples collected by FLOQ swabs compared to skin scrapings.

Conclusions/significance: This newly developed qPCR assay, combined with the use of an alternative non-invasive swab sampling technique offers the possibility of enhanced diagnosis of scabies. Further studies will be required to better define the diagnostic performance of these tests.
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http://dx.doi.org/10.1371/journal.pntd.0009149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7939366PMC
February 2021

Combined use of feature engineering and machine-learning to predict essential genes in .

NAR Genom Bioinform 2020 Sep 22;2(3):lqaa051. Epub 2020 Jul 22.

Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria 3010, Australia.

Characterizing genes that are critical for the survival of an organism (i.e. essential) is important to gain a deep understanding of the fundamental cellular and molecular mechanisms that sustain life. Functional genomic investigations of the vinegar fly, , have unravelled the functions of numerous genes of this model species, but results from phenomic experiments can sometimes be ambiguous. Moreover, the features underlying gene essentiality are poorly understood, posing challenges for computational prediction. Here, we harnessed comprehensive genomic-phenomic datasets publicly available for and a machine-learning-based workflow to predict essential genes of this fly. We discovered strong predictors of such genes, paving the way for computational predictions of essentiality in less-studied arthropod pests and vectors of infectious diseases.
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http://dx.doi.org/10.1093/nargab/lqaa051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7671374PMC
September 2020

OGEE v3: Online GEne Essentiality database with increased coverage of organisms and human cell lines.

Nucleic Acids Res 2021 01;49(D1):D998-D1003

Key Laboratory of Molecular Biophysics of the Ministry of Education, Hubei Key Laboratory of Bioinformatics and Molecular-imaging, Center for Artificial Biology, Department of Bioinformatics and Systems Biology, College of Life Science and Technology, Huazhong University of Science and Technology (HUST), 430074 Wuhan, Hubei, China.

OGEE is an Online GEne Essentiality database. Gene essentiality is not a static and binary property, rather a context-dependent and evolvable property in all forms of life. In OGEE we collect not only experimentally tested essential and non-essential genes, but also associated gene properties that contributes to gene essentiality. We tagged conditionally essential genes that show variable essentiality statuses across datasets to highlight complex interplays between gene functions and environmental/experimental perturbations. OGEE v3 contains gene essentiality datasets for 91 species; almost doubled from 48 species in previous version. To accommodate recent advances on human cancer essential genes (as known as tumor dependency genes) that could serve as targets for cancer treatment and/or drug development, we expanded the collection of human essential genes from 16 cell lines in previous to 581. These human cancer cell lines were tested with high-throughput experiments such as CRISPR-Cas9 and RNAi; in total, 150 of which were tested by both techniques. We also included factors known to contribute to gene essentiality for these cell lines, such as genomic mutation, methylation and gene expression, along with extensive graphical visualizations for ease of understanding of these factors. OGEE v3 can be accessible freely at https://v3.ogee.info.
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http://dx.doi.org/10.1093/nar/gkaa884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7779042PMC
January 2021

High-quality nuclear genome for Sarcoptes scabiei-A critical resource for a neglected parasite.

PLoS Negl Trop Dis 2020 10 1;14(10):e0008720. Epub 2020 Oct 1.

Cell and Molecular Biology Department, Infectious Diseases Program, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.

The parasitic mite Sarcoptes scabiei is an economically highly significant parasite of the skin of humans and animals worldwide. In humans, this mite causes a neglected tropical disease (NTD), called scabies. This disease results in major morbidity, disability, stigma and poverty globally and is often associated with secondary bacterial infections. Currently, anti-scabies treatments are not sufficiently effective, resistance to them is emerging and no vaccine is available. Here, we report the first high-quality genome and transcriptomic data for S. scabiei. The genome is 56.6 Mb in size, has a a repeat content of 10.6% and codes for 9,174 proteins. We explored key molecules involved in development, reproduction, host-parasite interactions, immunity and disease. The enhanced 'omic data sets for S. scabiei represent comprehensive and critical resources for genetic, functional genomic, metabolomic, phylogenetic, ecological and/or epidemiological investigations, and will underpin the design and development of new treatments, vaccines and/or diagnostic tests.
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http://dx.doi.org/10.1371/journal.pntd.0008720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7591027PMC
October 2020

First record of a tandem-repeat region within the mitochondrial genome of Clonorchis sinensis using a long-read sequencing approach.

PLoS Negl Trop Dis 2020 08 26;14(8):e0008552. Epub 2020 Aug 26.

Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria, Australia.

Background: Mitochondrial genomes provide useful genetic markers for systematic and population genetic studies of parasitic helminths. Although many such genome sequences have been published and deposited in public databases, there is evidence that some of them are incomplete relating to an inability of conventional techniques to reliably sequence non-coding (repetitive) regions. In the present study, we characterise the complete mitochondrial genome-including the long, non-coding region-of the carcinogenic Chinese liver fluke, Clonorchis sinensis, using long-read sequencing.

Methods: The mitochondrial genome was sequenced from total high molecular-weight genomic DNA isolated from a pool of 100 adult worms of C. sinensis using the MinION sequencing platform (Oxford Nanopore Technologies), and assembled and annotated using an informatic approach.

