MicroRNA-dependent metamorphosis in hemimetabolan insects

Abstract

How does a juvenile insect transform into an adult? This question, which sums up the wonder of insect metamorphosis, has fascinated mankind since ancient times. Modern physiology has established the endocrine basis regulating these transformations, which mainly depend on two hormone types: ecdysteroids, which promote molts, and juvenile hormones, which repress the transformation into the adult stage. The interplay of these two hormones regulates the genes involved in juvenile and adult programs and the shift from one to the other. microRNAs (miRNAs) are small noncoding RNAs, which participate in many biological processes, and we wondered whether they might be also involved in insect metamorphosis. In insects, Dicer-1 ribonuclease transforms miRNA precursors into mature miRNAs. Thus, using systemic RNA interference (RNAi) to silence the expression of Dicer-1 in the hemimetabolan insect Blattella germanica, we depleted miRNA contents in the last instar nymph. This practically inhibited metamorphosis after the next molt, as the resulting specimens showed nymphoid features and were able to molt again. The experiments show that miRNAs play a key role in hemimetabolan metamorphosis, perhaps regulating genes that are juvenile hormone targets.

Fig. 1.

Blattella germanica Dicer-1 (BgDcr1) and effects of RNAi. (A) Scheme of the organization of BgDcr1, indicating the regions targeted by dsBgDcr1-A and dsBgDcr1-B. (B) Expression profile of BgDcr1 during last instar nymph (n = 3). (C) BgDcr1 mRNA levels in dsBgDcr1-A-treated (RNAi) and dsMock-treated (Co) specimens (n = 3). In B and C, results are expressed as copies of Dicer-1 mRNA per 1,000 copies of actin-5C mRNA. In C, REST© statistical analysis indicates that Dicer-1 is down-regulated by a mean factor of 0.519, and that RNAi sample group is different to Co group (P(H1) = 0.048). (D) Northern blot analysis of miR-1 and let-7 and their corresponding precursors (premiR-1 and prelet-7) in dsBgDcr1-A-treated (RNAi) and dsMock-treated (Co) specimens; the small noncoding RNA U6 was used as a reference; the three lanes of RNAi and respective Co, represent three parallel experiments (the same as in C and E). (E) RT-PCR quantification of miR-1 and let-7 in dsBgDcr1-A-treated (RNAi) and dsMock-treated (Co) specimens (n = 3). In E, results are expressed as copies of miR-1 or let-7 per copy of U6. REST© statistical analysis indicates that miR-1 is down-regulated by a mean factor of 0.450, and let-7 by a mean factor of 0.232. Both miR-1 and let-7 sample groups (RNAi) are different to their respective control (Co) group (P(H1) = 0.0001).

Fig. 2.

Effects of dsBgDcr1-A in Blattella germanica. (A—H) Effects on metamorphosis in the experiments carried out in fifth instar nymphs; dorsal and ventral view of: normal sixth instar nymph (A and B), normal adult (C and D), seventh instar nymphoid (E and F) and adult with the wings not well stretched (G and H), females in all cases. (I—J) BgDcr1 mRNA levels in dsBgDcr1-A-treated (RNAi) and dsMock-treated (Co) specimens (n = 3) in the experiments carried out in fourth (I) and in sixth (J) instar nymphs; results are expressed as copies of Dicer-1 mRNA per 1,000 copies of actin-5C mRNA; both in I and J, REST© statistical analysis indicates that RNAi sample group is not different to Co group (P(H1) = 0.331 and P(H1) = 0.199, for I and J, respectively). (K) Rates of juvenile hormone III (JH) synthesis in control penultimate instar nymphs (N5) and in dsMock-treated (N6c) and dsBgDcr1-A-treated (N6t) last instar nymphs (day 4 of the respective stage in all experiments); results expressed as the mean ± SD (n = 7–9); different letters indicate statistically significant differences (one-way ANOVA, P < 0.0001).

Fig. 3.

Supplementary molt in Blattella germanica treated with dsBgDcr1-A. (A) Adult emerging after the molt of a seventh instar nymphoid, which resulted from Dicer-1 RNAi. (B–E) double cuticle structures in the same specimen, antennae (B), labium (C), maxillae (D), and mandible (E); the new and old cuticle structures are indicated by arrows and arrowheads, respectively; in C, the double structures have been separated by dissection. (F and G) Central portion of a PG from a 9-day-old seventh instar nymphoid (F) and from an untreated 7-day-old sixth instar nymph (G), stained with DAPI. (H) Detail of a PG from a 9-day-old seventh instar nymphoid stained with DAPI-rhodamine phalloidin, showing the polyploid glandular cells densely packed; the structures stained in red are the muscular axis of the gland. (I and J) Central portion of a PG from an untreated 1-day-old adult (I) and from a 9-day-old seventh instar nymphoid (J) stained with TUNEL method; the fluorescent green points (present in I and absent in J) correspond to fragmented DNA. (K) Ecdysteroid contents in untreated 7-day-old last instar nymph (N6), in 10-day-old seventh instar nymphoids (“N7”) and in 7-day-old adults (A); results expressed as the mean ± SD (n = 6–11); different letters indicate statistically significant differences (one-way ANOVA, P < 0.001).