MicroRNAs in metamorphic and non-metamorphic transitions in hemimetabolan insect metamorphosis

Metamorphosis has been a key innovation in insect evolution, which had a decisive influence on the dramatic diversification of this animal class, with around one million presently described species [1]. There are two types of metamorphosis: hemimetaboly, or gradual metamorphosis, and holometaboly or abrupt metamorphosis [2], the latter being evolutionarily the most successful [1]. In all cases, insect metamorphosis is hormonally regulated, and the most important hormones are the ecdysteroid 20-hydroxyecdysone (20E), which determines the successive moults, and the terpenoid juvenile hormone (JH), which represses the expression of adult features. Each hormone exerts its respective action through a cascade of transcription factors that transduce the hormonal signal to the effector genes [3, 4].

Recently, a number of reports have pointed to microRNAs (miRNAs) as important players in the metamorphic transition, in hemimetabolan as well as in holometabolan species [5]. miRNAs are small non-coding RNAs of about 21–22 nucleotides that modulate gene expression at a post-transcriptional level, often in the context of developmental and morphogenetic processes [6–8]. They undergo molecular processing before becoming mature miRNAs [9, 10]. First, miRNAs are transcribed as part of a primary transcript (pri-miRNA), which contains one or more miRNA precursors (pre-miRNAs). In the nucleus, the pri-miRNAs are processed into hairpin-structured pre-miRNAs by the ribonuclease Drosha, and exported to the cytoplasm. Once in the cytoplasm, the pre-miRNAs are cleaved by the ribonuclease Dicer-1 into an imperfectly-paired duplex, whose 5’- and 3’-strands can either give two respective miRNAs or only one. Generally, only one of the strands give rise to a mature miRNA, whereas the other, called miRNA* or the star strand, is degraded [9]. However, given that both strands can give mature miRNAs in some cases [11], the miRNAs are presently labelled as miRNA-5p or miRNA-3p according to the position of the original strand in the pre-miRNA. This is the convention used in the present paper.

One of the more dramatic demonstrations that miRNAs are involved in insect metamorphosis was reported by Gomez-Orte & Belles [12], who silenced dicer-1 expression by RNAi in the last nymphal instar of the cockroach Blattella germanica, and obtained supernumerary nymphs after the following moult, instead of adults [12]. These results showed that Dicer-1 and miRNAs are important factors in the mechanisms regulating metamorphosis. However, which particular miRNAs were involved remained unclear.

B. germanica is a polyneopteran exopterigote insect that shows a gradual morphological transformation during the life cycle, and is a good representative of the less modified hemimetaboly. This, and the fact that it is especially sensitive to gene silencing by RNAi [13], have made B. germanica a favourite hemimetabolan model to study metamorphosis. However, the genome of B. germanica is yet to be sequenced. The first step to approach the study of miRNAs involved in metamorphosis was therefore to establish a baseline miRNA catalogue using high-throughput sequencing [14]. To gain more information about which miRNAs may be important for B. germanica metamorphosis, we constructed two miRNA libraries, one with RNA extracted around the peak of 20E of the penultimate nymphal instar (N5), and the other constructed equivalently but during the last instar (N6). The endocrine context differs between these two libraries with the presence of JH in N5 and its absence in N6. We therefore aimed to identify miRNAs differentially expressed in N5 and N6. Those that were identified with this approach were further studied by establishing the expression pattern in N5 and N6, and by investigating the effect of 20E alone and that of 20E plus JH in their expression. Finally, we selected miR-252-3p, the most interesting miRNA emerging from these studies, to carry out a functional analysis using a specific anti-miRNA.

High throughput sequencing of whole body miRNAs around the peak of 20E in the N5 and N6 libraries of B. germanica gave a total of 61 canonical miRNAs that were present in both libraries, although in different proportions in each. In a previous whole body miRNA library prepared during N6, but representing all eight days of the stage, we obtained a total of 49 canonical miRNAs [14]. Therefore, the number of miRNAs recovered in the present study, using animals collected around the peak of 20E, is higher. This suggests that most miRNAs might be more efficiently expressed around the peak of 20E, and that there was a dilution effect in the former library, which had all the days of the instar represented. Practically all canonical miRNAs reported by Cristino et al. [14] are present (except miR-1-5p and miR-184-5p), and are the most abundant in the present libraries. Some of the miRNAs (miR-8, miR-9a, miR-10, miR-71, miR-252, miR-276, miR-281) are represented by both strands, -3p and -5p, as was also observed in the previous N6 library [14].

The comparative analysis of N5 and N6 libraries allowed the identification of three and 37 miRNAs significantly more expressed in N5 and N6 respectively (Figure 1). Subsequently, 12 of these 40 miRNAs were investigated further by qRT-PCR to establish their expression patterns during N5 and N6. Results indicate that miR-252-3p is the only clear representative of miRNA that is well expressed in N5 and with decreased expression in N6, whereas let-7-5p, miR-100-5p and miR-125-5p are clear representatives of the reverse pattern (Figure 2). Whereas the expression pattern of miR-252-3p had yet to be studied in other insects, those patterns of let-7, miR-100 and miR-125 have been thoroughly examined. In D. melanogaster, these three miRNAs cluster in the same primary transcript, as first revealed by Bashirullah et al. [31] and Sempere et al. [30], and later confirmed in this fly and other insects [5, 32]. Thus, the members of this miRNA cluster are expressed simultaneously, and studies in the holometabolan species, like D. melanogaster and B. mori have shown that expression concentrates in pre-metamorphic stages. In D. melanogaster, expression starts in late third (last) instar larvae, around the peak of 20E that triggers puparium formation, and continues until the imaginal moult [30, 33]. In B. mori, let-7 expression starts in the moult leading to the penultimate larval instar, and continues until the emergence of the adult [34]. In the hemimetabolan B. germanica, the present study shows that expression of let-7, miR-100 and miR-125 concentrates in the last nymphal instar, having an expression burst around the peak of 20E that triggers metamorphosis (Figure 2).

