In silico hybridization enables transcriptomic illumination of the nature and evolution of Myxozoa

Abstract

BackgroundThe Myxozoa, a group of oligocellular, obligate endoparasites, has long been poorly understood in an evolutionary context. Recent genome-level sequencing techniques such as RNA-seq have generated large amounts of myxozoan sequence data, providing valuable insight into their evolutionary history. However, sequences from host tissue contamination are present in next-generation sequencing reactions of myxozoan tissue, and differentiating between the two has been inadequately addressed. In order to shed light on the genetic underpinnings of myxozoan biology, assembled contigs generated from these studies that derived from the myxozoan must be decoupled from transcripts derived from host tissue and other contamination. This study describes a pipeline for categorization of transcripts asmyxozoan based on similarity searching with known host and parasite sequences, explores the extent to which host contamination is present in previously existing myxozoan datasets, and implements this pipeline on a newly sequenced transcriptome of Myxobolus pendula, a parasite of the common creek chub gill arch.MethodsThe insilico hybridization pipeline uses iterative BLAST searching and database-driven e-value comparison to categorize transcripts as deriving from host, parasite, or other contamination. Functional genetic analysis of M. pendula was conducted using further BLAST searching, Hidden Markov Modeling, and sequence alignment and phylogenetic reconstruction.ResultsThree RNA libraries of encysted M. pendula plasmodia were sequenced and subjected to the method. Nearly half of the final set of contiguous assembly sequences (47.3 %) was identified as putative myxozoan transcripts. Putative contamination was also identified in at least 1/3rd of previously published myxozoan transcripts. The set of M. pendula transcripts was mined for a range of biologically insightful genes, including taxonomically restricted nematocyst structural proteins and nematocyst proteins identified through mass tandem spectrometry of other cnidarians. Several novel findings emerged, including a fourth myxozoan minicollagen gene, putative myxozoan toxin proteins,and extracellular matrix glycoproteins.ConclusionsThis study serves as a model for the handling of next-generation myxozoan sequence. The need for careful categorization was demonstrated in both previous and new sets of myxozoan sequences. The final set of confidently assigned myxozoan transcripts can be mined for any biologically relevant gene or gene family without spurious misidentification of host contamination as a myxozoan homolog. As exemplified by M. pendula, the repertoire of myxozoan polar capsules may be more complex than previously thought, with an additional minicollagen homolog and putative expression of toxin proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2039-6) contains supplementary material, which is available to authorized users.

An evolutionary comparative analysis of the medusozoan (Cnidaria) exoskeleton

“…Medusozoa encompasses the classes Staurozoa, Cubozoa, Scyphozoa, and Hydrozoa, whose phylogenetic relationships have been explored using morphology, life cycles, and nuclear and mitochondrial molecular markers (Marques & Collins, ; Collins et al ., ; Van Iten et al ., , ). Recently, some molecular phylogenetic analyses indicated that Myxozoa is a cnidarian group (Chang et al ., ; Foox & Siddall, ; Foox et al ., ).…”

Comparative transcriptomics and host-specific parasite gene expression profiles inform on drivers of proliferative kidney disease

“…Although low, our Busco scores were comparable to previously reported CEG scores for myxosporeans 15 . It is well known that myxozoan genomes and transcripts are AT-rich (> 60% AT), and AT content can be used to identify transcripts of parasite versus host origin 15,18,23,32 . The increased representation of non-AT-rich contigs in the intersect may, therefore, reflect the presence of non-parasite sequences (see later discussion) but it may also reflect the presence of myxozoan transcripts that encode proteins rich in amino acids using GC rich codons, such as minicollagens 33 .…”

“…Non-parasite (< 60% AT) sequences were evident in the blob plots of both the fish kidneyderived and spore sac-derived transcriptomes. As highlighted by Foox and colleagues 23 , this illustrates the inadequacy of a single step depletion of host sequences by BLAST analysis against fish sequence resources. Notably the second peak in the blob plot of the spore sacderived transcriptome was not substantially reduced following filtering using a partial T. bryosalmonae genome assembly (H. Hartikainen pers comm).…”

“…Although dual RNA-Seq approaches and selective enrichment of parasite stages from host tissues can be attempted, most myxozoan genomes and transcriptomes still carry host contamination 23 . In situ expression experiments are hindered by low parasite to host tissue ratios in infected tissues and the subsequent very low coverage of parasite transcripts.…”

“…Indeed, It is likely that non-parasite sequences are universally present in existing myxosporean assemblies due to the presence of host cell/tissue contaminants 23 . We propose that in the absence of clean and complete host and parasite genomes to filter against, the intersect transcriptome approach can identify a key set of contigs for targeted analysis as shown in this study.…”