The Fasciola hepatica genome: gene duplication and polymorphism reveals adaptation to the host environment and the capacity for rapid evolution
© Cwiklinski et al.; licensee BioMed Central. 2015
Received: 10 November 2014
Accepted: 13 March 2015
Published: 3 April 2015
The liver fluke Fasciola hepatica is a major pathogen of livestock worldwide, causing huge economic losses to agriculture, as well as 2.4 million human infections annually.
Here we provide a draft genome for F. hepatica, which we find to be among the largest known pathogen genomes at 1.3 Gb. This size cannot be explained by genome duplication or expansion of a single repeat element, and remains a paradox given the burden it may impose on egg production necessary to transmit infection. Despite the potential for inbreeding by facultative self-fertilisation, substantial levels of polymorphism were found, which highlights the evolutionary potential for rapid adaptation to changes in host availability, climate change or to drug or vaccine interventions. Non-synonymous polymorphisms were elevated in genes shared with parasitic taxa, which may be particularly relevant for the ability of the parasite to adapt to a broad range of definitive mammalian and intermediate molluscan hosts. Large-scale transcriptional changes, particularly within expanded protease and tubulin families, were found as the parasite migrated from the gut, across the peritoneum and through the liver to mature in the bile ducts. We identify novel members of anti-oxidant and detoxification pathways and defined their differential expression through infection, which may explain the stage-specific efficacy of different anthelmintic drugs.
The genome analysis described here provides new insights into the evolution of this important pathogen, its adaptation to the host environment and external selection pressures. This analysis also provides a platform for research into novel drugs and vaccines.
The digenean trematode Fasciola hepatica is one of the most important pathogens of domestic livestock and has a global distribution [1-4]. The disease, fasciolosis, results in huge losses to the agricultural industry associated with poor food conversion, lower weight gains, impaired fertility and reduced milk (cattle) and wool (sheep) production. Heavy, acute infections can result in death, particularly in sheep and goats. Economic losses attributable to F. hepatica infection have been estimated at more than US$3 billion per annum worldwide [5,6], although even this estimate may be conservative as F. hepatica infection modulates its host’s immune response and its ability to resist or eliminate common microbial pathogens [7,8]. Fasciolosis is also an important zoonosis in regions where agricultural management practices are less advanced, particularly in South America and North Africa [3,9]. It is estimated that between 2.4 and 17 million people are infected with this liver fluke worldwide, with a further 91 million people living at risk, resulting in fasciolosis being included on the World Health Organization list of major neglected tropical diseases [1-3].
Here we provide a genome assembly for F. hepatica and assess genome-wide polymorphism and transcriptional profiles in order to identify key features of its genome that underlie its ability to migrate through different physiological environments, to parasitise different host species, and to respond rapidly to external selection pressures.
Results and discussion
A large genome with high gene polymorphism
Fasciola hepatica assembly statistics
204 Kbp (REAPR 155 Kbpa)
Number of scaffolds ≥3 Kbp
Number of scaffolds ≥1 Kbp
Contig N50 (≥100 bp)
9.7 K bp
Number of contigs (≥100 bp)
Total assembly length
Total length of gaps
Number of RNAseq-supported gene models
Mean number of exons/gene
Mean exon size (95% range)
303 bp (36 bp – 1,369 bp)
Mean intron size (95% range)
3.7 Kbp (33 bp - 17.5 Kbp)
Proportion CEGMA core eukaryotic genes found
We investigated levels of polymorphism among F. hepatica genes by re-sequencing the genomes of individual fluke from each of five isolates, all from the UK. Substantial polymorphism among isolates was observed; 48% of genes exhibited at least one non-synonymous SNP and the level of non-synonymous nucleotide diversity, pi, averaged across 21.8 Mbp of coding sequence, was 5.2 × 10-4 (that is, two randomly sampled sequences differed approximately every 1,900 bp). By comparison, this figure is higher than in humans , similar to most vertebrates  and, on limited data, smaller than some parasitic nematode populations . Although F. hepatica is a self-fertilising hermaphrodite, and so has the potential to inbreed and lose genetic diversity, our data show that F. hepatica populations, as a whole, harbour substantial genetic variation. A likely explanation is that parasite populations are typically large, often larger than that of their hosts, which greatly slows any enhanced effects of genetic drift caused by self-fertilisation .
