Dysregulation of global gene expression in the whole body in <i>BmAgo3</i> and <i>Siwi</i> KO.

(A) Transposon derepression by BmAgo3 and Siwi KO. RNA-seq libraries were prepared from total RNA extracted from the whole body of two third instar larvae. MAplots were created using the average of triplicate repeats compared to the WT. Each dot indicates a transposon; red dots indicate transposons at M (y-axis) > 1 and A (x-axis) > 0 in BmAgo3 KO females. The axes show: A (x-axis) = (log2(TPM in KO) + log2(TPM in WT))/2. M (y-axis) = log2(TPM in KO)–log2(TPM in WT). Derepressed transposons in BmAgo3 KO females were also depressed in Siwi KO females and in Siwi and BmAgo3 KO males. (B) Differentially expressed genes (DEGs) in BmAgo3 and Siwi KO larvae. MAplots were created using the average of triplicate repeats compared to the WT. Each dot indicates a gene; magenta dots indicate DEGs with variable expression at false discovery rate (FDR) of 56]. (C) Expression patterns of genes showing statistically significant expression changes in any comparisons (magenta dots in (B)). TPMs of all DEGs in BmAgo3 and Siwi KO larvae indicated by magenta dots in (B) were normalized by Z-score and clustered into six major clusters using Heatplus R package. (D) Plots of Z-scores of genes belonging to each of the six clusters. Three plots per gene are shown as they were analyzed in triplicate. The bar graph shows the average ± SD of all points.</p

Phenotypic characterization of <i>BmAgo3</i> and <i>Siwi</i> KO mutants.

(A) Hatching rate in crosses between BmAgo3 or Siwi heterozygous KO mutants. To obtain homozygous KO mutants, heterozygous male and female KO individuals were crossed. The average percentages of hatched, unhatched, and unfertilized eggs from 3 (WT and BmAgo3) or 4 (Siwi) batches are indicated by white, black, and gray bars, respectively. The crossing patterns are indicated in the figure. (B) Developmental delay in BmAgo3 and Siwi KO mutant larvae. Third instar larvae obtained from the parents carrying heterozygous mutations were photographed. Developmentally delayed larvae are indicated by arrowheads. Photographs of all larvae are shown in S2 Fig. (C) Detection of PIWI proteins. Whole cell extracts from the whole body of a day 0 third instar (III0) larva were separated by SDS-PAGE and immunoblotted with anti-BmAgo3, Siwi, and Tubulin antibodies. Developmentally delayed individuals were used as BmAgo3 and Siwi KO. (D) Survival curves of larvae obtained by crosses between BmAgo3 or Siwi heterozygous KO mutants. One hundred newly hatched larvae were reared using an artificial diet and the number of dead larvae was counted every day. (E) Timing of larval wandering in BmAgo3 and Siwi KO mutants. The larval wandering behavior that precedes pupation was delayed in BmAgo3 homozygous mutants by a few days. Pupae of Siwi homozygous mutants were not obtained in this experiment. The number indicates the sample size. (F) Phenotypes of BmAgo3 and Siwi KO mutant pupae. Impaired head and leg morphologies in both homozygous mutants. Female pupae of Siwi KO mutants were not obtained. Scale bars, 2 mm. (G) Abnormalities in internal tissues in BmAgo3 and Siwi KO mutant larvae. Ovaries (OV), testes (TES), and wing discs (WD) were dissected from day 4 fifth instar larvae. The positions connected to the duct are indicated by arrowheads. The margin of the wing disc is indicated by a dotted curve. Scale bars, 1 mm. (H) Developmental defect of gonads in BmAgo3 KO mutant pupae. Day 4 female pupae were dissected and the inside of the abdomen was observed under a microscope. No ovarian eggs were found in BmAgo3 KO mutant pupae. Testes were removed from day 4 male pupae and observed in phosphate-buffered saline under a microscope. Scale bars, 1 mm.</p

Up-regulated genes in <i>BmAgo3</i> KO tissues.

PIWI-interacting RNAs (piRNAs) guide PIWI proteins to target transposons in germline cells, thereby suppressing transposon activity to preserve genome integrity in metazoans’ gonadal tissues. Piwi, one of three Drosophila PIWI proteins, is expressed in the nucleus and suppresses transposon activity by forming heterochromatin in an RNA cleavage-independent manner. Recently, Piwi was reported to control cell metabolism in Drosophila fat body, providing an example of piRNAs acting in non-gonadal somatic tissues. However, mutant flies of the other two PIWI proteins, Aubergine (Aub) and Argonaute3 (Ago3), show no apparent phenotype except for infertility, blurring the importance of the piRNA pathway in non-gonadal somatic tissues. The silkworm, Bombyx mori, possesses two PIWI proteins, Siwi (Aub homolog) and BmAgo3 (Ago3 homolog), whereas B. mori does not have a Piwi homolog. Siwi and BmAgo3 are mainly expressed in gonadal tissues and play a role in repressing transposon activity by cleaving transposon RNA in the cytoplasm. Here, we generated Siwi and BmAgo3 loss-of-function mutants of B. mori and found that they both showed delayed larval growth and failed to become adult moths. They also exhibited defects in wing development and sexual differentiation. Transcriptome analysis revealed that loss of somatic piRNA biogenesis pathways results in abnormal expression of not only transposons but also host genes, presumably causing severe growth defects. Our results highlight the roles of non-gonadal somatic piRNAs in B. mori development.</div

GOs enriched in tissue-specific genes and corresponding expression changes.

GOs enriched in tissue-specific genes and corresponding expression changes.</p

GO analysis of differentially expressed genes in <i>BmAgo3</i> and <i>Siwi</i> KO mutants (Fig 4C cluster).

GO analysis of differentially expressed genes in BmAgo3 and Siwi KO mutants (Fig 4C cluster).</p

Top 10 genes with altered expression in <i>BmAgo3</i> and <i>Siwi</i> KO.

Top 10 genes with altered expression in BmAgo3 and Siwi KO.</p

GO analysis of differentially expressed genes in <i>BmAgo3</i> and <i>Siwi</i> KO third instar larvae.

(A and B) GO analysis for molecular function (A) and biological process (B). Bonferroni-adjusted p values (–log10) and selected gene numbers in each GO shown by gray bars and yellow circles, respectively. (TIF)</p

Detailed phenotypes of <i>BmAgo3</i> and <i>Siwi</i> KO mutant pupae.

(A) Pupal weight of BmAgo3 and Siwi KO mutants. No female pupae were obtained from the Siwi KO mutant. Bars indicate means ± SE. The number indicates the sample size. Asterisks indicate statistical significance in Mann-Whitney test (p BmAgo3 and Siwi KO mutant pupae. Scale bars, 2 mm. (TIF)</p

All delayed larvae possessed homozygous KO alleles as shown by genotyping.

All delayed larvae possessed homozygous KO alleles as shown by genotyping.</p

Phylogenetic tree of <i>B</i>. <i>mori CYP</i> genes.

Phylogenetic tree using amino acid sequences of B. mori Cytochrome P450. Genes in red indicate CYP genes commonly down-regulated in the absence of PIWI protein. Gene models annotated as Cytochrome P450 were aligned using the MUSCLE program [92], and a phylogenetic tree was built using MEGA X software with the maximum likelihood method [93]. (TIF)</p