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. 2023 Jan 9;3(1):47–63. doi: 10.1038/s43587-022-00329-2

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a, PCA of normalized read counts from the small RNA transcriptomes of the isolated germlines. b,c, Volcano plots of small RNA-seq transcriptome data of mated versus unmated germlines. Black dots, significantly differentially-expressed small RNAs (full list is available as Source Data); gray dots, small RNAs with no significant expression changes after mating. Significantly differentially-expressed miRNAs (b) or piRNAs (c) (P_adj ≤ 0.05) are highlighted in red (upregulation) and blue (downregulation). The pie chart (right) summarizes the number of significantly differentially-expressed and unchanged miRNAs (b) or piRNAs (c) in the mated germline compared with the unmated germline. d, Left, over 1,000 piRNAs express at a lower level in the distal germline compared with whole animals (regraphed using published data³⁵). Right, expression levels in mated and unmated germlines (small RNA-seq data from this study) for the same set of piRNAs as on the left. Axis unit, log₁₀(RPM). e, Mating does not change levels of main types of TE (Source Data). Seven biological replicates in total. LTR (left) unmated, 919 ± 200 RPM; mated, 1,044 ± 289 RPM; P = 0.3624; nonLTR (middle) unmated, 1,299 ± 444 RPM; mated, 1,556 ± 593 RPM; P = 0.3761; DNA (right) unmated, 4,360 ± 445 RPM; mated, 4,527 ± 1,240 RPM; P = 0.7432. Expression levels are presented as mean values ± s.d. Two-tailed t-test was used to determine statistical significance. NS, no statistical difference. f, The general piRNA sensor (SX1316) remains silenced in mated worms; 25 worms were checked for each condition. g,h, Mating does not affect the morphology or density of P granules. g, Representative images of mated and unmated Day 3 YY968 worms (P granules labeled by PGL-1::RFP). Mating started on Day 1 for 2 days. h, Quantification of P granule density (blindly scored) of Day 3 (left) and Day 7 (right) worms: Day 3 unmated, 123 ± 20 per 1,000 μm² (n = 55); mated, 123 ± 14 per 1,000 μm² (n = 63); P = 0.9363. Day 7 unmated, 97 ± 18 per 1,000 μm² (n = 16); mated, 95 ± 18 per 1,000 μm² (n = 16); P = 0.7217. Data are presented as mean values ± s.d.; n, number of biologically independent germlines. Two-tailed unpaired t-test was used to determine statistical significance. i, Mating-induced downregulated piRNAs are expressed at a significantly higher level in the unmated germline compared with the other over 1,000 unchanged piRNAs. ***P < 0.0001; two-tailed unpaired t-test was used to determine statistical significance.

Source data