Expression patterns from multi-gene families reveal important developmental host-parasite interactions
To explore novel processes associated with developmental changes, genes were clustered based on expression pattern (Figure 3b). Several biological processes were greatly downregulated in maturing and adult parasites relative to metacercariae (Cluster 12, Figure 3), including molecules involved in cell adhesion (cadherins, integrins) and cytoskeletal proteins (talins) that could play an important role in sensing changes in the physiological environment and rapidly initiating excystment following ingestion by the definitive host. Clusters showing strongest patterns of differential expression (both up- and downregulation) were markedly over-represented by peptidases and terms associated with regulation of peptidase activity (Clusters 2, 8, 10 and 11, Figure 3). Genes associated with F. hepatica structure, particularly protein polymerisation and microtubule based movement, such as tubulin, dynein and surface tegumental genes, were highly upregulated in immature and adult fluke relative to earlier stages (Clusters 1 and 3, Figure 3b and Additional file 9: Table S7, Additional file 10: Table S8 and Additional file 11: Table S9). Our data show that, across the transcriptome as a whole, the strongest changes in expression were observed among different members of the multigenic protease and tubulin families, as detailed below.
Unlike the FhCL peptidases, the FhCB family consists of a single clade of seven members. Nevertheless, these peptidases were also temporally regulated (Figure 4c) such that three members (FhCB1, FhCB2 and FhCB3) exhibited parallel expression patterns to FhCL3 and were thus highly expressed in the NEJs and downregulated as the parasites matured. These data suggest a concerted role for FhCLs and FhCBs in the early infection stage. Also, the constitution and specific expression of a family of peptidases, asparaginyl endopeptidases or legumains that are responsible for the processing of the inactive FhCL and FhCB zymogens to functional enzymes  suggest specific and important developmental roles for these peptidases (Figure 4d). Thus, legumain 1, which was the most highly expressed member in NEJs could be required for activation of the FhCL3 and FhCB 1/2/3 peptides at the time the parasite emerges from its encysted stage in the intestine and initiates infection. Furthermore, legumain 3, which was switched on late in parasite development, is the prospective candidate for activating the FhCL1, FhCL2 and FhCL5 in mature blood-feeding adult parasites.
Anti-oxidant and detoxification systems
We investigated the evolution of anti-oxidant systems in F. hepatica, which are essential for adaptation to the host environment. Thus, as the parasite rapidly develops and enters different aerobic/anaerobic environments, anti-oxidant systems are critical not only for the detoxification of reactive oxygen and nitrogen (ROS, RNS) generated by endogenous cellular metabolism but also as a frontline defence against superoxide and nitric oxide radicals released by host immune effector cells such as macrophages, eosinophils and neutrophils [43,44]. Parasitic platyhelminths express genes encoding superoxide dismutase (SOD) which dismutates superoxide radicals to H2O2 but they do not possess catalase, the enzyme responsible for converting H2O2 to water and oxygen (although a catalase gene is present in the genome of free-living flatworms, such as Schmidtea mediterranea). A catalase gene was not found in the F. hepatica genome, which is consistent with other parasitic platyhelminths, where the function of peroxide detoxification has been supplanted by the newly discovered thiol-dependent peroxiredoxin and its reducing partner thioredoxin . The presence of the gene encoding the recently described thioredoxin glutathione reductase (TGR; Additional file 14: Table S11), together with the absence of distinct thioredoxin reductase and glutathione reductase genes verified that TGR is the sole reductive enzyme that links the thioredoxin-dependent and glutathione-dependent anti-oxidant defence systems in this parasite as observed in other platyhelminths [43,46,47]. The pivotal position of TGR between these two essential anti-oxidant systems makes it a promising target for the development of new anti-trematode drugs . Included in the repertoire of F. hepatica anti-oxidants are genes encoding SOD, glutathione transferases (GSTs) and three fatty acid binding proteins (FABP; Additional file 14: Table S11). With the exception of the GSTs, we found that in F. hepatica each of these components is encoded by a single gene, which is in stark contrast to the expanded anti-oxidant gene families in the closely related trematodes, O. viverrini and C. sinensis [13,15]. Peroxiredoxin, GST and FABP are secreted by F. hepatica via non-classical secretory pathways, perhaps as cargoes of exosomes , into the host circulation where they influence host immune responses by recruiting and activating M2 macrophages [7,48,49] and suppressing dendritic cell activity . These immunomodulatory effects contribute to the establishment of an immune suppressive environment, which aids parasite survival and the development of chronic disease.
