Expression levels of the selected miRNAs in developmental stages of <i>S. litura</i>.

<p>The absolute expression levels of the selected miRNAs with respect to U6snRNA (endogenous control) were analyzed by TaqMan miRNA assays using the total RNA isolated from each stage of the insect, <i>S. litura</i>. The bar graphs represent the expression level of each miRNA at a particular stage in comparison to the expression level in 1^st instar larvae. The statistical significance of the qRT analysis was determined by P value < 0.05 while the error bars represent the SD.</p

Gene ontology classification of the target genes for the selected miRNAs.

<p>Gene ontology (GO) term was assigned to each target gene based on the annotation and were summarized into three main GO categories (biological process, cellular component, molecular function) while only the top 10 sub categories are presented.</p

Analysis of small RNA reads and conservation of homologous miRNAs.

<p>(A) The sequence length distribution of small RNA read tags in Sf21 cells showed majority of the population at 20 nt, a typical length of mature miRNAs followed by the read lengths around 27 nt which could be putative piRNA sequences. (B) Percentage of known miRNAs from <i>Spodoptera frugiperda</i> with homologs from other insects; <i>B. mori, D. melanogaster, T. castenum, A. aegypti, A. gambie</i> and <i>C. elegans</i>.</p

Analysis of novel miRNAs identified in the library.

<p>(A) Similarity between the precursor sequences of a miRNA family identified in the population. (B) (i) The largest miRNA cluster identified from the library using <i>Sf</i> draft genome assembly as a reference. (ii) Similarity in the nucleotide composition of the mature miRNA sequences present in the cluster, especially the seed region.</p

Homology analysis of <i>Spodoptera</i> miRNAs.

<p>(A) (i) Identity in the seed region of the <i>Spodoptera</i> miRNA with respect to its counterpart from other insects. (ii) Sequence conservation of the <i>Sf</i> miRNA, including the seed region over a wide range of insect species. (B) Similarity between the precursor sequences of <i>Sf</i> miRNA with its counterpart from <i>B. mori</i>. The nucleotide differences are only outside the mature region but in the stem loop sequence.</p

Northern validation of selected miRNAs.

<p>Both the known and novel miRNAs were checked by northern analysis for their expression in Sf21 cells using the total RNA isolated from them. All the miRNAs exhibited the expected sizes from the library.</p

Tissue distribution of selected miRNAs in <i>S. litura</i>.

<p>The absolute expression levels of the selected miRNAs with respect to U6snRNA (endogenous control) were analyzed by TaqMan miRNA assays using total RNA isolated from respective tissue parts of the larvae. The bar graphs (A, C) represent the expression level of each miRNA in a particular tissue part but in comparison to that of miR-305-5p. (B) The bar graph represents the relative distribution of expression levels of each miRNA in three segments of the whole gut, i.e., fore gut, mid gut and hind gut. The statistical significance of the qRT analysis was determined by P value < 0.05 while the error bars represent the SD.</p