Results: From > 93,500 long-reads, we assembled a 18,304 bp-mitochondrial genome for C. sinensis. Within this genome we identified a novel non-coding region of 4,549 bp containing six tandem-repetitive units of 719-809 bp each. Given that genomic DNA from pooled worms was used for sequencing, some variability in length/sequence in this tandem-repetitive region was detectable, reflecting population variation.

Conclusions: For C. sinensis, we report the complete mitochondrial genome, which includes a long (> 4.5 kb) tandem-repetitive region. The discovery of this non-coding region using a nanopore-sequencing/informatic approach now paves the way to investigating the nature and extent of length/sequence variation in this region within and among individual worms, both within and among C. sinensis populations, and to exploring whether this region has a functional role in the regulation of replication and transcription, akin to the mitochondrial control region in mammals. Although applied to C. sinensis, the technological approach established here should be broadly applicable to characterise complex tandem-repetitive or homo-polymeric regions in the mitochondrial genomes of a wide range of taxa.
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http://dx.doi.org/10.1371/journal.pntd.0008552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7449408PMC
August 2020

Marked mitochondrial genetic variation in individuals and populations of the carcinogenic liver fluke Clonorchis sinensis.

PLoS Negl Trop Dis 2020 08 19;14(8):e0008480. Epub 2020 Aug 19.

Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria, Australia.

Clonorchiasis is a neglected tropical disease caused by the Chinese liver fluke, Clonorchis sinensis, and is often associated with a malignant form of bile duct cancer (cholangiocarcinoma). Although some aspects of the epidemiology of clonorchiasis are understood, little is known about the genetics of C. sinensis populations. Here, we conducted a comprehensive genetic exploration of C. sinensis from endemic geographic regions using complete mitochondrial protein gene sets. Genomic DNA samples from C. sinensis individuals (n = 183) collected from cats and dogs in China (provinces of Guangdong, Guangxi, Hunan, Heilongjiang and Jilin) as well as from rats infected with metacercariae from cyprinid fish from the Russian Far East (Primorsky Krai region) were deep sequenced using the BGISEQ-500 platform. Informatic analyses of mitochondrial protein gene data sets revealed marked genetic variation within C. sinensis; significant variation was identified within and among individual worms from distinct geographical locations. No clear affiliation with a particular location or host species was evident, suggesting a high rate of dispersal of the parasite across endemic regions. The present work provides a foundation for future biological, epidemiological and ecological studies using mitochondrial protein gene data sets, which could aid in elucidating associations between particular C. sinensis genotypes/haplotypes and the pathogenesis or severity of clonorchiasis and its complications (including cholangiocarcinoma) in humans.
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http://dx.doi.org/10.1371/journal.pntd.0008480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7437864PMC
August 2020

Toward integrative 'omics of the barber's pole worm and related parasitic nematodes.

Infect Genet Evol 2020 11 11;85:104500. Epub 2020 Aug 11.

Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria, Australia. Electronic address:

Advances in nucleic acid sequencing, mass spectrometry and computational biology have facilitated the identification, annotation and analysis of genes, transcripts, proteins and metabolites in model nematodes (Caenorhabditis elegans and Pristionchus pacificus) and socioeconomically important parasitic nematodes (Clades I, III, IV and V). Significant progress has been made in genomics and transcriptomics as well as in the proteomics and lipidomics of Haemonchus contortus (the barber's pole worm) - one of the most pathogenic representatives of the order Strongylida. Here, we review salient aspects of genomics, transcriptomics, proteomics, lipidomics, glycomics and functional genomics, and discuss the rise of integrative 'omics of this economically important parasite. Although our knowledge of the molecular biology, genetics and biochemistry of H. contortus and related species has progressed significantly, much remains to be explored, particularly in areas such as drug resistance, unique/unknown genes, host-parasite interactions, parasitism and the pathogenesis of disease, by integrating the use of multiple 'omics methods. This approach should lead to a better understanding of H. contortus and its relatives at a 'systems biology' level, and should assist in developing new interventions against these parasites.
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http://dx.doi.org/10.1016/j.meegid.2020.104500DOI Listing
November 2020

Predicting gene essentiality in by feature engineering and machine-learning.

Comput Struct Biotechnol J 2020 15;18:1093-1102. Epub 2020 May 15.

Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria 3010, Australia.