The F. hepatica genome is one of the largest pathogen genomes sequenced to date but we found no evidence of genome duplication or repeat expansion to explain this. Why this large genome should have evolved is unclear, especially given that its replication may be energetically costly or slow cell division . For a parasite, such as Fasciola, that relies on the production of large numbers of eggs to facilitate transmission, one would expect strong selection against the accumulation of junk DNA if a large genome imposed a cost on egg production. It is possible that much of the non-coding portion of the F. hepatica genome is involved in gene regulation , and the size of F. hepatica’s genome may be related to its complex life cycle and variety of developmental morphs. If so, however, it would be difficult to explain why the F. hepatica genome is around three time the size of the Schistosoma genome , which has a similar life cycle. The large genome of F. hepatica therefore remains a paradox for which we may have to wait for comparative genome sequencing, currently underway , across other platyhelminth taxa to provide an answer.
The ability of F. hepatica to infect and survive in different tissue environments as it migrates from the intestine, through the liver and into the bile ducts is underpinned by gene duplication. Thus, our results show that, across the whole transcriptome, the strongest patterns of differential expression were observed among members of protease and tubulin gene families, and, in the case of proteases, these can be associated with changes in the active site and substrate specificity. While gene duplication appears to be a key process of adaptation to the parasitic life-style used by many helminths, it is notable that different helminth taxa have arrived at different evolutionary solutions. Comparison between F. hepatica and the bile-dwelling liver flukes C. sinensis and O. viverrini, shows the expansion of the anti-oxidant SOD families  and cathepsin F protease families [13,61] in C. sinensis and O. viverrini but not in F. hepatica, and conversely the expansion of the cathepsin L family in the latter species only, which suggests differences in how these related parasites tackle life within the same environment. Differences in the specificity or developmental expression of detoxification genes, such as members of the ABC and MATE families, or of the tubulin gene family, may be important in understanding why F. hepatica responds so markedly different to drugs at different stages of development compared to other digeneans. The exclusive activity of TCBZ against F. hepatica, and the potency of praziquantel to all these other digeneans except F. hepatica, points to a uniqueness in this parasite that needs to be resolved .
Fasciola hepatica is a highly adaptable parasite, evidenced by its ability to infect novel hosts, and it is notable that our results reveal high levels of polymorphism in genes specific to parasitic digeneans. Diversity within F. hepatica populations at genes important for the host-parasite interface may underpin a high evolutionary potential for F. hepatica to respond to changes in host availability or to other selection pressures. Similarly, the broad geographic range of F. hepatica would suggest that it is able to adapt to different climatic conditions and, in this respect, F. hepatica may also be able to respond to exploit changes in climate in temperate regions, such as the UK, where warmer and wetter winters favour transmission and increased prevalence . F. hepatica is seen to rapidly develop drug resistance to TCBZ  and the standing genetic diversity that we find across the genome suggests that it harbours the potential to evolve resistance to any novel drug treatment, which compounds the difficultly of controlling F. hepatica given the shortage of drugs effective against the juvenile stages. Nevertheless, the availability of a genome for F. hepatica that we provide, plus the characterisation of early molecular events in infection should help support the development of novel drugs and vaccines, particularly against the migrating juveniles that cause much of the pathology. For example, RNAi-mediated knockdown of cathepsin L and B expression has been shown to prevent NEJs from crossing the intestine  and vaccine trials with cathepsin L1 have provided partial protection against infection and pathology . The exploitation of a broader range of targets within the F. hepatica genome is now a priority given the widespread prevalence of resistance to TCBZ within F. hepatica populations. The commercial importance for agriculture to develop a replacement treatment to TCBZ may stimulate new treatments that could be translated to other important digenean parasites, including those of humans.
