Parasitic nematodes transition between dramatically different free-living and parasitic stages, with correctly timed development and migration crucial to successful completion of their lifecycle. However little is known of the mechanisms controlling these transitions. microRNAs (miRNAs) negatively regulate gene expression post-transcriptionally and regulate development of diverse organisms. Here we used microarrays to determine the expression profile of miRNAs through development and in gut tissue of the pathogenic nematode Haemonchus contortus. Two miRNAs, mir-228 and mir-235, were enriched in infective L3 larvae, an arrested stage analogous to Caenorhabditis elegans dauer larvae. We hypothesized that these miRNAs may suppress development and maintain arrest. Consistent with this, inhibitors of these miRNAs promoted H. contortus development from L3 to L4 stage, while genetic deletion of C. elegans homologous miRNAs reduced dauer arrest. Epistasis studies with C. elegans daf-2 mutants showed that mir-228 and mir-235 synergise with FOXO transcription factor DAF-16 in the insulin signaling pathway. Target prediction suggests that these miRNAs suppress metabolic and transcription factor activity required for development. Our results provide novel insight into the expression and functions of specific miRNAs in regulating nematode development and identify miRNAs and their target genes as potential therapeutic targets to limit parasite survival within the host.

Institute of Biodiversity Animal Health and Comparative Medicine College of Medical Veterinary and Life Sciences University of Glasgow Bearsden Road Glasgow G61 1QH UK

Institute of Biodiversity Animal Health and Comparative Medicine College of Medical Veterinary and Life Sciences University of Glasgow Bearsden Road Glasgow G61 1QH UK Department of Biochemical Sciences Faculty of Pharmacy Charles University Hradec Kralove Czech Republic

Institute of Biodiversity Animal Health and Comparative Medicine College of Medical Veterinary and Life Sciences University of Glasgow Bearsden Road Glasgow G61 1QH UK Wellcome Centre for Integrative Parasitology Institute of Infection Immunity and Inflammation College of Medical Veterinary and Life Sciences University Avenue Glasgow G12 8QQ UK

Institute of Biodiversity Animal Health and Comparative Medicine College of Medical Veterinary and Life Sciences University of Glasgow Bearsden Road Glasgow G61 1QH UK West of Scotland Genetic Services Level 2B Laboratory Medicine Queen Elizabeth University Hospital Govan Road Glasgow G51 4TF UK

Wellcome Sanger Institute Wellcome Genome Campus Hinxton Cambridgeshire CB10 1SA UK Global Station for Zoonosis Control Global Institution for Collaborative Research and Education Hokkaido University N20 W10 Kita ku Sapporo Japan

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$a Marks, Neil D $u Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Bearsden Road, Glasgow, G61 1QH, UK.

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$a Profiling microRNAs through development of the parasitic nematode Haemonchus identifies nematode-specific miRNAs that suppress larval development / $c ND. Marks, AD. Winter, HY. Gu, K. Maitland, V. Gillan, M. Ambroz, A. Martinelli, R. Laing, R. MacLellan, J. Towne, B. Roberts, E. Hanks, E. Devaney, C. Britton,

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$a Parasitic nematodes transition between dramatically different free-living and parasitic stages, with correctly timed development and migration crucial to successful completion of their lifecycle. However little is known of the mechanisms controlling these transitions. microRNAs (miRNAs) negatively regulate gene expression post-transcriptionally and regulate development of diverse organisms. Here we used microarrays to determine the expression profile of miRNAs through development and in gut tissue of the pathogenic nematode Haemonchus contortus. Two miRNAs, mir-228 and mir-235, were enriched in infective L3 larvae, an arrested stage analogous to Caenorhabditis elegans dauer larvae. We hypothesized that these miRNAs may suppress development and maintain arrest. Consistent with this, inhibitors of these miRNAs promoted H. contortus development from L3 to L4 stage, while genetic deletion of C. elegans homologous miRNAs reduced dauer arrest. Epistasis studies with C. elegans daf-2 mutants showed that mir-228 and mir-235 synergise with FOXO transcription factor DAF-16 in the insulin signaling pathway. Target prediction suggests that these miRNAs suppress metabolic and transcription factor activity required for development. Our results provide novel insight into the expression and functions of specific miRNAs in regulating nematode development and identify miRNAs and their target genes as potential therapeutic targets to limit parasite survival within the host.

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