Defining genes that are essential for life has major implications for understanding critical biological processes and mechanisms. Although essential genes have been identified and characterised experimentally using functional genomic tools, it is challenging to predict with confidence such genes from molecular and phenomic data sets using computational methods. Using extensive data sets available for the model organism , we constructed here a machine-learning (ML)-based workflow for the prediction of essential genes on a genome-wide scale. We identified strong predictors for such genes and showed that trained ML models consistently achieve highly-accurate classifications. Complementary analyses revealed an association between essential genes and chromosomal location. Our findings reveal that essential genes in tend to be located in or near the centre of autosomal chromosomes; are positively correlated with low single nucleotide polymorphim (SNP) densities and epigenetic markers in promoter regions; are involved in protein and nucleotide processing; are transcribed in most cells; are enriched in reproductive tissues or are targets for small RNAs bound to the argonaut CSR-1. Based on these results, we hypothesise an interplay between epigenetic markers and small RNA pathways in the germline, with transcription-based memory; this hypothesis warrants testing. From a technical perspective, further work is needed to evaluate whether the present ML-based approach will be applicable to other metazoans (including ) for which comprehensive data sets (i.e. genomic, transcriptomic, proteomic, variomic, epigenetic and phenomic) are available.
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http://dx.doi.org/10.1016/j.csbj.2020.05.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7251299PMC
May 2020

Elucidating the molecular and developmental biology of parasitic nematodes: Moving to a multiomics paradigm.

Adv Parasitol 2020 31;108:175-229. Epub 2020 Jan 31.

The University of Melbourne, Parkville, VIC, Australia. Electronic address:

In the past two decades, significant progress has been made in the sequencing, assembly, annotation and analyses of genomes and transcriptomes of parasitic worms of socioeconomic importance. This progress has somewhat improved our knowledge and understanding of these pathogens at the molecular level. However, compared with the free-living nematode Caenorhabditis elegans, the areas of functional genomics, transcriptomics, proteomics and metabolomics of parasitic nematodes are still in their infancy, and there are major gaps in our knowledge and understanding of the molecular biology of parasitic nematodes. The information on signalling molecules, molecular pathways and microRNAs (miRNAs) that are known to be involved in developmental processes in C. elegans and the availability of some molecular resources (draft genomes, transcriptomes and some proteomes) for selected parasitic nematodes provide a basis to start exploring the developmental biology of parasitic nematodes. Indeed, some studies have identified molecules and pathways that might associate with developmental processes in related, parasitic nematodes, such as Haemonchus contortus (barber's pole worm). However, detailed information is often scant and 'omics resources are limited, preventing a proper integration of 'omic data sets and comprehensive analyses. Moreover, little is known about the functional roles of pheromones, hormones, signalling pathways and post-transcriptional/post-translational regulations in the development of key parasitic nematodes throughout their entire life cycles. Although C. elegans is an excellent model to assist molecular studies of parasitic nematodes, its use is limited when it comes to explorations of processes that are specific to parasitism within host animals. A deep understanding of parasitic nematodes, such as H. contortus, requires substantially enhanced resources and the use of integrative 'omics approaches for analyses. The improved genome and well-established in vitro larval culture system for H. contortus provide unprecedented opportunities for comprehensive studies of the transcriptomes (mRNA and miRNA), proteomes (somatic, excretory/secretory and phosphorylated proteins) and lipidomes (e.g., polar and neutral lipids) of this nematode. Such resources should enable in-depth explorations of its developmental biology at a level, not previously possible. The main aims of this review are (i) to provide a background on the development of nematodes, with a particular emphasis on the molecular aspects involved in the dauer formation and exit in C. elegans; (ii) to critically appraise the current state of knowledge of the developmental biology of parasitic nematodes and identify key knowledge gaps; (iii) to cover salient aspects of H. contortus, with a focus on the recent advances in genomics, transcriptomics, proteomics and lipidomics as well as in vitro culturing systems; (iv) to review recent advances in our knowledge and understanding of the molecular and developmental biology of H. contortus using an integrative multiomics approach, and discuss the implications of this approach for detailed explorations of signalling molecules, molecular processes and pathways likely associated with nematode development, adaptation and parasitism, and for the identification of novel intervention targets against these pathogens. Clearly, the multiomics approach established recently is readily applicable to exploring a wide range of interesting and socioeconomically significant parasitic worms (including also trematodes and cestodes) at the molecular level, and to elucidate host-parasite interactions and disease processes.
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http://dx.doi.org/10.1016/bs.apar.2019.12.005DOI Listing
July 2020

Gene content evolution in the arthropods.

Genome Biol 2020 01 23;21(1):15. Epub 2020 Jan 23.

Department of Genetic Medicine and Development and Swiss Institute of Bioinformatics, University of Geneva, 1211, Geneva, Switzerland.

Background: Arthropods comprise the largest and most diverse phylum on Earth and play vital roles in nearly every ecosystem. Their diversity stems in part from variations on a conserved body plan, resulting from and recorded in adaptive changes in the genome. Dissection of the genomic record of sequence change enables broad questions regarding genome evolution to be addressed, even across hyper-diverse taxa within arthropods.

Results: Using 76 whole genome sequences representing 21 orders spanning more than 500 million years of arthropod evolution, we document changes in gene and protein domain content and provide temporal and phylogenetic context for interpreting these innovations. We identify many novel gene families that arose early in the evolution of arthropods and during the diversification of insects into modern orders. We reveal unexpected variation in patterns of DNA methylation across arthropods and examples of gene family and protein domain evolution coincident with the appearance of notable phenotypic and physiological adaptations such as flight, metamorphosis, sociality, and chemoperception.