Source of parasite material
Adult parasites from each of five isolates were used for genome sequencing: (1) FhepLivSP, from the laboratory maintained Shrewsbury isolate (Ridgeway Research, UK); (2) FhepLivS1, a clonal line derived from the Shrewsbury population; (3) and (4) FhepLivR1 and R3, clonal lines derived from two isolates recovered from sheep in Northern England naturally infected with F. hepatica; and (5) FhepLivR2, a clonal line derived from a F. hepatica population from naturally infected sheep in South West Wales. For RNA sequencing, the following were used: (1) metacercariae and newly excysted juveniles (NEJ) at 1, 3 and 24 h post excystment from a North American isolate (Baldwin Aquatics Inc., Monmouth, OR, USA). Twenty-one–day-old juvenile flukes were recovered from mice infected with the same isolate; (2) an adult parasite recovered from the bile ducts of cattle naturally infected with F. hepatica in Uruguay. All animal work was conducted with ethical approval from the Universities of Liverpool (UK) and McGill (Canada).
Approximately 10 μg of DNA from a single, adult fluke taken from the FhepLivS1 strain was used to prepare Illumina TruSeq fragment libraries and 26 Gbp of 2 × 250 bp reads generated on an Illumina MiSeq (mean insert sizes 470 bp and 580 bp). Further individuals of FhepLivS1 were used to prepare Nextera Mate-Pair libraries (3 Kbp and 10 Kbp) and approximately 60 m 2× 100 bp Illumina reads from each library were generated. Approximately 200 μg of DNA prepared from several fluke of the FhepLivSP isolate was used to construct 2 Kbp, 5 Kbp and 8 Kbp mate-pair libraries, which were sequenced either on an Illumina GAII or HiSeq 2000. For each of the parasite isolates FhepLivSP, FhepLivS1, FhepLivR1, FhepLivR2 and FhepLivR3, DNA was isolated from a single adult; a fragment library was prepared and sequenced on a single lane of an Illumina HiSeq 2000 to yield approximately 24 Gbp of sequence (Centre for Genomic Research, Liverpool, UK). For RNA sequencing, Illumina TruSeq RNA libraries were prepared from biological replicates of metacercariae (3 replicates), NEJ 1 h (2 replicates), NEJ 3 h (2 replicates), NEJ 24 h (2 replicates), Juveniles 21 days (1 replicate) and Adult (1 replicate) (Genome Quebec, Montreal, Canada).
Assembly and annotation
Illumina MiSeq reads were trimmed to Q ≥30 and adaptors removed using Sickle and Perl and assembled using Newbler (Roche GS-Assembler v2.6) with flags set for large genome and a heterozygote sample. Mate-pair reads were first mapped to these contigs using Bowtie2  to remove duplicates and wrongly orientated reads, and scaffolded into contigs using SSPACE . Gap filling was achieved using GapFiller for 2× 250 bp and 2× 100 bp paired-end reads and run for three iterations (available as ENA accessions LN627018-LN647175). RNAseq data were mapped to scaffolds within the assembled genome greater than 3 Kbp using TopHat2 to identify transcribed regions and splice junctions. These, together with RNAseq data assembled using Trinity and S. mansoni protein sequence, were passed to the MAKER pipeline  to predict genes. Repeatmasker, Windowmasker and Dustmasker were used to identify repetitive regions. CEGMA v2.4 , which searches for 248 highly conserved genes, was used to assess the completeness of genome with the settings for vertebrates to allow long introns. REAPR  was used to assess the quality of scaffolding within the assembly and to produce an alternative, more conservative assembly by splitting scaffolds at locations with lower support (available as ENA accessions LN736597-LN774150). Homologs of F. hepatica predicted protein sequences were identified within UniProt using BLAST and functional domains identified using InterPro. InParanoid and MultiParanoid  were used to identify ortholog clusters from Schistosoma mansoni (v3.1.16), Clonorchis sinensis (v3.7), Schmidtea mediterranea and Echinococcus multilocularis (v29042013) predicted proteins [11-15,70].