Of note, among the miRNAs selected to study the expression pattern, we included miR-252 and miR-276, which were represented by both the -3p and -5p strands. In principle, the two respective strands originate from the same precursor and both should presumably be expressed at similar rates through parallel patterns. But this was not found to be the case. In the case of miR-252, the -3p strand is expressed at much lower levels than those of the -5p strand, whereas in the case of miR-276, the situation is the reverse; therefore, in each case the pattern of expression of one strand is clearly different to the expression of the other (Figure 2). These observations suggest that the expression is regulated differently in both strands, and that they play distinct miRNA functions [5]. In support of this notion, Liu et al. [35] reported that both strands of miR-276 are differentially expressed in different tissues and stages of B. mori, although miR-276-3p is always the most highly expressed strand.

Among the patterns examined, most of the expression peaks, either in N5 or N6, occur close to a peak of 20E (Figure 2), which suggests cause-effect relationships. This possible hormonal regulation of miRNA expression was therefore tested experimentally. In general, the experiments of hormonal treatments suggested that 20E alone tends to stimulate miRNA expression, whereas JH attenuates, or even inhibits the 20E stimulatory effect (Figure 3). Among those miRNAs that increased in expression after 20E treatment, we found let-7, miR-100 and miR-125. The increase in expression of miR-100-5p after 20E treatment is statistically significant, whereas let-7-5p and miR-125-5p had a tendency to increase (Figure 3A). This is consistent with previous studies in D. melanogaster that revealed that 20E increases the levels of these miRNAs [30, 36] by inducing the expression of the polycistronic primary transcript that contains the let-7, miR-100 and miR-125 precursors [37]. In D. melanogaster, the burst of expression of let-7-5p in prepupae is important to ensure appropriate remodelling of the abdominal neuromusculature during metamorphosis [38]. Moreover, an independent work of Caygill and Johnston [39] showed that during D. melanogaster metamorphosis, the absence of let-7-5p and miR-125-5p results in temporal delays in the terminal cell-cycle exit in the wing, and in the maturation of neuromuscular junctions of imaginal abdominal muscles. The authors focused on the latter process by identifying the abrupt (ab) gene as a let-7-5p target, and by providing evidence that showed that let-7-5p regulates the maturation rate of abdominal neuromuscular junctions during metamorphosis by modulating ab expression [39].

Conversely, the significant stimulatory effect of 20E on miR-1-3p and the tendency of 20E to stimulate miR-34-5p that we observed in B. germanica contrasts with the results obtained in D. melanogaster, where 20E did not affect miR-1-3p and inhibited miR-34-5p [30]. We interpret these differences as reflections of different modes of regulation of these miRNAs in the two model species. The differences are also illustrated by the results obtained after JH treatment of B. germanica, which abolished the stimulatory effects of 20E on practically all miRNAs (Figure 3B), which was also the case for miR-34-5p. This contrasts again with the observations of Sempere et al. [30], which suggested that JH has a stimulatory effect on this miRNA in D. melanogaster.

Possibly the most interesting result of the experiments of hormonal treatments is that of miR-252-3p, which was the only miRNA that was inhibited by 20E alone (Figure 3A). Moreover, JH did not seem to counteract the 20E effect, as compared to other miRNAs (Figure 3B). Inhibition by 20E suggests that the decrease of miR-252-3p in N6, and the increase interrupted by a plateau around the 20E peak in N5 (Figure 2), might be due to the action of 20E. The other strand, classically considered the “mature” strand of miR-252, miR-252-5p, has been found in a number of insects [20], and its expression has been carefully studied in B. mori, where it shows a continuous high expression from the spinning larvae to pupal and adult stages [40]. Conversely, miR-252-3p has never been studied, nor has it been related with insect moulting and metamorphosis. It was for these reasons that we studied it functionally.

Depletion of miR-252-3p levels in the penultimate (N5) nymphal instar caused retarded growth and developmental delays within the instar (Figure 4). At the end, however, the treated insects were able to moult to the last (N6) nymphal instar and to the adult normally. Observed delays in N5 reached as long as 14 days in some cases, and levels of miR-252-3p measured in specimens on N5D13, which were presumably going to moult within the next one or two days, had somewhat recovered compared with levels measured on N5D6, although they were still significantly lower than levels of the miRNA of the controls (Figure 4). The data suggest that miR-252-3p regulates transcripts that are important for growth and development in N5, in the transition to the developmentally important N6 which precedes the imaginal moult. The fact that the treated specimens were finally able to moult to N6 and to the adult stage even with reduced levels of miR-252-3p, suggests that these levels, although still quite low, are above the operative threshold for this miRNA.

MR studied the miRNA expression patterns, the effect of hormonal treatments and the function of miR-252-3p, and drafted the manuscript. AdH carried out the comparison of the miRNA libraries. XB conceived and coordinated the study and wrote the final manuscript. All authors read and approved the final manuscript.