[
International Worm Meeting,
2021]
Nematoda is a diverse phylum that includes many free-living as well as parasitic species. The model nematode C. elegans has a diverse set of small RNAs and an expanded group of Argonautes, particularly the worm-specific clade Argonautes (WAGOs) and their endogenous siRNAs that are largely amplified by RNA-dependent RNA Polymerases (RdRPs). Our understanding of small RNA types, Argonautes, and pathways in other nematodes is limited. We carried out comparative studies of the small RNA pathways in the Clade III parasitic nematode Ascaris. Ascaris has 10 Argonautes. However, a PIWI Argonaute and piRNAs are absent. Five of the Argonautes are from the WAGO clade. We generated antibodies against all five Ascaris WAGOs as well as AsALG-1 (miRNAs) and AsALG-4 (26G-RNAs) and used them to identify their associated small RNAs in the early embryo, ovary, and testis including discrete developmental stages during spermatogenesis. We found that in general, AsALG-4, AsCSR-1 and AsWAGO-3 small RNAs target mRNAs while AsWAGO-1, AsWAGO-2 and AsNRDE-3 small RNAs target repetitive sequences. Notably, AsNRDE-3 small RNAs change their targets from repetitive sequences to mRNAs during male meiosis at the pachytene stage. RNA-seq data identified a group of genes expressed at pachytene during spermatogenesis which are rapidly degraded at the end of pachytene. Degradation of these mRNAs is associated with AsALG-4 26G-RNAs specifically expressed only in late pachytene and diplotene. The timing and expression of AsCSR-1 and AsALG-4 Argonautes and their small RNAs during spermatogenesis suggests they likely function independently, with AsCSR-1 and its 22-24G-RNAs fine-tuning expression of a broad set of transcripts and AsALG-4 and its 26G-RNAs down-regulating male meiosis-specific mRNAs. Genomic regions with transposons and their derivates are in general enriched for H3K9me3 and are targeted by an expansive set of 22G-RNAs associated with AsWAGO-1, AsWAGO-2 and AsNRDE-3 throughout development. Overall, there is clear conservation in the miRNA, 22G-RNA, and 26G-RNA pathways between the distantly related Ascaris and C. elegans nematodes. However, our data demonstrate the complexity and plasticity of small RNA pathways in a Clade III nematode without PIWI and a piRNA pathway and provide an in depth of analysis of the dynamics of small RNA pathways throughout spermatogenesis.