Gene expression analysis
RNAseq libraries were mapped to MAKER gene models using TopHat2  and read counts extracted using htseq-count. Genes with a sum of at least five reads across all libraries were analysed for differential expression in edgeR  using a negative binomial model of successive developmental stages relative to metacercariae and with tagwise dispersion estimated from all samples. Hierarchical clustering, based on model coefficients, was used to group differentially expressed genes by similarity of expression into 12 clusters and hypergeometric tests used to test for over-representation of gene ontology terms within each cluster relative to the whole gene-set.
Genetic diversity analysis
Reads for each isolate were mapped to the genome using Bowtie2 and resulting bam files passed together to the GATK pipeline  for local realignment and SNP calling. SNP calls within genes were filtered by score >100, FS <60 and combined coverage between 10 and 250. Because gene duplicates collapsed within the assembly might give erroneously high diversity for some genes, genes were excluded with a median coverage greater than 213 and by heuristic scoring of SNPs appearing as heterozygotes in all samples. IGV was used to manually assess the success of filtering parameters. Levels of nucleotide diversity for different classes of SNPs were calculated for all genes. To identify the most polymorphic genes, a generalised linear model with a Poisson distribution was fitted to the number of non-synonymous SNPs as the response variable versus number of non-synonymous sites as a quadratic function. This accounts for the distribution of SNP counts within a gene, the fact that longer genes have the potential to have more SNPs and the possibility that genes of different lengths may evolve at different rates. Genes were classified according to their conservation across platyhelminths by the presence of orthologs retained across increasing taxonomic scales. This was preferred to assessing conservation on the basis of non-synonymous versus synonymous substitution ratios between species, since synonymous sites were saturated by multiple substitutions at such broad taxonomic scales. The robustness of the generalised linear model was tested by randomly sampling equal proportions of genes from each quartile of the length distribution for each class of conserved gene, to ensure that no bias was introduced if the length of a gene was correlated to its level of conservation. From the generalised linear model, the most polymorphic genes were identified having residuals in the top 1% quantile and hypergeometric tests were used to test for over-representation of GO terms within these highly polymorphic genes relative to the gene-set as a whole.
Discovery and characterisation of gene families
Discovery of F. hepatica gene families was carried out using BLAST analysis (NCBI v2.2.27 and v2.2.29), with available published gene sequences of interest (Additional file 16: Table S13), followed by manual annotation. Comparative analysis was carried out using the closely related trematode genome sequence datasets: Clonorchis sinensis and Schistosoma mansoni. Sequence alignment and phylogenetic analysis was carried out using Clustal Omega  and MEGA5 , respectively.
Data are freely available from WormBase ParaSite and the European Nucleotide Archive under accessions LN627018-LN647175 (assembly data), PRJEB6687 (genomic read data) and PRJEB6904 (transcriptomic read data).
We are grateful to Katherine Allen for producing clonal lines of liver fluke and Catherine Hartley, University of Liverpool and Paula Martin and Oliver Gladstone, Ridgeway Research, UK for technical assistance in producing parasite material. We are grateful for sequencing support provided by Margaret Hughes and Suzanne Kay at the Centre for Genomic Research, University of Liverpool, and Mathieu Bourgey, Genome Quebec, Canada. This study was funded by grants from the Biotechnology and Biological Sciences Research Council, UK (BB1002480/1) to JH, SP, DW and JLC, and a Ministère Économie, Innovation et Exportation (MEIE), Québec, award and a European Research Council Advanced Grant (HELIVAC, 322725) to JPD.
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