Conclusions: These analyses demonstrate how large-scale comparative genomics can provide broad new insights into the genotype to phenotype map and generate testable hypotheses about the evolution of animal diversity.
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http://dx.doi.org/10.1186/s13059-019-1925-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6977273PMC
January 2020

The developmental phosphoproteome of Haemonchus contortus.

J Proteomics 2020 02 14;213:103615. Epub 2019 Dec 14.

Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria 3010, Australia. Electronic address:

Protein phosphorylation plays essential roles in many cellular processes. Despite recent progress in the genomics, transcriptomics and proteomics of socioeconomically important parasitic nematodes, there is scant phosphoproteomic data to underpin molecular biological discovery. Here, using the phosphopeptide enrichment-based LC-MS/MS and data-independent acquisition (DIA) quantitation, we characterised the first developmental phosphoproteome of the parasitic nematode Haemonchus contortus - one of the most pathogenic parasites of ruminant livestock. Totally, 1804 phosphorylated proteins with 4406 phosphorylation sites ('phosphosites') from different developmental stages/sexes were identified. Bioinformatic analyses of quantified 'phosphosites' exhibited distinctive stage- and sex-specific patterns during development, and identified a subset of phosphoproteins proposed to play crucial roles in processes such as spindle positioning, signal transduction and kinase activity. A sequence-based comparison of the phosphoproteome of H. contortus with those of two free-living nematode species (Caenorhabditis elegans and Pristionchus pacificus) suggested a limited number of common protein phosphorylation events among these species. Our findings infer active roles for protein phosphorylation in the adaptation of a parasitic nematode to a constantly changing external environment. The phosphoproteomic data set for H. contortus provides a basis to better understand phosphorylation and associated biological processes (e.g., regulation of signal transduction), and might enable the discovery of novel anthelmintic targets. SIGNIFICANCE: Here, we report the first phosphoproteome for a socioeconomically parasitic nematode (Haemonchus contortus). This phosphoproteome exhibits distinctive patterns during development, suggesting active roles of post-translational modification in the parasite's adaptation to changing environments within and outside of the host animal. This work sheds a light on the developmental phosphorylation in a parasitic nematode, and could enable the discovery of novel interventions against major pathogens.
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http://dx.doi.org/10.1016/j.jprot.2019.103615DOI Listing
February 2020

High-quality Schistosoma haematobium genome achieved by single-molecule and long-range sequencing.

Gigascience 2019 09;8(9)

Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Corner Flemington Road and Park Drive, Parkville, VIC 3010, Australia.

Background: Schistosoma haematobium causes urogenital schistosomiasis, a neglected tropical disease affecting >100 million people worldwide. Chronic infection with this parasitic trematode can lead to urogenital conditions including female genital schistosomiasis and bladder cancer. At the molecular level, little is known about this blood fluke and the pathogenesis of the disease that it causes. To support molecular studies of this carcinogenic worm, we reported a draft genome for S. haematobium in 2012. Although a useful resource, its utility has been somewhat limited by its fragmentation.

Findings: Here, we systematically enhanced the draft genome of S. haematobium using a single-molecule and long-range DNA-sequencing approach. We achieved a major improvement in the accuracy and contiguity of the genome assembly, making it superior or comparable to assemblies for other schistosome species. We transferred curated gene models to this assembly and, using enhanced gene annotation pipelines, inferred a gene set with as many or more complete gene models as those of other well-studied schistosomes. Using conserved, single-copy orthologs, we assessed the phylogenetic position of S. haematobium in relation to other parasitic flatworms for which draft genomes were available.

Conclusions: We report a substantially enhanced genomic resource that represents a solid foundation for molecular research on S. haematobium and is poised to better underpin population and functional genomic investigations and to accelerate the search for new disease interventions.
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http://dx.doi.org/10.1093/gigascience/giz108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736295PMC
September 2019

Dafachronic acid promotes larval development in Haemonchus contortus by modulating dauer signalling and lipid metabolism.

PLoS Pathog 2019 07 23;15(7):e1007960. Epub 2019 Jul 23.

Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria, Australia.

Here, we discovered an endogenous dafachronic acid (DA) in the socioeconomically important parasitic nematode Haemonchus contortus. We demonstrate that DA promotes larval exsheathment and development in this nematode via a relatively conserved nuclear hormone receptor (DAF-12). This stimulatory effect is dose- and time-dependent, and relates to a modulation of dauer-like signalling, and glycerolipid and glycerophospholipid metabolism, likely via a negative feedback loop. Specific chemical inhibition of DAF-9 (cytochrome P450) was shown to significantly reduce the amount of endogenous DA in H. contortus; compromise both larval exsheathment and development in vitro; and modulate lipid metabolism. Taken together, this evidence shows that DA plays a key functional role in the developmental transition from the free-living to the parasitic stage of H. contortus by modulating the dauer-like signalling pathway and lipid metabolism. Understanding the intricacies of the DA-DAF-12 system and associated networks in H. contortus and related parasitic nematodes could pave the way to new, nematode-specific treatments.
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http://dx.doi.org/10.1371/journal.ppat.1007960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6677322PMC
July 2019

An Evaluation of Machine Learning Approaches for the Prediction of Essential Genes in Eukaryotes Using Protein Sequence-Derived Features.

