Abstract
BackgroundNext generation sequencing (NGS) technologies are extensively used to dissect the molecular mechanisms of host-parasite interactions in human pathogens. However, ecological studies have yet to fully exploit the power of NGS as a rich source for formulating and testing new hypotheses.MethodsWe studied Eurasian perch (Perca fluviatilis) and its eye parasite (Trematoda, Diplostomidae) communities in 14 lakes that differed in humic content in order to explore host-parasite-environment interactions. We hypothesised that high humic content along with low pH would decrease the abundance of the intermediate hosts (gastropods), thus limiting the occurrence of diplostomid parasites in humic lakes. This hypothesis was initially invoked by whole eye RNA-seq data analysis and subsequently tested using PCR-based detection and a novel targeted metabarcoding approach.ResultsWhole eye transcriptome results revealed overexpression of immune-related genes and the presence of eye parasite sequences in RNA-seq data obtained from perch living in clear-water lakes. Both PCR-based and targeted-metabarcoding approach showed that perch from humic lakes were completely free from diplostomid parasites, while the prevalence of eye flukes in clear-water lakes that contain low amounts of humic substances was close to 100%, with the majority of NGS reads assigned to Tylodelphys clavata.ConclusionsHigh intraspecific diversity of T. clavata indicates that massively parallel sequencing of naturally pooled samples represents an efficient and powerful strategy for shedding light on cryptic diversity of eye parasites. Our results demonstrate that perch populations in clear-water lakes experience contrasting eye parasite pressure compared to those from humic lakes, which is reflected by prevalent differences in the expression of immune-related genes in the eye. This study highlights the utility of NGS to discover novel host-parasite-environment interactions and provide unprecedented power to characterize the molecular diversity of cryptic parasites.
Highlights
Generation sequencing (NGS) technologies are extensively used to dissect the molecular mechanisms of host-parasite interactions in human pathogens
A typical bioinformatics analysis pipeline involves a step where reads from DNA or RNA sequencing are aligned to the target species genome; those that do not align are discarded
Initial insights from eye transcriptomes Altogether, 94% of the reads from the 14 RNA sequencing (RNA-seq) libraries generated from whole-eye tissue were mapped to the reference perch genome (Additional file 2: Table S3)
Summary
Generation sequencing (NGS) technologies are extensively used to dissect the molecular mechanisms of host-parasite interactions in human pathogens. A typical bioinformatics analysis pipeline involves a step where reads from DNA or RNA sequencing are aligned to the target species genome; those that do not align are discarded. Given that parasite and pathogen RNA typically represent only a tiny proportion of the total RNA of the host, very deep sequencing is necessary to obtain comprehensive understanding of the pathogen transcriptomes and genetic diversity. This means that by using untargeted sequencing of the host transcriptome it is rarely possible to obtain enough power for pathogen community composition analyses. A targeted amplicon-based high-throughput sequencing, known as metabarcoding, has become an essential tool for monitoring biodiversity [19, 20] and increasingly used for understanding parasite diversity in host tissues and environmental samples [e.g
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