Deep sequencing of organ- and stage-specific microRNAs in the evolutionarily basal insect Blattella germanica (L.) (Dictyoptera, Blattellidae)

Abstract

Background: microRNAs (miRNAs) have been reported as key regulators at post-transcriptional level in eukaryotic cells. In insects, most of the studies have focused in holometabolans while only recently two hemimetabolans (Locusta migratoria and Acyrthosiphon pisum) have had their miRNAs identified. Therefore, the study of the miRNAs of the evolutionarily basal hemimetabolan Blattella germanica may provide valuable insights on the structural and functional evolution of miRNAs.

Methodology/principal findings: Small RNA libraries of the cockroach B. germanica were built from the whole body of the last instar nymph, and the adult ovaries. The high throughput Solexa sequencing resulted in approximately 11 and 8 million reads for the whole-body and ovaries, respectively. Bioinformatic analyses identified 38 known miRNAs as well as 11 known miRNA*s. We also found 70 miRNA candidates conserved in other insects and 170 candidates specific to B. germanica. The positive correlation between Solexa data and real-time quantitative PCR showed that number of reads can be used as a quantitative approach. Five novel miRNA precursors were identified and validated by PCR and sequencing. Known miRNAs and novel candidates were also validated by decreasing levels of their expression in dicer-1 RNAi knockdown individuals. The comparison of the two libraries indicates that whole-body nymph contain more known miRNAs than ovaries, whereas the adult ovaries are enriched with novel miRNA candidates.

Conclusions/significance: Our study has identified many known miRNAs and novel miRNA candidates in the basal hemimetabolan insect B. germanica, and most of the specific sequences were found in ovaries. Deep sequencing data reflect miRNA abundance and dicer-1 RNAi assay is shown to be a reliable method for validation of novel miRNAs.

Figure 1. Length distribution of sequences.

Length distribution of sequence reads obtained in the WB-6 and in the Ov-A libraries of Blattella germanica. Values are based on the raw data from Solexa sequencing.

Figure 2. The most abundant known miRNAs identified in Blattella germanica.

Known miRNAs with more than 100 reads found in the WB-6 and Ov-A Blattella germanica libraries.

Figure 3. Quantitative value of Solexa reads.

The number of reads of Solexa sequencing and fold change of qPCR analysis (miRNA copies per 1000 copies of U6) are correlated. The two Blattella germanica miRNA libraries, WB-6 and Ov-A are indicated. Squares are known miRNA with more than 100 Solexa reads. Diamonds are miRNAs with less than 100 reads. Solid lines are trend line for correlation with 100-more Solexa reads. Dashed lines are trend line for all miRNAs tested.

Figure 4. miRNA candidates conserved in other insect species.

Conserved miRNA candidates found in the WB-6 and Ov-A Blattella germanica libraries with more than 500 reads. They have been reported in small RNA databases from other insects such as Locusta migratoria (Lmi), Bombyx mori (Bmo), Acyrthosiphon pisum (Api) and Culex quinquefasciatus (Cqu) (see also Table S5).

Figure 5. miRNA precursors validated by PCR amplification and sequencing.

(A) The most abundant miRNA, bge-miR-cand1, in the cockroach ovaries is also conserved in the aphid A. pisum. (B) Secondary structure of miR-cand1 hairpin in both cockroach and aphid. (C) Other four miRNA precursors showing the location of novel miRNAs in B. germanica.

Figure 6. Effect of dicer-1 knockdown on miRNA expression.

Effect of dicer-1 knockdown in the expression of known miRNAs (A) and miRNA candidates (B) of Blattella germanica. Data showed are the values obtained in dicer-1 knockdown animals normalized against the control (reference value = 1). Differences with respect to control were statistically significant in all cases (p<0.05), according to the REST© software tool (see the Table S6). The error-bars show standard deviation for three biological replicates.

Figure 7. Comparison of WB-6 (A) and Ov-A (B) libraries.

A–B: Distribution of small RNAs in the WB-6 (A) and Ov-A (B) Blattella germanica libraries. “Other small RNAs” states for rRNAs, tRNAs, ncRNAs, piRNAs-like and Blattabacterium sp. sequences. (C) The Shannon diversity index (H′) estimated from WB-6 (A) and Ov-A (B) libraries considering the known miRNAs, conserved candidates and specific candidates.