Molecular alterations during larval development of Haemonchus contortus in vitro are under tight post-transcriptional control

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Highlights

Abstract

In this study, we explored the molecular alterations in the developmental switch from the L3 to the exsheathed L3 (xL3) and to the L4 stage of Haemonchus contortus in vitro using an integrated transcriptomic, proteomic and bioinformatic approach. Totals of 9,754 mRNAs, 88 microRNAs (miRNAs) and 1,591 proteins were identified, and 6,686 miRNA-mRNA pairs inferred in all larval stages studied. Approximately 16% of transcripts in the combined transcriptome (representing all three larval stages) were expressed as proteins, and there were positive correlations (r = 0.39–0.44) between mRNA transcription and protein expression in the three distinct developmental stages of the parasite. Of the predicted targets, 1,019 (27.0%) mRNA transcripts were expressed as proteins, and there was a negative correlation (r = −0.60 to −0.50) in the differential mRNA transcription and protein expression between developmental stages upon pairwise comparison. The changes in transcription (mRNA and miRNA) and protein expression from the free-living to the parasitic life cycle phase of H. contortus related to enrichments in biological pathways associated with metabolism (e.g., carbohydrate and lipid degradation, and amino acid metabolism), environmental information processing (e.g., signal transduction, signalling molecules and interactions) and/or genetic information processing (e.g., transcription and translation). Specifically, fatty acid degradation, steroid hormone biosynthesis and the Rap1 signalling pathway were suppressed, whereas transcription, translation and protein processing in the endoplasmic reticulum were upregulated during the transition from the free-living L3 to the parasitic xL3 and L4 stages of the nematode in vitro. Dominant post-transcriptional regulation was inferred to elicit these changes, and particular miRNAs (e.g., hco-miR-34 and hco-miR-252) appear to play roles in stress responses and/or environmental adaptations during developmental transitions of H. contortus. Taken together, these integrated results provide a comprehensive insight into the developmental biology of this important parasite at the molecular level in vitro. The approach applied here to H. contortus can be readily applied to other parasitic nematodes.

Introduction

The free-living nematode Caenorhabditis elegans is one of the best-characterised metazoan organisms (Corsi et al., 2015). Integrated analyses of molecular data sets for this nematode have made it possible to gain deep, global insights into areas such as the developmental and reproductive biology, physiology, biochemistry, genetics and neurobiology of metazoans (Piano et al., 2006, van Assche et al., 2015). By comparison, little is known about these areas for parasitic nematodes. Since C. elegans is relatively closely related to the socioeconomically important parasitic nematodes of the Order Strongylida (Blaxter et al., 1998), the information and data sets available for C. elegans can significantly assist molecular investigations of strongylid nematodes (Gilleard, 2004, Howe et al., 2016a, Howe et al., 2016b). There have been major advances in the genomics and transcriptomics of worms such as Haemonchus contortus (barber’s pole worm), Necator americanus (hookworm) and Oesophagostomum dentatum (nodule worm) (see Laing et al., 2013, Schwarz et al., 2013, Tang et al., 2014, Tyagi et al., 2015), allowing novel insights into the systems biology of strongylid nematodes.

Haemonchus contortus is one of the most important parasitic nematodes of livestock animals worldwide (cf. Gasser and von Samson-Himmelstjerna, 2016). This parasite is transmitted orally from contaminated pasture to the host through a relatively complex, 3 week life cycle (Veglia, 1915): eggs are excreted in host faeces; the L1s develop inside the eggs and then hatch (within 1 day) and moult to the L2 and L3 stages within 1 week; the infective L3s are then ingested by the host, exsheath and, following a histotropic phase, develop through the L4 stage to dioecious adults which both feed on blood from vessels in the stomach wall.

Although some studies have explored changes or differences in mRNAs or proteins in H. contortus (see Campbell et al., 2008, Cantacessi et al., 2010, Laing et al., 2013, Schwarz et al., 2013, Wang et al., 2016), no investigation has yet integrated or compared transcriptomic and proteomic profiles during developmental transitions in this nematode or explored regulatory molecular processes. For instance, while small RNAs, such as microRNAs (miRNAs) (Kim, 2005, Grosshans and Filipowicz, 2008), play key roles in regulating genes via mRNA silencing or chromatin modification (Kim, 2005, Grosshans and Filipowicz, 2008), limited information is available for H. contortus (see Winter et al., 2012, Gillan et al., 2017, Gu et al., 2017), particularly in relation to this parasite’s developmental biology. Here, we explored mRNA, miRNA and protein alterations in H. contortus during the developmental transition from ensheathed L3 to xL3 and then to the L4 stage in vitro, utilising a combination of transcriptomic, proteomic and bioinformatic tools. The goal was to identify key molecular changes associated with this crucial phase of this nematode’s development using a data integration approach.

Section snippets

Procurement of parasite and storage

Haemonchus contortus (Haecon-5 strain) was maintained in experimental sheep (cf. Schwarz et al., 2013), in accordance with the institutional animal ethics guidelines (permit no. 1613878; The University of Melbourne, Australia). In brief, helminth-free Merino sheep (8 weeks of age) were inoculated with 7,000 L3s of H. contortus. Four weeks p.i., faecal samples were collected each day; L3s were produced by incubating faeces at 27 °C for 1 week (cf. Schwarz et al., 2013), purified through two

Transcriptome, miRNAome and proteome of L3s, xL3s and L4s of H. contortus

Three datasets (i.e. transcriptome, miRNAome and proteome) were produced for each L3, xL3 and L4 of H. contortus. For the transcriptome, a total of 9,754 mRNA transcripts were identified amongst >700 million pair-end reads for all three developmental stages (Supplementary Table S1). The numbers of mRNA transcripts detected in the L3, xL3 and L4 stages were 9,492, 9,581 and 9,487, respectively (Supplementary Table S1). Small RNA libraries yielded >400 million reads, with 88 miRNAs identified (

Discussion

Using transcriptomic, miRNA and proteomic datasets, we explored molecular alterations in the developmental switch from L3 to L4 of H. contortus in vitro. Key changes were seen in mRNA, miRNA and protein profiles, and plausible relationships were established between the transcription (mRNA and/or miRNA) and protein expression.

Qualitative and quantitative analyses performed in this study identified substantial molecular changes in the developmental switch of H. contortus. The molecular changes

Acknowledgements

Funding from the National Health and Medical Research Council (NHMRC) of Australia, the Australian Research Council, Melbourne Water Corporation and The University of Melbourne (BIP) is gratefully acknowledged (R.B.G.). Support from the Melbourne Bioinformatics Platform is gratefully acknowledged. P.K.K. holds an NHMRC Early Career Researcher Fellowship (ECRF), and N.D.Y. holds an NHMRC Career Development Fellowship (CDF1).

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