Comput Struct Biotechnol J 2019 8;17:785-796. Epub 2019 Jun 8.

Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria 3010, Australia.

The availability of whole-genome sequences and associated multi-omics data sets, combined with advances in gene knockout and knockdown methods, has enabled large-scale annotation and exploration of gene and protein functions in eukaryotes. Knowing which genes are essential for the survival of eukaryotic organisms is paramount for an understanding of the basic mechanisms of life, and could assist in identifying intervention targets in eukaryotic pathogens and cancer. Here, we studied essential gene orthologs among selected species of eukaryotes, and then employed a systematic machine-learning approach, using protein sequence-derived features and selection procedures, to investigate essential gene predictions within and among species. We showed that the numbers of essential gene orthologs comprise small fractions when compared with the total number of orthologs among the eukaryotic species studied. In addition, we demonstrated that machine-learning models trained with subsets of essentiality-related data performed better than random guessing of gene essentiality for a particular species. Consistent with our gene ortholog analysis, the predictions of essential genes among multiple (including distantly-related) species is possible, yet challenging, suggesting that most essential genes are unique to a species. The present work provides a foundation for the expansion of genome-wide essentiality investigations in eukaryotes using machine learning approaches.
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http://dx.doi.org/10.1016/j.csbj.2019.05.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6607062PMC
June 2019

Comparative genome analysis indicates high evolutionary potential of pathogenicity genes in Colletotrichum tanaceti.

PLoS One 2019 31;14(5):e0212248. Epub 2019 May 31.

Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria, Australia.

Colletotrichum tanaceti is an emerging foliar fungal pathogen of commercially grown pyrethrum (Tanacetum cinerariifolium). Despite being reported consistently from field surveys in Australia, the molecular basis of pathogenicity of C. tanaceti on pyrethrum is unknown. Herein, the genome of C. tanaceti (isolate BRIP57314) was assembled de novo and annotated using transcriptomic evidence. The inferred putative pathogenicity gene suite of C. tanaceti comprised a large array of genes encoding secreted effectors, proteases, CAZymes and secondary metabolites. Comparative analysis of its putative pathogenicity gene profiles with those of closely related species suggested that C. tanaceti likely has additional hosts to pyrethrum. The genome of C. tanaceti had a high repeat content and repetitive elements were located significantly closer to genes inferred to influence pathogenicity than other genes. These repeats are likely to have accelerated mutational and transposition rates in the genome, resulting in a rapid evolution of certain CAZyme families in this species. The C. tanaceti genome showed strong signals of Repeat Induced Point (RIP) mutation which likely caused its bipartite nature consisting of distinct gene-sparse, repeat and A-T rich regions. Pathogenicity genes within these RIP affected regions were likely to have a higher evolutionary rate than the rest of the genome. This "two-speed" genome phenomenon in certain Colletotrichum spp. was hypothesized to have caused the clustering of species based on the pathogenicity genes, to deviate from taxonomic relationships. The large repertoire of pathogenicity factors that potentially evolve rapidly due to the plasticity of the genome, indicated that C. tanaceti has a high evolutionary potential. Therefore, C. tanaceti poses a high-risk to the pyrethrum industry. Knowledge of the evolution and diversity of the putative pathogenicity genes will facilitate future research in disease management of C. tanaceti and other Colletotrichum spp.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0212248PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6544218PMC
January 2020

Long-read sequencing reveals a 4.4 kb tandem repeat region in the mitogenome of Echinococcus granulosus (sensu stricto) genotype G1.

Parasit Vectors 2019 May 16;12(1):238. Epub 2019 May 16.

Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC, Australia.

Background: Echinococcus tapeworms cause a severe helminthic zoonosis called echinococcosis. The genus comprises various species and genotypes, of which E. granulosus (sensu stricto) represents a significant global public health and socioeconomic burden. Mitochondrial (mt) genomes have provided useful genetic markers to explore the nature and extent of genetic diversity within Echinococcus and have underpinned phylogenetic and population structure analyses of this genus. Our recent work indicated a sequence gap (> 1 kb) in the mt genomes of E. granulosus genotype G1, which could not be determined by PCR-based Sanger sequencing. The aim of the present study was to define the complete mt genome, irrespective of structural complexities, using a long-read sequencing method.

Methods: We extracted high molecular weight genomic DNA from protoscoleces from a single cyst of E. granulosus genotype G1 from a sheep from Australia using a conventional method and sequenced it using PacBio Sequel (long-read) technology, complemented by BGISEQ-500 short-read sequencing. Sequence data obtained were assembled using a recently-developed workflow.

Results: We assembled a complete mt genome sequence of 17,675 bp, which is > 4 kb larger than the complete mt genomes known for E. granulosus genotype G1. This assembly includes a previously-elusive tandem repeat region, which is 4417 bp long and consists of ten near-identical 441-445 bp repeat units, each harbouring a 184 bp non-coding region and adjacent regions. We also identified a short non-coding region of 183 bp, which includes an inverted repeat.

