Transcriptional patterns of sexual dimorphism and in host developmental programs in the model parasitic nematode Heligmosomoides bakeri

Abstract

Background Heligmosomoides bakeri (often mistaken for Heligmosomoides polygyrus) is a promising model for parasitic nematodes with the key advantage of being amenable to study and manipulation within a controlled laboratory environment. While draft genome sequences are available for this worm, which allow for comparative genomic analyses between nematodes, there is a notable lack of information on its gene expression. Methods We generated biologically replicated RNA-seq datasets from samples taken throughout the parasitic life of H. bakeri. RNA from tissue-dwelling and lumen-dwelling worms, collected under a dissection microscope, was sequenced on an Illumina platform. Results We find extensive transcriptional sexual dimorphism throughout the fourth larval and adult stages of this parasite and identify alternative splicing, glycosylation, and ubiquitination as particularly important processes for establishing and/or maintaining sex-specific gene expression in this species. We find sex-linked differences in transcription related to aging and oxidative and osmotic stress responses. We observe a starvation-like signature among transcripts whose expression is consistently upregulated in males, which may reflect a higher energy expenditure by male worms. We detect evidence of increased importance for anaerobic respiration among the adult worms, which coincides with the parasite’s migration into the physiologically hypoxic environment of the intestinal lumen. Furthermore, we hypothesize that oxygen concentration may be an important driver of the worms encysting in the intestinal mucosa as larvae, which not only fully exposes the worms to their host’s immune system but also shapes many of the interactions between the host and parasite. We find stage- and sex-specific variation in the expression of immunomodulatory genes and in anthelmintic targets. Conclusions We examine how different the male and female worms are at the molecular level and describe major developmental events that occur in the worm, which extend our understanding of the interactions between this parasite and its host. In addition to generating new hypotheses for follow-up experiments into the worm’s behavior, physiology, and metabolism, our datasets enable future more in-depth comparisons between nematodes to better define the utility of H. bakeri as a model for parasitic nematodes in general. Graphical Abstract

A single-cell transcriptomics atlas for the parasitic nematodeHeligmosomoides bakeri: Extrapolating model organism information to non-model systems

“…While no obvious batch effects are apparent here from the use of liberase vs pronase during cell dissociation, or between libraries of the same worm sex, a pronounced batch effect is seen between the male and female worm samples (Figure1D). In our previous analysis of bulk RNA-seq of whole worms (Pollo et al, 2023), the male and female worms at 10 days post-infection (the same time point as used here) were found to statistically significantly differently express 70% of their transcripts. Part of that is due to the different gametes each sex produces and differences in gene expression related to reproduction.…”

“…The Ascaris suum ortholog of this gene was found to be highly expressed in bulk RNA-seq analysis of dissected intestine (Rosa, Jasmer & Mitreva, 2014). Of note, whole worm expression of this transcript in H. bakeri puts it among the top 51% of transcripts in worms of the same age as used here (Pollo et al, 2023).…”

Genome assembly variation and its implications for gene discovery in nematode species

“…Protein coding genes (gene models) in the generated assemblies were predicted using Braker 3 (Stanke et al, 2006, 2008; Hoff et al, 2016, 2019; Brůna et al, 2021) installed using the Singularity container (available at https://hub.docker.com/r/teambraker/braker3). For H. contortus and H. bakeri , we used short-read RNA-seq data from the Sequence Read Archive (Leinonen, Sugawara & Shumway, 2011) as external evidence for the gene prediction (BioProjects: PRJEB506 (Laing et al, 2013) and PRJNA750155 (Pollo et al, 2023) respectively). The RNA-seq data was first aligned using the STAR v.2.7.10a aligner (Dobin et al, 2013) and the generated alignments, in bam-format, used to produce a training set for AUGUSTUS, using GeneMark-ET (Hoff et al ., 2016; Li et al ., 2009; Barnett et al ., 2011) and DIAMOND (Buchfink, Xie & Huson, 2015), to filter redundant training gene structures.…”

“…Protein coding genes (gene models) in the generated assemblies were predicted using Braker 3 (Stanke et al, 2006(Stanke et al, , 2008Hoff et al, 2016Hoff et al, , 2019Brůna et al, 2021) installed using the Singularity container (available at https://hub.docker.com/r/teambraker/braker3). For H. contortus and H. bakeri, we used short-read RNA-seq data from the Sequence Read Archive (Leinonen, Sugawara & Shumway, 2011) as external evidence for the gene prediction (BioProjects: PRJEB506 (Laing et al, 2013) and PRJNA750155 (Pollo et al, 2023)…”

“…Currently, the five autosomes and one X chromosome of the H. contortus genome is assembled into seven scaffolds, with a total length of ∼283Mb. Finally, H. bakeri is an intestinal parasite of mice that serves as a model for studying host-parasite interactions and testing anthelmintic efficacy (Pollo et al ., 2023; Reynolds et al ., 2012; Maizels et al ., 2012). Please note, that the Chow assembly paper described the species sequenced as Heligmosomoides polygyrus , a long-standing miss-classification of the study species which has been recently corrected through genomic comparisons (Stevens et al, 2023).…”