Conclusions: We report what we consider to be the first complete mt genome of E. granulosus genotype G1 and characterise all repeat regions in this genome. The numbers, sizes, sequences and functions of tandem repeat regions remain to be studied in different isolates of genotype G1 and in other genotypes and species. The discovery of such 'new' repeat elements in the mt genome of genotype G1 by PacBio sequencing raises a question about the completeness of some published genomes of taeniid cestodes assembled from conventional or short-read sequence datasets. This study shows that long-read sequencing readily overcomes the challenges of assembling repeat elements to achieve improved genomes.
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http://dx.doi.org/10.1186/s13071-019-3492-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6521400PMC
May 2019

High throughput LC-MS/MS-based proteomic analysis of excretory-secretory products from short-term in vitro culture of Haemonchus contortus.

J Proteomics 2019 07 6;204:103375. Epub 2019 May 6.

Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria 3010, Australia. Electronic address:

Parasitic nematodes of humans, animals and plants have a major, adverse impact on global health and agricultural production worldwide. To cope with their surrounding environment in and the immune attack from the host, excretory-secretory (ES) proteins are released by nematodes to orchestrate or regulate parasite-host interactions. In the present study, we characterised the ES products from short-term (12 h) in vitro culture of different developmental stages/sexes of Haemonchus contortus (one of the most important parasitic nematodes of livestock animals worldwide) using a high throughput tandem mass-spectrometry, underpinned by the most recent genomic dataset. In total, 878 unique proteins from key developmental stages/sexes (third-stage and fourth-stage larvae, and female and male adults) were identified and quantified with high confidence. Bioinformatic analyses showed noteworthy ES protein alterations during the transition from the free-living to the parasitic phase, especially for proteins which are likely involved in nutrient digestion and acquisition as well as parasite-host interactions, such as proteolytic cascade-related peptidases, glycoside hydrolases, C-type lectins and sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7 (= SCP/TAPS) proteins. Our findings provide an avenue to better explore interactive processes between the host and this highly significant parasitic nematode, to underpin the search for novel drug and vaccine targets. SIGNIFICANCE: The present study represents a comprehensive proteomic analysis of the secretome of key developmental stages/sexes of H. contortus maintained in short-term in vitro culture. High throughput LC-MS/MS analysis of ES products allowed the identification of a large repertoire of proteins (secretome) and the establishment of a new proteomic database for H. contortus. The secretome of H. contortus undergoes substantial changes during the nematode's transition from free-living to parasitic stages, suggesting a constant adaptation to different environments outside of and within the host animal. Understanding the host-parasite relationship at the molecular level could assist significantly in the development of intervention strategies (i.e. novel drugs and vaccines) against H. contortus and related nematodes.
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http://dx.doi.org/10.1016/j.jprot.2019.05.003DOI Listing
July 2019

Dauer signalling pathway model for Haemonchus contortus.

Parasit Vectors 2019 Apr 29;12(1):187. Epub 2019 Apr 29.

Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, VIC, 3010, Australia.

Background: Signalling pathways have been extensively investigated in the free-living nematode Caenorhabditis elegans, but very little is known about these pathways in parasitic nematodes. Here, we constructed a model for the dauer-associated signalling pathways in an economically highly significant parasitic worm, Haemonchus contortus.

Methods: Guided by data and information available for C. elegans, we used extensive genomic and transcriptomic datasets to infer gene homologues in the dauer-associated pathways, explore developmental transcriptomic, proteomic and phosphoproteomic profiles in H. contortus and study selected molecular structures.

Results: The canonical cyclic guanosine monophosphate (cGMP), transforming growth factor-β (TGF-β), insulin-like growth factor 1 (IGF-1) and steroid hormone signalling pathways of H. contortus were inferred to represent a total of 61 gene homologues. Compared with C. elegans, H. contortus has a reduced set of genes encoding insulin-like peptides, implying evolutionary and biological divergences between the parasitic and free-living nematodes. Similar transcription profiles were found for all gene homologues between the infective stage of H. contortus and dauer stage of C. elegans. High transcriptional levels for genes encoding G protein-coupled receptors (GPCRs), TGF-β, insulin-like ligands (e.g. ins-1, ins-17 and ins-18) and transcriptional factors (e.g. daf-16) in the infective L3 stage of H. contortus were suggestive of critical functional roles in this stage. Conspicuous protein expression patterns and extensive phosphorylation of some components of these pathways suggested marked post-translational modifications also in the L3 stage. The high structural similarity in the DAF-12 ligand binding domain among nematodes indicated functional conservation in steroid (i.e. dafachronic acid) signalling linked to worm development.

Conclusions: Taken together, this pathway model provides a basis to explore hypotheses regarding biological processes and regulatory mechanisms (via particular microRNAs, phosphorylation events and/or lipids) associated with the development of H. contortus and related nematodes as well as parasite-host cross talk, which could aid the discovery of new therapeutic targets.
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http://dx.doi.org/10.1186/s13071-019-3419-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6489264PMC
April 2019

Common workflow language (CWL)-based software pipeline for de novo genome assembly from long- and short-read data.

Gigascience 2019 04;8(4)

Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria 3010, Australia.

Background: Here, we created an automated pipeline for the de novoassembly of genomes from Pacific Biosciences long-read and Illumina short-read data using common workflow language (CWL). To evaluate the performance of this pipeline, we assembled the nuclear genomes of the eukaryotes Caenorhabditis elegans (∼100 Mb), Drosophila melanogaster (∼138 Mb), and Plasmodium falciparum (∼23 Mb) directly from publicly accessible nucleotide sequence datasets and assessed the quality of the assemblies against curated reference genomes.

Findings: We showed a dependency of the accuracy of assembly on sequencing technology and GC content and repeatedly achieved assemblies that meet the high standards set by the National Human Genome Research Institute, being applicable to gene prediction and subsequent genomic analyses.

Conclusions: This CWL pipeline overcomes current challenges of achieving repeatability and reproducibility of assembly results and offers a platform for the re-use of the workflow and the integration of diverse datasets. This workflow is publicly available via GitHub (https://github.com/vetscience/Assemblosis) and is currently applicable to the assembly of haploid and diploid genomes of eukaryotes.
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http://dx.doi.org/10.1093/gigascience/giz014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451199PMC
April 2019

Somatic proteome of Haemonchus contortus.

Int J Parasitol 2019 03 14;49(3-4):311-320. Epub 2019 Feb 14.

Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria 3010, Australia. Electronic address:

Currently, there is a dearth of proteomic data to underpin fundamental investigations of parasites and parasitism at the molecular level. Here, using a high throughput LC-MS/MS-based approach, we undertook the first reported comprehensive, large-scale proteomic investigation of the barber's pole worm (Haemonchus contortus) - one of the most important parasitic nematodes of livestock animals worldwide. In total, 2487 unique H. contortus proteins representing different developmental stages/sexes (i.e. eggs, L3s and L4s, female (Af) and male (Am) adults) were identified and quantified with high confidence. Bioinformatic analyses of this proteome revealed substantial alterations in protein profiles during the life cycle, particularly in the transition from the free-living to the parasitic phase, and key groups of proteins involved specifically in feeding, digestion, metabolism, development, parasite-host interactions (including immunomodulation), structural remodelling of the body wall and adaptive processes during parasitism. This proteomic data set will facilitate future molecular, biochemical and physiological investigations of H. contortus and related nematodes, and the discovery of novel intervention targets against haemonchosis.
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http://dx.doi.org/10.1016/j.ijpara.2018.12.003DOI Listing
March 2019

Molecular evidence for distinct modes of nutrient acquisition between visceral and neurotropic schistosomes of birds.

Sci Rep 2019 02 4;9(1):1347. Epub 2019 Feb 4.

Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic.

Trichobilharzia species are parasitic flatworms (called schistosomes or flukes) that cause important diseases in birds and humans, but very little is known about their molecular biology. Here, using a transcriptomics-bioinformatics-based approach, we explored molecular aspects pertaining to the nutritional requirements of Trichobilharzia szidati ('visceral fluke') and T. regenti ('neurotropic fluke') in their avian host. We studied the larvae of each species before they enter (cercariae) and as they migrate (schistosomules) through distinct tissues in their avian (duck) host. Cercariae of both species were enriched for pathways or molecules associated predominantly with carbohydrate metabolism, oxidative phosphorylation and translation of proteins linked to ribosome biogenesis, exosome production and/or lipid biogenesis. Schistosomules of both species were enriched for pathways or molecules associated with processes including signal transduction, cell turnover and motility, DNA replication and repair, molecular transport and/or catabolism. Comparative informatic analyses identified molecular repertoires (within, e.g., peptidases and secretory proteins) in schistosomules that can broadly degrade macromolecules in both T. szidati and T. regenti, and others that are tailored to each species to selectively acquire nutrients from particular tissues through which it migrates. Thus, this study provides molecular evidence for distinct modes of nutrient acquisition between the visceral and neurotropic flukes of birds.
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http://dx.doi.org/10.1038/s41598-018-37669-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6362228PMC
February 2019

Whole-genome sequence of the bovine blood fluke Schistosoma bovis supports interspecific hybridization with S. haematobium.

PLoS Pathog 2019 01 23;15(1):e1007513. Epub 2019 Jan 23.

The University of Queensland Diamantina Institute, The University of Queensland, Brisbane, QLD, Australia.

Mesenteric infection by the parasitic blood fluke Schistosoma bovis is a common veterinary problem in Africa and the Middle East and occasionally in the Mediterranean Region. The species also has the ability to form interspecific hybrids with the human parasite S. haematobium with natural hybridisation observed in West Africa, presenting possible zoonotic transmission. Additionally, this exchange of alleles between species may dramatically influence disease dynamics and parasite evolution. We have generated a 374 Mb assembly of the S. bovis genome using Illumina and PacBio-based technologies. Despite infecting different hosts and organs, the genome sequences of S. bovis and S. haematobium appeared strikingly similar with 97% sequence identity. The two species share 98% of protein-coding genes, with an average sequence identity of 97.3% at the amino acid level. Genome comparison identified large continuous parts of the genome (up to several 100 kb) showing almost 100% sequence identity between S. bovis and S. haematobium. It is unlikely that this is a result of genome conservation and provides further evidence of natural interspecific hybridization between S. bovis and S. haematobium. Our results suggest that foreign DNA obtained by interspecific hybridization was maintained in the population through multiple meiosis cycles and that hybrids were sexually reproductive, producing viable offspring. The S. bovis genome assembly forms a highly valuable resource for studying schistosome evolution and exploring genetic regions that are associated with species-specific phenotypic traits.
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http://dx.doi.org/10.1371/journal.ppat.1007513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6361461PMC
January 2019

Comparative bioinformatic analysis suggests that specific dauer-like signalling pathway components regulate Toxocara canis development and migration in the mammalian host.

Parasit Vectors 2019 Jan 14;12(1):32. Epub 2019 Jan 14.

Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria, 3010, Australia.

Background: Toxocara canis is quite closely related to Ascaris suum but its biology is more complex, involving a phase of arrested development (diapause or hypobiosis) in tissues as well as transplacental and transmammary transmission routes. In the present study, we explored and compared dauer-like signalling pathways of T. canis and A. suum to infer which components in these pathways might associate with, or regulate, this added complexity in T. canis.

Methods: Guided by information for Caenorhabditis elegans, we bioinformatically inferred and compared components of dauer-like signalling pathways in T. canis and A. suum using genomic and transcriptomic data sets. In these two ascaridoids, we also explored endogenous dafachronic acids (DAs), which are known to be critical in regulating larval developmental processes in C. elegans and other nematodes, by liquid chromatography-mass spectrometry (LC-MS).

Results: Orthologues of C. elegans dauer signalling genes were identified in T. canis (n = 55) and A. suum (n = 51), inferring the presence of a dauer-like signalling pathway in both species. Comparisons showed clear differences between C. elegans and these ascaridoids as well as between T. canis and A. suum, particularly in the transforming growth factor-β (TGF-β) and insulin-like signalling pathways. Specifically, in both A. suum and T. canis, there was a paucity of genes encoding SMAD transcription factor-related protein (daf-3, daf-5, daf-8 and daf-14) and insulin/insulin-like peptide (daf-28, ins-4, ins-6 and ins-7) homologues, suggesting an evolution and adaptation of the signalling pathway in these parasites. In T. canis, there were more orthologues coding for homologues of antagonist insulin-like peptides (Tc-ins-1 and Tc-ins-18), an insulin receptor substrate (Tc-ist-1) and a serine/threonine kinase (Tc-akt-1) than in A. suum, suggesting potentiated functional roles for these molecules in regulating larval diapause and reactivation. A relatively conserved machinery was proposed for DA synthesis in the two ascaridoids, and endogenous Δ4- and Δ7-DAs were detected in them by LC-MS analysis. Differential transcription analysis between T. canis and A. suum suggests that ins-17 and ins-18 homologues are specifically involved in regulating development and migration in T. canis larvae in host tissues.

Conclusion: The findings of this study provide a basis for functional explorations of insulin-like peptides, signalling hormones (i.e. DAs) and related nuclear receptors, proposed to link to development and/or parasite-host interactions in T. canis. Elucidating the functional roles of these molecules might contribute to the discovery of novel anthelmintic targets in ascaridoids.
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http://dx.doi.org/10.1186/s13071-018-3265-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6332619PMC
January 2019

A TGF-β type I receptor-like molecule with a key functional role in Haemonchus contortus development.

Int J Parasitol 2018 11 26;48(13):1023-1033. Epub 2018 Sep 26.

State Key Laboratory of Agricultural Microbiology, Key Laboratory for the Development of Veterinary Products, Ministry of Agriculture, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, China. Electronic address:

Here we investigated the gene of a transforming growth factor (TGF)-β type I receptor-like molecule in Haemonchus contortus, a highly pathogenic and economically important parasitic nematode of small ruminants. Designated Hc-tgfbr1, this gene is transcribed in all developmental stages of H. contortus, and the encoded protein has glycine-serine rich and kinase domains characteristic of a TGF-β family type I receptor. Expression of a GFP reporter driven by the putative Hc-tgfbr1 promoter localised to two intestinal rings, the anterior-most intestinal ring (int ring I) and the posterior-most intestinal ring (int ring IX) in Caenorhabditis elegans in vivo. Heterologous genetic complementation using a plasmid construct containing Hc-tgfbr1 genomic DNA failed to rescue the function of Ce-daf-1 (a known TGF-β type I receptor gene) in a daf-1-deficient mutant strain of C. elegans. In addition, a TGF-β type I receptor inhibitor, galunisertib, and double-stranded RNA interference (RNAi) were employed to assess the function of Hc-tgfbr1 in the transition from exsheathed L3 (xL3) to the L4 of H. contortus in vitro, revealing that both galunisertib and Hc-tgfbr1-specific double-stranded RNA could retard L4 development. Taken together, these results provide evidence that Hc-tgfbr1 is involved in developmental processes in H. contortus in the transition from the free-living to the parasitic stage.
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http://dx.doi.org/10.1016/j.ijpara.2018.06.005DOI Listing
November 2018
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